Vdoc - Pub - Brazilian Medicinal Plants

Brazilian Medicinal Plants
Natural Products Chemistry of Global Plants

Editor: Raymond Cooper
This unique book series focuses on the natural products chemistry of botanical medicines from
different countries such as Sri Lanka, Cambodia, Brazil, China, Africa, Borneo, Thailand and Silk
Road countries. These fascinating volumes are written by experts from their respective countries.
The series will focus on the pharmacognosy, covering recognized areas rich in folklore as well as
botanical medicinal uses as a platform to present the natural products and organic chemistry. Where
possible, the authors will link these molecules to pharmacological modes of action. The series
intends to trace a route through history from ancient civilizations to the modern day showing the
importance to man of natural products in medicines, foods and a variety of other ways.
Recent Titles in this Series:
Traditional Herbal Remedies of Sri Lanka

Viduranga Y. Waisundara
Medicinal Plants of Bangladesh and West Bengal

Botany, Natural Products, and Ethnopharmacology
Christophe Wiart

Luzia Modolo and Mary Ann Foglio
https://www.crcpress.com/Natural-Products-Chemistry-of-Global-Plants/book-series/CRCNPCGP
Edited by
Luzia Valentina Modolo
Mary Ann Foglio
Cover Image: © 2019 Dr. João Renato Stehmann. Used with Permission.
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Library of Congress Cataloging-in-Publication Data
Names: Modolo, Luzia V., author.

Title: Brazilian medicinal plants / Luzia V. Modolo, Mary Ann Foglio.
Description: Boca Raton, Florida : CRC Press, 2019. | Series: Natural products chemistry of global plants |
Includes bibliographical references and index. | Summary: “The vast and exciting Brazilian flora biodiversity
is still underexplored. Several research groups are devoted to the study of the chemical structure richness
found in the different Biomes. This volume presents a comprehensive account of the research collated on
natural products produced from Brazilian medicinal plants and focuses on various aspects of the field. The
authors describe the key natural products and their extracts with emphasis upon sources, an appreciation
of these complex molecules and applications in science. Many of the extracts are today associated
with important drugs, nutrition products, beverages, perfumes, cosmetics and pigments, and these are
highlighted”-- Provided by publisher.
Identifiers: LCCN 2019031670 | ISBN 9781138093751 (hardback) | ISBN 9781315106427 (ebook)
Subjects: LCSH: Medicinal plants--Brazil. | Medicinal plants--Brazil--Identification.
Classification: LCC QK99.B6 M63 2019 | DDC 581.6/340981--dc23
LC record available at https://lccn.loc.gov/2019031670
Visit the Taylor & Francis Web site at

http://www.taylorandfrancis.com
and the CRC Press Web site at

http://www.crcpress.com
Contents
Introduction to Book Series..............................................................................................................vii
Preface...............................................................................................................................................ix
Editors................................................................................................................................................xi
Contributors.................................................................................................................................... xiii
Chapter 1 Unraveling the Complexities of Brazilian Regulations for

Medicinal Plants and Herbal Medicinal Products........................................................1
Ana Cecília Bezerra Carvalho, Melina Cossote Kumoto,
and João Paulo Silvério Perfeito
Chapter 2 Physico-Chemical Methods for the Quality Control of Medicinal Plants,

Plant Derivatives and Phytomedicines in Brazil......................................................... 13
Paula Carolina Pires Bueno and Alberto José Cavalheiro
Chapter 3 The Widening Panorama of Natural Products Chemistry in Brazil........................... 33

Maria Fátima das Graças Fernandes da Silva, João Batista Fernandes,
Moacir Rossi Forim, Michelli Massaroli da Silva,
and Jéssica Cristina Amaral
Chapter 4 Molecular Biology Tools to Boost the Production of Natural Products:

Potential Applications for Brazilian Medicinal Plants................................................ 71
Luzia Valentina Modolo, Samuel Chaves-Silva, Thamara Ferreira da Silva,
and Cristiane Jovelina da-Silva
Chapter 5 Diversity of Endophytes and Biotechnological Potential............................................ 91

Daiani Cristina Savi and Chirlei Glienke
Chapter 6 Environmental Factors Impacting Bioactive Metabolite Accumulation

in Brazilian Medicinal Plants.................................................................................... 109
Camila Fernanda de Oliveira Junkes, Franciele Antonia Neis,
Fernanda de Costa, Anna Carolina Alves Yendo,
and Arthur Germano Fett-Neto
Chapter 7 Brazilian Bryophytes and Pteridophytes as Rich Sources of

Medicinal Compounds.............................................................................................. 135
Adaíses Simone Maciel-Silva and Lucas Vieira Lima
Chapter 8 Chemical and Functional Properties of Amazonian Fruits...................................... 173

Elaine Pessoa, Josilene Lima Serra, Hervé Rogez, and Sylvain Darnet
v
vi Contents
Chapter 9 Plant Species from the Atlantic Forest Biome and

Their Bioactive Constituents..................................................................................... 217
Rebeca Previate Medina, Carolina Rabal Biasetto,
Lidiane Gaspareto Felippe, Lilian Cherubin Correia,
Marília Valli, Afif Felix Monteiro, Alberto José Cavalheiro,
Ângela Regina Araújo, Ian Castro-Gamboa, Maysa Furlan,
Vanderlan da Silva Bolzani, and Dulce Helena Siqueira Silva
Chapter 10 Plants from the Caatinga Biome with Medicinal Properties.................................... 257
Maria da Conceição Ferreira de Oliveira, Mary Anne Sousa Lima,
Francisco Geraldo Barbosa, Jair Mafezoli, Mary Anne Medeiros Bandeira,
and Wellyda Rocha Aguiar
Chapter 11 Natural Products Structures and Analysis of the Cerrado Flora in Goiás................ 281
Lucilia Kato, Vanessa Gisele Pasqualotto Severino,
Aristônio Magalhães Teles, Aline Pereira Moraes, Vinicius Galvão Wakui,
Núbia Alves Mariano Teixeira Pires Gomides, Rita de Cássia Lemos Lima,
and Cecilia Maria Alves de Oliveira
Chapter 12 Total Synthesis of Some Important Natural Products from

Brazilian Flora.......................................................................................................... 305
Leonardo da Silva Neto, Breno Germano de Freitas Oliveira,
Wellington Alves de Barros, Rosemeire Brondi Alves,
Adão Aparecido Sabino, and Ângelo de Fátima
Index............................................................................................................................................... 329
Introduction to Book Series – Natural
Products Chemistry of Global Plants
CRC Press is publishing a new book series on the Natural Products Chemistry of Global Plants. This
new series will focus on pharmacognosy, covering recognized areas rich in folklore and botanical
medicinal uses as a platform to present the natural products and organic chemistry and, where possi-
ble, link these molecules to pharmacological modes of action. This book series on the botanical medi-
cines from different countries include, but are not limited to, Brazil, Bangladesh, Borneo, Cambodia,
Cameroon, Iran, Madagascar, The Silk Road, Sri Lanka, Thailand, Turkey, Uganda, Vietnam, Western
Cape (South Africa) and Yunnan Province (China), written by experts from each country. The inten-
tion is to provide a platform to bring forward information from under-represented regions.
Medicinal plants are an important part of human history, culture and tradition. Plants have been
used for medicinal purposes for thousands of years. Anecdotal and traditional wisdom concerning
the use of botanical compounds is documented in the rich histories of traditional medicines. Many
medicinal plants, spices and perfumes changed the world through their impact on civilization, trade
and conquest. Folk medicine is commonly characterized by the application of simple indigenous
remedies. People who use traditional remedies may not understand in our terms the scientific ratio-
nale for why they work but know from personal experience that some plants can be highly effective.
This series provides rich sources of information from each region. An intention of the series of
books is to trace a route through history from ancient civilizations to the modern day showing the
important value to humankind of natural products in medicines, in foods and in many other ways.
Many of the extracts are today associated with important drugs, nutrition products, beverages, per-
fumes, cosmetics and pigments, which will be highlighted.
The series is written both for chemistry students who are at university level and for scholars
wishing to broaden their knowledge in pharmacognosy. Through examples of the chosen botanicals,
herbs and plants, the series will describe the key natural products and their extracts with emphasis
upon sources, an appreciation of these complex molecules and applications in science.
In this series the chemistry and structure of many substances from each region are presented
and explored. Often books describing folklore medicine do not describe the rich chemistry or the
complexity of the natural products and their respective biosynthetic building blocks. By drawing
on the chemistry of these functional groups to show how they influence the chemical behavior of
the building blocks, which make up large and complex natural products, the story becomes more
fascinating. Where possible it will be advantageous to describe the pharmacological nature of these
natural products.
In this book on medicinal plants in Brazil, the great diversity of the Brazilian flora is documented
and presented for the unique biological and chemical diversity. The book emphasizes the biodiver-
sity and the critical importance of the fragile ecosystem and can contribute to a better understand-
ing of Brazilian diversity, as well as the effect of the anthropological and environmental conditions
in this ecosystem.
R. Cooper, Ph.D., Editor-in-chief

Dept. Applied Biology & Chemical Technology
The Hong Kong Polytechnic University
Hong Kong
rcooperphd@aol.com
vii
Preface
According to the Brazilian Institute for Geography and Statistics (IBGE) the country is divided
into six biomes: Amazon, Pantanal, Cerrado, Caatinga, Atlantic Forest and Pampa. The different
biomes reveal an impressive variety of plants, microorganisms and animals. Indeed, Brazil has an
outstanding vast biodiversity. The country has approximately 20% of the world’s known plant spe-
cies. With such an immense territory and complex ecosystems, many other species remain yet to be
discovered. Overall, Brazil is in a privileged situation in terms of natural products, with biodiversity
being one of the major bases for the progressive discovery of new compounds with great potential
for both biological and non-biological applications, such as biofuels, textiles and others.
When did this all begin? Some will say with Adam and Eve, others with the native Indian popu-
lations, who indeed deserve credit for all the knowledge that they have passed on from one genera-
tion to the next, giving rise to extremely important data. The country’s rich cultural and biological
diversity is a priceless treasure to be wisely used.
Many scientists have given their share of time to explore and understand the wonders that the
ecosystems have to offer. Among the many important contributions throughout time are those of
Otto Gottlieb, who was an enthusiastic pioneer on the study of natural products, together with
Ernest Wenkert, Jayr de Paiva Campello and Giuseppe Cilento, who paved the way for many others.
The awareness of the importance of studying these natural resources permitted the expansion of the
Brazilian scientific community triggering the work by Walter B. Mors, Mauro Taveira Magalhães,
Carl Djerassi, Frederick Sanger, Margaret Joan Sanger, Raimundo Braz-Filho, Alaíde Braga de
Oliveira, Anita Marsaioli, Francisco José de Abreu Matos, Maria Iracema Lacerda Machado
Madruga, David W. Cochran, Hugo E. Gottlieb, Edward W. Hagaman, Arnaldo Felisberto Imbiriba
da Rocha, Angelo da Cunha Pinto, Nidia Franca Roque, Sebastião Ferreira Fonseca, José Rego de
Sousa, Afrânio Aragão Craveiro, Tanus Jorge Nagem and so many others. Now the new science
generations have the obligation to address aspects that will guarantee equilibrium of wildlife with
mankind for all to benefit from these ecosystems.1
Brazilian Medicinal Plants gives a snapshot of the marvelous research work being performed by
Brazilian scientists. Among the chapters represented herein, we cover regulation issues, different
biome treasures and microorganisms of medicinal plants found in the Brazilian biomes. Some concepts
behind forward and reverse genetics and potential use of genome editing technologies on Brazilian
medicinal plants and an integrated view of plant-environment interaction strategies to improve target
metabolite yields are presented. Also, aspects of physicochemical methods for the quality control of
medicinal plants, plant derivatives and herbal medicines in Brazil and some approaches used for the
synthesis of natural products of Brazilian medicinal plants origin are discussed.
Brazilian flora is undoubtedly one of the most plentiful sources of inspiration for the devel-
opment of new drugs. However, various natural products that have therapeutic properties are not
always available in sufficient amounts for sustainable use and/or for the development of new deriva-
tives by modifying such substances. Moreover, obtaining a renewable supply of active compounds
from biological sources may be problematic, especially with respect to perennial plant species. The
complexity of many natural products can also limit the scope of chemical modifications necessary
to optimize therapeutic use. Despite these barriers, the total synthesis of various bioactive natural
products and analogs has proven that organic synthesis is a powerful tool for increasing the avail-
ability of valuable natural products of limited supply or with very complex structures.
Plant natural products evolved as key elements in adaptive responses to stress, both biotic and
abiotic, in close connection with the sessile habit. During this process, an intricate relationship
1 Angelo C. Pinto; Fernando de C. da Silva; Vitor F. Ferreira; Otto R. Gottlieb e as conexões com o Brasil de Ernest
Wenkert; Quím. Nova vol.35 no.11 São Paulo 2012
ix
x Preface
between dedicated metabolic pathways and structural features of plants was established, affording
high efficiency in metabolic competence and generating great metabolite diversity. This metabolic
array has proven a major reservoir of bioactive compounds for treating and preventing human dis-
eases. Considering the defense-related role of natural products in plants and the signaling pathways
that trigger their biosynthesis upon stress exposure, it may be advantageous to use environmental
signals or their transduction elements for enriching biomass with pharmacologically interesting
metabolites. Among the environmental factors that promote natural product accumulation when
applied at moderate intensity are the following: heat, cold, drought, herbivory, pathogens, UV radia-
tion, osmotic stress and heavy metals.
Some specific regions are described. The Caatinga biome is a semi-arid ecosystem found exclu-
sively in Brazil, referred to as “a mosaic of scrubs and patches of seasonally dry forest.” This unique
biome occupies a large geographic area of the country, spread from the state of Ceará (CE) to the
north of the state of Minas Gerais (MG). Despite being one of the largest Brazilian biomes, the
scientific knowledge of this biodiversity is poorly understood.
Also, a review of the natural products from the Goiás Cerrado provides the importance of ethno-
botany and Brazilian traditional medicine, used as a strategy to focus on isolation, structural eluci-
dation and biological evaluation and use of modern mass spectrometry (MS) techniques to analyze
plants from this region.
The biogeographic region of the Atlantic Forest covers a part of the Brazilian coast and parts of
Paraguay, Argentina and Uruguay. This biome is considered an important hotspot and a priority for
biodiversity conservation. The huge plant diversity accounts for 5% of the world’s flora. Although
only small fragments of the Atlantic Forest remain due to intense deforestation across the past five
centuries, this area is biologically and chemically very rich.
Among challenges to consider for the next 20 years are food security and nutrition issues
worldwide. Insights into the sustainable use of biodiversity are crucial factors to provide new food
products from unused fruits and vegetables with high nutritional gain. Four Amazon fruits from
trees, Spondias mombin, Myrciaria dubia, Genipa americana and the well-known Brazilian nut
(Bertholletia excelsa) are presented. Also palm tree fruits from Astrocaryum vulgare, Mauritia
flexuosa, Bactris gasipaes and the well-known açai (Euterpe oleracea) are described. These fruits
and nuts are the most abundant sources of bioactive compounds with antioxidant activity as a result
of phenolic compounds, carotenoids, tocopherols, vitamin C, unsaturated fatty acids (UFA), terpe-
noids and steroids. Characteristic compounds, present in a higher amount, are a highlight for some
fruits, such as vitamin C in camu-camu fruit, carotenoids in the peach palm and tucuma fruits,
iridoids in genipap, and selenium and UFA in the Brazil nut. The synergistic effect of these com-
pounds leads to many health benefits.
Many research groups are working on different aspects of natural products in Brazil. This
volume presents an important account of ongoing research on natural products produced from
Brazilian medicinal plants. The vast and exciting Brazilian flora biodiversity is still underexplored,
yet several research groups are devoted to the study of the chemical structural richness found in the
different biomes. The authors described the key natural products and their extracts with emphasis
upon sources, an appreciation of these complex molecules and applications in science. Many of the
extracts are today associated with important drugs, nutrition products, beverages, perfumes, cos-
metics and pigments, and these are highlighted. Specifically, Brazilian biodiversity, its flora, its peo-
ple and its research are described. With an emphasis on the increasing global interests in botanical
drugs, this volume may help the international natural product communities to better understand the
herbal resources in Brazil and the regulations and legislation to work with native plants. Recent
achievements on plant research of regionally different groups are presented to give the reader the
tip of the iceberg, recognizing that much more research and funding is required.
Mary Ann Foglio

Brazil
Editors
Luzia Valentina Modolo received her PhD degree in Functional and
Molecular Biology in 2004 from the State University of Campinas
(SP, Brazil). She is a faculty member of the Department of Botany at the
Federal University of Minas Gerais (MG, Brazil) and currently holds
Associate Professor Position. Dr. Modolo is the coordinator of the Network
for the Development of Novel Urease Inhibitors (www.redniu.org) and her
research interests include plant nutrition and secondary metabolism and sig-
nalling processes in plant tissues triggered by environmental stress.
ary Ann Foglio is Senior Researcher at the Faculty of Pharmaceutical

M
Science (FPS) at the University of Campinas (UNICAMP). Her early training
was in chemistry with a Bachelor’s degree in Chemistry (1982), master’s (1987)
and PhD (1996) at UNICAMP in Organic Chemistry. From 1987–2015 she
was a researcher at the Multidisciplinary Chemistry, Biology and Agricultural
Research Center. Thereafter she took a position as full professor at FPS School
of Pharmacy. Her work focuses on translational research of natural products.
Over the years her work has involved standardization of plant inputs and the
development of products that meet safety, efficacy and reproducibility criteria,
resulting in fifteen patent deposits. She supervises undergraduate and graduate students, and post-docs
on bioactive natural product research. Her lab is sponsored by CNPq, CAPES, FINEP, and FAPESP
governmental agencies.
xi
Contributors
Wellyda Rocha Aguiar Vanderlan da Silva Bolzani
Medicinal Herb Garden “Francisco José Universidade Estadual Paulista
de Abreu Matos” Núcleo de Bioensaios, Biossíntese e
Federal University of Ceará, Campus do Pici Ecofisiologia de Produtos
Fortaleza, Ceará, Brazil Naturais – NuBBE
Araraquara, São Paulo, Brazil
Rosemeire Brondi Alves
Grupo de Estudos em Química Orgânica Paula Carolina Pires Bueno
e Biológica (GEQOB) Instituto de Química
Departamento de Química, Instituto Universidade Estadual Paulista (UNESP)
de Ciências Exatas Araraquara, São Paulo, Brazil
Universidade Federal de Minas Gerais
(UFMG) Ana Cecília Bezerra Carvalho
Belo Horizonte, Minas Gerais, Brazil General Office of Drugs and Biological
Products
Jéssica Cristina Amaral Brazilian Health Regulatory
Chemistry Department Agency – ANVISA
São Carlos Federal University Brasília, DF, Brazil
São Carlos, São Paulo, Brazil
Ian Castro-Gamboa
Ângela Regina Araújo Universidade Estadual Paulista
Universidade Estadual Paulista Núcleo de Bioensaios, Biossíntese
Núcleo de Bioensaios, Biossíntese e Ecofisiologia de Produtos Naturais – NuBBE
e Ecofisiologia de Produtos Araraquara, São Paulo, Brazil
Naturais – NuBBE
Araraquara, São Paulo, Brazil Alberto José Cavalheiro
Instituto de Química
Mary Anne Medeiros Bandeira Universidade Estadual Paulista (UNESP)
Medicinal Herb Garden “Francisco José Araraquara, São Paulo, Brazil
de Abreu Matos”
Federal University of Ceará Samuel Chaves-Silva
Campus do Pici Departamento de Botânica
Fortaleza, Ceará, Brazil Instituto de Ciências Biológicas
Universidade Federal de Minas Gerais
Francisco Geraldo Barbosa Belo Horizonte, Minas Gerais, Brazil
Department of Organic and Inorganic
Chemistry Lilian Cherubin Correia
Federal University of Ceará Universidade Estadual Paulista
Campus do Pici Núcleo de Bioensaios, Biossíntese
Fortaleza, Ceará, Brazil e Ecofisiologia de Produtos
Naturais – NuBBE
Carolina Rabal Biasetto Araraquara, São Paulo, Brazil
Universidade Estadual Paulista
Núcleo de Bioensaios, Biossíntese Maria Fátima das Graças Fernandes da Silva
e Ecofisiologia de Produtos Chemistry Department
Naturais – NuBBE São Carlos Federal University
Araraquara, São Paulo, Brazil São Carlos, São Paulo, Brazil
xiii
xiv Contributors
Michelli Massaroli da Silva Lidiane Gaspareto Felippe

Chemistry Department, São Carlos Federal Universidade Estadual Paulista
University Núcleo de Bioensaios, Biossíntese e
São Carlos, São Paulo, Brazil Ecofisiologia de Produtos Naturais – NuBBE
Thamara Ferreira da Silva
Departamento de Botânica João Batista Fernandes
Instituto de Ciências Biológicas Chemistry Department
Universidade Federal de Minas Gerais São Carlos Federal University
Belo Horizonte, Minas Gerais, Brazil São Carlos, São Paulo, Brazil
Sylvain Darnet Arthur Germano Fett-Neto

Centre for Valorization of Amazonian Plant Physiology Laboratory
Bioactive Compounds & Federal University Center for Biotechnology and Department of
of Pará Botany
Belém, Pará, Brazil Federal University of Rio Grande do Sul
(UFRGS)
Cristiane Jovelina da-Silva Campus do Vale
Departamento de Botânica Porto Alegre, Rio Grande do Sul, Brazil
Instituto de Ciências Biológicas
Universidade Federal de Minas Gerais Moacir Rossi Forim
Belo Horizonte, Minas Gerais, Brazil Chemistry Department
São Carlos Federal University
Wellington Alves de Barros São Carlos, São Paulo, Brazil
Grupo de Estudos em Química Orgânica
e Biológica (GEQOB) Maysa Furlan
Departamento de Química, Instituto Universidade Estadual Paulista
de Ciências Exatas Núcleo de Bioensaios, Biossíntese e
Universidade Federal de Minas Gerais (UFMG) Ecofisiologia de Produtos Naturais – NuBBE
Belo Horizonte, Minas Gerais, Brazil Araraquara, São Paulo, Brazil
Fernanda de Costa Chirlei Glienke

Plant Physiology Laboratory Federal University of Paraná
Center for Biotechnology and Department Department of Genetics
of Botany Curitiba, Paraná, Brazil
Federal University of Rio Grande do Sul (UFRGS)
Campus do Vale Núbia Alves Mariano Teixeira Pires
Porto Alegre, Rio Grande do Sul, Brazil Gomides
Unidade Acadêmica Especial
Ângelo de Fátima de Biotecnologia
Grupo de Estudos em Química Orgânica Universidade Federal de Goias
e Biológica (GEQOB) Catalão, Goiás, Brazil
Departamento de Química, Instituto
de Ciências Exatas Camila Fernanda de Oliveira Junkes
Universidade Federal de Minas Gerais (UFMG) Plant Physiology Laboratory
Belo Horizonte, Minas Gerais, Brazil Center for Biotechnology and Department
of Botany
Cecilia Maria Alves de Oliveira Federal University of Rio Grande do Sul
Instituto de Química (UFRGS)
Universidade Federal de Goias Campus do Vale
Goiânia, Goiás, Brazil Porto Alegre, Rio Grande do Sul, Brazil
Contributors xv
Lucilia Kato Luzia Valentina Modolo

Instituto de Química Departamento de Botânica
Universidade Federal de Goias Instituto de Ciências Biológicas
Goiânia, Goiás, Brazil Universidade Federal de Minas Gerais
Belo Horizonte, Minas Gerais, Brazil
Melina Cossote Kumoto
General Office of Drugs and Biological Afif Felix Monteiro
Products Universidade Estadual Paulista
Brazilian Health Regulatory Núcleo de Bioensaios, Biossíntese
Agency – ANVISA e Ecofisiologia de Produtos
Brasília, DF, Brazil Naturais – NuBBE
Lucas Vieira Lima
Universidade Federal de Minas Gerais Aline Pereira Moraes
Laboratório de Sistemática Vegetal Instituto de Química
Departamento de Botânica Universidade Federal de Goias
Instituto de Ciências Biológicas Goiânia, Goiás, Brazil
Franciele Antonia Neis
Mary Anne Sousa Lima Plant Physiology Laboratory
Department of Organic and Inorganic Center for Biotechnology and Department of
Chemistry Botany
Federal University of Ceará Federal University of Rio Grande do Sul
Campus do Pici (UFRGS)
Fortaleza, Ceará, Brazil Campus do Vale
Porto Alegre, Rio Grande do Sul,
Rita de Cássia Lemos Lima Brazil
Department of Drug Design and
Pharmacology Leonardo da Silva Neto
University of Copenhagen Grupo de Estudos em Química Orgânica
Copenhagen, Denmark e Biológica (GEQOB)
Adaíses Simone Maciel-Silva de Ciências Exatas
Universidade Federal de Minas Gerais Universidade Federal de Minas Gerais
Laboratório de Sistemática Vegetal (UFMG)
Departamento de Botânica Belo Horizonte, Minas Gerais,
Instituto de Ciências Biológicas Brazil
Instituto Federal Farroupilha
Jair Mafezoli Alegrete, Rio Grande do Sul,
Department of Organic and Inorganic Brazil
Chemistry
Federal University of Ceará Breno Germano de Freitas Oliveira
Campus do Pici Grupo de Estudos em Química Orgânica
Fortaleza, Ceará, Brazil e Biológica (GEQOB)
Rebeca Previate Medina de Ciências Exatas
Universidade Estadual Paulista Universidade Federal de Minas Gerais
Núcleo de Bioensaios, Biossíntese (UFMG)
e Ecofisiologia de Produtos Naturais – NuBBE Belo Horizonte, Minas Gerais,
Araraquara, São Paulo, Brazil Brazil
xvi Contributors
Maria da Conceição Ferreira de Oliveira Josilene Lima Serra

Department of Organic and Inorganic Centre for Valorization of Amazonian
Chemistry Bioactive Compounds & Federal University
Federal University of Ceará of Pará
Campus do Pici Belém, Pará, Brazil
Fortaleza, Ceará, Brazil
Vanessa Gisele Pasqualotto Severino
João Paulo Silvério Perfeito Instituto de Química
General Office of Drugs and Biological Universidade Federal de Goias
Products Goiânia, Goiás, Brazil
Brazilian Health Regulatory
Agency – ANVISA Dulce Helena Siqueira Silva
Brasília, DF, Brazil Univ. Estadual Paulista
Núcleo de Bioensaios, Biossíntese
Elaine Pessoa e Ecofisiologia de Produtos Naturais – NuBBE
Centre for Valorization of Amazonian Araraquara, São Paulo, Brazil
Bioactive Compounds & Federal University
of Pará Aristônio Magalhães Teles
Belém, Pará, Brazil Instituto de Ciências Biológicas,
Universidade Federal de Goias
Hervé Rogez Goiânia, Goiás, Brazil
Centre for Valorization of Amazonian
Bioactive Compounds & Federal University Marília Valli
of Pará Universidade Estadual Paulista
Belém, Pará, Brazil Núcleo de Bioensaios, Biossíntese e
Ecofisiologia de Produtos Naturais – NuBBE
Adão Aparecido Sabino Araraquara, São Paulo, Brazil
Grupo de Estudos em Química Orgânica
e Biológica (GEQOB) Vinicius Galvão Wakui
Departamento de Química, Instituto Instituto de Química
de Ciências Exatas Universidade Federal de Goias
Universidade Federal de Minas Gerais Goiânia, Goiás, Brazil
(UFMG)
Belo Horizonte, Minas Gerais, Anna Carolina Alves Yendo
Brazil Plant Physiology Laboratory
Center for Biotechnology and Department
Daiani Cristina Savi of Botany
Federal University of Paraná Federal University of Rio Grande do Sul (UFRGS)
Department of Genetics Campus do Vale
Curitiba, Paraná, Brazil Porto Alegre, Rio Grande do Sul, Brazil
1 Unraveling the Complexities
of Brazilian Regulations
for Medicinal Plants and
Herbal Medicinal Products
Ana Cecília Bezerra Carvalho,
Melina Cossote Kumoto, and João Paulo Silvério Perfeito
General Office of Drugs and Biological Products.
Brazilian Health Regulatory Agency – ANVISA.
Brasília, DF, Brazil
CONTENTS
1.1 Introduction�� 1
1.2 Various Definitions and Approaches�� 2
1.2.1 Definitions�� 2
1.2.2 Medicinal Plants Trade, as Established by Law 5,991/1973�� 4
1.2.3 Compounding of HMPs�� 4
1.2.4 Industrialized HMPs�� 5
1.3 Conclusion�� 10
References�� 11
1.1 INTRODUCTION
The regulation of medicinal plants and herbal medicinal products (HMPs) is quite complex since
it involves several factors and rules. There are several steps to be considered, such as cultivation,
harvesting, manufacturing or manipulation in compounding pharmacies, commercialization and
prescription, being essential that adequate control be performed so that safe and effective products
are available to the population.
The laws involved in this regulation include those published at the federal government
level, such as the National Congress, the Presidency of the Republic, ANVISA (Brazilian
Health Regulatory Agency), the National Health Council, the Ministry of Health and the
Ministry of the Environment, among other governmental bodies, as well as state and munici-
pal legislatures. This legislative framework is often complex to follow because it is based on
the legal definitions of medicines, which encompass rigid rules of control, production and
evidence of safety and efficacy. Conversely, there is a lot of informal trade in HMPs in Brazil,
where there are minimal controls at best to guarantee plant identification and the safe level
of contaminants.
Unfortunately, a large part of the Brazilian population does not understand that these products
require a license before being prescribed or sold, and this fact generates doubts and uncertainties
for producers and consumers. Thus, discussion of all possible regulatory paths of product release,
1
2 Brazilian Medicinal Plants
from the simplest one to the most complex – the latter being industrial production, which encom-
passes more steps and is more regulated is important to discuss.
Before starting the technical-regulatory discussion, the roles of normative acts in the Brazilian
sanitary regulation discussed in this chapter need to be clarified:
• Law: published at the federal level, by the National Congress, or in the state or municipal
sphere, aims to introduce a new subject to be regulated;
• Decree: published by the head of the Executive Power – in the federal, state or municipal
sphere. Usually regulates and complements a law;
• Ordinance: published by public administration bodies, such as ministries, to guide compli-
ance with legal provisions. In the case of HMP, ordinances have legal force and are usually
issued by the Ministry of Health;
• Resolution- Resolução da Diretoria Colegiada (RDC) (Collegiate Board Resolution): This
is the highest act published by ANVISA, after consideration from its board of directors and
public discussion of the matter. The RDC can be accompanied by Normative Instructions (IN),
which complement and detail it. ANVISA may also publish Specific Resolutions (RE)
usually for concessions of manufacturing and marketing authorization.
ANVISA was created in 1999, through Law 9,782 (Brazil, 1999). Nowadays, the Agency is the main
public body responsible for regulating medicines, including HMP. Thus, since 1999, RDC, RE and
IN have been observed in these products’ regulations. Before that, the norms were published by the
Ministry of Health, mainly through ordinances.
1.2 VARIOUS DEFINITIONS AND APPROACHES

1.2.1 Definitions
Products obtained from plant species can have a different classification, depending on the process-
ing level: medicinal plant, herbal drug, herbal preparation or HMP.
Medicinal plant is defined as the fresh or dry plant species, usually collected at the time of use,
used for therapeutic purposes.
Herbal drug consists of a plant that has gone through collection/harvesting, stabilization, when
applicable, and drying, that can be found whole, cut or powdered form. The term “drug”, as defined
by law, gives the product a medicinal or sanitary purpose (Ministério da Saúde, 2018a).
Herbal preparation is the product obtained from the extraction of fresh medicinal plant or herbal
drug, which contains the substances responsible for the therapeutic action, and may occur in the
form of extract, fixed and volatile oil, wax, exudate and others (Ministério da Saúde, 2018a).
The herbal raw material that can be used in the production of HMPs comprises the medicinal
plant, the herbal drug and the herbal preparation (Ministério da Saúde, 2018a).
Finally, HMPs are medicines, technically elaborated, which use herbal raw materials as the active
ingredient, for prophylactic, curative, palliative or diagnostic purposes. These are divided into two
categories: herbal medicine (HM) and traditional herbal product (THP) (Ministério da Saúde, 2018a).
HMPs can be single or in combination, when elaborated with one or more plant species, respec-
tively. The isolated active substances of any origin (plant or synthetic) and their association with
herbal preparations are not considered HMPs; instead, these types of assets are registered in Brazil
in a different class of medicines in accordance with RDC 24/2011 (Ministério da Saúde, 2011a).
The commercial establishments legally involved in the production chain of medicinal plants and
HMPs are ervanaria, pharmacy, drugstore, pharmaceutical ingredient suppliers and pharmaceuti-
cal industry.
Ervanaria is an herbal shop, where the dispensation of medicinal plants is carried out. This
establishment does not need to have a pharmacist authorized by the Regional Pharmacy Council
Complexities of Brazilian Regulations 3
(CRF) responsible for the dispensary, which is not the case with the others listed. However, it can-
not dispense medicines or any product other than medicinal plants. Also, the dispensed medicinal
plants cannot have therapeutic claims, and the correct storage and botanical classification must be
respected, as determined by Law 5,991/1973 (Brazil, 1973).
Drugstore is the establishment authorized to sell and dispense medicines, special foods and
health products directly to the consumer (Brazil, 1973).
Pharmacy is where compounded medications are made, according to prescription formula
(issued by a professional allowed to prescribe drugs) or officinal formula (included on national
forms or international forms recognized by ANVISA). It is authorized to trade drugs, medicines
and pharmaceutical ingredients, as well as carrying dispensation of medicines in hospital units or
other equivalent healthcare facility.
Both pharmacy and drugstore need an authorization for regular operation. (Autorização de
Funcionamento de Empresas [AFE]).
In the context of pharmacies, in 2010, the Farmácia Viva project was established by the Brazilian
Ministry of Health, under SUS (the acronym for Brazil’s public health system ‘Único de Saúde’),
for the exclusive compounding of medicinal plants and HMPs, in order to provide pharmaceutical
social assistance (Brazil, 2010). With the intention of regulating this institution, ANVISA published
RDC 18/2013, which addresses the good practices of processing and storage of medicinal plants,
preparation and dispensing of magistral and officinal HMPs (Ministério da Saúde, 2013). This
approach brings all the requirements for the public compounding of HMPs, according to the claims
brought by the Brazilian Ministry of Health and set out in the National Policy on Medicinal
Plants and Herbal Medicines (PNPMF) (Ministério da Saúde, 2006a).
Although several categories of health professionals regulate the prescription of medicinal plants
and HMPs, only the council of each specific category determine regulations that determine their
professionals’ attributions. Even an HMP considered exempt from medical prescription can only be
prescribed by a professional qualified by the respective professional council.
The pharmaceutical ingredient supplier is important in the supply chain of the pharmaceuti-
cal ingredients, both active and excipient, used in the production of medicines by the industry
and compounding pharmacies. They need to receive an AFE before producing the raw materials
(Ministério da Saúde, 2018a).
Pharmaceutical industries, in turn, are establishments that produce large amounts of drugs,
in batches, according to the good manufacturing practices (GMP). They must have authoriza-
tion for manufacturing medicines, an AFE and certificate of GMP (Ministério da Saúde, 2018a).
There is a specific authorization to produce THP, which is issued according to RDC 13/2013
(Ministério da Saúde, 2018a).
In 2006, two important public policies were published to expand the access of the Brazilian pop-
ulation to medicinal plants and HMPs and to increase their use: the National Policy on Integrative
and Complementary Practices (PNPIC) and the National Policy on Medicinal Plants and Herbal
Medicines (PNPMF).
The PNPIC was published by Ordinance 971/2006, with the goal to introduce, in the public
health system – SUS – services and products related to traditional knowledge, such as Phytotherapy,
Acupuncture, Homeopathy and Social thermotherapy, guaranteeing integral coverage in health
care, through practices that previously were only accessible in private care. PNPIC brought actions
to be implemented in SUS and in other government healthcare bodies, such as the Ministry of
Health, municipal and state health secretariats, ANVISA and Fiocruz (Ministério da Saúde, 2006b).
In 2018, the number of practices available to the population was expanded, reaching 29 (Ministério
da Saúde, 2018b).
The PNPMF was published by Decree 5.813/2006 establishing the guidelines of priority
actions to promote the safe and effective use of medicinal plants and HMPs, aiming to consolidate
relevant initiatives in the country and national and international recommendations on the subject.
The PNPMF promotes transversal actions involving different areas such as health, the environment
and economic and social development. This broadens the therapeutic options available for the pre-
vention and treatment of diseases; values the knowledge of traditional communities; encourages
the sustainable use of Brazilian biodiversity; and stimulates the expansion and strengthening of the
productive chain and the national industry (Ministério da Saúde, 2006c).
As a regulatory agency, ANVISA plays an important role in PNPMF, executing activities like
monitoring and overseeing the commercialization, compounding and distribution of herbal raw
materials and HMPs. The Agency also grants marketing authorization to new HMPs, through
registration or notification (Ministério da Saúde, 2006c).
The publication of these policies urged ANVISA to renew its legislative framework, in order to
adapt to the new national and international scenarios that were emerging. In consideration, several
guidelines were published in subsequent years, as discussed below.
There are several ways in which to produce and regulate HMPs in Brazil. According to Brazilian
legislation, medicines can be compounded or industrialized. They also can be aimed at human or
veterinary use, thus, are regulated by ANVISA or by the Ministry of Agriculture, Livestock and
Supply (MAPA), respectively.
The following different types of production and commercialization of HMPs are recognized for
human use:
1.2.2 Medicinal Plants Trade, as Established by Law 5,991/1973

The trade of medicinal plants is regulated by Law 5,991/1973, and article 7 establishes that their dis-
pensation is exclusive to pharmacies and ervanarias and that the plants must be properly identified,
in botanical terms, and in adequate packing. So, the rules settled by this law do not address medi-
cines, but only medicinal plants. The latter cannot have therapeutic indications on their packaging
or advertising material (Ministério da Saúde, 2018a). Although this article was published more than
40 years ago, it has not been regulated yet, leaving open the requirements of quality, safety and
efficacy for medicinal plants.
1.2.3 Compounding of HMPs
In Table 1.1 the main rules related to the compounding of HMPs in Brazil are presented.
Nowadays there are two types of compounding pharmacies authorized to compound HMP in
Brazil: the compounding pharmacy and the Farmácia Viva. Compounding pharmacies are regu-
lated by RDC 67/2007, which was updated by RDC 87/2008. This type of establishment is autho-
rized to handle a wide range of medicinal products, depending on the authorization granted, such
as low therapeutic substances, hormones, antibiotics, cytostatics, substances under special control,
sterile products and homeopathic medicines. Parenteral nutrition, enteral and polyelectrolyte con-
centrate for hemodialysis (Concentrado polieletrolítico para hemodiálise [CPHD]) solutions are
TABLE 1.1
Main Rules Related to the Compounding of Herbal Medicinal Products (HMPs) in Brazil
Resolution Addresses the Following Issue
RDC 67/2007 Good compounding practices of magistral and officinal preparations for human use in
pharmacies
RDC 18/2013 Good practices of processing and storage of medicinal plants, preparation and dispensing of
magistral and officinal products of medicinal plants and HMPs in Farmácia Viva under SUS
RDC 87/2008 Good practices of magistral and officinal preparations for human use in pharmacies, updating
RDC 67/2007
excluded from its scope. The Farmácia Viva, in turn, is a public establishment, set up under the
aegis of SUS, and has the scope of compounding only medicinal plants and HMPs.
Compounded HMPs for human use can be obtained in two different ways: magistral or officinal
preparation. The magistral preparation is the one prepared in the pharmacy, from a prescription of a
qualified professional, aimed at an individual patient, and that establishes in detail its composition,
pharmaceutical form and posology. The official preparation must follow the formula registered in
the Herbal Medicines National Formulary of the Brazilian Pharmacopoeia (FFFB) – or other codes
recognized by ANVISA.
The FFFB was initially published in 2011 and updated in 2018, through the publication of its
first supplement. The first publication of the FFFB contained 47 monographs on herbal drugs for
infusions and decoctions, 17 tinctures, 1 syrup, 5 gels, 5 balms, 1 soap and 2 creams. With the
publication of the first supplement, the chapter on tinctures was updated with the inclusion of new
monographs, totaling 40, and a chapter on capsules with herbal preparations (containing 28 new
monographs) was added. All formulations presented in the FFFB are considered officinal and may
be handled in the compounding pharmacy or the Farmácia Viva, without the need for any individu-
alized prescription (Ministério da Saúde, 2011b).
Of note, a pharmacy can maintain a minimum stock of preparations as listed in the FFFB, duly
identified, according to the technical and management needs of the establishment, since the quality
and stability of the herbal drugs and their preparations can be assured. Quality control when pro-
ducing a minimum stock is similar to that stipulated for the pharmaceutical industry, because it is a
batch production (Ministério da Saúde, 2011b, 2018a).
Compounded medicines have their quality controlled during their production in the pharmacy
facilities and are exempted from registration in ANVISA.
The Farmácia Viva has somewhat different regulations from compounding pharmacies, since it
must be regulated by both ANVISA, within the scope of compounding medicines, and the Ministry
of Agriculture, which has the competence to regulate cultivation and harvesting of herbs. However,
at the time of writing, the regulations of the Ministry of Agriculture have not yet been published.
Lastly, the Farmácia Viva is a compounding pharmacy which is authorized to produce its own
inputs; that is, it carries out the cultivation, harvesting, processing and storage of native or acclima-
tized medicinal plants (Ministério da Saúde, 2018c). Moreover, it is authorized to dispense HMPs in
other health facilities of the SUS network, such as outpatient clinics, hospitals and healthcare units,
while other compounding pharmacies can only dispense in their own facilities.
1.2.4 Industrialized HMPs
In Table 1.2 the main rules applied for industrialized HMPs in Brazil (Ministério da Saúde, 2018a)
are presented.
Together with the rising use of industrialized herbal products, a concern has emerged about
updating this subject in the Brazilian regulatory framework. The first Brazilian legislation for
the registration of HMs was established in 1967 (Perfeito, 2012); this normative has already been
revised four times through RDC 17/2001, RDC 48/2004, RDC 14/2010 and RDC 26/2014. It is
noticeable that the Brazilian regulation on the topic has been improving and evolving, mainly in
the last decade, in a process of international convergence that resulted in the publication of a new
regulatory milestone in 2014 – RDC 26.
Nowadays, the normative ruling registration and post-registration of HMPs are RDC 26/2014,
RDC 38/2014, IN 2/2014 and IN 4/2014 – their subjects are detailed in Table 1.2. The ruling
GMPs are RDC 17/2010 (regarding medicines), RDC 13/2013 (regarding THP) and RDC 69/2014
(regarding active pharmaceutical ingredient) (Brazil, 2018a).
Besides these normatives, there are other regulations common to all medicines licensed in
Brazil, such as the rules for leaflets, packaging, labeling, clinical research, validation of analytical
methodologies, stability studies and others (Brazil, 2018a). They are mentioned also in Table 1.2.
TABLE 1.2
Main Rules Applied for Industrialized Herbal Medicinal Products (HMPs) in Brazil
(Ministério da Saúde, 2018a)
Resolution Addresses the Following Issue
RDC 26/2014 Registration of HMs and registration and notification of THPs
RDC 38/2014 Post-approval changes in HMs and THPs
IN 2/2014 “List of HMs for simplified licensing” and “List of THPs for simplified licensing”
IN 4/2014 Guide for registration of HMs and registration and notification of THPs
RDC 13/2013 Good manufacturing practices for THPs
RDC 69/2014 Good manufacturing practices for active pharmaceutical ingredient
Law 6,360/1976 Measures on sanitary surveillance of medicines, drugs, pharmaceutical ingredient and
related materials, cosmetics, sanitizers and other products
Decree 8,077/2013 Regulates the conditions for the operation of companies subject to sanitary licensing, and
the registration, control and monitoring, within the sanitary surveillance, of the products
referred to in Law No. 6,360/1976
Law 5,991/1973 Sanitary control of trade in drug, medicine and pharmaceutical ingredients
RDC 17/2010 Good manufacturing practice of medicines
RDC 47/2009 Elaboration, harmonization, updating, publication and availability of medicine leaflet for
patients and health professionals
RDC 71/2009 Rules for labeling of medicines
RE 1/2005 Guide for conducting stability studies
RDC 166/2017 Criteria for the validation of analytical methods
RDC 37/2009 Admissibility of foreign pharmacopoeias
RDC 59/2014 Medicine name, its complements, and medicine family
RDC 234/2018 Outsourcing of production steps, analysis of quality control, transportation and storage of
medicines and biological products
RDC 4/2009 Pharmacovigilance of medicinal products for human use
RDC 9/2015 Rules for conducting clinical trials in Brazil
RDC 98/2016 Provides for the criteria and procedures for the framework of medicinal products
classified as over the counter (OTC)
RDC 26/2014 divided HMP into two subclasses: HM, which is the product that should demon-
strate its safety and efficacy through non-clinical and clinical studies; and THP, when the proof of
safety and effectiveness occurs through the traditional use. Thus, when the term HMP is used, both
HMs and THPs are covered.
HMPs produced on an industrial scale must have authorization provided by ANVISA before
their commercialization, and this can be granted by registration or notification. In the case of HMs,
registration is necessary, which is the regulatory process that depends on evaluation and favorable
manifestation of ANVISA, prior to releasing the product. THPs, in turn, can be registered or noti-
fied. Notification is a simplified process as explained below. Thus, ANVISA’s licensing authoriza-
tion is essential to confirm the quality of an HMP, since it is preceded by a technical evaluation, in
which the safety and efficacy of the product must have been demonstrated.
Notification is a simplified manufacturing and marketing authorization process, implemented
by ANVISA, to reduce bureaucracy in the process of licensing products. Since they are produced
within predefined technical criteria, by authorized companies that comply with GMP, this offers a
low health risk to patients. In this process, the product release to the market can occur immediately
after the communication to ANVISA, and this is accomplished simply through a specific electronic
system, with the control provided later through regulatory inspections in the companies’ sites.
RDC 26/2014 determines that the notification procedure is only applicable to THP in the follow-
ing manner: (a) they are obtained from an herbal active pharmaceutical ingredient (IFAV) listed in
the latest edition of the FFFB; and (b) they have a specific monograph of quality control published
in a pharmacopoeia recognized by ANVISA (Ministério da Saúde, 2018a).
As required by RDC 26/2014, all THPs should be nonprescription products (over the counter
[OTC]). Therefore, for the notification of THPs, it is necessary that the therapeutic indication cited
in the FFFB is considered a nonprescription one, in accordance with current regulations, namely
RDC 98/2016 and IN 11/2016.
The registration of HMs and THPs, on the other hand, constitutes a more detailed process, in
which the requesting company presents all the technical-scientific evidence related to the efficacy/
effectiveness, safety and quality of the HM for evaluation, aiming at obtaining authorization for its
production and marketing.
The registration dossier of an HMP consists of a documentary part, a technical report, a stabil-
ity report, a production and quality control report, and a safety and efficacy (for HM)/effectiveness
(for THP) report.
Regarding the safety and efficacy of HMPs, the traditional use was already stipulated in RDC
17/2000, but this was not a usual path adopted by companies. Since the advent of RDC 26/2014, this
alternative path has been expanded, and now it is much more detailed, basing much of its require-
ments on international legislation, mainly the European, Canadian and Australian.
To be considered a THP, the herbal product must prove a continued safe use for a period longer
than 30 years; the administration route cannot be injectable or ophthalmic; the claims must be
coherent with the traditional usage and must be appropriate for use without the supervision of a phy-
sician for diagnosing, prescribing and monitoring. It is important to note that a THP cannot com-
prise ingredients which have a known toxic hazard or toxic chemical substances above safe limits.
HMs, on the other hand, should demonstrate their safety and efficacy through the presentation
of non-clinical and clinical studies. Since there are no specific rules for conducting these studies
in HMPs, these should be conducted according to the general regulations of ANVISA and the
Brazilian National Health Council.
According to RDC 26/2014, when there are no non-clinical tests proving the safety of the HMP,
they should be carried out in accordance with the latest version of the ANVISA Guide for conduct-
ing non-clinical studies of toxicology and pharmacological safety necessary to the development of
medicinal products, where applicable to HMs. Also, when there are no clinical trials demonstrating
the HMP’s safety and efficacy, these should be performed following good clinical practice (GCP);
RDC 9/2015 – the current standard for conducting clinical trials; the Guide “Operational guid-
ance: information needed to support clinical trials of herbal products” published in 2008, by WHO/
Ministry of Health (Ministério da Saúde [MS]); and the determinations of the National Health
Council (CNS), established through Resolution 446/2011, and Resolution 251/1997 (Ministério da
Saúde, 2018a).
RDC 26/2014 also determines that when there are non-clinical and clinical trials available in
scientific and technical literature, these can be presented to ANVISA for evaluation of their quality
and representativeness. If valid, it is not necessary to carry out new studies by the company which
intends to register the HM. The company should send to ANVISA copies of all technical and scientific
documentation corresponding to them. The studies presented must have been carried out with the
same herbal drug (when this is the finished product) or herbal preparation, in the same dose and
therapeutic indications presented by the registrant of the HMP (Ministério da Saúde, 2018a).
Besides the notification and ordinary registration, there is the procedure of simplified regis-
tration. In this modality, it is not necessary to present a safety and efficacy report for the HMP,
since these items have been previously evaluated by ANVISA or by the European Community and
European Medicines Agency (EMA), for a particular HMP. Hence, there are two possibilities for
simplified registration: presence of the HMP in the lists of HMs or THPs, published by IN 2/2014;
or among those published by the European Community (Community herbal monographs based on
well-established or traditional use) and elaborated by the Committee on Herbal Medicinal Products –
HMPC of the EMA.
RDC 26/2014 has improved the technical requirements for quality assurance, which are better
suited to the control of raw materials and complex products such as IFAV and HMPs, bringing
the Brazilian legislation requirements closer to the international framework. It is noteworthy that
regarding quality control, regulatory requirements are the same for HMs and THPs.
A primary requirement to ensure the quality of HMs and THPs is the compliance with GMP of
IFAV, whose requirements are specified in RDC 69/2014.
Manufacturers of HMPs can also produce the IFAV for themselves. But for this, they must fol-
low the norm applied to this activity, as well as possessing an AFE for the manufacturing of active
pharmaceutical ingredients (APIs).
RDC 26/2014 also defines that the production of HMs and THPs must follow the GMP, regulated
by RDC 17/2010 or by RDC 13/2013, the latter being applied to THPs. Therefore, HM manufactur-
ers must be GMP-certified by RDC 17/2010, and THP manufacturers must be certified by RDC
17/2010 or by RDC 13/2013.
HMP quality control comprises assessments of the herbal raw material (the herbal drug, the
herbal preparation) and the finished product (HM and THP), as well as the stability of the medicine
during the proposed shelf life time.
For the herbal drug, it is necessary to confirm its botanical identity; its integrity; organoleptic
characteristics; humidity; ash content and presence of foreign material, such as micro- and macro-
scopic contaminants, including fungi, bacteria, mycotoxins and heavy metals. The harvesting site
and methods for eliminating contaminants, if used, must be stated, together with the investigation
of possible residues. Finally, the qualitative and quantitative analysis of markers must be presented,
and quantitative control can be replaced by biological control of the therapeutic activity.
For the herbal preparation, the solvents, excipients and vehicles used in its extraction should be
reported; the extraction methods employed; the part(s) of the plant used; approximate drug: extract
ratio and presence of residual solvents. The results of the physical-chemical tests of the herbal
preparation are also requested, as described in RDC 26/2014 and IN 4/2014.
Regarding the finished product (HM and THP), the control requirements depend on the dosage
form and are focused in the evaluation of the HMP’s integrity and stability, which requires the chro-
matographic profile, marker assays and control of microbiological contamination, among others.
The methods used to control HMP quality should be present in the Brazilian Pharmacopoeia,
current edition, or one of the pharmacopoeias recognized by ANVISA, according to RDC 37/2009
(German, American, Argentinian, British, European, French, International, Japanese, Mexican and
Portuguese). Another option is to validate the methodology according to the provisions stated by
RDC 166/2017. The source of the method (internal development or compendial) must be properly
indicated.
Considering the absence of a methodology in an official pharmacopoeia, validation should be
carried out in order to demonstrate that the method is appropriate for the intended purpose, and this
can be a qualitative, quantitative or semi-quantitative determination. To do so, the methodology
must be challenged according to the validation parameters explained in RDC 166/2017.
Regarding the validation of HMP methods, specific caveats are made in the evaluation of the
parameter accuracy. In order to perform the tests related to this parameter, the chemical reference
substance (CRS) must be added to a diluted solution of the finished product, allowing the complex
matrix effect to be considered. This matrix effect should also be evaluated by comparing the angu-
lar coefficients of the calibration curves constructed with the CRS of the analyte in the solvent and
with the sample fortified with the CRS of the analyte. The parallel approach of these lines indicates
an absence of interference from the matrix constituents.
In addition to the control of raw materials and finished product, stability studies are required in
order to verify if the physical, chemical, biological and microbiological characteristics of the HMP
remain within the specifications, during the period of shelf life proposed. The results are used to
establish or confirm this period and recommend storage conditions. The tests to be carried out dur-
ing this study are determined by RE 1/2005 and comprise, among others, the product’s physical
characteristics, chromatographic profile, qualitative and quantitative analysis of markers and micro-
biological control.
Stability studies and validation of analytical procedures for HMs and THPs follow the general
regulations established for medicines by ANVISA. However, due to the complexity of HMP com-
position, specific guidelines have been adopted and are detailed in IN 4/2014.
In addition to the possibility of a company producing and controlling the HMP quality in its own
facilities, it is also possible to outsource these activities, as recently established by RDC 234/2018
(Ministério da Saúde, 2018d).
All registered or notified HMPs must renew their marketing authorization every five years, in
order to demonstrate that the product remains safe and effective, according to market data (Brazil,
2015a). Also, after a registration has been granted, the requesting company may need to make
changes in its product, and this can be done using RDC 38/2014. This norm guides the submis-
sion of post-approval changes in HMs and THPs. For notified THPs, post-approval changes do not
apply, so if any change in the product is required, the notification must be canceled and resubmitted,
including the proposed change in the new application.
The regulations already discussed and presented in Table 1.2 are constantly improved and
revised. Since the establishment of PNPIC and PNPMF, virtually all the HMP regulatory frame-
work was republished, aiming to promote and develop the HMP national market.
In 2017, there were 359 HMPs licensed in Brazil, of which 332 were single HMPs and 27 in
combination. At that time there were no THPs notified. Between 2006 and 2012, there was a 31%
decrease in the number of HMPs registered in Brazil (a reduction of 159 products). The number
of HMPs that left the market during this period is equivalent to about half the number of products
registered today. This decrease took place specifically between 2008 and 2011. These products were
withdrawn from the market due to lack of technical sanitary requirements (Perfeito, 2012) or lack
of commercial interest.
There are 101 plant species with herbal preparations registered as HMP actives in Brazil: 35 are
native, naturalized or exotic plants cultivated in Brazil; that is, only about 35% of the total registered
species are obtained on Brazilian soil. Thus, most HMPs produced in Brazil have as active ingredi-
ents plant species that come from abroad. One possible reason for this is that there is more published
scientific information about these exotic species (Santos et al., 2011), both in terms of their safety
and efficacy as well as quality control. Clinical studies on native Brazilian herbal species are rare,
and even if there is a long traditional use of some species, this tradition in human use is not well
documented (Carvalho et al., 2018).
Another aspect that could have contributed to the reduction of HMPs on the Brazilian market
is related to sales restrictions. Prescription medicines, unlike nonprescription (or OTC), cannot be
advertised to people other than prescribers and cannot be displayed over the counter. Considering
the 359 HMPs registered in Brazil, 214 (59.6%) are OTCs and 145 (40.4%) are prescription medi-
cines. The number of HMPs classified as under prescription is considerably higher in Brazil than
in most other countries (WHO, 2005, 2011). Many products designated as for sale under prescrip-
tion in Brazil are sold in other countries as OTCs or supplements. It is worth noting that there
are no specific rules on sales restrictions of HMPs, so the general determination for medicines,
established by RDC 98/2016, must be followed. This regulation states that a list will be published
specifying the therapeutic indications and sales restrictions for each API. This list is still under
development, and the best classification for each HMP will be discussed individually (Ministério
da Saúde, 2016a).
In 2017, 77 companies had market authorization in Brazil. These companies are distributed in
11 Brazilian states, with most of them concentrated in the southeastern region. There is no company
producing HMPs in the northern region, despite the biodiversity of this area, showing how this
market niche is so little exploited in Brazil (Carvalho et al., 2018).
It is essential to emphasize that together with the current reduction in HMPs on the market there
is also a decrease in the production of herbal raw material. National companies of API are not
complying with the GMP to produce herbal drugs and herbal preparations; hence a high number of
IFAVs used in the country are imported (Branco, 2015).
Many companies that hold licenses for HMP products in Brazil do not follow the changes in
legislation and do not adapt to them. Also, they rarely take advantage of the financing offered by
governmental programs and barely invest in research and technological development. Often, reg-
istration requests are rejected because of technical problems that have been repeated for years and
should already have been overcome.
Unfortunately, there is little interaction between companies and national research centers; thus,
many studies carried out in Brazilian universities do not reach the possibility of pharmaceutical
development and registration as a regulated product. There is a lack of investigation with native
plants, and a dearth of professionals specialized in the production of drugs from complex sources,
such as HMPs, which requires equally complex control techniques (Araújo et al., 2013; Perfeito,
2012).
Although the number of HMP companies has decreased, there was no reduction in the turnover
values in the sector. This is because there is no price regulation of HMPs in Brazil, so it is up to the
companies to define the prices, unlike the practice in synthetic medicines. Nevertheless, Brazilian
values, in terms of product numbers and market value, are very low when compared to other
markets: 80% of Germans, 70% of Canadians and 49% of French people use HMs (Carvalho et al.,
2018; WHO, 2011). Despite this, according to the Ministry of Health, between 2013 and 2015, there
was a 161% increase in demand for herbal and medicinal treatments in SUS (Ministério da Saúde,
2018e).
There are few registered products containing native species in Brazil, as mentioned above, but
taking into account informal trade the number of native products is quite large. It is necessary
to raise awareness in the Brazilian population about the need for HMPs to be evaluated for their
quality, safety and efficacy, prior to commercialization. It is rare for users to verify whether the
herbal product is authorized, and people assume that if it is on the market it must be legal, or if it is
“natural” it does not pose any danger to health. From January 2015 to June 2018, ANVISA published
440 resolution – RE related to drug irregularities, of which 14% were related to HMP, 80% of
which were unauthorized products (Ministério da Saúde, 2018f). Additionally, there are few cases
of adverse events related to the use of HMPs, and the number of adverse events reported with regard
to medicinal plants is very low (Balbino and Dias, 2010).
It is important to note that, in a similar manner to the international legislation, according to
Brazilian law, the same herbal species can be licensed for human use not only in medicines, but
also in food, cosmetic and health products, often with effects in a similar way to those approved for
drugs and often coexisting on the market with the same dosages (Minghetti et al., 2016). Examples
of products obtained from medicinal plants that are regulated in foods are Allium sativum and iso-
flavones from soybean (Glycine max). Several products of topical use found in Brazilian pharmacies
are regulated in Brazil in the area of cosmetics, such as gels and shampoos of Calendula officinalis
or Matricaria recutita. There are also products registered in the category of health products, such as
vegetable oils, including sunflower (Helianthus annuus) and orange (Citrus aurantium) (Ministério
da Saúde, 2016b). Regulation for these other classes of products is more lenient than that required
for medicines, causing many products that have lost their registration as medicines to migrate to
these other categories.
1.3 CONCLUSION
Brazilian sanitary regulation has changed considerably over recent years in order to deal with the
peculiarities and complexity of herbal materials, such as the herbal active ingredient and HMPs.
The new regulations have brought internationally harmonized concepts of quality control, safety
and efficacy to the national framework. Since 2010, a procedure of medicinal notification has been
put in place, which allows the rapid release of products into the market. Also, with the expansion
of the scope of official compendia, like the FFFB, the notification should be expanded. So, the new
directions established by ANVISA, together with public policies, converge to foster the develop-
ment of the national HMP production chain, as well as encouraging financial support for research
on the topic.
The requirements for using Brazil’s rich socio-biodiversity are now better delineated, both
in health legislation and in the new biodiversity law. Now, it is up to the commercial sector to
dedicate efforts on the development of new products containing new medicinal plants, taking
into consideration the traditional knowledge of Brazilian communities and the wide acceptance
of natural products in the treatment or prevention of diseases, by the population. Therefore, it
is expected that all these factors will contribute to increase the number of safe and effective
HMPs traded in Brazil and will also promote a viable and successful alternative to the national
industry.
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2 Physico-Chemical Methods
for the Quality Control
of Medicinal Plants,
Plant Derivatives and
Phytomedicines in Brazil
Paula Carolina Pires Bueno and Alberto José Cavalheiro
Instituto de Química, Universidade Estadual Paulista
(UNESP), Araraquara, São Paulo, Brazil
CONTENTS
2.1 Introduction.............................................................................................................................. 14
2.2 Important Publications............................................................................................................. 14
2.3 Receiving and Sampling Plant Raw Materials and Plant Derivatives...................................... 17
2.4 Physico-Chemical Tests for the Quality Control of Plant Raw Materials............................... 18
2.4.1 Identification Tests..................................................................................................... 18
2.4.1.1 Macroscopic and Microscopic Botanical Examination.............................. 18
2.4.1.2 Identification by Phytochemical Prospection
or Chemical Identification........................................................................... 19
2.4.1.3 Identification by Chromatographic Fingerprinting.....................................20
2.4.2 Determination of Water and Volatile Material.......................................................... 21
2.4.3 Determination of Total Ash Content......................................................................... 21
2.4.4 Determination of Acid-Insoluble Ash Content.......................................................... 21
2.4.5 Determination of Foreign Matter.............................................................................. 21
2.4.6 Determination of Essential Oils Content................................................................... 22
2.4.7 Extractable Material.................................................................................................. 22
2.4.8 Quantitative Analysis of the Active Principles and/or Chemical Markers................ 22
2.4.9 Determination of Heavy Metals................................................................................ 23
2.4.10 Determination of Pesticides Residues....................................................................... 23
2.4.11 Radioactivity Contamination.....................................................................................24
2.4.12 Determination of Aflatoxins......................................................................................24
2.5 Physico-Chemical Tests for the Quality Control of Plant Derivatives: Liquid Extracts,
Soft Extracts and Dry Extracts................................................................................................24
2.5.1 Determination of Dry Residue (or Soluble Solids)....................................................25
2.5.2 Determination of Solvent Residues...........................................................................25
2.5.3 Relative Density.........................................................................................................26
2.5.4 Apparent Density (or Tapped Density)......................................................................26
2.5.5 Granulometry.............................................................................................................26
2.5.6 Solubility....................................................................................................................26
13
2.5.7 pH................................................................................................................................ 27
2.5.8 Viscosity....................................................................................................................... 27
2.5.9 Alcohol Content........................................................................................................... 27
2.6 Physico-Chemical Tests for the Quality Control of Essential Oils..........................................28
2.6.1 Determination of the Refraction Index........................................................................ 29
2.6.2 Determination of the Optical Rotatory Power............................................................. 29
2.6.3 Determination of the Relative Density........................................................................ 29
2.7 Physico-Chemical Tests for the Quality Control of the Fixed Oils......................................... 29
2.8 Physico-Chemical Tests for the Quality Control of Phytomedicines...................................... 30
References�� 32
2.1 INTRODUCTION
Worldwide, the population uses a plethora of plant species as homemade medicines for the treat-
ment and prevention of several diseases. Therefore, they represent an important niche of the global
drug market and health care system, which emphasizes the need to ensure their quality, safety
and efficacy. Many factors impact directly or indirectly on the quality of plant raw materials and
their derived products. These factors include the selection of matrices for cultivation, domesti-
cation, establishment of cultivation conditions, harvesting, storage and the operation procedures
for the production of extracts and other derivatives. Regardless of being widely used in different
disciplines, such as medicinal, alimentary or industrial, many publications in international high
impact journals have brought attention to the need of performing a critical analyses, considering
the variation of weather and soils in each cultivation area, the seasonality, the time of harvesting, the
conditions of the drying and stabilization process, and the accurate botanical identification of the
plant species.
For this reason, in the last decades, there has been a growing concern with the standardization
for this type of product in order to guarantee a safe and reliable use. For this purpose, the World
Health Organization (WHO), several international regulatory agencies and the Brazilian National
Health Surveillance Agency (ANVISA) have been emphasizing the importance of guaranteeing
the quality of the plant material, using modern and adequate techniques of analysis and suitable
standards.
In order to efficiently characterize these factors while conducting the quality control of herbal
materials, an important aspect is for the analysts to take a multidisciplinary approach and pursue
areas of pharmacognosy, natural products chemistry, botany, plant morphology, plant physiology,
organic and analytical chemistry. Thereafter, during the development and definition of the final
pharmaceutical preparation, aspects of pharmaceutical technology and industrial physics also are
essential.
Therefore, the objective of this chapter is to (a) introduce the regulatory framework in Brazil,
(b) present the understanding of the main physico-chemical tests for assessing the quality of plant
raw materials, intermediate products (for example, essential oils and extracts) and phytomedicines
in a comprehensive and integrated way and (c) their potential applications to the important com-
merce of natural products use in foods and medicines in Brazil.
2.2 IMPORTANT PUBLICATIONS

The World Health Organization (WHO) provides free access to the Quality control methods for
herbal materials (World Health Organization, 2011), a wide-ranging publication concerning the
applicable analytical procedures to products of herbal origin (available at http://www.who.int/iris/
handle/10665/44479). Another free-access WHO publication, the WHO monographs on selected
medicinal plants, provides detailed information on selected medicinal plants, with to date, 116
monographs, distributed in 4 volumes (available at http://apps.who.int/medicinedocs/en/d/Js2200e/).
Quality Control: Physico-Chemical Methods 15
These monographs present chemical and pharmacological data, parameters and specifications of
quality control for each plant species described there. Regarding the international context, other
monographs and procedures available at the American and European Pharmacopoeias are also very
useful and important to the understanding and harmonization of analytical methods described in
this chapter.
In Brazil, the National Health Surveillance Agency (ANVISA), through the Resolução da
Diretoria Colegiada (RDC) Resolution, n. 26 from May 13, 2014 establishes the registration of
herbal medicines, and the registration and notification of traditional herbal medicines. The resolu-
tion defines the required tests that must compose the analysis report of the plant raw material, the
corresponding derivatives and final products. Along with the results, the report must also indicate
the methods used and the technical specification defined for each batch.
Other ANVISA publications include requirements for quality control, registration and good
manufacturing practices (GMP) applicable to phytomedicines. Among them, there is the Normative
Instruction IN n. 04, from June 18, 2014 that determines the publication of the Orientation guide to
the registration of phytomedicines and registration and notification of a traditional phytotherapeutic
product; the RDC n. 13, from March 14, 2013, which displays the GMP for traditional phytothera-
peutic products; and the RDC n. 69, from December 08, 2014 that relates to the GMP for active
pharmaceutical ingredients. Accordingly, the Brazilian Pharmacopoeia, another ANVISA publica-
tion, provides the methods, general analysis procedures, official reference substances and mono-
graphs applicable to plant materials and phytomedicines, some of which are native from Brazil.
Looking at the definitions available at RDC n. 26 from March 13, 2014, the understanding of
three of them is crucial for the correct interpretation of the nomenclature used in the process of
registration and division of the physico-chemical tests presented in this chapter: (i) plant crude drug
(or plant raw material), (ii) plant derivatives, which are plant active raw materials (or plant active
pharmaceutical ingredients) and (iii) herbal medicine product (or phytomedicine).
Plant crude drug or plant raw material is the medicinal plant or its parts, which contain com-
pounds responsible for the therapeutic action, after collection, harvesting, stabilization and drying
processes. The plant can be whole, fragmented, crushed or pulverized. Plant derivative is the prod-
uct obtained from the extraction of the fresh medicinal plant, or plant raw material that contains
the substances responsible for the therapeutic action. The product can be in the form of extract,
fixed and volatile oil, wax, exudate and others. Herbal medicine product or phytomedicine is the
product obtained from an active plant raw material, except isolated substances, with prophylactic,
curative or palliative finalities, including herbal medicines and traditional herbal products. The
product can be simple, when the active ingredient originates from a single medicinal plant species,
or combined, when the active ingredient originates from more than one plant species.
The physico-chemical tests established for the quality control testing routine of the plant raw
material, the corresponding derivatives and phytomedicines can be basically divided into four parts:
(I) identification tests, (II) quantitative analysis, (III) purity and integrity tests and (IV) charac-
terization tests, which are described in Table 2.1. Such tests, when interpreted together, supply a
comprehensive physico-chemical profile and the overall quality of the product under evaluation.
Except for the purity and integrity tests, and the characterization tests, which are applicable to
any plant species, some determinations are very specific, for example, the qualitative and quantita-
tive analysis of a certain chemical marker or active principle of a plant species. If such a species
does not yet have an official monograph, the development and validation of the method for chemi-
cal analysis is necessary. Because of the high chemical complexity of such a matrix, the analyst or
researcher must have good multidisciplinary knowledge, and an accurate critical sense to select the
best sample preparation, as well as to interpret the final results.
Apart from being necessary for the reliability of the results in the context of the quality sys-
tem, the validation of analytical methods is justified for legal, technical and commercial rea-
sons. The international bodies and agencies, such as IUPAC (International Union of Pure and
Applied Chemistry), FDA (Food and Drug Administration) and ICH (International Conference
TABLE 2.1
List of the Main Physico-Chemical Tests Applicable to the Quality of Plant Raw Materials
and Plant Derivatives
Plant Raw Liquid Dry Essential Fixed
Type Tests Material Extracts Extracts Oils Oils
I Organoleptic analysis X X X X X
Macroscopic botanical identification X
Microscopic botanical identification X
Chemical identification X
Chromatographic fingerprinting X X X X X
II Quantitative analysis X X X X X
III Water and volatile material X X
Total ash X
Acid-insoluble ash X
Foreign material X
Extractable material X
Content of essential oils X
Pesticide residues X X X X X
Heavy metals X X X X X
Radioactivity contamination X
Aflatoxins X X X X X
Solvent residues X X X X
Dry residue X
IV pH X
Relative density X X X
Apparent density X
Alcohol content X
Refraction index X
Optical rotatory power X
Solubility X
Viscosity X X
Saponification value X
Acidic value X
Ester value X
Iodine value X
(I) identification tests; (II) quantitative analysis; (III) purity and integrity tests; (IV) characterization tests.
on Harmonization), and the Brazilian organizations such as INMETRO (National Institute of

Metrology, Quality and Technology) and ANVISA, provide guidelines and recommendations for
the execution of validation procedures, a fundamental requirement for demonstration of process
technical competence.
In Brazil, ANVISA displays the resolution RDC n. 166 from July 24, 2017, which establishes
criteria for the validation of analytical methods applicable to pharmaceutical ingredients, medi-
cines and biologic products in every production step. Among the parameters to be evaluated, are
selectivity, linearity, accuracy, precision (repeatability and intermediate precision), detection limit,
quantification limit, linear response range and robustness. The experimental procedures, limits and
criteria of acceptation must be defined according to the method to be validated, and according to the
values recommended in the guidelines mentioned above.
2.3 RECEIVING AND SAMPLING PLANT RAW

MATERIALS AND PLANT DERIVATIVES
Usually, plant raw materials are dehydrated, which allows for better handling and storage, together
with improving the conservation and durability of the crude drug. Liquid extracts can be commer-
cialized concentrated or diluted. In both cases, in the act of receiving, the analyst must pay attention
to the packaging conditions, verifying the conformity regarding the type of material used (plastic,
paper, cardboard), hygiene (presence of dirt and stains in the external part) and sealing. When
opening a crucial aspect is to pay attention to the level of division and compaction of the plant
(in the case of plant raw materials), the presence of physical (shards of glass, pieces of paper, sand
and rocks) and biologic contaminants (mold, insects, etc.). Only then, the following initial organo-
leptic analysis, verifying the aspect, texture, color and smell of the material should take place.
The next step is the sampling, which should be executed carefully, so that the results express
representative values of the total quality of the batch. Accordingly, establishing a sampling plan
according to the norms of the defined quality system, considering the procedures of the pharmaco-
poeia or those described in publications such as ISO 2859 is recommended– Sampling procedures
for inspection by attributes, the WHO Guidelines for Sampling of Pharmaceutical Products and
Related Materials, or the NBR 5426 from 1985 – Sampling plans and procedures in the inspection
of attributes, among others.
For plant raw materials, the Brazilian Pharmacopoeia 5th edition suggests a sampling plan,
which considers the amount of sample available, the numbers of containers and the level of division
of the drug (Table 2.2). Overall, for drugs finely fragmented, pulverized or with dimensions smaller
than 1.0 cm, the sample must be at least 250.0 g, considering a total amount batch up to 100 kg,
and/or at least an amount, which is sufficient for the execution of every physico-chemical test with
further amount for sample. For drugs with dimensions greater than 1.0 cm, the sample must be at
least 500.0 g. If the total amount of the received batch is smaller than 10 kg, the final sample must
be at least 125.0 g. The samples must be taken in similar amounts from the superior part, middle
and inferior part of each container to be sampled. In the end, every portion must be joined, homog-
enized and divided in four equal parts, separating the sample over a square area. Then, the two
diagonal parts must be put together followed by the remaining parts. If necessary, the process must
be repeated until there is no accentuated difference in the dimensions of the fragments in question.
Finally, a part of this sampled and homogenized material must be retained as a reference sample,
which might be used in any retesting procedures if necessary.
The physico-chemical analysis of the plant sample properly homogenized, standardized and labeled
must be initiated by an organoleptic analysis, verifying the aspect, color and smell of the sample. Also,
before initiating the assays, the plant material must be pulverized and the size of the particle must be
standardized with a standard sieve, aiming to guarantee reproducible and reliable results.
TABLE 2.2
Sampling Plan for Plant Raw Materials Preconized by the
Brazilian Pharmacopoeia, 5th Edition
Number of Packages (units) Number of Packages to Be Sampled
1–3 All
4 – 10 3
11 – 20 5
21 – 50 6
51 – 80 8
81 – 100 10
More than 100 10% of the total of packages
2.4 PHYSICO-CHEMICAL TESTS FOR THE QUALITY

CONTROL OF PLANT RAW MATERIALS
The physico-chemical tests for the quality control of plant raw materials can be divided into iden-
tification tests, quantitative analysis purity and integrity tests (Table 2.1), so that the compilation
of the results allows a very careful evaluation on the authenticity and quality of the material under
investigation.
The identification tests aim to determine the authenticity of the plant material. Among the most
important tests are the macroscopic and microscopic botanical examination, the qualitative analy-
sis by phytochemical prospection (chemical identification) and qualitative analysis of the chemical
profile by chromatographic fingerprinting.
The purity and integrity tests may be interpreted as general tests, since the methodologies and
principles are applicable to any sample, regardless of the species concerned. Such tests provide
information on the drying process, cultivation and harvesting conditions, pesticides contamination,
storage, possibility of adulteration and others. Among the physico-chemical tests recommended,
there are the determination of the water and volatile matter, total ash content, acid-insoluble ash
content, presence of foreign matter, essential oils content (if applicable), determination of extract-
able matter, radioactive contamination, determination of pesticide residues, heavy metals and
aflatoxins.
Finally, the quantitative analysis of metabolites groups or chemical markers depends on the
expression of the biosynthetic apparatus characteristic of each plant species. For this purpose, spec-
trophotometric methods for total determinations (such as determination of total flavonoids or total
phenols) and various chromatographic methods for quantitative determinations are very useful and
valuable for the complete characterization of the plant raw material.
2.4.1 Identification Tests
2.4.1.1 Macroscopic and Microscopic Botanical Examination
The botanical identification of the raw plant material is based on the comparison of the macroscopic
and microscopic botanical features of the sample with an authentic material, or with information
presented in monographs or reference literature. The macroscopic identification of the plant raw
material, if whole depends on the plant organ (flowers, leaves, skin, stem, roots and seeds) and
is based on the analysis of the flavor and smell (if possible), color, size (length, width, thickness),
surface, texture, fracture and appearance of the fracture or other characteristics described in mono-
graphs. Because of the subjective and probably misleading because of the presence of similar or
contaminating materials, the association of the analysis with the microscopic analysis is crucial,
being indispensable if the material is broken or powdered.
The procedure for the microscopic examination of plant raw materials consists in the exam
of histologic cuts of the plant material, whether intact or fragmented, or directly in the sample
if the plant is pulverized. For this purpose, microscopes equipped with 4×, 10× and 40× mag-
nifying lenses are typically used. Briefly, the preparation of the slides consists in (a) softening
or re-hydrating the dehydrated material (using water for fine tissues such as flowers and leaves,
or mixtures of water, ethanol and glycerol for rigid and dense tissues such as barks and stems);
(b) execution of the histological cuts (freehand or with the help of a microtome); (c) clarification
with chloral hydrate test solution (TS) or hypochlorite TS; (d) staining (simple or combined using
reagents such as safranin 1% (w/v) solution for observation of cutin, lignin and suberin; iodinated
zinc chloride TS for observation of cellulosic cell wall; phloroglucinol TS, for lignified cell wall
staining, among many others available); (e) slide assembly. Finally, the botanical features observed
in the microscope must be compared with slides prepared with authentic material, or with informa-
tion available in monographs of pharmacopoeias, specialized literature or in virtual databases of
images and illustrations.
2.4.1.2 Identification by Phytochemical Prospection or Chemical Identification

The phytochemical prospection is a fast, simple and low-cost test, which allows the identification of
chemical compounds or functional groups characteristic of the plant species. Briefly, a small portion
of the plant material is pulverized and extracted, aiming to obtain an extract rich in substances of
interest, such as alkaloids, anthraquinones, phenolic compounds, steroids, etc. Thereafter, specific
reagents solutions are added to the extract, in which color changes or precipitation will evidence
the class of the existing substances. Some of the most characteristic reactions and observed results
follow, which are described in Table 2.3.
TABLE 2.3
Common Tests for Phytochemical Prospection of Plant Raw Materials
Alkaloids
Acid/base extraction of the dewaxed material and partition with chloroform
Dragendorff (potassium iodide and bismuth subnitrate): orange or orange/red precipitate indicates the presence of alkaloids
Mayer (potassium iodide and mercuric chloride): white precipitate indicates the presence of alkaloids
Wagner/Bouchardat (iodine in potassium iodide): brown precipitate indicates the presence of alkaloids
Bertrand (silico-tungstic acid): white precipitate indicates the presence of alkaloids
Hager (saturated solution of picric acid): yellow precipitate indicates the presence of alkaloids
Vitali-Morin reaction (acetone and potassium hydroxide in methanolic solution): violet color develops in the presence of
tropane alkaloids
Wasicky reaction (p-dimethylaminobenzaldehyde in concentrated sulfuric acid): red color develops in the presence of
tropane alkaloids
Mandelin reaction (ammonium vanadate in concentrated sulfuric acid): violet to red color develops in the presence of
indole alkaloids
Otto reaction (potassium dichromate in concentrated sulfuric acid): violet color indicates the presence of indole alkaloids
Murexide reaction (potassium chlorate and concentrated hydrochloric acid, followed by evaporation and exposition to
ammonia vapor): pink/violet color indicates the presence of purine alkaloids (methylxanthines)
Anthraquinones
Hot hydroalcoholic extraction, followed by hydrolysis and partitioning with chloroform or n-hexanes
Bornträger reaction (diluted ammonium hydroxide): red color indicates the presence of 1,8-hydroxy-anthraquinones;
violet/blue color indicates the presence of 1,2-hydroxyl-anthraquinones
Cardiac Glycosides
Hydroalcoholic extraction followed by partition with chloroform
Keller-Killiani reaction (glacial acetic acid, ferric chloride and concentrated sulfuric acid): brown-reddish color in the
contact zone of the two liquids and bluish-green (gradual) in the layer containing acetic acid indicates the presence of
deoxy sugars from cardiac glycosides
Pesez reaction (glacial acetic acid, xanthydrol and heating): red color indicates the presence of deoxy sugars
Kedde reaction (3,5-dinitrobenzoic acid in ethanolic solution and alkaline environment): dark red/violet color indicates the
presence of the lactonic ring
Baljet reaction (picric acid solution in alkaline environment): orange color indicates the presence of the lactonic ring
Legal reaction (sodium nitroprusside in alkaline environment): dark red color indicates the presence of the lactonic ring of
cardenolides
Raymond-Marthoud reaction (m-dinitrobenzene in alkaline environment): orange or violet color indicates the presence
of the lactonic ring of cardenolides
Coumarins
Alkaline extraction with re-acidification and recovery in organic solvents
Detection by UV light (360 nm): a shiny blue or green fluorescence develops in the presence of coumarins
Reaction with alkaline solution (potassium hydroxide in methanol): yellow color develops due to the rupture of the lactonic ring
(Continued)
TABLE 2.3 (Continued)

Common Tests for Phytochemical Prospection of Plant Raw Materials
Flavonoids
Hydroalcoholic or alcoholic extraction
Shinoda reaction (concentrated hydrochloric acid and magnesium): orange/red color develops in the presence of flavonoids
Pew reaction (concentrated hydrochloric acid and zinc in methanol): red color develops in the presence of flavonoids
Boric acid complexation (boric acid and oxalic acid in acetone followed by evaporation, resuspension in ethyl ether and
observation under UV light): a greenish-yellow fluorescence indicates the presence of flavonoids
Reaction with aluminum chloride (alcoholic solution of aluminum chloride and observation under UV light):
intensification of the fluorescence/formation of a greenish-yellow color indicate the presence of flavonoids
Saponins
Hot aqueous extraction for general detection; acid hydrolysis of the aqueous extract and extraction with chloroform for
detection of sapogenins
Frothing Test: formation of stable persistent foam after the agitation indicates the presence of saponins
Haemagglutination test (red blood cell suspension): precipitation indicates the presence of saponins
Liebermann-Burchard reaction (acetic anhydride and concentrated sulfuric acid in chloroform): blue/green color
develops in the presence of steroid nucleus and dark pink/red color develops in the presence of triterpene nucleus
Salkowski reaction (concentrated sulfuric acid): red or reddish-brown ring indicates positive result for triterpene nucleus,
while a pink /violet ring indicates the presence of steroid nucleus
Steroids and Triterpenes
Alcoholic extraction followed by partition with chloroform or ether
Liebermann-Burchard reaction (acetic anhydride and concentrated sulfuric acid in chloroform): blue/green color
develops in the presence of steroid nucleus and dark pink/red color develops in the presence of triterpene nucleus
Salkowski reaction (concentrated sulfuric acid): red or reddish-brown ring indicates positive result for triterpene nucleus,
while a pink/violet ring indicates the presence of steroid nucleus
Tannins
Hot aqueous extraction
Haemagglutination test (red blood cell suspension): precipitation indicates the presence of tannins
Reaction with gelatin (gelatin solution or skin powder): precipitation indicates the presence of tannins
Reaction with alkaloids (quinine sulfate in acid environment): white precipitate indicates the presence of tannins
Reaction with copper acetate (copper acetate solution): precipitation or turbidity indicates the presence of tannins
Lead acetate reaction (solution of lead acetate in acid environment): precipitation or turbidity indicates the presence of
hydrolysable tannins
Reaction with bromine water: yellow or red precipitate indicates the presence of condensed tannins
Reaction with ferric chloride (aqueous solution of ferric chloride): blue color indicates the presence of hydrolysable
tannins and green color indicates the presence of condensed tannins
Stiasny reaction (concentrated hydrochloric acid and formaldehyde): red precipitate indicates the presence of condensed
tannins; hydrolysable tannins in solution can be detected with the addition of sodium acetate and methanolic solution of
ferric chloride (formation of blue color)
2.4.1.3 Identification by Chromatographic Fingerprinting

The chromatographic fingerprinting is a very versatile test, which supports the chemical identifica-
tion of the plant raw material. The analysis must be performed not only for plant raw materials, but
fundamentally also for the characterization of plant derivatives (extracts and essential oils) and fin-
ished products (phytomedicines). Because of the inherent characteristics of each type of matrix, the
first step is to pay attention to the sample preparation, which should include an efficient extraction
procedure, using the adequate solvents and, if necessary, followed by a pre-concentration step. Apart
from helping in the authenticity and characterization of the plant material, the chromatographic
fingerprinting allows the detection of eventually contamination or adulteration, which can occur by
the presence of other plant species or even by the addition of isolated substances. Among the most
used techniques, high performance liquid chromatography (mainly HPLC-UV, HPLC-UV-DAD
or HPLC-MS), gas chromatography (GC-FID or GC-MS) and thin-layer chromatography (TLC
or HPTLC) are important due to the methods low complexity, easy accessibility, dissemination
in several pharmacopoeias and low-cost. Besides, in the case of TLC the use of adequate staining
solutions (which can be general or specific) is important as well as making sure the bands have good
separation and resolution for the calculation of the Rf values. Regardless of the chosen chromato-
graphic technique, the comparison of the results with reference standards or materials is important,
which will allow the correct authentication of the plant material concerned.
2.4.2 Determination of Water and Volatile Material

Usually medicinal plants are dehydrated, a practice that prevents enzymatic reactions responsible
for the modification of the original chemical constitution, deterioration, and microbial and myco-
toxin contamination. In quality control, the determination of the water content allows to verify if the
batch was dehydrated properly and to calculate the content of active principles on a dry weight basis.
Methods of analysis such as the azeotropic (distillation with toluene) and the volumetric meth-
ods (also known as Karl Fischer method, which is based on the reaction between water and an
anhydrous solution of iodine and sulfur dioxide in methanol) can be used for the determination of
water content. However, the most used method is the determination of water by loss on drying. For
that, 2.0–5.0 g of the herbal material accurately weighed is placed in a previously dried and tared
weighing flask, and then dried in an oven at 100–105°C. Alternatively, a protocol using balances
attached to an infrared irradiation drying system is feasible. However, aside the water percentage,
the gravimetric method also considers the loss of volatile material, as essential oils. For plants that
contain resins or volatile substances that are altered in high temperatures, the measurementof loss
by desiccation in a desiccator containing phosphorus pentoxide, under atmospheric pressure, or
reduced pressure at room temperature is recommended.
2.4.3 Determination of Total Ash Content

The determination of total ash content allows the measurement of inorganic compounds content,
which can be originated from the plant tissue itself, such as carbonates, phosphates, chlorides, or
originated by exogenous adulterants such as sand, rocks and soil attached to the surface of the plant.
Furthermore, this test provides information about the hygiene conditions during the harvesting and
drying process. In general, the method of analysis consists of transferring 2.0–4.0 g of the pulver-
ized plant material accurately weighed to a previously ignited and tared crucible. Then the material
is ignited in an oven at 500–600°C, until obtaining a whitish residue, which indicates the absence
of organic matter. The percentage of remaining ashes in the sample is calculated by gravimetry.
2.4.4 Determination of Acid-Insoluble Ash Content

The determination of acid-insoluble ash provides the silica content (especially sand), present in the
sample. The procedure consists in boiling the residue obtained in the total ash determination with
diluted hydrochloric acid, filtration and ignition in an oven at 500–600°C. In the same way, the
determination of the acid-insoluble ash percentage is calculated by gravimetry.
2.4.5 Determination of Foreign Matter

The determination of foreign matter may be divided in three main levels: (i) if there are parts of the
herbal material other than those described in the monograph of a given plant or if they are present
above the limits specified by the monograph; (ii) if there is the presence of any organisms, parts or
sub-products other than those specified in the plant monograph; (iii) if there are impurities of min-
eral or inorganic nature, such as rocks, sand, soil, etc. The determination can be made by weighing
the sample before and after the visual inspection and manual separation (with the help of tweezers
and magnifying lenses, if necessary) of any foreign material present. The results must be registered
in terms of percentage. For example, the Brazilian native species Espinheira Santa (Maytenus
ilicifolia Mart. ex Reissek – Celastraceae, Brazilian Pharmacopoeia, 5th edition) must be consti-
tuted by dry leaves and might contain the maximum of 2.0% of foreign material, whether they are
branches or pieces of stems, insects, parts of other plants, rocks, sand, etc.
2.4.6 Determination of Essential Oils Content

For those plants rich in essential oils the determination of the essential oils content is recommended,
since frequently they are responsible for the pharmacological effect, for example Cordia verbenacea
DC (Boraginaceae), whose essential oil is the active principle of a phytomedicine in Brazil recom-
mended as a topical anti-inflammatory agent. If the amount of essential oil is lower than specified in
the monographthat can be an indication that the plant material was dehydrated at high temperatures,
impairing the quality of the raw material and the corresponding derivatives. Besides the negative
impact in the essential oil yield, the qualitative and quantitative chemical profile are considerably
modified, since lower boiling point mono- and sesquiterpenes volatilize more quickly than the higher
boiling point ones. Also, if a plant rich in essential oils is dehydrated under high temperatures, this
can lead to the formation of undesirable products through oxidation or degradation. The most used
method for the determination of the essential oil’s content in plants is steam distillation, which allows
the separation and determination of the percentage of oil in relation to the dehydrated crude drug.
2.4.7 Extractable Material
This method determines the quantity of active compounds that can be extracted with a certain
solvent from a given amount of the plant raw material on a laboratory scale. Although considered
a characterization test, the results provide information regarding the quality from the performance
point of view and, consequently, about the expected yield on the industrial scale.
The extraction can be made using portions of 2.0–4.0 g of the plant material and 100 mL of
solvent applying at least three methods: (i) hot extraction, using water; (ii) by cold maceration using
ethanol or other solvent specified in the drug monograph; (iii) Sohxlet extraction using ethanol.
Also, for research and industrial scaling up purposes, the processes of maceration and percolation
using all the different proportions of hydroalcoholic mixtures (or other solvents) can be used. The
result is obtained through the determination of the dry residue of the obtained extract by gravim-
etry, using an oven at 100–105°C. The final result is calculated in mg/g or in % w/w.
2.4.8 Quantitative Analysis of the Active Principles and/or Chemical Markers

The quantification of the active principles or chemical markers (which may or may not be respon-
sible for the pharmacologic effect) is an obligatory quality requirement, being mandatory to be
performed for plant raw materials, plant derivatives (extracts, essential oils or others) and for the
evaluation of the final phytomedicine.
Such analysis is considered very complex due to the chemical variability of these phytochemi-
cals, the inherent biological variability of most of the species, and the dynamic limitations of the
different methodologies that can be applied. The great diversity of existing metabolites requires,
most times, the use of separation techniques coupled with detectors that provide some type of
spectral information. Also, the sample preparation must allow the extraction of the substances of
interest with good recovery. The quantification itself is achieved using analytical curves prepared
with reference standards in known concentrations.
For that reason, the best analytical technique should be considered for the development of an
analytical methodology for the quantification of phytochemicals, which must be sensitive and selec-
tive for the compound or compounds of interest. Several chromatographic arrays, colorimetric,
spectrophotometric, titrimetric and densitometric techniques are often used. Techniques such as
GC-FID or GC-MS and liquid chromatography (HPLC-UV, HPLC-UV-DAD, HPLC-MS) are
widespread and allow a very advantageous detection, in terms of both specificity and selectivity,
lowering the interference of other compounds. However, the use of other techniques like infrared
spectroscopy (IR), nuclear magnetic resonance (NMR), sequential mass spectrometry (MS/MS),
high performance liquid chromatography hyphenated to NMR or fluorescence detectors, capillary
electrophoresis (CE) and others may be very useful, especially if very little is known about the
chemical profile of the plant species concerned.
Total quantification is another approach for the quantitative analysis, which does not require a
chromatographic separation step. However, the sum of all substances belonging to a given chemi-
cal class, such as flavonoids, alkaloids, tannins, anthraquinones, etc. is considered. Total flavonoids
expressed in quercetin equivalents (or expressed in rutin) and total polyphenols expressed in gallic
acid equivalents are good examples of widespread methodologies. A classic example of this type
of quantification is described in the pharmacopeia monograph of Barbatimão (Stryphnodendron
adstringens Mart. Coville – Fabaceae; Brazilian Pharmacopoeia, 5th edition). The plant raw mate-
rial, composed by the dry stem barks, must contain, at least, 8.0% of total tannins expressed in pyro-
gallol equivalents, determined by spectrophotometry in the visible region of the spectrum (760 nm).
Another similar example is described in Espinheira Santa (Maytenus ilicifolia Mart. ex Reissek –
Celastraceae, Brazilian Pharmacopoeia, 5th edition) monograph. The plant raw material, composed
by the dry leaves, must contain, at least, 2.0% of total tannins expressed in pyrogallol equivalents,
of which at least 2.8 mg/g equals epicatechin. Except for differences in the sample preparation, the
methodology for the determination of total tannins is the same as that used in the determination of
total tannins of Barbatimão. However, the determination of epicatechin is achieved by HPLC-UV,
with the aid of an analytical curve of epicatechin.
2.4.9 Determination of Heavy Metals

Naturally, plants accumulate minerals as part of the nutritional or as a detoxification mechanism.
Elements such as aluminum, arsenic, cadmium, cobalt, lead and mercury are very common.
However, the over-accumulation is caused mainly due to the presence of metals in contaminated
soils, sediments or air, by cross contamination and sequestering of the pesticides. In general, heavy
metals are toxic not only for the plant organism, but also for human beings. Therefore, regulatory
agencies worldwide, including in Brazil, recommend the verification and control of the presence of
heavy metals in medicinal plants.
The determination of heavy metals can be made in terms of limit tests for specific metals or
total metals, using techniques such as colorimetry, voltammetry or atomic absorption. However,
the heavy metal determination using atomic absorption spectrometry provides more accurate and
reliable results.
2.4.10 Determination of Pesticides Residues

Pesticide residues include insecticides, herbicides, fungicides, fumigants for pest control during
storage and viral disease control agents. Because of the high toxicity for human beings and envi-
ronment, the use of these agents has been restrained worldwide. In Brazil, the use of pesticides in
medicinal plants plantations is prohibited. However, control is required due to the possibility of
cross- or accidental-contamination originating from other close plantations or from contaminated
water, air and soil. Therefore considering the history of pesticides’ persistent use in the area of cul-
tivation and to promote alternative actions for the pest control in agriculture is needed.
Often, the determination of pesticide residues is achieved by liquid or gas chromatography with
mass spectrometry hyphenation techniques preferably. The sample preparation procedure requires
a standard protocol in which impurities are removed by partition or adsorption (clean up) while
residues of a wide range of pesticides are concentrated.
2.4.11 Radioactivity Contamination
The radioactivity determination in plant raw materials must be performed when the plant species is culti-
vated in a place of probable radioactive contamination or in the proximity. Also the radioisotope activity
concentration and the types of radioactive contamination that might be present must be considered. For
this purpose, the measurements must be carried out by official laboratories in accordance with the inter-
national organizations recommendations, such as the Codex Alimentarius, the International Atomic
Energy Agency (IAEA), the Food and Agriculture Organization of the United Nations (FAO) and the
World Health Organization (WHO). In Brazil, the Institute of Radiation Protection and Dosimetry
(IRD) of the Brazilian Nuclear Energy Commission (under the authority of the Ministry of Science,
Technology, Innovation and Communications) is the official government reference body and the center
of expertise in radiation protection, dosimetry and metrology of ionizing radiation in the country.
2.4.12 Determination of Aflatoxins

Aflatoxins are secondary metabolites belonging to difurano-coumarins class, mainly produced by
Aspergillus flavus and A. parasiticus, fungi that contaminate foods and herbal medicines under
high temperature and humidity conditions. Since they are highly carcinogenic, their control is
required by the regulatory agencies worldwide. Among the aflatoxins found in plant matrices,
aflatoxins, B1, B2, G1 and G2 are the most important and must be controlled. Aflatoxins from
group B (B1 and B2) contain a cyclopentane ring and show a blue fluorescence under ultraviolet
light. Whereas aflatoxins from group G (G1 and G2) contain a lactone ring and exhibit a green and
yellow green fluorescence, respectively. Such fluorescence capacity allows the identification and
differentiation between aflatoxins from G and B groups.
According to the American Pharmacopoeia, herbal materials with history of contamination by
mycotoxins must be tested regarding the presence of aflatoxins. The maximum limit for the pres-
ence of aflatoxins according to this pharmacopoeia is 5 µg of aflatoxin B1 per kg of plant material
or 20 µg/kg for the sum of the aflatoxins B1, B2, G1 and G2. Also, the American Pharmacopoeia
highlights three possible methods for the quantitative analysis of aflatoxins, which are described
in Chapter 561 (Articles of Botanical Origin). The method of choice should be Method I, which
applies thin layer chromatography and the use of standard solutions for the aflatoxins. Alternatively,
Methods II (HPTLC) or III (HPLC) can also be used (The United States Pharmacopoeia, 2014).
The European Pharmacopoeia recommends the use of high performance liquid chromatography
and the maximum limit of aflatoxin B1 is 2 µg/kg, or 4 µg/kg for the sum of the aflatoxins B1, B2,
G1 and G2, unless there is a specific value in the monograph (European Directorate for the Quality
of Medicines and Health Care, 2007). The Brazilian legislation follows the same limits and proce-
dures recommended by the European Pharmacopoeia.
2.5 PHYSICO-CHEMICAL TESTS FOR THE QUALITY CONTROL OF PLANT

DERIVATIVES: LIQUID EXTRACTS, SOFT EXTRACTS AND DRY EXTRACTS
The physico-chemical tests for the quality control of liquid extracts (fluid extracts and tinctures),
soft extracts and dry extracts can be divided in identification tests, purity and integrity tests, quan-
titative analysis and, additionally, characterization tests.
Fluid extracts and tinctures are preparations of liquid consistency that are usually obtained from
dried plant parts by maceration or percolation. They concentrate the active ingredients extracted
from the plant matrix and the main difference between them is the extraction ratio: fluid extracts
contain a 1:1 ratio of dried plant raw material/extract and tinctures contain a 1:5 or 1:10 ratio dried
plant raw material/extract. Because of the liquid nature, the organoleptic analysis must evaluate the
following: aspect (presence of particles in the liquid, turbidity level, transparency), color (usually
amber, yellowish or greenish, light or dark), smell and flavor (characteristic of the plant, being bitter
or sweet, spicy or astringent, etc.).
Soft extracts are semi-solid preparations obtained by partial solvent evaporation of liquid
extracts. The color is usually dark amber, and the smell and flavor are characteristic of the plant
species. On the other hand, dry extracts are solid preparations obtained by total evaporation of the
solvent used for their production, usually with the aid of a drying agent or excipient. The analysis
of the aspect refers to homogeneity of the sample, presence of particles or other foreign matters, the
color, smell and flavor if applicable.
For extracts, the identification tests are the same as those used for raw plant materials, except for
the macroscopic and microscopic botanical examination. One of the most used methods is chro-
matographic fingerprinting by TLC, HPLC or GC, as previously described in the identification tests
of the raw plant material. However, in some cases, this test does not require a previous and exhaus-
tive extraction step since the active principles or chemical markers are already extracted from the
plant matrix, facilitating the procedure.
Likewise, the quantitative analysis of the active principles/chemical markers is considered one
of the main tests for assessing the quality of such plant derivatives and should be performed using
the same methodologies developed for the plant raw material, except for any inadequacies regarding
sample preparation. Thereby, the spectrophotometric and titrimetric methods are often used for total
determinations (as for example, determination of total flavonoid or total phenolic content), as well as
chromatographic methods, such as GC-FID, GC-MS, HPLC-UV-DAD or HPLC-MS, mainly.
The purity and integrity tests determine the dry residue (also denominated soluble solids) for liq-
uid and soft extracts. For dry extracts, the water and volatile material can be efficiently determined
by loss on drying. The determination of solvents residues other than hydroalcoholic solutions must
be performed if hazardous extraction solvents are used in the extraction process. Also, depending
on the need, the determination of the presence of pesticide residues, heavy metals and aflatoxins, as
described for raw plant materials is required.
Finally, the characterization tests of liquid extracts contemplate the determination of the relative
density, pH, viscosity and alcohol content. For dry extracts the characterization tests comprise the
granulometry, apparent density and solubility.
2.5.1 Determination of Dry Residue (or Soluble Solids)

The classic extraction methods for the production of fluid extracts and tinctures apply a given amount
of the plant raw material, which can be fresh or dried, for a certain final product volume, usually
using hydroalcoholic solutions. Aiming to guarantee a minimum content of active principles per
milliliter of product, the determination of soluble solids content is an important tool for the extrac-
tion process monitoring and to assess the quality of the final product. Moreover, the determination
of the dry residue is very useful when the content of active principles or chemical markers present
in the final extract is not possible to quantify.
The simplest method of analysis is the evaporation of the extract solvent in an oven at 100–105°C
and calculation of the percentage of dry residue by gravimetry. Results are usually expressed in
mg/mL or in % w/v.
2.5.2 Determination of Solvent Residues

Liquid extracts are usually produced with the use of hydroalcoholic solutions. However, obtain-
ing some standardized extracts requires the use of less friendly solvents, such as methanol, ethyl
acetate, n-hexanes or acetone, among others. The use of such solvents can be necessary, depending
on the characteristics of polarity and solubility of certain classes of plant metabolites, or to increase
the efficacy of the manufacturing process.
However, even if the final product has the solvent removed, as in the case of dry extracts, there
is the difficulty of trace residues. Therefore, regulatory agencies worldwide have established limits
of tolerance of solvent residues in liquid or dry plant extracts. For the qualitative and quantitative
determination of these residues, the most applied method is gas chromatography attached to flame
ionization detector (GC-FID) or mass detector (GC-MS): the latter being the more advantageous
regarding specificity and selectivity.
2.5.3 Relative Density
The determination of the relative density of an extract can be an indicator for the type of extraction
solvent used in the process of maceration and percolation. When hydroalcoholic combinations are
used, the relative density values are lower than 1.0 (which is the relative density value of water at
20°C) and tend to be lower, as the ethanol percentage increases. Whereas, when propylene glycol or
glycerin is used as extraction solvents, the values of relative density are higher than 1.0 and decrease
if water or ethanol is added.
The most common methods used to determine the relative density of an extract include the use
of hydrostatic balance, densimeter or pycnometer in which the latter is simpler and less expensive.
2.5.4 Apparent Density (or Tapped Density)

The apparent density is an important parameter for the characterization of dry extracts. By defini-
tion, this corresponds to the volume occupied by a determined mass of solid, powder or grainy mate-
rial, considering the total volume of the sample, including any empty space between the particles.
This differs from the real density, which is the real volume occupied by a determined solid, not taking
into consideration the porosity. The apparent density is influenced by the powder’s particle shape,
size, and size distribution, among others. The analytical procedure for the determination is a quick,
cost-effective and standardized method. The test consists in measuring the volume occupied by a
given amount of the sample introduced in a test tube after tapping the tube against a rigid bulkhead
until little further volume change is observed. The measured value is generally expressed in g/mL.
2.5.5 Granulometry
The granulometry is also a characterization test of samples in solid state (powder or grains) that is
determined with the help of sieves operated by mechanical devices. Depending on the characteris-
tics of the sample, a set of sieves is assembled so that the ones with the greatest opening overlap
the sieve with the smaller opening. Approximately 25.0 g of the sample (depending on the nature
of the material, density and diameter of the sieves to be used) is transferred to the superior sieve
and the set is subjected to mechanical vibration. After this process ends, the sample resting in each
sieve is removed, weighed and the retaining percentage is calculated.
2.5.6 Solubility
The determination of the solubility of a dry extract in any given solvent and temperature consti-
tutes another characterization test. This information is important both to assess the quality of the
received batch and as a parameter in formulation studies. The analytical procedure is simple and
consists adding increasing portions of the sample to constant volumes of solvent in which the extract
must be analyzed. The total amount of solute is determined in the supernatant liquid. Results are
calculated by gravimetry and usually expressed in mg/g.
2.5.7 pH
The determination of the pH is important, not only as a characterization parameter of liquid

or solid extracts, but also as a crucial factor for their use in formulations, as this affects both
solubility and stability. The pH measures the acidity of a substance, which depends on the con-
centration [H+] or [H3O +] ions dissociated in the solution. Depending on the extract, the pH
can be measured directly with the insertion of the electrode of the pH meter in the sample.
Alternatively, a previous water dilution step may be required, especially for highly concentrated
extracts and dry extracts.
2.5.8 Viscosity
The viscosity test expresses the resistance of a liquid to the flow: in a given temperature, the less
viscous the liquid, more fluidly . If the temperature increases, the kinetic energy of the molecules
also increases, decreasing the time in which the molecules remain cohesive. Consequently, viscosity
is reduced. Therefore, since this is a property related to all liquids, this determination is a very use-
ful parameter for the quality control of liquid extracts and tinctures. The results provide indications
regarding the type of extraction solvent used in the percolation (for example, extractions performed
with 70% ethanol provide extracts less viscous than extractions made with propylene glycol). Also,
depending on the plant species used, the final extract may be more or less viscous. For example,
plants rich in mucilage provide more viscous extracts than those deficient in this chemical group.
Finally, rapid information concerning the concentration of soluble solids in the extract can also be
obtained: an extract at 2.5% w/v is less viscous than an extract at 20.0% w/v.
The simplest and most cost-effective method measures the time required for the test liquid to
flow through capillary tubes (using for example the viscometers of Ostwald, Ubbelohde, Baumé and
Engler) or Ford cup orifice viscometer. The result is expressed in seconds (s). Similarly, the sphere
viscometer measures the velocity of falling of a sphere inside a tube containing the sample. The
greater the viscosity of the sample, the lower the velocity of falling. Finally, other more sophisti-
cated methods determine the viscosity measuring the resistance of the rotation movement in metal-
lic axes when immerse in the liquid (for example using the Brookfield viscometer), with results
expressed in centipoise (cP) or centistokes (cSt).
2.5.9 Alcohol Content
Most of the liquid extracts produced for medicinal purposes are obtained using hydroalcoholic com-
binations. The choice of the alcohol concentration of the extraction solvent is made according to the
class of the compounds that is going to be extracted, and the intended use. On that account, concen-
tration standardization is important in order to guarantee the quality and efficacy of the final product.
The determination of the alcohol content, along with other tests, provide important information
regarding the physico-chemical profile of the extract. The greater the ethanol proportion in the
extraction solvent combination, the greater the alcohol content, the lower the relative density and
consequently the lower viscosity. Furthermore, deviations in the alcohol content in the final prod-
uct may indicate problems in the extraction process, such as temperature out of control, moisture
content above the specified limits for the plant raw material, errors during the preparation of the
extraction solvent, among others.
The simplest and most inexpensive method consists in determining the alcohol content by direct
measurement using an alcoholmeter. This is a simple and direct method, very useful for process
control. However, depending on the alcohol content of the extract, at levels lower than 30%, the
distillation method is more precise. This method consists in distilling a portion of the sample along
with an equal quantity of water and determining the density of the distilled liquid at 20°C. The
results are calculated with the aid of an alcoholometric table.

CONTROL OF ESSENTIAL OILS
Essential or volatile oils are products obtained from parts of the plant raw material (leaves, fruits,
flowers, inflorescences or stem barks) and constitute complex combinations of volatile, lipophilic,
odoriferous and liquid substances. They are obtained especially through steam distillation, by
pressing citric fruit pericarps, or even by lipophilic extraction using organic solvents (with the dis-
advantage of also extracting fixed oils and other lipophilic substances). They can also be obtained
through procedures of extraction such as Enfleurage (very common for the extraction of essen-
tial oils of flowers) or still by supercritical CO2 extraction. Chemically, most of the essential oils
are composed by derivatives of phenylpropanoids (especially phenols and its ethers), constituting a
group of metabolites which have very peculiar physical and physico-chemical characteristics.
The common tests for the quality control of essential oils include the identification tests
(organoleptic analysis and chromatographic fingerprinting), purity and integrity tests (solvents
residues, heavy metals, radioactivity, pesticides residues) and quantitative analysis. The main
characterization tests include the determination of the refraction index, rotatory power and rela-
tive density, which also contribute to the identification and determination of purity and integrity
of essential oils.
The identification of the essential oils is enhanced by the organoleptic characteristics. They
have an acrid and spicy flavor and a smell that is very characteristic of each plant species from
which the essential oil was extracted. Frequently, an expert analyst is able to differentiate essential
oils obtained from different plant species from the same genera. For example: the essential oil
of Eucalyptus globulus Labill (Myrtaceae) has a strong smell of camphor, totally different from
the essential oil of another species from the Eucalyptus genera, E. citriodora Hook (Myrtaceae),
which is characteristic of citronellal. Regarding coloration, normally they are colorless or slightly
yellowish, apart from some exceptions, as is the case of the essential oil of Matricaria recutita
L. Rauschert (Asteraceae), which is blue due to its azulene content.
A noteworthy aspect of essential oils is the volatility. Further feature, when evaporated, they
should not leave residues. This factor is important when one suspects that the sample was adulter-
ated with fixed oils, which leaves oil residues when subjected to evaporation. In practical terms, a
drop of the sample is applied on a dry paper filter and left to stand at room temperature or in an oven
at controlled temperature. If a translucent stain remains, this is an indication of the adulteration by
fixed oils.
The identification using chromatographic techniques as TLC allows a quick, simple and afford-
able analysis, also allowing the detection of contaminants and adulterants. For this purpose, the
identification of some compounds can be made through staining and Rf calculation of the bands.
The results are compared with reference standards, photographs. Semi-quantitative analysis is also
possible to be performed by TLC, using standard solutions prepared in known concentrations, fol-
lowed by densitometry.
The use of gas chromatography, although of higher cost, is extremely useful and advantageous for
the analysis of essential oils. The method is performed using capillary columns, which allows great
separation of compounds and consequently excellent efficiency. The most used detection systems
are the flame ionization detection (GC-FID) and the mass spectrometry detection (GC-MS), the last
providing precise information regarding the identity and purity of the peaks. Approaches like the
comparison with the retention times of reference standards, the calculation of retention index, or the
comparison with databases and libraries containing information regarding the molecular mass and
fragmentation profile, allow a very fast and reliable qualitative analysis of essential oils.
For the quantitative analysis, as well as for other quantitative methods, the need to establish
analytical curves using reference standards prepared in known concentrations is necessary. The
calculations are made as a function of the areas, which are directly proportional to the quantity of
the corresponding compounds in the sample.
2.6.1 Determination of the Refraction Index

The refraction index of a pure substance under given temperature and pressure conditions is an
indicative parameter for identification. For the essential oils, the refraction index varies from 1.450
to 1.590 when compared to water at 20°C, which is 1.333. If the sample is adulterated with fixed
oils, ethanol, water or other solvent, the refraction value index is modified, indicating the presence
of adulterants or contaminants.
2.6.2 Determination of the Optical Rotatory Power

Essential oils show optical activity due to their chemical constitution: most of the compounds have
stereogenic centers. They have the capability to rotate the plane of polarized light in a way that the
transmitted light is deviated in a certain angle in relation to the incident one. For a certain sub-
stance, if one of the enantiomers deflects the plane of polarized light to the right (+), this is called
dextrorotatory, while the other, deflecting the plane of polarized light to the left (−) is called levo-
rotatory. The angle of this reflection is the same in magnitude for the enantiomers, however with
opposite signs.
The determination of the rotational power in essential oils is performed by polarimetry, and the
resulting value is equal to the sum of all substances present in the oil. The analysis of the experi-
mental values when compared with the reference values allows to obtain information about the
authenticity of the essential oil concerned, possible adulterations, or even if the sample is originated
from a natural or synthetic source.
2.6.3 Determination of the Relative Density

The determination of the relative density is a simple procedure, as described for the evaluation of
liquid extracts, and can be an indicative of the quality of the sample. For essential oils, the relative
density varies from 0.690 to 1.118, so that this is characteristic to each type of oil. The most used
method is the determination of the relative density by pycnometry.

CONTROL OF THE FIXED OILS
Fixed oils consist mainly of triacylglycerols, which are esters of long-chain fatty acids (identical or
different) and glycerol, and their corresponding derivatives. Such compounds can be biosynthesized
by both animals and plants, and, along with fats and waxes, they compose the group of lipids, whose
main biological function is the storage of energy. In general, oils can be extracted by cold pressing
or extraction with organic solvents from many parts of the plant; however, the seeds contain greater
quantities.
The importance of studying and controlling the quality of such compounds is explained by the
great economic value for the food, energy, pharmaceutical and cosmetic industry. Classic examples
of the high-value commercial products can be highlighted: olive oil (Olea europaea L.; Oleaceae),
soy oil (Glycine max L. Merril, Fabaceae), castor oil (Ricinus communis, Euphorbiaceae), argan oil
(Argania spinosa L.; Sapotaceae), almond oil (Prunus amygdalus Batsch, Rosaceae), among others.
Physico-chemically these oils are liquids at room temperature, non-volatiles (fixed), insoluble in
water and soluble in organic solvents. Exceptions may occur, such as the coconut oil (Cocos nucifera L,
Arecaceae), which tends to be solid at room temperature. According to the chemical composition of
the fatty acids present in each oil, they may be classified as saturated fixed oils, which are mainly
composed by glyceryl esters of saturated fatty acids, such as lauric, myristic, palmitic and stearic
acids; monounsaturated fatty acids, mainly composed by glyceryl esters of monounsaturated fatty
acids, such as oleic and euric acids; and polyunsaturated fatty acids, mainly consisting of glyceryl
TABLE 2.4
Main Physico-Chemical Tests for the Characterization of Fixed Oils
Test Definition Finality
Saponification value Amount of potassium hydroxide (mg) The saponification value provides indication about
required to neutralize the free acids and adulterations of the fats with unsaponifiable
saponify the esters present in 1.0 g of sample substances (mineral oil, for example)
Acid value Amount of potassium hydroxide (mg) High acid value suggests hydrolysis of the esters
required to neutralize the free fatty acids in which constitute the fatty matter. It may be
1.0 g of sample caused by chemical industrial treatments during
extraction and purification, bacterial activity,
catalytic action (heat, light), inappropriate
storage and presence of impurities such as
humidity, among others
Ester value Amount of potassium hydroxide (mg) Ester value equal or very close to the
required to saponify the esters present in saponification value indicates a good quality oil
1.0 g of sample. It can be calculated by the
difference between the saponification value
and the acid value
Iodine value Amount of iodine (g) absorbed by 100 g of Provides the level of unsaturation of the esterified
substance and free fatty acids. Suggests the level of purity
and highlights the presence of adulterants
esters of unsaturated fatty acids, such as linoleic and linolenic acids. Therefore, the degree of unsat-
uration will directly reflect the distinct physico-chemical characteristics of each oil.
The physico-chemical tests required for the quality control of such products are the same as
recommended for the analysis of essential oils and include the identification tests such as TLC
and gas chromatography (the last requiring a previously sample derivatization step), quantitative
analysis, and purity and integrity tests. However, the characterization of fixed oils includes the
determination of the saponification value, acid value, ester value, and iodine value, as summarized
in Table 2.4. Furthermore, the pharmacopoeia characterization tests include the determination of
the melting temperature, solidification temperature, refraction index, unsaponifiable matter, fatty
acid composition, peroxide value and hydroxyl value, which should also be performed according to
the monograph of each oil and destination of the final product.

CONTROL OF PHYTOMEDICINES
Pharmaceutical formulations can be classified in solid formulations (powders, granules, tablets,
capsules, suppositories), semi-solids (ointments, gels, creams, pastes) and liquids (syrup, suspen-
sion, solutions). The physical and physico-chemical tests applicable are defined according to the
pharmaceutical formulation in which the extract of natural origin was incorporated. Therefore, the
quality control of phytomedicines is based on the same general methods for assessing the confor-
mity of formulations of any other medicines containing synthetic drugs. Such tests are required
not only for the routine quality control, but also for the registration and stability studies. Table 2.5
summarizes the main physical and physico-chemical tests for the quality control of very common
phytomedicines formulations while Table 2.6 lists all plant species exemplified in this chapter.
Although such tests are exhaustively described in the pharmacopoeias, special attention must be
given to the qualitative and quantitative analysis of the active principles or chemical markers, since
they are specific to the plant material responsible for the pharmacological action. Very often, for
pharmacopoeial formulations, these methods are described in the respective monographs. However,
TABLE 2.5
List of the Main Physico-Chemical Tests for the Quality Control of Phytomedicines
Ointments, Creams, Syrups, Solutions
Tests Tablets Capsules Suppositories Liniments and Gels and Suspensions
Aspect, colour, smell, dimensions, texture X X X X X
Qualitative analysis (chromatographic X X X X X
profile)
Content of active ingredients or chemical X X X X X
markers
Average mass X X X X
Determination of volume X
Uniformity of the unitary doses X X X X X
Desintegration X X X
Dissolution X X X
Hardness X
Friability X
Content of water X X
Softening temperature X
pH X X X
Relative density X
Viscosity X X
in the absence of official monographs, the method for the qualitative and quantitative analysis must
be developed and validated according to the same validation standards suggested for medicines
containing synthetic drugs.
Finally, the importantance of regulations procedures and monographs, both in Brazil and world-
wide are highlighted, which are available and must be practiced extensively to ensure quality of
phytomedicines. For a country such as Brazil which has such a rich biodiversity and depends on the
commerce of phyto-medicinal products, the quality control of medicinal plants, plant derivatives
and phytomedicines is very important.
TABLE 2.6
Summary of the Names of All Plant Species, Their Respective Family and Common Names
Discussed in This Chapter
Scientific Name Family Common Names
Argania spinosa (sin. Sideroxylon argan) Sapotaceae Argan tree, tree of iron
Cocos nucifera Arecaceae Coconut palm
Cordia verbenaceae (sin. Cordia curassavica) Boraginaceae Erva baleeira and maria-milagrosa
Eucalyptus citriodora (sin. Corymbia citriodora) Myrtaceae Lemon-scented gum
Eucalyptus globulus Myrtaceae Southern blue gum
Glycine max Fabaceae Soybean
Matricaria recutita (sin. Matricaria chamomilla) Asteraceae German chamomile
Maytenus ilicifolia Celastraceae Espinheira santa
Olea europaea (sin. Phillyrea lorentii) Oleaceae Wild olive, brown olive, Indian olive, olienhout
Prunus amygdalus Rosaceae Almond
Ricinus communis Euphorbiaceae Castor bean
Stryphnodendron adstringens (sin. Stryphnodendron Fabaceae Barbatimão
barbatimam)
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inspeção por atributos. NBR 5426. http://www.saude.rj.gov.br/comum/code/MostrarArquivo.php?C=
Njg1Nw%2C%2C.
Batalha, M. O.; Ming, L. C. 2003. Plantas medicinais e aromáticas: um estudo de competitividade no Estado
de São Paulo. São Paulo: Sebrae.
Calixto, J. B. 2000. Efficacy, safety, quality control, marketing and regulatory guidelines for herbal medicines
(phytotherapeutic agents). Brazilian Journal of Medical and Biological Research, 33, 179–189.
Di Stasi, L. C. 1996. Plantas medicinais: arte e ciência. Um guia de estudo interdisciplinar. São Paulo: Editora
da Universidade Estadual Paulista.
European Directorate for the Quality of Medicines and Health Care. 2007. European Pharmacopeia, 6th ed.
Strasbourg: European Pharmacopoeia Commission.
Evans, W. C. 1996. Trease and Evans’ Pharmacognosy, 14th ed. London: WB Saunders Company.
Instituto Nacional de Metrologia – INMETRO. 2016. Orientação sobre validação de métodos de ensaios químicos,
DOQ-CGCRE-008. http://www.inmetro.gov.br/Sidoq/Arquivos/CGCRE/DOQ/DOQ-CGCRE-8_05.pdf.
International Organization for Standardization. 1999. ISO 2859-1 – Sampling procedures for inspection
by attributes. Sampling schemes indexed by acceptance quality limit (AQL) for lot-by-lot inspection.
Geneva: British Standard BS 6001-1.
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C2%BA+49-2010.pdf/06bfd0e9-c2d2-4fa5-8c9e-bce42fe92592.
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3 The Widening Panorama
of Natural Products
Chemistry in Brazil
Maria Fátima das Graças Fernandes da Silva,
João Batista Fernandes, Moacir Rossi Forim,
Michelli Massaroli da Silva, and Jéssica Cristina Amaral
Chemistry Department, São Carlos Federal
University, São Carlos, Brazil
CONTENTS
3.1 Introduction and an Overview of Natural Products Chemistry and
Chemosystematics in Brazil.................................................................................................... 33
3.2 Structural Diversity and Distribution of Secondary Metabolites
in Brazilian Plants.................................................................................................................... 41
3.2.1 Alkaloids...................................................................................................................... 45
3.2.2 Terpenes....................................................................................................................... 51
3.2.3 Phenylpropanoids........................................................................................................ 56
3.3 Impact on Brazilian Ecosystems.............................................................................................. 56
References�� 65
3.1 INTRODUCTION AND AN OVERVIEW OF NATURAL PRODUCTS

CHEMISTRY AND CHEMOSYSTEMATICS IN BRAZIL
Secondary metabolites (natural products) from different sources such as plants and microorgan-
isms have been and will continue to be one of the most important and promising sources in the
discovery of raw materials for the development of pharmaceuticals, agrochemicals and other bio-
products. Over thousands of years, mankind has used natural products for the treatment and pre-
vention of various diseases. Earliest reports of the use of natural products date back to 2600 BC,
in Mesopotamia. Among the various plant-derived substances described Cupressus sempervirens
(cypress), Commiphora species (myrrh) and Papaver somniferum (poppy juice) were mentioned,
which are indeed still used nowadays in the treatment of parasitic infections, colds and inflamma-
tions (Cragg and Newman, 2005; Cragg and Newman, 2013; Dias et al., 2012). To date, nature in
its vast biodiversity has proven to be a continuing and rich source of substances with great potential
not only for the treatment of countless diseases but also for non-biological purposes, highly relevant
to the world economy. However, it is estimated that only 15% of the existing higher plants (about
300,000 to 500,000) have already been studied phytochemically and even more dramatically, only
6% of them have undergone pharmacological investigation (Cragg and Newman, 2013). The value
of natural products to humanity is invaluable. From the knowledge acquired from generation to
generation and the exchange of experiences between different peoples, mankind uses bio-derived
and bio-inspired products in the most diverse areas ranging from food conservation and tinctures
33
FIGURE 3.1 Structures of dyes isolated from C. echinata (Pau-Brasil).
to prevention and treatments of diseases since the sixteenth century. This fact, together with other
existing organisms on the planet, clearly demonstrates the potential that nature has in providing new
substances with high added value.
Brazil stands out especially for the vast biodiversity. Different biomes, such as Caatinga, Pantanal,
Atlantic Forest, Cerrado, Pampa and Amazon, reveal an impressive variety of plants, microorgan-
isms and animals (Silva and Rodrigues, 2014). An estimation of 20% of the world-known species
are in Brazil, and due to the immense territory and complex ecosystems, many other species remain
yet to be discovered (Silva et al., 2010; Silva and Rodrigues, 2014). Overall, Brazil is in a privileged
situation in terms of natural products, with a biodiversity that is one of the major basis for the pro-
gressive discovery of new compounds with great potential for both biological and non-biological
applications, such as biofuels, textiles, and others.
Since the sixteenth century, Brazil has drawn attention to the immense and extraordinary
biodiversity. Among examples is the red dye Braziline, 1 and the oxidation product, Brazileine 2,
(Figure 3.1) extracted from Caesalpinia echinata (also known as Fabaceae; Pau-Brasil) that was
the main export product of colonial Brazilian time (Pinto, 1995; Pinto et al., 2002; Viegas Jr and
Bolzani, 2006).
During the scientific expeditions, the first Portuguese physicians soon recognized the impor-
tance of local plants used by indigenous tribes in healing numerous diseases (Pinto et al., 2002). The
zoologist Johann Baptist Spix and botanist and physician Carl Friederich von Martius carried out
systematic studies of the Brazilian fauna and flora (Pinto et al., 2002). Von Martius was crucial in
the first steps of Brazilian phytochemistry, and through him that the pharmacist Theodoro Peckolt
came to Brazil in 1847 to study the Brazilian flora. Due to his exceptional contributions, many con-
sider Peckolt the father of Brazilian phytochemistry (Pinto et al., 2002).
Among several important investigations, the isolation of the first iridoid from Plumeria lanci-
folia Mart, agoniadin, stands out today known as plumierid (3, Figure 3.2). The chemical structure
FIGURE 3.2 Structures of some natural products from Brazilian plants.

Natural Products Chemistry in Brazil 35
was elucidated 88 years after the compound was isolated, by Halpern and Schmid (Halpern and
Schmid, 1958; Pinto et al., 2002; Silva et al., 2010; Santos et al., 1998). In 1838, Ezequiel Correia
dos Santos isolated the first alkaloid from Brazilian plants, namely pereirine from Geissospermum
laeve (Vell.) Baill (“Pau Pereira”). The structure was elucidated many years later and was called
geissoschizoline (4, Figure 3.2) (Santos, 2007). When discovered, this plant had already long been
used by indigenous people for the treatment of various diseases such as inflammation, malaria
and others (Almeida, 2017; Bolzani et al., 2012; Silva and Rodrigues, 2014). Another example of a
substance, isolated from Brazilian plants, is tubocurarine (5, Figure 3.2), found in Chondodendron
tomentosum (Menispermaceae), widely used as a muscle relaxant during the preoperative period.
Furthermore, South American Indians have used it in the past for subsistence purposes such as
hunting and fishing. Over the years, several side effects were observed with the use of tubocurarine,
leading to the replacement with new synthesized anesthetics inspired in that compound (Bolzani
et al., 2012; Silva et al., 2010).
In Brazil, modern phytochemistry was introduced in the 1950s mainly by Walter Baptist Mors
and Otto Richard Gottlieb (Pinto et al., 2002; Valli et al., 2018). The former was responsible for the
creation of the current Research Center for Natural Products in Rio de Janeiro, one of the most pres-
tigious in Brazil. From the extensive work list done by Mors, it is worth highlighting the isolation
and identification of the active substance, 14,15-epoxygeranylgeraniol, from the oil obtained from
the fruits of Ptedoron pubescens Benth, which protects the mammalian skin from the penetration
of the cercariae of Schistosoma mansoni. In addition, he contributed intensively to the training of
numerous researchers in the field of natural products. Otto R. Gottlieb was considered the great-
est twentieth century phytochemist in Latin America due to his numerous contributions in terms
of articles, books and patents published, as well as in the training of numerous researchers. His
trajectory can be translated in more than 700 published articles, some patents on neolignans and
lignans of Lauraceae and Myristicaceae and several books (Caparica, 2012; Gottlieb, 1996; Silva
and Bolzani, 2012; Valli et al., 2018). He created a chemosystematics system that classifies plants
by their chemical characteristics, bringing rational methods for the search of bioactive compounds
in plants (Gottlieb, 1992; Silva and Bolzani, 2012). His group studied lignans and neolignans under
the chemosystematics, phytochemical and biological aspects (Gottlieb and Mors, 1980; Gottlieb and
Yoshida, 1989), leading to numerous publications, which correspond to approximately 75% of all
literature on these substances of the Brazilian flora. Lignan and neolignan derivatives have a broad
spectrum of biological activities such as antileishmanial, antitumor, antichagasic, and antimalarial
(Gottlieb and Mors 1980; Gottlieb, 1988; Silva Filho et al., 2008; Souza, 2012).
Gottlieb’s chemosystematic studies also show that there is a tendency for morphological diver-
sification to occur in parallel with chemical diversification. Thus, the morphological classification
of a genus is questioned, and characterizes it as a strong candidate for the search of substances dif-
ferent from those of the family chemical profile and with greater probability of being unpublished.
An example is the Brazilian species of Hortia, which is a neotropical genus of the Rutaceae, tradi-
tionally included in the subfamily Toddalioideae, subtribe Toddaliinae (Engler, 1931; ibid. Severino
et al., 2012). Historically, De Candolle (1824, ibid. Severino et al., 2012) described H. brasiliana
Vand., which was collected in the southeastern part of the state of Minas Gerais and in the state of
Rio de Janeiro, and Saint-Hilaire (1824, ibid. Severino et al., 2012) described a shrub collected in
Goiás and in western Minas Gerais, and also named it H. brasiliana. Engler (1874, ibid. Severino
et al., 2012) also attributed the name H. brasiliana to the shrubby species of central Brazil. In the
same study, he described a new arborescent species, H. arborea Engl. Recently, Groppo et al. (2005,
ibid. Severino et al., 2012) showed that H. brasiliana and H. arborea represent the same arborescent
species, and that H. brasiliana Vand. ex DC. is the correct name. Albeit well known, the shrubby
species found in central Brazil remained unnamed, so Groppo et al. (2005, ibid. Severino et al.,
2012) called it H. oreadica. The taxonomic interest in the Rutaceae motivated an investigation of
the taproots and stems of H. oreadica, and the result of this study and literature data showed that
Hortia produce highly specialized limonoids (6–16) that are similar to those from the Flindersia
(Flindersioideae). The taxonomy of Hortia has been debatable, with most authors placing this sub-
family in the Toddalioideae. Considering the complexity of the isolated limonoids, Hortia does not
show any close affinity to the genera of Toddalioideae (Severino et al., 2012; Severino et al., 2014).
Indeed, many chemists were perhaps interested in alkaloids from the Rutaceae and rarely identify
all potentially systematic important classes of compounds present in the plant. The low concentra-
tion of limonoids is also likely to be responsible for the complex limonoids having remained undis-
covered for many years in the genera of the Toddalioideae. Thus, complex limonoids appear to be
of little value in resolving the taxonomic situation of Hortia (Figure 3.3).
A well-known example of alkaloids in Rutaceae is pilocarpine, which is isolated from Pilocarpus sp.
(17, Figure 3.4), that is used in the relief of xerostomia (dry mouth), one of the side effects of radio-
therapy for the treatment of neck and head cancers (Almeida, 2010; Valli et al., 2018).
Among other taxonomic markers, we can mention the indole monoterpene alkaloids from
Apocynaceae and Rubiaceae, which may reveal evolutionary clues and help in the delimitation of
subfamilies (da Silva et al., 2010; Bolzani et al., 2012). These are some examples of several other
studies that demonstrate the importance of Brazil’s natural products in chemosystematics.
FIGURE 3.3 Typical limonoids of Hortia species (Rutaceae).

FIGURE 3.4 Structure of pilocarpine (17).
Currently, research groups in Brazil have been tackling many different scientific puzzles
in several fields. Among them are the following: chemical ecology, chemosystematics, bio-
logical activity, biosynthesis, analytical methodology, biotechnology, green chemistry, natural
products with added value, natural products of marine origin, chemical products of natural
micro-organisms. One of the factors that led to the diversification of research lines was the
Brazilian researchers’ awareness of the value of Brazilian biodiversity, which holds tremen-
dous potentiality for the discovery of new substances with added value and of biological inter-
est. One of the extremely relevant initiatives to advance the chemistry of natural products in
Brazil was the implementation of the BIOTA-FAPESP program supported by the Foundation
for Research Support of the State of São Paulo (FAPESP). Created in 1999, with the aims to
report the diversity and distribution of the fauna and flora of the State of São Paulo, as well as a
tool to evaluate the possibilities of sustainable exploitation with economic potential (Joly et al.,
2008, 2010). Another very important initiative was the development of a free access database of
natural products coming from the Brazilian biodiversity created in 2013, through a collaborative
project between the Nuclei of Bioassays, Biosynthesis and Ecophysiology of Natural Products
(NuBBE, São Paulo State University UNESP – Araraquara) and Computational and Medicinal
Chemistry of São Paulo University (São Carlos, SP) groups (NuBBeDB). Since its inception,
more than 2,000 compounds isolated from microorganisms, plants, animals, marine organisms,
biotransformation and semi-synthetic products have been deposited in the database. In addition
to the compounds’ structures, biological and pharmacological information, spectroscopic and
physico-chemical properties are also compiled (Pilon et al., 2017; Valli et al., 2013). The infor-
mation contained in NuBBeDB help researchers and those interested in bioprospecting secondary
metabolites, chemosystematics, metabolomics, among others.
In recent decades, there has been a significant advance in the study of natural products derived
from different biological sources. A search conducted in the NuBBe database shows that of the
2,218 compounds of Brazilian biodiversity, 78.6% were isolated from plants, 4.9% from micro-
organisms, 0.36% from marine organisms and 0.18% from animals (Figure 3.5). Other 15.6% are
semi-synthetic products and 1.53% are biotransformation products.
Another project worth highlighting is the development of the global natural products social
molecular networking (GNPS) platform. This project was created at the University of San Diego by
several researchers, among them some Brazilians such as Norberto Peporine Lopes of the Nucleus
of Research in Natural and Synthetic Products (Faculty of Pharmaceutical Sciences of Ribeirão
Preto, USP) (Wang et al., 2016). In this open access platform, any researcher in the world can enter
their data enlarging the library. This type of information will be extremely useful for those who
work in the field of natural products.
These data demonstrate that the main biological source of research in natural products is still
from plants. However, in recent years there has been a considerable increase in the diversifica-
tion of these sources. Research on microorganisms is the second most explored source and the
trend is that this number will increase even more. Interest in other biological sources is due,
in part, to the structural diversity found in other sources, such as microorganisms and marine
organisms.
FIGURE 3.5 Sources of substances isolated from Brazilian biodiversity and derivatives. Data available in
NuBBE database.
An example from Brazilian researchers was the isolation and/or UPLC-HRMS detection
of bromotyrosine-derived alkaloids, 11-hydroxyaerothionin (18), fistularin-3 (19), verongidoic
acid (20), aplysinamisine II (21), aerothionin (22), purealidin L (23), homopurpuroceratic acid
B (24) (Figure 3.6) from the culture of Pseudovibrio denitrificans Ab134, bacterium isolated
from the tissue of the marine sponge Arenosclera brasiliensis. These substances are known only
in sponges of the order Verongida, making this publication pioneer in the literature (Nicacio
et al., 2017).
Silva-Junior et al. (2018), aiming to understand the relationship between the bacterium Serratia
marcescens 3B2 and cutter ants of the genus Atta sexdens rubropilosa, carried out the chemical
study of the bacteria and isolated several pyrazines (25–30, Figure 3.7). Some of these compounds
were identified as alarm and trail pheromones of leafcutter ants. This work will help in the elabora-
tion of future studies that aim at the control of these ants.
Fruit waste provides an opportunity to obtain useful and valuable chemicals. For example,
15 million tons of citrus waste accumulates annually from the food and drink processing industry and
other minor contributors. Often simply disposed of or incorporated into animal feed, this huge and
naturally occurring resource is not being fully taken advantage of, although the means to do so have
been successfully demonstrated. It has long been recognized that orange peel represents a promising
source of hesperidin, a flavonoid. One million metric tons of peel residues are generated as a result of
fruit processing, and thus, an extract of this residue could be considered for the isolation of hesperi-
din, other flavonoids, and many other compounds. Bellete et al. (2018) showed that several flavonoids
present in citrus waste can be isolated using a faster and greener methodology. They observed the
presence of flavonoids hesperitin (31), narigenin (32), nobiletin (33), chrysoerythol (34), sinensetin
(35), isosakuranetin (36), 3,5,6,7,3′,4′- hexamethoxylflavone (37), 3,5,6,7,8,3′,4′-heptamethoxyflavone
FIGURE 3.6 Bromotyrosine derivatives isolated from P. denitrificans.
FIGURE 3.7 Substances belonging to the pyrazine family produced by S. marcescens 3B2.
FIGURE 3.8 Flavonoids present in Brazilian citrus fruit waste.
(38), 3,5,6,7,4′-pentamethoxyflavone (39) and 5-methoxysalvigenin (40) (Figure 3.8). This class of
compounds presents a broad spectrum of biological activities as antioxidant, antimicrobial, antiviral,
anti-inflammatory, etc. (Cao et al., 1997; Cushnie and Lamb, 2005). Lani et al. (2016) showed the
potential of certain flavonoids against the Chikungunya virus, making this study a good example of
the potential of agro-industrial waste to produce bioactive substances.
Recently, the Quézia Cass group has shown the importance of determining the chirality of isolated
natural products, which is often very difficult. An example of the structural complexity is palytoxin
(41, Figure 3.9) isolated from Palythoa sp. corals, which contains 64 stereogenic centers (Viegas Jr
and Bolzani, 2006). A significant amount of racemates or enantiomerically enriched mixtures has
been reported from natural sources. This number is estimated to be even larger according to the Cass
group, since the enantiomeric purity of secondary metabolites is rarely checked. This latter fact may
have significant effects on the evaluation of the biological activity of chiral natural products. A second
bottleneck is the determination of their absolute configurations. Despite the widespread use of opti-
cal rotation and electronic circular dichroism, most of the stereochemical assignments are based on
empirical correlations with similar compounds reported in the literature. As an alternative, the Cass
group suggest the combination of vibrational circular dichroism and quantum chemical calculations,
FIGURE 3.9 Palytoxin (41) structure isolated from Palythoa sp.
which has emerged as a powerful and reliable tool for both conformational and configurational analy-
sis of natural products, even for those lacking UV-Vis chromophores (Batista et al., 2018).
It is well-known that nature produces countless secondary metabolites at different concentrations,
for instance a single plant can produce 5,000 to 25,000 metabolites (Leme et al., 2014). To identify
and quantify the set of secondary metabolites produced by an organism as well as to understand the
role of these substances in the relationships and interactions of these organisms with the environ-
ment is practically impossible using traditional techniques. However, with more sensitive equip-
ment of nuclear magnetic resonance (NMR), mass spectrometry (MS), and chemometrics applied
to Liquid Chromatography (LC)-MS and/or LC-NMR data we can evaluate the similarities and
differences between the chromatographic profiles and perform a metabolomics analysis. Oliviera.
used mass spectrometry and molecular networking to identify 63 flavonoids from 19 extracts of
Adenocalymma imperatoris-maximilianni (Oliveira et al., 2017). Angolini et al. (2016) after investi-
gation of Streptomyces wadayamensis A23 genome, actinobacteria isolated from Citrus reticulata,
showed that this lineage has biosynthetic machinery capable of generating various antibiotics. The
metabolic profile from this lineage analyzed by mass spectrometry showed the production of sev-
eral bioactive substances already predicted by the genome mining. The pioneer results presented by
Angolini et al. (2016) open up exciting opportunities for different research fields from genomics to
the production of bioactive substances.
3.2 STRUCTURAL DIVERSITY AND DISTRIBUTION OF

SECONDARY METABOLITES IN BRAZILIAN PLANTS
Analyzing all the chemical data recorded in the NuBBe database for the species of a plant family, we
verified that the Rutaceae were the most studied in Brazil with 12.5% of the total metabolites reported,
followed by Meliaceae (9.1%), and a large part of the work was developed by da Silva’s group. There is
a general agreement between taxonomists that both these families should be placed in the same order,
Sapindales (da Silva et al., 2010). The Rutaceae family includes about 150 genera with more than
1,500 species, which are distributed throughout the tropical and temperate regions of the world, being
most abundant in tropical America, South Africa, Asia and Australia. Rutaceae constitute the largest
group of Sapindales and are characterized by a great diversity of secondary metabolites not common
in other families of the order. The most representative are alkaloids derived from anthranilic acid, cou-
marins, flavonoids and limonoids. There are 32 genera with about 200 species currently recognized in
Brazil, and species of 24 genera have already been studied by da Silva’s group. Meliaceae consists of
about 550 species distributed between approximately 51 genera. There are six genera currently recog-
nized in Brazil, and species of all genera have already been studied by the da Silva group. Limonoids,
biosynthetically related compounds, are found in Meliaceae and Rutaceae. There are consistent differ-
ences between the limonoids of the Rutaceae and those of the Meliaceae.
In agreement with the NuBBE database the other families most studied in Brazil are in order of
their decreasing total metabolites reported: Lauraceae (8.9%), Rubiaceae (7.5%), Fabaceae (6.8%),
Euphorbiaceae (4.2%), Piperaceae (4.1%), Myrtaceae (2.2%), Verbenaceae (1.5%), Anarcadiaceae
and Celastraceae (1.2%), Annonaceae; Asteraceae; Moraceae and Sapindacea (<1%) and others cor-
responding to 38.5%. These families and others discussed in this chapter, their respective species
and common names can be seen in Table 3.1.
Regarding the class of compounds reported in the database of chemical and biological infor-
mation of Brazilian biodiversity, the most representative are terpenes (34%). Another 15.3% are
TABLE 3.1
Summary of the Names of All Plant Species, Their Respective Family and Common
Names Presented in This Chapter
Species Family Common Names
Cupressus sempervirens Cupressaceae Cipreste-dos-cemitérios, cipreste, cipreste-comum,
cipreste-de-Itália, falso-cedro
Commiphora species (sin. Commiphora Burseraceae –
voensis)
Papaver somniferum Papaveraceae Papoula, papoula do ópio, dormideira
Caesalpinia echinata (sin. Guilandina Fabaceae Pau-brasil, ibirapitanga, orabutã, brasileto, ibirapiranga,
echinata) ibirapita, ibirapitã, muirapiranga, pau-rosado,
pau-de-pernambuco
Plumeria lancifolia Apocynaceae Agonia, agonium, arapou, arapuê, arapuo, colônia,
guina-mole, jasminmanga, quina-branca, quina-mole,
sacuíba, sucuba, sucuriba, sucuúba, tapioca, tapouca,
tapuoca
Geissospermum laeve (sin. Geissospermum Apocynaceae Pau-pereiro, pinguaciba, pau-de-pente e
martianum) pau-para-toda-obra
Chondrodendron tomentosum (sin. Menispermaceae Curare, pareira-brava, pareira, uva-da-serra, uva-do-mato
Chondrodendron hypoleucum)
Pterodon emarginatus (sin. Pterodon Fabaceae Sucupira branca
pubescens)
Hortia oreadica Rutaceae Quina-do-campo, para-tudo, quina
Citrus reticulata Rutaceae Tangerina, mexerica, laranja-mimosa, mandarina,
fuxiqueira, poncã, manjerica, laranja-cravo, mimosa,
bergamota, clementina
Swinglea glutinosa (sin. Chaetospermum Rutaceae Limão swinglea, limão ornamental
glutinosum)
Maytenus ilicifolia (sin. Maytenus Celastraceae Espinheira-santa,
aquifolium)
(Continued)

Salacia campestris Hippocrateaceae Bacupari-do-campo, japicuru, capirucu
Citrus sinensis (sin. Citrus aurantium) Rutaceae Laranja, laranja-doce, laranja-de-umbigo, laranja-
bahianinha, laranja-lima, laranja-natal, laranja-pera-do-
Rio, laranja-rubi, laranja-valência, laranja-hamlin,
laranja-bahia, laranja-sangüinea, laranja-pêra, laranjeira
Citrus limonia Rutaceae Limão-rosa, limão-cavalo, limão-egua, limão-francês,
limão-china, limão-vinagre, limão tambaqui
Alternanthera littoralis (sin. Achyranthes Amaranthacea Periquito-da-praia
maritima)
Erythrina verna (sin. Erythrina mulungu) Fabaceae Suinã, suiná, sapatinho-de-judeu, canivete, amansa-
-senhor, bico-de-papagaio, comedoi, molongo, murungu,
corticeira, sananduva, pau-imortal
Annona hypoglauca (sin. Annona Annonaceae Biribá
tessmannii)
Annona crassiflora (sin. Annona rodriguesii) Annonaceae Acanga, araticum, araticum do mata, tapanahuacanga
Duguetia furfuracea (sin. Duguetia Annonaceae Ata-brava, ata-de-lobo, pinha do campo, pinha brava
hemmendorffii)
Croton grandivelum (sin. Croton Euphorbiaceae Sangra d’água, sangue-de-drago
echinocarpus)
Chimarrhis turbinata (sin. Rubiaceae Pau de remo
Pseudochimarrhis diformis)
Tabernaemontana catharinensis (sin. Apocynaceae Leiteiro, leiteiro-de-folha-fina, leiteiro de vaca
Tabernaemontana australis)
Psychotria laciniata (sin. Psychotria kleinii) Rubiaceae Gandiúva-dánta, café-dánta, pimenteira-do-mato, buta,
cravo-de-negro, erva-de-anta, pasto-de-anta
Piper tuberculatum (sin. Piper arboreum Piperaceae Pimenta longa, pimenta-d’arda
subsp. tuberculatum)
Senna spectabilis (sin. Cassia spectabilis) Fabaceae Cássia, Cássia-do-nordeste, Cássia-macranta, Cássia-
macrantera, Fedegoso, Fedegoso do Rio, Macrantera
Senna multijuga (sin. Cassia multijuga) Fabaceae Pau-cigarra, aleluia-amarela, árvore-da-cigarra,
canafístula, canudeiro.
Hippeastrum psittacinum (sin. Hippeastrum Amaryllidaceae Amarílis, açucena, flor-da-imperatriz
illustre)
Crotalaria retusa (sin. Crotalaria Fabaceae Amendoim bravo, chique-chique, guizo de cascavel,
retusifolia) chocalho de cascavel
Calycophyllum spruceanum Rubiaceae Pau-mulato, mulateiro, mulateiro-da-várzea, escorrega-
macaco, pau-mulato-da-várzea, capirona, pau-marfim
Laurencia dendroidea (sin. Laurencia Rhodomelaceae –
obtusa var. dendroidea)
Vellozia graminifolia Velloziaceae –
Stemodia foliosa (sin. Stemodiacra foliosa) Scrophulariaceae Meladinha
Croton cajucara (sin. Oxydectes cajucara) Euphorbiaceae Sacaca, cajuçara, casca-sacaca, muirasacaca
Casearia sylvestris (sin. Casearia Flacourtiaceae Guaçatonga, baga-de-pomba, bugre-branco, café-bravo,
subsessiliflora) cafezeiro-bravo, cafezeiro-do-mato, carvalhinho,
pau-de-bugre, erva-de-pontada, erva-de-bugre,
erva-de-lagarto, erva-de-teiú, gaibim
Casearia rupestris Flacourtiaceae Pururuca, pururuba
(Continued)

Guarea macrophylla (sin. Guarea Meliaceae Pau-de-arco, camboatá, catiguá-morcego
demerarana)
Baccharis retusa (sin. Baccharis affinis) Asteraceae Carqueja
Cedrela fissilis (sin. Cedrela elliptica) Meliaceae Cedro, cedro-rosa, cedro-cetim, acaiacá, acaiacatinga,
acajá-catinga, acajatinga, acaju, acaju-caatinga, capiúva,
cedrinho
Cabralea canjerana (sin. Cabralea Meliaceae Canjarana, canjerana, canjerana de prego, cajarana,
lacaziana) canharana, cedro canjerana, pau de santo, caieira,
canjarana do litoral, caja espúrio
Typha domingensis (sin. Typha salgirica) Typhaceae Taboa, bucha, capim-de-esteira, erva-de-esteira,
espadana, paina, paineira-de-flecha, paineira-do-brejo,
partasana, pau-de-lagoa, tabebuia, tabuca
Pterocaulon alopecuroides (sin. Pterocaulon Asteraceae –
latifolium)
Pterocaulon balansae (sin. Pterocaulon Asteraceae –
paniculatum)
Pterocaulon polystachyum (sin. Pterocaulon Asteraceae Quitoco
polystachyum)
Polygala sabulosa Polygalaceae Timutu-pinheirinho
Conchocarpus fontanesianus (sin. Angostura Rutaceae Pitaguará
fontanesiana)
Helietta apiculata (sin. Helietta cuspidata) Rutaceae Canela-de-veado
Fridericia platyphylla (sin. Arrabidaea Bignoniaceae Cervejinha-do-campo
brachypoda)
Bryophyllum pinnatum (sin. Kalanchoe Crassulaceae Flores-da-fortuna, folha-da-costa, erva-da-costa,
pinnata) folha-grossa, folha-da-vida, coirama, coirama-branca,
coirama-brava, roda-da-fortuna, saião, saião-roxo,
amor-verde, paratudo, planta-do-amor, sempre-viva,
Passiflora quadrangularis (sin. Passiflora Passifloraceae –
macrocarpa)
Croton betulaster (sin. Oxydectes betulaster) Euphorbiaceae –
Dimorphandra mollis Fabaceae Barbatimão-falso
Amburana cearensis (sin. Amburana claudii) Fabaceae Cerejeira, cumaru-do-cerá-, cumaré, cumaru-das-
caatingas, imburana-de-cheiro, umburana, amburana-
de-cheiro, imburana, cerejeira-rajada,
cumaru-de-cheiro
Neoraputia alba Rutaceae Arapoca, arapoca branca, arapoca verdadeira, banha-de-
galinha, mucanga, sucanga.
Neoraputia paraensis (sin. Raputia Rutaceae –
paraensis)
Cenostigma macrophyllum Fabaceae Caneleiro, canela de Velho, maraximbé, fava do Campo
Garcinia brasiliensis (sin. Garcinia Clusiaceae Bacupari, bacupari do achado, bacupari mirim
gardneriana)
Phyllanthus amarus (sin. Diasperus nanus) Euphorbiaceae Quebra-pedra, arrebenta-pedra, erva-pombinha
Piper solmsianum Piperaceae –
Ocotea fasciculata (sin. Ocotea duckei) Lauraceae Louro-de-cheiro
Zanthoxylum petiolare (sin. Zanthoxylum Rutaceae Juva, tembetari, espinho
naranjillo)
(Continued)

Combretum fruticosum (sin. Combretum Combretaceae Escova-de-macaco-alaranjada, escovinha, flor-de-fogo,
loeflingii) limpa-garrafa-laranja, bugio, escovinha-raspadeira,
escovinha-flor-de-fogo
Aniba burchellii Lauraceae Abacaterana
Ocotea cymbarum (sin. Licaria cymbarum) Lauraceae Louro inamuí
Nectandra hihua (sin. Nectandra glabrescens) Lauraceae –
Styrax ferrugineus (sin. Styrax ferrugineus Styracaceae Laranjinha do cerrado
var. grandifolius)
alkaloids, 14.16% flavonoids, 12.3% aromatic derivates, 9.6% lignoids, 5.2% coumarins, 3.5% phen-
ylpropanoids and others (Figure 3.10).
In the chosen set of topics below, the known distributions of the above class of compounds in
Brazilian plants are revised and the data were obtained from Chemical Abstracts (SciFinder) and
Web of Science from 1998 to 2018.
3.2.1 Alkaloids
The most practical classification of alkaloids, due to their structural diversity, is in accordance with
their known or hypothetical biogenesis. Thus, the reported alkaloids from the Brazilian plants are
derivatives of anthranilic acid, tryptophan, phenylalanine and/or tyrosine, histidine, nicotinic acid,
ornithine, and lysine, or in some cases, they are formed from two amino acid precursors (Dewick
P. M., 2002; Wink M., 2016) (Figure 3.11).
From Brazilian plants, 34 alkaloids were isolated from 1998 to 2018: these were isolated from 15
species belonging to 14 genera and 9 families (Table 3.2, Figure 3.12). Alkaloids from phenylalanine
and/or tyrosine are the most widespread in the Brazilian plants studied, such as isoquinolines
(42, 43); tetrahydroisoquinoline (45), aporphines (46, 47, 48, 49, 50) and protoberberine (51). They
are distributed in Annonaceae, Fabaceae and Amaranthaceae.
The alkaloids derived from anthranilic acid are the second group most isolated, for example,
a simple 2-quinolone (44) and acridones (52–59). They were found in Amaranthaceae (simple
2-quinolone) and Rutaceae (acridones). The tryptophan derivative alkaloids, as terpene indole alka-
loids (60–65) occur in Apocynaceae and Rubiaceae. Lysine derivatives piperidine (66–69) and
pyridine (70) alkaloids were reported in Fabaceae and Piperaceae.
Amaryllidaceae alkaloids are also derived from phenylalanine and tyrosine; however, they are
classified according to their main skeleton structure, as galanthamine-type (71), montanine-type
(72), homolycorine-type (73) and tazettine-type (74).
Alkaloids derived from ornithine, such as pyrrolizidine alkaloid (75), were reported only in
Fabaceae. This family is characterized by a diversity of alkaloids.
The interest in alkaloids has always been very great because these compounds exhibit marked
biological activities such as leishmanicidal, antimalarial, antiprotozoal, antimicrobial and antican-
cer activities. However, a wide spectrum of other biological properties for the above alkaloids has
been discovered (Table 3.2). One example is they act as inhibitors of acetylcholinesterase (AChE),
which are currently one of the few therapies approved for the treatment of Alzheimer’s disease
(Table 3.2). The enzyme AChE acts in the central nervous system and rapidly hydrolyzes the active
neurotransmitter acetylcholine into the inactive compounds choline and acetic acid. Low levels of
acetylcholine in the synaptic cleft are associated with a decrease in cholinergic function character-
izing Alzheimer’s disease, which is the most common cause of dementia among the elderly.
46
FIGURE 3.10 Statistics of the distribution of isolated and/or identified metabolites of Brazilian biodiversity according to (A) the families of plants and (B) metabolic
classes. Data available in NuBBE database.
Natural Products Chemistry in Brazil
FIGURE 3.11 Biosynthesis of alkaloids. (Based on Wink M. (2016).)
47
TABLE 3.2
Occurrence of Alkaloids Identified from Brazilian Plants over the Last 20 Years
Occurrence
(plant part)
Code Alkaloid Substituents Activity (references)
42 (R)-1-(3,4-dihydroxyphenyl)- Antiprotozoal and Alternanthera littoralis
1,2,3,4-tetrahydroisoquinoline- antioxidant Amaranthacea (Aerial
6,7-diol parts)
43 6,7-dihydroxy-3,4- (Koolen H. H. F. et al.,
dihydroquinoline-1-one 2016).
44 7,8-dihydroxy-1,2,4,5-
tetrahydro-3H-1,5-ethano[c]
azepin-3-one
45 (+)-11α-Hydroxyerythravine R1 R2 Anxiolytic Erythrina mulungu
H OH Fabaceae (flowers)
(+)-Erythravine H H (Flausino O. et al., 2007).
(+)-α-Hydroxyerysotrine CH3 OH
46 R1 R2 R3 R4 Antimicrobial Annona hypoglauca
Isoboldine CH3 OH OCH3 OH Annonaceae (bark)
Nornuciferine H H H OCH3 (Rinaldi et al., 2017).
47 Stephalagine Citotoxic Annona crassiflora
Annonaceae (fruits)
(Pereira M. N. et al.,
2017).
48 Duguetine Antitrypanocidal and Duguetia furfuracea
49 Dicentrinone antileishmanial Annonaceae (bark)
50 N-methylglaucine (da Silva B. D. et al.,
51 N-methyltetrahydropalmatine 2009).
52 Citrusinine-II R1 R2 R3 Inhibition of Swinglea glutinosa
H OH OCH3 cathepsin V Rutaceae (roots)
Citrusinine-I H OCH3 OCH3 (Severino R. P. et al.,
Citibrasine OCH3 OCH3 OCH3 2011).
53 3,4-dihydro-3,5,8-trihydroxy-6-
ethoxy-2,2,7-trimethyl-2H-
pyrano[2,3-]
acridin-12(7H)-one
54 Corydine R1 R2 R3 Anti-HIV Croton echinocarpus
CH3 OCH3 H Euphorbiaceae (leaves)
Norisoboldine H H OH Ravanelli N. et al., 2016.
55 Glycocitrine-IV Cytotoxic Swinglea glutinosa
56 5-Dihydroxyacronycine Rutaceae (fruits)
57 bis-5-Hydroxyacronycine (Braga P. A. C. et al.,
2007).
58 2,3-dihydro-4,9-dihydroxy-2- Antimalarial Swinglea glutinosa
(2-hydroxypropan-2-yl)-11- Rutaceae (stem bark)
methoxy10-methylfuro[3,2-b] (dos Santos D. A. P. et al.,
acridin-5(10H)-one 2009).
59 5-hydroxynoracronycine
60 Strictosidine R1 Antioxidant Chimarrhis turbinata
H Rubiaceae (bark)
5α-Carboxystrictosidine CO2H (Cardoso C. L. et al., 2004,
Cardoso C. L. et al., 2008).
(Continued)

Occurrence of Alkaloids Identified from Brazilian Plants over the Last 20 Years
Occurrence
(plant part)
Code Alkaloid Substituents Activity (references)
61 Coronaridine R1 R2 R3 Inhibition of Tabernaemontana
H COOCH3 H acetylcholinesterase australis
Voacangine OCH3 COOCH3 H Apocynaceae (bark)
62 Voacangine hydroxyindolenine (Andrade M. T. et al.,
63 Rupicoline 2005).
64 Prunifoleine Inhibition of Psychotria laciniata
65 14-Oxoprunifoleine cholinesterases and Rubiaceae (leaves)
monoamine (Passos C. S. et al., 2013).
66 (Z)-piplartine Antifungal and Piper tuberculatum
67 Piperine antitrypanossomal Piperaceae
(seeds and leaves)
(Cotinguiba F. et al.,
2009, Navickiene H. M.
D. et al., 2000).
68 R1 Antioxidant and Cassia spectabilis
(-)-Spectaline H anti-inflamatory Fabaceae (fruits)
(-)-3-O-acetylspectaline CH3 (Viegas JR. C. et al., 2004).
69 (+)-3-O-feruloylcassine Viegas JR. C. et al., 2007.
70 7′-multijuguinone R1 Inhibition of Senna multijuga
CH3 acetylcholinesterase Fabaceae (leaves)
12′-hydroxy-7′-multijuguinone OH (Serrano M. A. R. et al.,
2010).
71 Galanthamine Inhibition of Hippeastrum psittacinum
72 Montanine acetylcholinesterase Amaryllidaceae (bulbs)
73 Hippeastrine (Pagliosa L. B. et al.,
74 Pretazettine 2010).
75 Usaramine Antibacterial Crotalaria retusa
Fabaceae (seeds)
(Neto T. S. N. et al., 2016).
The low enzymatic stability that is associated with the high cost of purification makes offline,
in-solution enzymes assays impractical for high-throughput screening. However, the achievement
of online assays using immobilised enzymes is a valuable alternative. Immobilized enzyme reac-
tors (IMERs) are highly stabile in the presence of organic solvents and temperature variations; fur-
thermore, these reactors enable the use of small amounts of enzyme and enzyme reuse. The use of
IMERs for online screening using different formats and settings is extended because a high number
of different immobilization approaches can be allied with a wide range of available chromatographic
media. IMERs have been efficiently used in selective affinity chromatography methods and have been
exploited through frontal and zonal (linear and nonlinear) chromatography. The optimized prepara-
tion of capillary enzyme reactors (ICERs) based on AChE for the screening of selective inhibitors
was developed recently by a Brazilian group (Silva et al., 2013). The AChE-ICERs were prepared
using the homo-bifunctional linker glutaraldehyde through a Schiff base linkage. The enzyme was
anchored onto a modified fused silica capillary and used as an LC bio-chromatography column for
online studies with UV-vis detection. Not only did the tailored AChE-ICER maintain the activity
of the immobilized enzyme, but it also significantly improved the stability of the enzyme in the
presence of organic solvents. In addition, kinetic studies demonstrated that the enzyme retained its
FIGURE 3.12 Structure of alkaloids identified from Brazilian plants over the last 20 years, based on
information outlined in Table 3.2.
activity with high stability, preserving its initial activity over ten months. The absence of nonspe-
cific matrix interactions, the immediate recovery of the enzymatic activity and the short analysis
time were the main advantages of this AChE-ICER. The use of AChE-ICER in the ligand recogni-
tion assay was validated by the evaluation of four known reversible inhibitors (galantamine, tacrine,
propidium and rivastigmine), and the same order of inhibitory potencies as described in the litera-
ture was found.
The immobilized enzyme was used to screen 21 synthetic coumarin derivatives. In this library,
two new potent inhibitors were identified: [3-ethylcarboxylate-7-(2-piperidine-ethoxyl)coumarin]
(IC50 17.14 ± 3.50 μM) and [3-ethylcarboxylate-7-hydroxy-8-(1-piperidine-methoxyl)coumarin]
(IC50 6.35 ± 1.20 μM), or piperidine coumarins, which were compared to the standard galantamine
(IC50 12.68 ± 2.40 μM). Considering the high inhibitory activities of these compounds with respect
to the AChE-ICER, the mechanism of action was investigated. Both coumarins exhibited a competi-
tive mechanism of action, producing Ki values of 8.04 ± 0.18 and 2.67 ± 0.18 μM, respectively. The
results revealed that the AChE-ICER is useful for the biological screening of inhibitor candidates
and evaluating the mechanism of action.
A second example of alkaloids acting as enzyme inhibitors is the acridones, which have been
shown to be potent inhibitors of cathepsin V. Several natural products have been investigated for
their inhibitory effects on the catalytic activity of cathepsins, and Brazilian researchers developed
bioassay methodologies with these enzymes (Severino et al., 2011). Cathepsins, also known as lyso-
somal cysteine peptidases, are members of the papain-like peptidase family, which are implicated
in many pathological conditions. These enzymes have been intensively studied as valuable targets
for drug discovery and development. Although the major role of cathepsins is related to the terminal
protein degradation in lysosomes, it has been shown that these enzymes are also involved in other
relevant biochemical pathways, acting at selective and controlled processes with specific functions
associated to their restricted tissue localization. In addition, evidence has indicated that cysteine
cathepsins have specific intra- and extracellular functions, being involved in a number of diseases
including cancer, osteoarthritis, osteoporosis, autoimmune disorders and viral infections. Cathepsin V
was identified as a lysosomal cysteine protease specifically expressed in thymus, testis and corneal
epithelium. It is believed that cathepsin V plays a role in cancer progression, thus becoming a valu-
able drug target for oncology.
Eleven acridone alkaloids were isolated from Swinglea glutinosa (Bl.) Merr. (Rutaceae), with
eight of them being identified as potent and reversible inhibitors of cathepsin V (IC50 values ranging
from 1.2 to 3.9 µM ). Detailed mechanistic characterization of the effects of these compounds on the
cathepsin V-catalyzed reaction showed clear competitive inhibition with respect to substrate, with
dissociation constants (Ki) in the low micromolar range. The most potent inhibitor citibrasine (52)
(Table 3.2) has a Ki value of 200 nM (Severino et al., 2011).
3.2.2 Terpenes
The sesqui-, di- and triterpenes appear to be common in Brazilian plants. During the last few years
several accounts have been published on the biosynthesis of terpenes. The literature clearly has
shown that all compounds of the family are derived from the two building blocks isopentenyl diphos-
phate (IPP) and dimethylallyl diphosphate (DMAPP) and are biosynthesized either by the mevalon-
ate (MVA) or the 2-C-methyl-D-erythritol 4-phosphate (MEP) pathway (Figure 3.13). Nevertheless,
no studies of the biosynthesis of this class have apparently been reported in Brazilian plants. Until
recently the only work in this series is due to Maysa Furlan and her colleagues, who showed that
enzymatic extracts obtained from leaves and/or root bark of Maytenus aquifolium (Celastraceae)
and Salacia campestris (Hippocrateaceae) displayed cyclase activity with conversion of the sub-
strate oxidosqualene to the triterpenes, 3-friedelanol and friedelin. In addition, administration of
(±)5-3H mevalonolactone in leaves of M. aquifolium seedlings produced radio labeled friedelin in
the leaves, twigs and stems, while the root bark accumulated labeled maytenin and pristimerin (89).
FIGURE 3.13 Biosynthesis of terpenes through the mevalonate pathways (MVA) and 2-C-methyl-D-
erythritol 4-phosphate (MEP). Based on Lange B. M., 2015.
These experiments indicated that the triterpenes once biosynthesized in the leaves are translocated
to the root bark and further transformed to the quinonemethide triterpenoids (Corsino et al., 2000).
Three secoiridoids were found in Brazilian Rubiaceae (skeleton 76), which are common in
this family. A sesquiterpene complex, named triquinane type (77), was mentioned in Asteraceae,
whose class is characteristic of the family. The diterpenes were found in greater numbers: five
labdanes (skeletons 78, 79), nine clerodanes (skeletons 80–83), two isopiparane (skeleton 86) and
three kaurene (skeleton 87). These were isolated from Brazilian plants of families Asteraceae,
Euphorbiaceae, Flacourtiaceae, Meliaceae, Scrophulariaceae and Velloziaceae (Table 3.3). The trit-
erpenes were the most abundant in the Brazilian plants mentioned in Table 3.3: a total of 13. Among
these the tirucallanes/euphanes (88, 89) and their derivatives, the limonoids (90–92), were found in
Meliaceae, which is well characterized by such compounds. The cycloartane series is very common
in higher plant families, and they (84, 85) were also found in Meliaceae. Friedelanes (93) are also
common in higher plant families, and one was isolated from Malpighiaceae, however, their bioge-
netic derivatives quinonemethides (94) are restricted to Celastraceae and Hippocrateaceae. Two
quinonemethides are cited for Brazilian plants of the latter family (Table 3.3).
The above terpenes (Figure 3.14) exhibit a variety of biological activities, such as antitumoral,
antibiotic, antimalarial, antileishmanial and trypanocidal activities (Table 3.3).
TABLE 3.3
Occurrence of Terpenes Identified from Brazilian Plants over the Last 20 Years
Occurrence
(plant part)
Code Terpene Substituents Activity (references)
76 7-Methoxydiderroside R1 R2 R3 Antitrypanosomal Calycophyllum spruceanum
CH3 CH3 H Rubiaceae
6′-Acetyl-β-D-glucopyranosyldiderroside H CH3 OCH3 (wood bark)
8-O-Tigloyldiderroside H (CH3)C=CH(CH)3 H (Zuleta L. M. C. et al., 2003).
77 Triquinane Antileishmanial Laurencia dendroidea
Rhodomelaceae
(Machado F. L. S. et al., 2011).
78 ent-3b-Hydroxylabd-8(17)-en-15-oic acid R1 R2 Vellozia graminifolia
βOH,H CO2H Velloziaceae (roots, steam and leaves)
3-oxo-Labd-8(17)-en-15-oate O CO2CH3 (Branco A. et al., 2004).
ent-3b-Hydroxylabd-8(17)-ene-15-ol βOH,H CH2OH
79 6α-Acetoxymanoyl oxide R1 Antibacterial Stemodia foliosa
COCH3 Scrophulariaceae
6α-Malonyloxymanoyl oxide COCH2CO2H (aerial parts)
(da Silva L. L. D. et al., 2008).
80 trans-Dehydrocrotonin Antileishmanial Croton cajucara
81 Crotonin Euphorbiaceae (bark steam)
(Lima G. S. et al., 2015).
82 Casearin L R1 R2 R3 Cytotoxic and Casearia sylvestris
CH3O CH3CO2 OH Anticancer Flacourtiaceae (leaves)
Casearin O CH3O CH3CO2 n-C3H7CO2H (Ferreira P. M. P.
Casearin X n-C3H7CO2H OH H et al., 2010).
83 Casearupestrin A R1 R2 R3 Citotoxic Casearia rupestres
OAc a OH Flacourtiaceae (leaves) (Vieira-Júnior
Casearupestrin B OAc OH A G. M. et al., 2011).
Casearupestrin C OCH3 OH A
Casearupestrin D OAc OAc A
R1
53
(Continued)
54
Occurrence of Terpenes Identified from Brazilian Plants over the Last 20 Years
Occurrence
(plant part)
Code Terpene Substituents Activity (references)
84 Cycloart-23E-ene-3β,25-diol Antitumoral Guarea macrophylla
85 (23S*,24S*)-Dihydroxycicloart-25-en-3-one Meliaceae (leaves)
86 Isopimara-7,15-diene-2α,3β-diol H (Conserva G. A. A. et al., 2017).
Isopimara-7,15-dien-3β OH
87 ent-15β-Senecioyloxy-kaur-16-en-19-oic R1 R2 Antitrypanosomal Baccharis retusa
acid CH2 O2CH=C(CH3)2 Asteraceae (aereal parts)
ent-Kaur-16-en-19-oic acid CH2 H (Ueno A. K. et al., 2018).
ent-16-oxo-17-nor-Kauran-19-oic acid O H
88 Hispidol A R1 Trypanocidal Cedrela fissilis
A Meliaceae (steam)
Pentaol B (Leite A. C. et al., 2008).
Iso-odoratol C
Odoratone D
89 Odoratol Antitumor Cabralea canjerana
90 Gedunin Meliaceae (fruits)
91 6α-acetoxy-14β,15β-epoxyazadirone (Cazal C. M. et al., 2010).
92 Cedrelona
93 6α, 7α, 15β, 16β, 24-Pentacetoxy-22α- Antileishmanial Lophanthera lactescens
carbometoxy-21β,22β-epoxy-18β−hydroxy- Malpighiaceas (stems)

27,30-bis nor-3,4-secofriedela-1,20 (Danelli M. G. M.
(29)-dien-3,4 R-olide et al., 2009).
94 Salacin R1 R2 R3 R4 Antioxidant Salacia campestres
H CH3 OH OH Hippocrateaceae (roots)
Pristimerin CH3 CO2CH3 H H (Carvalho P. R. F. et al., 2005).
FIGURE 3.14 Structure of terpenes identified from Brazilian plants over the last 20 years, according to code
of Table 3.3.
3.2.3 Phenylpropanoids
Several phenylpropanoids have recently been described from Brazilian plants; these are coumarins,
flavonoids, lignans and neolignans (Table 3.4). Simple coumarins (95–98; see Figure 3.16) occur
widely in the plant kingdom, but the proliferation of a wide range of complex furo- and pyrano-
coumarins is a feature that is largely confined to Rutaceae and to Apiaceae. Trans-p-coumaric acid
is the general precursor of 7-oxygen coumarins synthesized in plants (Figure 3.15). Prenylation of
umbelliferone at C-6 leads to linear pyrano and furocoumarins and at C-8 to pyrano- and furo-
angular coumarins (99–101; Figure 3.16). This class of compounds showed anti-inflammatory, anti-
microbial and anticholinesterase properties, in Brazilian studies.
Flavonoids 102–111 (Figure 3.17) are formed by a mixed pathway, the C6C3 moiety being derived
from the shikimic acid and the A ring from malonate (Figure 3.15). Flavonoids are widely occurring
in nature and may suggest they are present in all angiosperms (Table 3.4). They showed antibacte-
rial, antifungal, anticancer and other properties in Brazilian bioassays (Table 3.4).
Lignans and neolignans are a diverse group of compounds formed by the coupling of two phen-
ylpropanoid (C6C3) units (Figure 3.15). The nomenclature recommended by International Union of
Pure and Applied Chemistry (IUPAC) in 2000 respects the first definition of lignan introduced by
Haworth, for whom the term “lignan” was introduced for the structures where the two units C6C3
are linked by a bond between positions 8 and 8′-linked (or β,β′) (Moss, 2000). For nomenclature
purposes the C6C3 unit is treated as propylbenzene and numbered from 1 to 6 in the ring, starting
from the propyl group, and with the propyl group numbered from 7 to 9, starting from the ben-
zene ring. When the two are coupled in other ways they are called neolignans. This group is also
considered to include examples where the two units are joined by an ether oxygen atom which for
nomenclature purposes is treated as linking oxygen of an assembly. In addition, the class names
lignan and neolignan are spelled in the conventional way without a terminal “e”. The structures are
spelled with a terminal “e” to indicate a saturated side chain unless modified to show unsaturation.
The compounds (Figure 3.18) encountered may be classified into lignane: 3′,4′,5,9,9′-pentamethoxy-
3,4-methylenedioxy-lignane (112), 3,4,5,5′-tetramethoxy-3′,4′-methylenedioxy-7,7′-epoxy-lignane
(113), 5,5′-dimethoxy-3,4,3′,4′-dimethylenedioxy-7,7′-epoxy-lignane (114), 3,4,5,3′,4′,5′-hexamethoxy-
7,9′:7′,9-diepoxy-lignane (115), 9-hydroxy-3,4,3′,4′-dimethylenedioxy-9,9′-diepoxy-lignane (116),
4′,8-dihydroxy-3,3′,4′-trimethoxy-lignano-9,9′-lactone (117); neolignane: 3′-methoxy-3,4-
methylenedioxy-2 ′,7-epoxy-4′H-8,1′-neolign-8′-en-4′-one (118), 4-hydroxy-3,3′-dimethoxy-
4′,7-epoxy-8,3′-neolign-7′-ene (119), 9-hydroxy-3′,4′,5-trimethoxy-4,7′-epoxy-3,8′-9′norneolignan
(120), 9-hydroxy-5-methoxy-3′,4′-methylenedioxy-4,7′-epoxy-3,8′-9′norneolignan (120). In Table 3.3
the name given to these compounds in the cited reference was considered, and in several of them
they considered the Gottlieb nomenclature.
According to Gottlieb the difference between both these subclasses, rather than structural features,
is the presence (lignans) or absence (neolignans) of oxygen functions at terminal carbon of the C3 side
chains. This difference is due to different biosynthetic pathways; cinnamyl alcohol or less commonly
cinnamic acid is the precursors of lignans, while propenylphenol or allyphenol are the precursors of
neolignans. However, the tendency is for the IUPAC proposal to be universally accepted as it has been
discussed with representatives of numerous countries and respects the first definition.
Many lignans show physiological activity as tumor-inhibiting, some exhibit antibiotic, antima-
larial, antileishmanial and trypanocidal activities (Table 3.4).
3.3 IMPACT ON BRAZILIAN ECOSYSTEMS

Finally, this survey indicates that the families most studied in Brazil are, in order of their decreasing
total metabolites reported: Rutaceae, Meliaceae, Lauraceae, Rubiaceae, Fabaceae, Euphorbiaceae,
Piperaceae, Myrtaceae, Verbenaceae, Anarcadiaceae, Celastracea, Annonaceae, Asteraceae,
Moraceae and Sapindacea, and they offer considerable potential for the discovery of new or known
compounds with significant and possibly valuable biological activities.
TABLE 3.4
Occurrence of Phenylpropanoids Identified from Brazilian Plants over the Last 20 Years
Occurrence (plant part)
Code Phenylpropanoids Substituents Activity (references)
Coumarins Anti inflamatory Typha domingensis
95 Umbelliferone Typhaceae (aerial parts)
(Vasconcelos J. F. et al., 2009).
96 5-Methoxy-6,7-ethylenedioxycoumarin OCH3 Antifungal Pterocaulon alopecuroides, Pterocaulon
Ayapin H balansae, Pterocaulon polystachyum
97 R1 R2 Asteraceae (leaves)
Prenylatin OH CH2CHC(CH3)2 (Stein A. C. et al., 2006).
Prenylatin-methyl-ether OCH3 CH2CHC(CH3)2
7-(2′,3′-Epoxy-3′-methylbutyloxy)-6- OCH3 CH2CHOC(CH3)2
methoxycoumarin
98 7-Prenyloxy- R1 Antinociceptive Polygala sabulosa
6-methoxycoumarin CH2CHC(CH3)2 Polygalaceae (whole plant)
Scopoletin H (Meotti F. C. et al., 2006).
Acetylscopoletin COCH3
Benzoylscopoletin C6H6
99 Marmesin Anticholinesterase Conchocarpus fontanesianus
Rutaceae (stems)
(Cabral R. S. et al., 2011).
100 (+)-3-(1′-Dimethylallyl)-decursinol Antileishmanial Helietta apiculate
101 (-)-Heliettin Rtaceae (stem bark)
(Ferreira M. E. et al., 2010).
Flavonoids R1 R2 R3 Antimicrobial Arrabidaea brachypoda
102 3′,4′-Dihydroxy-5,6,7- CH3 CH3 OH Bignoniaceae (leaves)
trimethoxyflavone (Alcerito T. et al., 2002).
Cirsiliol H CH3 OH
Cirsimaritin H CH3 H
Hispidulin H H H
(Continued)
57
58
103 R1 R2 Citotoxic Kalanchoe pinnata
Kaempferol 3-O-α-L-arabinopyranosyl H 3-O-α-L-arabinopyranosyl Crassulaceae (leaves)
(1 → 2) α-L-rhamnopyranoside (1→2) α-L-rhamnopyranoside (Muzitano M. F. et al., 2006).
Quercetin 3-O-α-L-arabinopyranosyl OH 3-O-α-L-arabinopyranosyl
(1 → 2) α-L-rhamnopyranoside (1→2) α-L-rhamnopyranoside
Quercitrin OH α-L-rhamnopyranose
Apigenin R1 R2 Sedative Passiflora quadrangulares
H H Passifloraceae (fruits)
(Gazola A. C. et al., 2015).
H H Antitumoral Croton betulaster
Euphorbiaceae
(Santos B. L. et al., 2015).
104 Rutin R1 Antitumoral Dimorphandra mollis
3-O-rutinoside Fabaceae
Quercetin OH (Santos B. L. et al., 2015).
105 Odoratin R1 R2 R3 Inhibition of DNA Amburana cearenses
H OH OCH3 topoisomerase II Fabaceae (resin)
Calycosin OH H H (de Oliveira G. P. et al., 2017).
106 Erycibenin D
107 Penduletin R1 R2 Antitumoral Croton betulaster

H OH Euphorbiaceae
Casticin OH OCH3 (aerial parts)
(Coelho P. L. C et al., 2016).
(Continued)
108 3′,4′,5,7,8-Pentamethoxyflavone R1 Antitrypanossomal Neoraputia alba Neoraputia paraensis
Rutaceae (leaves)
OCH3 (Moraes V. R. S., et al., 2003).
3′,4′,5′,5,7-Pentamethoxyflavone H
109 3′,4′,7,8-Tetramethoxy-5,6-(2″,2″-
dimethylpyrano)-flavone
110 Agathisflavone Antiviral Cenostigma macrophyllum
Fabaceae
(stem and leaves)
(de Sousa R. L. F. et al., 2015).
111 R1 Antioxidant Garcinia brasiliensis
Amentoflavone OH Clusiaceae (leaves)
Podocarpusflavone OCH3 (Arwa P. S. et al., 2015).
112 Lignans and neolignans Anti-inflammatory Phyllanthus amarus
Niranthin and antiallodynic Euphorbiaceae
(aerial parts)
(Kassuya C. A. L. et al., 2006).
113 rel-(7R,8R,7′R,8′R)-3′,4′- Antitrypanossomal Piper solmsianum
Methylenedioxy -3,4,5,5′ Piperaceae (inflorescence)
-tetramethoxy-7,7′-epoxylignan Martins R. C. C. et al., 2003.
114 rel-(7R,8R,7′R,8′R)-3,4,3′,4′-
dimethylenedioxy-5,5′-dimethoxy-
7,7′-epoxylignan
115 Yangambin Antileishmanial Ocotea duckei
Lauraceae (leaves)
(Neto R. L. M. et al., 2011).
116 Cubebin Anti-inflammatory Zanthoxyllum naranjillo Rutaceae
(leaves)
(Bastos J. K. et al., 2001).
59
(Continued)
60
117 (−)-Trachelogenin Antitumoral Combretum fruticosum
Combretaceae (stalks)
(Moura A. F. et al., 2018).
118 Burchellin Antidiuretic and Aniba burchelli Lauraceae (leaves),
antitrypanossomal Ocotea cymbarum Lauraceae (bark)
(Cabral M. M. O. et al., 2000, Cabral
M. M. O. et al., 2010).
119 Licarin A Antitrypanossomal Nectandra glabrescens Lauraceae
(fruits)
(Cabral M. M. O. et al., 2010).
120 R1 R2 Antibacterial and Styrax ferrugineus
antifungal Styracaceae (leaves)
5-(3″-Hydroxypropyl)-7-methoxy-2- -CH2- (Pauletti P. M. et al., 2000).
(3′ ,4′-ethylenedioxyphenyl)
benzofuran
5-(3″-Hydroxypropyl)-7-methoxy-2- CH3 CH3

(3′,4′-dimethoxyphenyl)benzofuran

61
FIGURE 3.15 Biosynthesis of phenylpropanoids through shikimate pathway (taken from Cheynier et al., 2013; Ferrer et al., 2008)
FIGURE 3.16 Structure of coumarins identified from Brazilian plants over the last 20 years, based on
information in Table 3.4.
For over 20 years, Brazil has become the largest consumer of pesticides worldwide. The increased
farming of biofuel crops and the use of genetically modified seeds caused the pesticide sales in
Brazil to more than double recently, with the country surpassing the United States to become the
largest market in the world. In spite of intensive research on plant natural products over the past
three decades, only one type of botanical insecticides has been commercialized with any success in
the past 15 years. It is based on neem seed extracts (limonoid azadirachtin, Meliacee) (da Silva
et al., 2010). In Brazil, few groups are working to search for agricultural pesticides; this review showed
that the vast majority have the objective of research to search for potential drugs. The data discussed
above show that it is urgent to stimulate the scientific community to use scientific knowledge
and expertise to improve pest management practices for the benefit of Brazil and the environment.
Brazil recently had an initiative to address this problem. The National Institutes of Science and
Technology Program, which was launched in July 2008 by the Ministry of Science and Technology –
National Council for Scientific and Technological Development (CNPq), with the collaboration of São
Paulo Research Foundation (FAPESP), recruited scientists to work in networks in research areas that
are strategic to the sustainable development of the country. Thus, the São Carlos Federal University
(UFSCar, Brazil) aggregated in networks the best research groups in chemical ecological areas from
five states and seven institutions to transform Brazil into the model country for the control of insects
with a low environmental impact and created the National Institute of Science and Technology for the
Biorational Control of Pest-Insect (NIST-BCPI). The NIST-BCPI is involved in teaching, research
and extension oriented for the development of skilled researchers and the generation of knowledge
and agrochemical products through the following areas: (i) Natural products as sources for new pes-
ticides; (ii) Semisynthetic modifications; (iii) The mode of action of natural and synthetic pesticides
via the inhibition of enzymes: immobilized enzymes reactors; (iv) Nanotechnology to improve activ-
ity, solubility and stability and (v) Citrus diseases and resistance mechanisms.
The example below should serve as a stimulus for the young Brazilian scientific community to
start their research line with a focus on agriculture.
Citrus trees can exhibit a host of symptoms reflecting various disorders that can affect their
health, vigour and productivity to varying degrees. Correctly identifying symptoms is an impor-
tant aspect of management, as inappropriate remedial applications or actions can be costly and
sometimes detrimental. Many diseases are difficult to distinguish from one another. Early disease
detection and management are essential to ensuring the continued viability of the citrus industry.
The rapid communication of new diseases, significant outbreaks and accurate information are vital.
One of the major biotic diseases in Citrus is citrus variegated chlorosis (CVC). Xylella fastidiosa, a
Gram-negative bacterium, colonizes plants xylem, thereby causing CVC in sweet orange. Flavonoids
are the most bioactive secondary metabolites of citrus; however, only a few references to the role of
FIGURE 3.17 Structure of flavonoids identified from Brazilian plants over the last 20 years, based on infor-
mation in Table 3.4.
these compounds in citrus tolerance to the CVC bacterium could be found. Alves and collaborators
(2009) studied how X. fastidiosa colonizes and spreads within the xylem vessels of the sweet orange
C. sinensis cultivar (cv.) Pêra (Alves et al., 2009). The authors reported that X. fastidiosa initially
attached to the cell wall followed by an increase in the number of bacteria, the production of strand-
like material and the formation of biofilm. Needle-like crystallized material was often present in
xylem vessels of C. sinensis that were infected with X. fastidiosa. One hypothesis was that the
needle-like crystal was hesperidin (121; Figure 3.19). These crystals were not observed in healthy
plants. Hesperidin (121) is most likely involved in natural defence or in resistance mechanisms
against X. fastidiosa in sweet orange varieties. A HPLC-UV method was developed to quantify hes-
peridin in the leaves and stems of C. limonia. This quantification method was applied to C. sinensis
grafted onto C. limonia with and without CVC symptoms after X. fastidiosa infection. The total
FIGURE 3.18 Structure of lignans and neolignans identified from Brazilian plants over the last 20 years,
based on information in Table 3.4.
FIGURE 3.19 Structure of the flavonoid hesperidin (121).
content of hesperidin significantly increases in symptomatic leaves. Scanning electron microscopy

studies on leaves with CVC symptoms showed vessel occlusion by biofilm and revealed crystal-
lized material. Considering the impossibility of isolating these crystals for analysis, tissue sec-
tions were analyzed by Matrix-Assisted Laser Desorption/Ionization Mass Spectrometric Imaging
(MALDI-MSI) to confirm the presence of hesperidin at the site of infection. The images that were
constructed from Mass Spectrometry/Mass Spectrometry (MS/MS) data with a specific diagnostic
fragment ion also showed higher concentrations of hesperidin in infected plants than in healthy
ones, mainly in the vessel regions. These data suggest that hesperidin plays a role in plant-pathogen
interactions, most likely as a phytoanticipin. This HPLC-UV method is simple and accurate for the
determination of hesperidin in C. sinensis, C. limonia and their respective grafts. Citrus producers
in Brazil have undertaken several control measures, including the eradication of diseased plants
to remove the inoculum, spraying insecticides to reduce the population of transmission vectors
(sharpshooters), and producing seedlings in greenhouses that are covered by plastic and laterally pro-
tected by screens. These measures have increased production costs that could reach US$ 286 to 322
million per year. The HPLC-UV method that was developed and applied to 60 citrus plants showed
a 22% increase in the hesperidin content in asymptomatic plants. This increase may indicate the
presence of the bacteria. Plants without CVC symptoms, also known as asymptomatic, are the plants
which showed test positive by polymerase reaction chain (PCR) for X. fastidiosa, and the negative
controls are the plants that were not inoculated with the bacteria. Therefore, the HPLC-UV method
has become a powerful tool for detecting CVC in citrus before symptoms appear, thereby informing
the citrus producers in advance when the plant should be removed from the orchard. This method
could prevent the disease from being transmitted to other plants by insects and represents signifi-
cant savings in pesticide application costs. In addition, it is less expensive to detect CVC disease in
asymptomatic sweet orange trees using HPLC-UV than using other methods, such as PCR, and many
samples can be screened per hour using approximately 1 mg of leaves (Soares et al., 2015).
Several flavonoids were tested for in vitro activity on the growth of X. fastidiosa, with hesperidin
showing moderate activity (MIC 3.3 μM), suggesting that it can act as a good barrier for small-sized
colonies from X. fastidiosa (Ribeiro et al., 2008). The main problem with natural products is gener-
ally low solubility in aqueous media when performing the tests, which has been and will continue to
be a challenge. Therefore, the search for novel semisynthetic modifications of some flavonoids has
attracted attention. One strategy would be to promote the metal chelation of hesperidin and study
the potential biological relevance of these interactions. Magnesium is a metal of interest because of
its biological importance as an essential metal for life, participating in a variety of metabolic and
physiological functions. Moreover, compounds with magnesium (II) are good models for semisyn-
thetic modifications of hesperidin because these species readily react with O-heterocyclic ligands,
yielding stable compounds, and may provide a water-soluble compound. Because hesperidin activ-
ity is attributed to the generation of reduced metabolites that are involved in its antioxidant activity,
it is expected that the Mg-hesperidin complex, in which the hesperidin ligand would be more acces-
sible to oxidation, exhibits greater antioxidant activity than free hesperidin. Using this strategy and
aiming to develop a more water-soluble compound that is bioactive and has reduced toxicity and
luminescent diagnostic properties, the [Mg(hesp)2(phen)] complex, where hesp is hesperidin
and phen is 1,10′-phenanthroline were prepared (Oliveira et al., 2013).
The complex [Mg(hesp)2(phen)] is more hydrosoluble (S = 472 ± 3.05 μg mL−1) and liposoluble
(log P = −0.15 ± 0.01) than free hesperidin (S = 5.92 ± 0.49 μg mL−1, log P = 0.30). This complex
is a better radical scavenger for superoxide radical (IC50 = 68.3 μM at pH 7.8) than free hesperidin
(IC50 = 116.68 μmol L−1) and vitamin C (IC50 = 852 μmol L−1). Hesperidin and its complex were
assayed on the growth of X. fastidiosa in vitro, and the complex showed a better MIC than hesperi-
din, 0.34 and 3.3 μM, respectively (in vivo bioassay is in development).
Finally, the study of these interactions has led to new potential models for pesticides, suggesting
that this line of research addresses the needs of Brazilian agriculture and should continue.
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4 Molecular Biology Tools
to Boost the Production
of Natural Products
Potential Applications for
Luzia Valentina Modolo, Samuel Chaves-Silva,

Thamara Ferreira da Silva, and Cristiane Jovelina da-Silva
Departamento de Botânica, Instituto de Ciências Biológicas,
Universidade Federal de Minas Gerais, Belo Horizonte, Brazil
CONTENTS
4.1 Introduction.............................................................................................................................. 71
4.2 Forward Genetics..................................................................................................................... 72
4.3 Genome Editing and Reverse Genetics.................................................................................... 72
4.4 Gene Edition Based on Crispr/Cas9 Technology..................................................................... 75
4.5 Synthetic Biology.................................................................................................................... 76
4.6 Molecular Biology in the Context of Brazilian Medicinal Plants............................................ 78
4.7 Use of Molecular Biology to Improve the Production of Valuable Metabolites in
Medicinal Plants...................................................................................................................... 79
References�� 84
4.1 INTRODUCTION
The ability to synthesize a variety of chemically diverse metabolites makes plants a potential
source of inspiration to produce valuable compounds. Natural products have been used since
ancient times in fragrances as pigments, as pesticides and as therapeutics (Facchini et al.,
2012). Even though, potentially, many more secondary metabolites are yet to be discovered,
of 200,000–1,000,000 bioactive substances estimated to occur in the plant kingdom (Afendi
et al., 2012; Dixon and Strack, 2003).
A known fact is that many plants synthesize valuable compounds in amounts that are usually
not enough to meet the various commercial demands, thus, molecular biology is an interesting
approach for the large-scale production of structurally complex substances. In fact, the advance
in sequencing technologies has boosted research on plant genomics in recent years. The develop-
ment of bioinformatic tools for the analysis of transcriptional data has been of considerable help
in studies on evolution, organization and gene expression regulation. The generation of metabo-
lite libraries and elucidation of species-specific biosynthetic pathways beyond those reported for
model plants such as Arabidopsis thaliana (Brassicaceae), Medicago truncatula (Fabaceae) or
71
Oryza sativa (Poaceae) serve to expand the knowledge on the biosynthesis of natural products in
medicinal plants (Unamba et al., 2015). Once identified, the genes involved in the biosynthesis of
a valuable substance, new biotechnological approaches such as synthetic biology, genome editing
and reverse genetics can then assist the manipulation of biosynthetic pathways in planta or in a
host (e.g. bacteria, yeast, insect cells, cell culture, hairy root culture, etc.). This approach can lead
to improve the production of desired compounds. The following topics provide details on some
technological approaches based on molecular biology that are useful to enrich the accumulation of
natural products in living cells.
4.2 FORWARD GENETICS

The biosynthesis of secondary metabolites in plants is a complex process controlled by genetics
and environment (Kessler and Kalske, 2018). The identification of genes involved in a pathway is
the very first step to rationally modulate plant secondary metabolism (Yang et al., 2014). Prior to
the advent of whole genome sequencing, forward genetics was predominantly used to identify the
molecular basis associated with a trait of interest (Tierney and Lamour, 2005). From the observa-
tion of a particular phenotype originating from a naturally occurring mutation, one can identify
the genes involved in that trait. This approach is based on “phenotype to gene” determinations.
The observable variation (phenotype) may also be purposely induced in cells or an organism using
a DNA mutagen. The investigator eventually ends up sequencing the gene or genes thought to be
involved in a certain biosynthetic pathway related to the observable trait (Figure 4.1). The main
strategies used in forward genetics are map-based cloning and mutational breeding. The former
consists of mapping of a biparental population based on recombination frequency during meiosis
and identifies the underlying genetic cause of a mutant phenotype. Mutational breeding, on the other
hand, induces mutation to generate various phenotypes to identify candidate genes. This technique
is used to obtain new genetic combinations, without changing the major total genetic setup of an
organism (Abbai et al., 2017). Both techniques are widely used in the study of medicinal plants:
recognizing there is the disadvantage that only one trait can be analyzed at a time, some genes
may be missed in the screening and the procedure is feasible only in plants that are amenable
to Agrobacterium transformation. Examples of mutagens used in forward genetics include X-rays
and ethylmethanesulfonate (EMS), in which the former may generate large deletions in the chro-
mosomes or chromosomes rearrangements (it induces point mutation while the latter causes point
mutation – changes at a single nucleotide position).
4.3 GENOME EDITING AND REVERSE GENETICS

In the early 2000s, a large repository of sequence data was created with the disclosure of A. thaliana
and O. sativa (rice) genomes (Arabidopsis Genome Initiative, 2000; Goff et al., 2002; Yu et al.,
2002), among others. Nowadays, at the post-genomic stage, technology has advanced, the cost to
generate omics data has been reduced and a set of computational tools for data analysis has been
developed, leading to the annotation and identification of thousands of genes (Abbai et al., 2017).
Although nucleotide sequences of several plant genes have been revealed, the function of most of
them and the involvement in specific networks remains to be elucidated. Thus, the current challenge
in plant research is the analysis of gene function. Therefore, genome editing techniques is a useful
tool to achieve this goal.
Genome editing is a resource used to make changes in specific regions of a genome (e.g. inser-
tion, substitution or deletion of DNA fragments) of a cell or organism for several purposes. This
technique is based on the cleavage of the DNA double strand in targeted regions followed by the
use of the own cell repair system to introduce precise mutations (Tan et al., 2018). In this scenario,
reverse genetics (from gene to mutant phenotype) arises as a powerful tool to unravel gene func-
tion (Alonso and Ecker, 2006). In reverse genetics, a specific gene or gene product is disrupted or
Applications for Brazilian Medicinal Plants 73
FIGURE 4.1 Some differences between forward and reverse genetics. Reproduced from Alonso and Ecker
(2006) with permission by Springer Nature.
modified, and the plant phenotype is consequently determined (Figure 4.1; Tierney and Lamour,
2005). Some strategies of reverse genetics are described in the literature. Among them are included
those that rely on the use of zinc-finger nucleases (ZFNs), transcription activator-like effector nucle-
ases (TALENs), homologous recombination, RNAi, T-DNA insertional mutagenesis, targeting
induced local lesions in genome (TILLING) and clustered, regularly interspaced, short palindromic
repeat-Cas9 (CRISPR-Cas9) (Abbai et al., 2017).
The first technologies developed to break down DNA focused on the use of ZFNs and TALENs,
artificial enzymes generated by the fusion of DNA-binding domains to a nonspecific cleavage
domain of the bacterial endonuclease FokI produced by Flavobacterium okeanokoites (Li et al.,
2011). Such enzymes, however, were shown to introduce additional mutations in regions of the DNA
different from the target one (Zych et al., 2018). Furthermore, the process of generating customized
enzymes by specialized enterprises is time consuming, which makes the first-generation technol-
ogy expensive. TALEN was first applied to plants to generate Solanum tuberosum lines with lower
levels of both cholesterol and the toxic glycoalkaloids, such as α-solanine and α-chaconine (Sawai
et al., 2014).
Homologous recombination is a technique based on the similarity of sequence between the host
gene and the gene to be replaced. This method is simple and site-specific, but nevertheless works
better for less complex organisms (Abbai et al., 2017). Few studies highlighted the success of homol-
ogous recombination in crops (Iida and Terada, 2004), but no work using this technique on medici-
nal plants has been reported up to date. Nevertheless, homologous recombination can be considered
an important tool for synthetic biology in the coming years (Alonso and Ecker, 2006).
The RNA interference (RNAi) or posttranscriptional gene silencing technology has become an
important tool to speed up the breeding of medicinal plants, from which a conventional mutation
breeding approach was shown to fail (Allen et al., 2004). The RNAi works by knocking down the
expression of the target gene (Abbai et al., 2017). This provides an alternative to block the activity
of enzymes that are encoded by a multigene family and are expressed in different plant tissues at
distinct developmental stages. This technique has been employed to modulate the biosynthesis of
morphine-like alkaloids (psychoactive drugs) by interfering with the activity of codeinone reductase
(Allen et al., 2004). The gene that encodes for codeinone reductase was knocked down in Papaver
somniferum plants (opium poppy) through DNA-directed RNAi, which resulted in the accumulation
of (S)-reticuline, the precursor of isoquinoline alkaloid biosynthesis, at the expense of morphine,
codeine, oripavine and thebaine (Allen et al., 2004). This same technique was used to block the
activity of the berberine bridge enzyme in California poppy culture cells, also resulting in the accu-
mulation of (S)-reticuline (Fujii et al., 2007). Recently, RNAi technology was used to elucidate the
role of cytochrome CYP76AH1 in the metabolism of hairy roots of Salvia miltiorrhiza. The silenc-
ing of the CYP76AH1 gene affected the production of tanshinones (Ma et al., 2016). Therefore, this
gene is a potential target for metabolic engineering in medicinal plants.
The T-DNA vector is used to insert DNA fragments, in a completely random way, into the tar-
get genome. During transformation, there is a differential loss of several T-DNA genes (Abbai
et al., 2017). This loss can affect the growth and morphological patterns of hairy roots, expression
of biosynthetic pathway genes and accumulation of specific metabolites. In addition, the variabil-
ity in different insertion lines can be used to select the lines that are better producers of a desired
metabolite. One disadvantage of this technique is that it can be applied only for transformation
and tissue culture friendly plant species (Abbai et al., 2017). However, the T-DNA insertional
mutagenesis technique is most commonly used for reverse genetics to produce secondary metabo-
lites. This approach increased the production of alkaloids in Solanaceae (Moyano et al., 1999),
ginkgolides in Ginkgoaceae (Ayadi and Trémouillaux-Guiller, 2003), isoflavones in Fabaceae
(Shinde et al., 2009), plumbagin in Droseraceae (Putalun et al., 2010) and phenols in Lamiaceae
(Sitarek et al., 2018) plants.
Targeting induced local lesions in genome (TILLING) or chemical mutagenesis is a transformation-
free functional genomic technique. This technique comprises an alternative to apply on plants that
are not conducive to transformation or production of tissue cultures (Abbai et al., 2017). Two of
the most widely used mutagens in this technique are EMS (also used in forward genetics) and
ethylnitrosourea (ENU). The EMS is the most common mutagen applied to plants that functions
by alkylating guanine bases. The alkylated guanine will then pair with thymine instead of the pre-
ferred cytosine base, ultimately resulting in a G/C to A/T transition (Tierney and Lamour, 2005).
The ENU is a more potent mutagen than SEM and induces point mutations. It is also an alkylat-
ing agent that transfers an ethyl group to oxygen or nitrogen atoms present in the DNA structure.
Such alkylation leads to mispairing, base pair substitutions and even base pair losses (Tierney and
Lamour, 2005). A variant of TILLING is the EcoTILLING, in which the natural population is the
starting material (Abbai et al., 2017). The TILLING technology has already been used to increase
production of triterpenoid saponins in soybean (Glycine max) by a loss-of-function mutation in a
gene that encodes for the cytochrome P450 CY72A69 (Yano et al., 2017). The production of
cyanogenic glucosides decreased in Sorghum bicolor by using this same technique (Blomstedt et al.,
2012). Similarly, the EMS TILLING was useful to generate Catharanthus roseus plants with new
alkaloid profiles, accumulating mainly intermediates of vindoline biosynthesis (Edge et al., 2018).
The main current disadvantage of using reverse genetics in medicinal plant research is that not
all techniques related to this approach can be applied to all organisms. This is because many valu-
able secondary metabolites are produced by exotic plant species or in some cases by woody spe-
cies. Hence, the available methods of reverse genetics may not be suitable for such species due to
unavailability of transgenesis protocols, besides unreasonable time scales (Gandhi et al., 2015). For
organisms that are difficult to be transformed, TILLING appears to be an interesting alterative,
unless the genome of the target plant is riddled with mutations that make hard the detection of
mutational phenotypes (Tierney and Lamour, 2005). Despite the drawback, reverse genetics can be
improved since it is promising for boosting the production of metabolites of pharmacological inter-
est. It may be used to activate naturally silenced routes: the “silence part” of a pathway to allow for
the accumulation of intermediate valuable substances and/or silence routes to obtain cleaner and
contaminant-free desired metabolites.
A new generation genome editing technology called clustered, regularly interspaced, short pal-
indromic repeat-Cas9 (CRISPR-Cas9) has been recently developed and does not rely on the use of
customized restriction enzymes. More details about this technology will be given in the next topic
of this chapter since this know-how can be used for both forward and reverse genetics.
The current challenge for the broad application of available genome editing technologies to boost
natural products production is the establishment of protocols for transformation and regeneration of
plant species other than the model ones.
4.4 GENE EDITION BASED ON CRISPR/CAS9 TECHNOLOGY

In CRISPR/Cas9 technology, the cleavage of double-strand DNA is assisted by a single nuclease
(Cas9) that is guided to the target by a small RNA through Watson–Crick base pairing (Gasiunas
et al., 2012). This method is of relatively low cost, versatile, easy to perform, highly specific and
efficient that can be used for both forward and reverse genetics. This gene editing system enables the
generation of an organism with a “clean” modified genome, which would essentially make it a non-
genetically modified organism (Abbai et al., 2017). CRISPR-Cas9 technique was developed from
the observation of the defense system, naturally occurring in bacteria. One of the defense mecha-
nisms exhibited by prokaryotes is the regularly interspaced short palindromic repeats (CRISPR).
The locus received this name because it is constituted of sequence repeats of 29 base pairs separated
by variable sequences of 32 nucleotides referred to as spaces (Ishino et al., 1987). The sequencing of
some bacteria and virus genomes shed light on the compatibility of the CRISPR spaces with phages
and plasmids. Currently, it is known that CRISPR is involved in a protection system highly con-
served among prokaryotes: 45% of bacteria and 90% of Archaea possess CRISPR locus (Nemudryi
et al., 2014). For instance, bacteria capture DNA fragments from an invading virus to generate small
DNA sequences called CRISPR arrays that will function as a “memory” of the pathogen attack for
future self-defense, in case the virus and related pathogens try to infect the bacteria again. The bac-
terial enzyme Cas9, breaks down the viral DNA to prevent its action (Barrangou et al., 2007). The
system CRISPR-Cas9, for the sake of genome editing, works in a similar way. First, it is synthesized
as a short RNA sequence (about 20 base pair long) containing a guiding sequence that is comple-
mentary to a specific sequence of the DNA to be edited. A complex formed between Cas9 and the
short RNA sequence then “search” for the complementary region in the target DNA and intercalates
the DNA double strand at that point to indicate the region where Cas9 is required to break down
(Hsu et al., 2014). The host cell repairing machinery can take care of the damaged DNA, adding or
suppressing DNA fragments or even substituting DNA fragments for customized DNA. The DNA
repair may occur by two mechanisms: (1) nonhomologous end-joining or (2) homology-directed
repair (Zych et al., 2018). In the first mechanism, the DNA ends become adjacent to recombine
without a template, which in turn can lead to insertions/deletions, altering the gene open reading
frame. As for the homology-directed repair strategy, disrupted sequences are resynthesized, using
as a template a homologous sequence throughout the genome (Wyman and Kanaar, 2006). Thus,
homology-directed repair is useful to promote site-directed genome editions, while nonhomologous
end-joining is used to rearrange chromosomes or generate functional knockouts (Montano et al.,
2018; Tan et al., 2018).
The most common employed CRISPR/Cas9 system is the one that uses the endonuclease Cas9
from Streptococcus pyogenes and a chimeric single guide RNA (sgRNA) to direct the endonuclease
to the target. The cleavage sites need to be near a sequence termed PAM (Protospacer Adjacent
Motif; 5′-NGG-3′). This can be achieved by customizing the 5′ region of sgRNA so that it can reach
any genome sequence near PAM (Cong et al., 2013). In addition, multiples genes can be edited
simultaneously by using several sgRNAs.
Since the beginning of the 2010s, the CRISPR/Cas9 system has efficiently edited specific genes
in bacteria (Jiang et al., 2013), mice (Yin et al., 2014), human cells (Cong et al., 2013) and plants
(Ito et al., 2015). In the scope of plants, CRISPR/Cas9 has been used to edit genes in crops such as
O. sativa, Solanum lycopersicum, Zea mays and Triticum aestivum (Mishra and Zhao, 2018). This
technology was employed to increase the levels of γ-aminobutyric acid (GABA) in S. lycopersicum
(Nonaka et al., 2017). GABA is a non-proteinogenic amino acid that possesses hypotensive properties.
Such success opens a window for the application of CRISPR/Cas9 on medicinal plants as well.
The efficiency of CRISPR/Cas9 system on medicinal plants was first demonstrated in a study with
opium poppy. CRISPR-Cas9 system was used to knock out an O-methyltransferase gene (4′OMT2)
involved in the biosynthesis of benzylisoquinoline alkaloids. This significantly decreased the pro-
duction of alkaloids, such as thebaine, codeine, noscapine and papaverine, in the transgenic plants.
Furthermore, a novel uncharacterized alkaloid was observed only in CRISPR/Cas9 edited plants,
demonstrating how this technique is useful for metabolic engineering and the discovery of new
compounds in genome-edited plants (Alagoz et al., 2016). Furthermore, the CRISPR-Cas9 technol-
ogy efficiently knocked out a diterpene synthase gene (SmCPS1) involved in a committed step of
tanshinones biosynthesis in S. miltiorrhiza (Chinese medicinal plant), such as cryptotanshinone,
tanshinone IIA and tanshinone I. The use of this technique decreased the amount of the target
metabolites, without interfering with the biosynthesis of other phenolic acid metabolites (Li et al.,
2017). Likewise, the gene SmRAS, which encodes for rosmarinic acid synthase, was silenced in the
same species using the CRISPR/Cas9 system, causing disruption in the production of rosmarinic
acid and lithospermic acid B (Zhou et al., 2018).
4.5 SYNTHETIC BIOLOGY

Synthetic biology is a transdisciplinary science based on the knowledge on biology, chemistry,
engineering, physics and informatics used to further understand how living systems function to
redesign them for specific purposes (Moses and Goossens, 2017). A milestone article on synthetic
biology was published in 2010, in which scientists successfully created the bacteria Mycoplasma
mycoides JCVI-syn1.0 controlled by a synthetic genome (Gibson et al., 2010). This was the begin-
ning of a most ambitious goal – to transform bacteria, yeast, algae and virus in synthetic organisms
(bearing synthetic genomes) to perform specific functions such as the sustainable production of
valuable molecules and biomaterials. Researchers who work on this branch of science use and/or
modify techniques related to genetic engineering, microbiology and bioinformatics to design, syn-
thesize and transfer DNAs to microorganisms (van der Helm et al., 2018). In a manner analogous
to a computer, the microorganisms would be the hardware, while the synthetic DNA, the software.
Idealized in a virtual environment, the synthetic DNA contains “scripts”, a series of programmable
commands, that once integrated, will result in several responses by the microorganism that host the
synthetic DNA (Wohlsen, 2011). Synthetic biology has the potential to revolutionize the production
of plant-derived natural products from unicellular organisms (Moses and Goossens, 2017). Once a
biosynthetic pathway is well characterized from the genetic point of view, synthetic biology can be
used to introduce such pathways in heterologous expression systems such as Saccharomyces cerevisiae
or Escherichia coli. Such organisms are of relatively easy maintenance in the laboratory, thus the
shorter life cycle when compared, for instance, with whole medicinal plants. In this context, micro-
organisms are more advantageous when taking into account the production of natural products on a
large scale (Moses and Goossens, 2017).
One of the most notable examples of a successful application of synthetic biology to produce
plant metabolites in microorganisms was the production of artemisinic acid (precursor of the anti-
malarial agent, artemisinin) in S. cerevisiae (Paddon et al., 2013). Briefly, the genes that encode for
the enzymes of the mevalonate pathway were over-expressed in yeast cells together with the gene of
Artemisia annua that encodes for amorphadiene synthase. In A. annua, amorphadiene formed from
the activity of amorphadiene synthase is further converted to artemisinic acid through an oxidative
process that involves three steps. Based on this work, the following genes were inserted in the yeast:
CYP71AV1, CPR1 and CYB5 (they encode for enzymes involved in the production of artemisinic
alcohol from amorphadiene); ADH1 (it encodes for an enzyme that oxidizes artemisinic alcohol to
artemisinic aldehyde) and ALDH1 (it encodes for an enzyme that oxidizes artemisinic aldehyde to
artemisinic acid).
By using this yeast strain, engineered via synthetic biology, the global biopharmaceutical
company Sanofi started the large scale production of artemisinic acid in 2013/2014 (Paddon and
Keasling, 2014; Peplow, 2013). Notably, the use of such a biotechnological approach can increase by
over 30% the production of artemisinic acid to meet global demand for the antimalarial artemisinin.
The pharmacological proprieties of plant natural products belonging to the class of benzyliso-
quinoline alkaloids (BIAs) have caught the attention of synthetic biologists. For instance, BIAs
such as oxycodone, hydrocodone and hydromorphine are opioid analgesics supplied by pharma-
ceutical companies using semi-synthesis approaches. It was recently estimated that P. somniferum
(opium poppy) was cultivated in approximately 100,000 hectares to obtain 800 tons of thebaine
or morphine (natural precursors of semisynthetic BIAs) to meet the medical demand of analgesic
opiods) (Galanie et al., 2015). Although some synthetic routes to provide morphine and derivatives
are disclosed, none of them are commercially competitive or viable in large scale compared to the
semisynthetic approach (Reed and Hudlicky, 2015). Efforts in synthetic biology have been made
since the end of the 2000s to produce BIAs in microorganisms. S. cerevisiae was genetically modi-
fied to produce reticuline, a key intermediate of BIA’s biosynthesis, from the commercially avail-
able (R,S)-norlaudanosoline (Hawkins and Smolke, 2008). A few years later, a fermentation system
constituted from E. coli was developed to produce reticuline from simpler and cheaper carbon
sources (Nakagawa et al., 2012). In 2014, researchers achieved the introduction of ten plant genes
in S. cerevisiae, which in turn, resulted in the production of dihydrosanguinarine and sanguinarine,
BIAs of notable antimicrobe and antineoplasic activities (Fossati et al., 2014). Additionally, 21 and
23 genes (of plants, mammalians and bacteria origin) were introduced to yeast strains to make them
competent to produce thebaine and hydrocodone, respectively, from sugar (Galanie et al. 2015).
Another natural product group of medicinal interest contains the monoterpene indole alkaloids
(MIAs), represented, but not limited to, the anticancer agents vinblastine, vincristine and vinflunine
(Leggans et al., 2013). Strictosidine is the common intermediate to produce the structurally diverse
MIAs. The insertion of 21 genes (from which 14 are of known to participate in the biosynthesis of
MIAs) in S. cerevisiae and deletion of three genes from the yeast genome resulted in the production
of strictosidine by the altered yeast cells (Brown et al., 2015).
Other plant natural products such as glycyrrhetinic acid (Seki et al., 2011), taxadien (precursor of
the anticancer agent paclitaxel; Ajikumar et al., 2010), sapogenins and saponins (Moses et al., 2014)
were successfully produced in genetically engineered yeast or E. coli.
The upcoming challenges will be the improvement of tools to make viable the synchronized
expression of multiple genes, reduction of metabolic loads on the host and the development of
microorganisms with increased efficiency to produce xenobiotics (Moses and Goossens, 2017).
Although E. coli and S. cerevisiae have been used as the host to produce valuable phytochemicals,
other organisms might prove to be more efficient for large scale production purposes. Furthermore,
one might consider that a great microbial system is the one that transforms substrates of renew-
able sources or industrial byproducts to metabolites of multiple interests (Eisenstein, 2016; Rai
et al., 2017).
4.6 MOLECULAR BIOLOGY IN THE CONTEXT

OF BRAZILIAN MEDICINAL PLANTS
According to the Convention on Biological Diversity, Brazil hosts the greatest number of endemic
species on the planet, sheltering an estimated biota of over 170 thousand species, which cor-
responds to about 13.1% of the world’s known wealth. Endemism rates as high as 55% of plant
biodiversity are reported from Brazilian biomes (Stehmann and Sobral, 2017). Many natural
products originating from Brazilian medicinal plants have been disclosed. They are either used
as phytoterapics or as inspiration source for the design of valuable substances of pharmacologi-
cal, cosmetic, agronomic and supplemental food interests (Fougat et al., 2015). Indeed, over 60%
of the anticancer drugs are derived from natural products (Cragg et al., 1997). Among them,
β-lapachone and lapachol, used for the treatment of various neoplasms, are extracted from the
bark of Handroanthus impetiginosus, native to Brazil (Melo et al., 2011). Many other commercial
phytopharmaceuticals are known to be extracted from the Brazilian native flora. Quercetin, used
in treatment of heart disease, is extracted from Dimorphandra mollis, a native tree of Cerrado.
One of the main drugs used for the treatment of chronic glaucoma is pilocarpine, a phytophar-
maceutical used in treatment of glaucoma and extracted from Pilocarpos spp. Pilocarpine is also
used to treat xerostomia in patients undergoing radiotherapy for head and neck cancer, since it
stimulates the secretion of large amounts of saliva and sweat (Nogueira et al., 2010). Some of
these compounds, such as d-tubocurarine (extracted from Chondrodendron tomentosum) and
emetine (isolated from Carapichea ipecacuanha) are supplied by foreign pharmaceutical compa-
nies as anesthetics and vomiting inducer, respectively (Figure 4.2; Nogueira et al., 2010). Other
examples include, but are not limited to, emetine, pilocarpine, rupununine, d-tubocuranine and
vitexin (Figure 4.2).
Despite the great biodiversity, only 8% of Brazilian plant species were so far investigated for
bioactive compounds and relatively few of them had their medicinal properties evaluated (Simões
et al., 2003). Additionally, most medicinal plants used in the preparation of medicines are exotic
species brought to Brazil during the country’s colonization (Brandão et al., 2009). Nevertheless,
many efforts have been made to voucher native species for the purpose of valuing and prioritiz-
ing the research with Brazilian flora wealth. Indeed, a database and samples of aromatic, medici-
nal and toxic plants namely DATAPLAMT was created in the beginning of the 2000s, in which
a lot of information about native medicinal plants was compiled, mainly, for species located in
the Minas Gerais (Southeast Brazil). Some examples of bioactive compounds identified in plants
FIGURE 4.2 Structure of some notable pharmaceuticals produced by plant species native to Brazil.
The indicated pharmacological properties are described elsewhere (He et al., 2016; Nogueira et al., 2010;
www.thoughtco.com/drugs-and-medicine-made-from-plants-608413).
species belonging to the different Brazilian biomes are shown in Table 4.1, while Figure 4.3 shows
representative images of Brazilian medicinal plants that occurs in Amazon Forest, Atlantic Forest,
Caatinga, Cerrado, Pampa and Pantanal.
4.7 USE OF MOLECULAR BIOLOGY TO IMPROVE THE PRODUCTION

OF VALUABLE METABOLITES IN MEDICINAL PLANTS
The great advance in genomics integrated with high-resolution metabolomics became a powerful
tool to investigate structural aspects and the regulation of secondary metabolism in organisms (Kim
and Buell, 2015; Scossa et al., 2018). These approaches allow increasing the number of cells that pro-
duce a valuable natural product, increasing the carbon flux toward a particular biosynthetic pathway
by overexpressing key genes, blocking feedback inhibition, among others (Karuppusamy, 2009).
This can be better exemplified by the improvement of the accumulation of the alkaloid chemothera-
peutics, vinblastine and vincristine in C. roseus hairy roots (Wang et al., 2010). Both vinblastine
and vincristine are produced in very low levels in C. roseus leaves. However, the overexpression of
G10H and ORCA3 that encodes for geraniol 10-hydroxylase and octadecanoid-derivative responsive
TABLE 4.1
Some Bioactive Compounds Isolated from Plant Species Native to the Brazilian Amazon
Forest, Atlantic Forest, Caatinga, Cerrado, Pampa and Pantanal
Brazilian
Common Bioactive Phytogeographical
Family Scientific Name Names Compounds Domains Reference
Adoxaceae Sambucus australis Sabugueiro, Ursolic acid Atlantic Forest Rao et al.
(sin. Sambucus acapora (2011)
pentagynia)
Anacardiaceae Anacardium Cajueiro Catechin, Amazon and Trox et al.
occidentale (sin. epicatechin Atlantic Forests, (2011)
Anacardium Cerrado, Caatinga
microcarpum)
Anacardiaceae Myracrodruon Aroeira Artemiseole, Atlantic Forest, Figueredo et al.
urundeuva bergamotene, Caatinga, Cerrado (2014)
terpinolene
Annonaceae Annona crassiflora (sin. Araticum Epicatechin, Cerrado Lage et al.
Annona macrocarpa) peltatoside, (2014)
quercetin
Aquifoliaceae Ilex paraguariensis Erva-mate Theobromine, Atlantic Forest, Heck and de
(sin. Ilex curitibensis) theophylline Caatinga, Cerrado Mejia (2007)
Asteraceae Achyrocline Marcela Quercitrin, Atlantic Forest, de souza et al.
satureioides (sin. isoquercitrin, Cerrado, Pampa (2007)
Gnaphalium luteolin
saturejaefolium)
Asteraceae Mikania laevigata Guaco Campestero, Atlantic Forest, Ferreira and
taraxasterol Cerrado, Pampa Oliveira
(2010)
Asteraceae Baccharis Carqueja Bicyclogermacrene, Atlantic Forest, de Oliveira
genistelloides var. caryophyllene, Pampa, Cerrado et al. (2012)
trimera (sin. germacrene D
Baccharis trimera)
Asteraceae Eremanthus arboreus Candeeiro Bisabolol Caatinga Matos et al.
(sin. Vanillosmopsis (1988)
arborea)
Bignoniaceae Handroanthus Ipê-roxo Lapachol, Cerrado Gupta et al.
impetiginosus (sin. β-lapachona (2002)
Tabebuia impetiginosa)
Boraginaceae Cordia curassavica Erva-baleeira Sabinene, Amazon Forest, Nizio et al.
(sin. Varronia δ-elemene, Atlantic Forest, (2015)
curassavica) α-gurjunene, Caatinga, Cerrado
hellandrene
Bromeliaceae Bromelia antiacantha Gravatá Chrisin, hesperidin, Atlantic Forest Santos et al.
(sin. Hechtia hyperoside, Pampa (2009)
longifolia) orientin, quercetin,
rutin, vitexin
Celastraceae Maytenus ilicifolia (sin. Espinheira-santa Erythrodiol, Caatinga Ohsaki et al.
Monteverdia truncata) oxotingenol, (2004)
pristimerin
Cucurbitaceae Wilbrandia ebracteata Taiuiá Cucurbitacin Atlantic Forest, Peters et al.
(sin. Wilbrandia Cerrado (1999)
ebracteata var.
ebracteata)
(Continued)

Brazilian
Equisetaceae Equisetum giganteum Cavalinha Caffeic acid, Atlantic Forest, Jabeur et al.
(sin. Equisetum kaempferol Cerrado (2017)
bolivianum)
Euphorbiaceae Manihot esculenta (sin. Mandioca Esculentoic acids, Amazon and Idibie et al.
Manihot flexuosa) linamarin Atlantic Forests, (2007),
Caatinga, Cerrado, Chaturvedula
Pantanal et al. (2003)
Euphorbiaceae Croton grewioides (sin. Cunha Anethole, Caatinga Donati et al.
Croton zehntneri) caryophyllene, (2015)
estragole, myrcene
Fabaceae Copaifera langsdorffii Copaíba, Kaurenoic acid Atlantic Forests, Costa-Lotufo
(sin. Copaifera nitida) copaibeira Cerrado, Caatinga et al. (2002)
pau-de-óleo
Fabaceae Hymenaea courbaril Jatobá Halimane, Amazon and Abdel-Kader
(sin. Hymenaea diterpenoids Atlantic Forests, et al. (2002)
multiflora) Cerrado, Caatinga,
Pantanal
Fabaceae Stryphnodendron Barbatimão Epigallocatechin, Caatinga de Mello et al.
adstringens (sin. gallocatechin, Cerrado (1996)
Stryphnodendron
barbatimam)
Fabaceae Dimorphandra mollis Faveiro Rutin Amazon Forest, Lucci and
(sin. Ocotea rodiei) Cerrado, Pantanal Mazzafera
(2009)
Lauraceae Aniba rosaeodora (sin. Pau-rosa Linalool Amazon Forest d’Acampora-
Aniba duckei) Zellner et al.
(2006)
Lauraceae Chlorocardium rodiei Bibiri Rupununine Amazon Forest Gorinsky
(sin. Ocotea rodiei) (1996)
Lauraceae Ocotea odorifera (sin. Canela-sassafraz Camphor, safrole Atlantic Forest, Mossi et al.
Ocotea pretiosa) Cerrado (2013)
Meliaceae Trichilia catigua (sin. Catuaba Flavalignans Amazon and Pizzolatti et al.
Trichilia alba) Atlantic Forests, (2002)
Caatinga, Cerrado,
Pantanal
Menispermaceae Chondrodendron Curare, d-Tubocurarin Amazon Forest King (1940)
tomentosum (sin. parreira-brava,
Chondrodendron uva-da-serra,
hypoleucum) uva-do-mato
Menispermaceae Cissampelos Jarrinha, Desmethylroraime, Amazon and de Lira et al.
sympodialis orelha-de-onça, roraimine Atlantic Forests, (2002)
abuteira, Caatinga
milona
Moraceae Brosimum glaziovii Leiteira Campesterol, Atlantic Forest Coqueiro et al.
(sin. Alicastrum lupenone, (2014)
glaziovii) stigmasterol
(Continued)

Brazilian
Nyctaginaceae Boerhavia diffusa (sin. Pega-pinto Boeravinones, Amazon and Ahmed-
Boerhavia caespitosa) punarnavine Atlantic Forests, Belkacem
Caatinga, Cerrado et al. (2007),
Manu and
Kuttan (2009)
Piperaceae Piper hispidum (sin. Pimenta-longa Safrole Amazon and Estrela et al.
Piper hispidinervum) Atlantic Forests, (2006)
Caatinga, Cerrado
Plumbaginaceae Plumbago zeylanica Caataia, Plumbagin Amazon and Paiva et al.
(sin. Plumbago folha-de-louro, Atlantic Forests, (2004)
scandens) queimadura Caatinga
Rubiaceae Carapichea Ipecacuanha Emetine, ephaeline Amazon and Alves et al.
ipecacuanha (sin. Atlantic Forests, (2005)
Psychotria Caatinga, Cerrado
ipecacuanha)
Rubiaceae Genipa americana (sin. Genipapo Iridoids, genipin, Amazon and Ono et al.
Genipa venosa) gardendiol, Atlantic Forests, (2007)
shanzhiside Caatinga, Cerrado,
Pantanal
Rubiaceae Uncaria tomentosa Unha-de-gato Isopteropodine, Amazon Forest Laus et al.
(sin. Ourouparia speciophylline, (1997)
tomentosa) Akuammigine,
hirsuteine
Rutaceae Pilocarpus Jaborandi Pilocarpine Amazon Forest Sawaya et al.
microphyllus (2011)
Salicaceae Casearia sylvestris (sin. Guaçatonga Casearvestrins Amazon and Oberlies et al.
Casearia Atlantic Forests, (2002)
subsessiliflora) Caatinga, Cerrado,
Pampa, Pantanal
Sapindaceae Paullinia cupana Guaraná Caffeine, catechin, Amazon Forest Marques et al.
(sin. Paullinia epicatechin (2016)
sorbilis)
Solanaceae Solanum mauritianum Fumo-bravo Caulophyllumine Atlantic Forest Jayakumar
(sin. Solanum et al. (2016)
tabacifolium)
Urticaceae Cecropia pachystachya Embaúba Orientin, apigenin, Amazon and Cruz et al.
(sin. Cecropia luteolin Atlantic Forests, (2013)
catarinensis) Caatinga, Cerrado,
Pantanal
Verbenaceae Lippia origanoides (sin. Alecrim- Thymol, carvacrol Amazon and Botelho et al.
Lippia pimenta Atlantic Forests, (2007)
schomburgkiana) Caatinga, Cerrado
Winteraceae Drimys brasiliensis Casca-d’anta Polygodial, Atlantic Forest, Malheiros et al.
(sin. Drimys retorta) drimanial Caatinga, Cerrado (2005)
FIGURE 4.3 Brazilian medicinal plant species Anacardium occidentale, Bromelia antiacantha and
Hymenaea courbaril. A. occidentale is reported in Caatinga biome, B. antiacantha occurs in the Pampa
and Atlantic Forest biomes, whereas H. courbaril is found in Amazon and Atlantic Forests, Cerrado and
Pantanal. A. occidentale and H. courbaril images were kindly provided by Dr. João Renato Stehmann (Federal
University of Minas Gerais, Brazil). Dr. Mara Rejane Ritter (Federal University of Rio Grande do Sul, Brazil)
kindly provided the B. antiacantha image. Pictures are reproduced with permission by the owners.
Catharanthus AP2-domain, respectively, yielded higher amounts of catharanthine in hairy roots,

a precursor of vinblastine and vincristine biosynthesis (Wang et al., 2010). As a result, much larger
amounts of these anticancer compounds accumulated in C. roseus hairy roots. The biosynthesis of
the terpene artemisinin, a frontline antimalarial drug, was increased by roughly 80% in A. annua
due to overexpression of the transcription factor gene AaNAC1 that, in turn, led to the increased
expression of the genes that encode amorphous-4,11-diene synthase (ADS), artemisinic aldehyde
Δ11 (13) reductase (DBR2) and aldehyde dehydrogenase 1 (ALDH1) (Lv et al., 2016). The trans-
genic plants were also found to exhibit tolerance to drought and the fungus Botrytis cinerea
(Lv et al., 2016). Similarly, the simultaneous overexpression of the genes that encode for putrescine
N-methyltransferase (PMT) and hyoscyamine 6β-hydroxylase (H6H) in Hyoscyamus niger hairy
roots increased the accumulation of the alkaloid anticholinergic agent scopolamine by nine-fold
in comparison with the wild-type hairy roots (Zhang et al., 2004). The biosynthesis of resveratrol,
anti-inflammatory and antioxidant stilbene, was considerably stimulated in cell cultures of Vitis
amurensis overexpressing VaCPK29, a member of a multigene family of calcium-dependent protein
kinases (Aleynova et al., 2015).
Some efforts have been made to identify genes involved in synthesis of bixin, an apocarotenoid
produced by the South American native species Bixa orellana. Bixin is widely used in pharmaceuti-
cal, food, cosmetic and dye industries (Teixeira da Silva et al., 2018). Therefore, genetic engineering
could be an interesting approach for the heterologous expression of B. orellana genes in hairy roots
system, for instance, as an alternative for the large-scale production of such an important pigment.
Although the establishment of protocols to produce cell or hairy root cultures of some plant species
is quite challenging, there are still some approaches to be considered for the development of bioreac-
tor systems based on medicinal plants. Additionally, one can consider the heterologous expression of
genes from species native to Brazil in plant systems that are already used as models in the scope of
molecular biology. In this sense, the Nucleus of Bioassays, Biosynthesis and Ecophysiology of Natural
Products (NuBBE) created a database that became a source of eligible molecules that can be targeted
for the improvement of their production in heterologous systems. Conceived in 1998, the NuBBE
database (nubbe.iq.unesp.br/portal/nubbe-search.html) comprises a catalog of bioactive molecules,
in which the chemical, pharmacological and toxicological features of metabolites and derivatives are
disclosed and identified in species from the Brazilian biodiversity. The NuBBE database serves as
a useful tool for studies on multidisciplinary interfaces related to chemistry and biology, including
virtual screening, dereplication, metabolomics and medicinal chemistry. Such a database certainly is
contributing to a more sustainable development of the Cerrado and Atlantic Forest.
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5 Diversity of Endophytes and
Biotechnological Potential
Daiani Cristina Savi and Chirlei Glienke
Federal University of Paraná, Department of Genetics,
Curitiba, Brazil
CONTENTS
5.1 Endophytes – General Aspects................................................................................................. 91
5.2 Exploring Endophytes with Biotechnological Potential.......................................................... 91
5.3 Isolation of Endophytes........................................................................................................... 93
5.4 Diversity of Endophytes...........................................................................................................94
5.5 Exploring the Biotechnological Potential of the Isolated Endophytes....................................97
5.6 Diaporthe terebinthifolii: A Promising Species to Control the Citrus
Phytopathogen Phyllosticta citricarpa.................................................................................. 101
5.7 Conclusion............................................................................................................................. 103
References....................................................................................................................................... 104
5.1 ENDOPHYTES – GENERAL ASPECTS

The term “endophyte” is derived from the Greek whereby “endon” means within, and “phyton”
means plant. Thus, endophytes comprise microorganisms that for part of their life colonize plants
tissues without causing any damage to the host or disease symptoms (Hardoim et al., 2015). However,
the interaction between endophytes and their hosts depends on several aspects, such as environ-
mental, biological, chemical and physiological characteristics (Jia et al., 2016; Rajamanikyam
et al., 2017). Some studies have shown that the host plant and the interactions between other micro-
organisms can balance this symbiotic relationship (Jalgaonwala et al., 2012), which can be dis-
rupted, and the symptoms of the disease can occur due to endophytic virulence factors or due to
mechanisms of host defense (Kusari et al., 2012).
Endophytes colonize the intercellular space of various parts of the plant including roots, leaves,
stems, flowers and seeds (Liu et al., 2017). However, fungi are more commonly isolated from leaves
and stems, and bacteria in the roots (Roy and Sharma, 2015; Savi et al., 2015; Wang et al., 2016),
colonizing intercellular space in the plant, mainly because these areas have an abundance of car-
bohydrates, amino acids and inorganic nutrients (Kandel et al., 2017). In view of the specificity of
chemical compounds found in the host, one or two endophytic species are frequently predominant
in a specific host, while other isolates are considered rare (Bernardi-Wenzel et al., 2010). Thus,
studies of endophytes isolated from hotspot biomes can contribute to a better understanding of
Brazilian diversity, as well as the effect of the anthropological and environmental conditions in this
ecosystem.
5.2 EXPLORING ENDOPHYTES WITH BIOTECHNOLOGICAL POTENTIAL

Our key interest is to ask what makes medicinal plants found in Brazil a unique source for exploring
endophytes with biotechnological potential? Brazil has an inestimable biodiversity of plants, being at the
top of 17 megadiversity countries in the world (Mittermeier et al., 1997), with more than 55,000 species of
91
plants (http://www.unesco.org/new/en/brasilia/natural-ciences/environment/biodiversity/). These plants

are distributed in six biomes, Amazonian rainforest, Caatinga, Cerrado (Savanna), Atlantic Rainforest,
Pampa and Pantanal (Swampland) (Myers et al., 2000), based on the adaptation to biotic and abiotic
factors (Antonelli and Sanmartín, 2011; BFG, 2015; Forzza et al., 2010; 2012).
Two Brazilian biomes are recognized as biodiversity hotspots: Cerrado and Atlantic Rainforest
(Mittermeier et al., 1998; Myers et al., 2000). However, the diversity of some other biomes, such as
the Pantanal, remains relatively unexplored (Alho, 2011). Considering that some endophytes may
be host specific, the plant diversity found in Brazil represents an extraordinary variety of habitats,
life forms and biological associations confined to particular environments at different geographical
scales (Forzza et al., 2010).
Medicinal plants in Brazil have been widely documented and investigated for their unique biologi-
cal and chemical diversity (Atanasov et al., 2015). The great diversity of the Brazilian flora has been
used for pharmaceutical purposes and is known in the global market as the origin of many products
(Castro et al., 2014). We have been studying the biological potential of the endophytic community of
four medicinal plants in Brazil (Table 5.1), Vochysia divergens from the Pantanal (Gomes et al., 2013;
Gos et al., 2017; Hokama et al., 2017; Noriler et al., 2018; Savi et al., 2015), Stryphnodendron adstringens
from the Cerrado (Noriler et al., 2018), Maytenus ilicifolia and Schinus terebinthifolius from the Atlantic
forest (Figueiredo et al., 2018; Gomes-Figueiredo et al., 2007; Lima et al., 2012; Tonial et al., 2016).
The Pantanal is the largest wetland in the world, and it is subject to different climatic conditions
during the year, experiencing flooding and a dry season (Alho, 2008). As a consequence, the Pantanal
harbors its own flora (Arieira et al., 2006), especially the medicinal plant Vochysia divergens used
in the treatment of colds, coughs, fever, pneumonia and other diseases (Pott et al., 2004). Extracts
of stem and leaves of V. divergens have been reported to possess considerable anti-inflammatory,
antibacterial and molluscicidal activities (Corrêa et al., 2018; Dos Santos and Sant’Ana, 2000; Hess
et al., 1995). Chemical analysis revealed the presence of 3-sitosterol, betulinic acid, sericic acid
(Hess et al., 1995), 5-methoxyluteolin-7-O-β-glucopyranoside, rutin, galloyl-HHDP-glucopyran-
oside, 3′,5-dimethoxyluteolin-7-O-β-glucopyranoside (Corrêa et al., 2018), divergioic acid (Hess
et al., 1999) and tormentic acid (Bortalanza et al., 2002). Among the compounds identified, tormentic
acid has been reported as a promising metabolite due to its strong anticancer, anti-inflammatory and
antiatherogenic properties (Bortalanza et al., 2002; Ma et al., 2015; Yang et al., 2018).
In contrast to the Pantanal, which is subject to flooding, the Cerrado is characterized by periods
of natural burning due to fires affecting the vegetation in this biome (Felfili and Fagg, 2007). Due to
this peculiar condition, the Cerrado is considered one of the most diverse places in the world (Myers
et al., 2000), with approximately 6000 species of plants (Felfili and Fagg, 2007; Oliveira-Filho and
Ratter, 2002). For example, S. adstringens is a medicinal plant commonly found in the Cerrado,
and has been used in the treatment of cutaneous and mucosal lesions, diseases of genitourinary sys-
tem, such as STDs, and as anti-inflammatory and antiseptic (Ferrão et al., 2014; Morey et al., 2016;
Pinto et al., 2015; Rodrigues and Andrade, 2014). Several studies have evaluated the toxicity of the
S. adstringens extract using rats and Drosophila melanogaster as models, and these evaluations
TABLE 5.1
Taxonomic Classification of the Medicinal Plants Used for the Isolation
of Endophytes in the BIOGEMM Laboratory – UFPR
Scientific Name Common Name Family
Maytenus ilicifolia (sin. Celastrus spinifolius) Espinheira Santa Celastraceae
Schinus terebinthifolia (sin. Schinus mellisii) Peppertree, Aroeira Anacardiaceae
Vochysia divergens Cambará Vochysiaceae
Stryphnodendron adstringens (sin. Stryphnodendron barbatimam) Barbatimão Fabaceae
Endophytes and Biotechnological Potential 93
have shown that the plant extract has no genotoxicity or mutagenic activity (Costa et al., 2010;
De Sousa et al., 2003). In addition, a clinical study has demonstrated the efficacy of S. adstringens
in the healing of decubitus ulcers in 51 patients (Ricardo et al., 2018).
The Atlantic Forest is considered a hotspot biome, but most of this biodiversity has been reduced
to less than 8% of its original coverage, being replaced primarily by sugarcane, coffee, cocoa and
Eucalyptus forest for cellulose and pulp production (Colombo et al., 2010). M. ilicifolia is widely distrib-
uted in the Atlantic Forest and is commonly used in folk medicine in the treatment of gastric diseases
(Sá et al., 2017). In addition, foliar extracts of M. ilicifolia showed antinociceptive, antioxidant, anti-
inflammatory and antiulcerogenic activities (Cipriani et al., 2009; Jorge et al., 2004; Sá et al., 2017), with
active compounds: polygalacturonic acid, catechin, friedelan-3β-ol, friedelin, and several phenolic and
flavonoids (Cipriani et al., 2009; Queiroga et al., 2000; Sá et al., 2017; Tiberti et al., 2007).
Among the four plants studied by our group, S. terebinthifolius has been more exploited due
to its biotechnological potential and chemical proprieties (Bernardes et al., 2014; Fedel-Miyasato
et al., 2014; Richter et al., 2010; Rosas et al., 2015; Sereniki et al., 2016; Silva et al., 2017; Salem
et al., 2018). It has been reported that the extract of S. terebinthifolius acts in Parkinson’s disease,
as an anti-inflammatory, immunomodulatory, chemopreventive, wound healing, antioxidant, anti-
mycobacterial and antiproliferative (Bernardes et al., 2014; Fedel-Miyasato et al., 2014; Richter
et al., 2010; Rosas et al., 2015; Sereniki et al., 2016; Silva et al., 2017; Salem et al., 2018).
Based on the specificity of the biomes, in the use of these plants in folk medicine, biotechnologi-
cal potential and in the absence of extensive studies on the endophytic community, we have tried to
catalog and explore the diversity of endophytes, as well as to understand the endophyte-host associ-
ation in these four medicinal plants (Figueiredo et al., 2018; Gomes-Figueiredo et al., 2007; Gomes
et al., 2013; Gos et al., 2017; Hokama et al., 2017; Lima et al., 2012; Medeiros et al., 2018; Noriler
et al., 2018; Savi et al., 2015; Savi et al., 2016; Savi et al., 2018; Tonial et al., 2016; Tonial et al., 2017).
5.3 ISOLATION OF ENDOPHYTES

Upon selection of the medicinal plant, choosing the methodology for the isolation of endophytes is
necessary. A convenient and common method accepted by many researchers for the isolation of endo-
phytes is the adaptation of the method developed by Petrini (1982). The method consists of superfi-
cially disinfecting the plant samples (Figure 5.1A) using 70% alcohol for about 30 seconds, followed
by immersion in 0.5–3.5% sodium hypochlorite for 1-10 minutes, 70% alcohol for about 30 seconds
again and finally the sample is rinsed in sterile doubly distilled water (Figure 5.1B). Exposure to
alcohol eliminates bacteria living on the tissue surface (Kampf et al., 2008), and exposure to sodium
hypochlorite acts on fungi (Amirabadi and Sasannejad, 2016), so the remaining microorganisms
will be those that live inside the plant tissue. Variations in the exposure time to sodium hypochlorite
can be applied, depending on the composition of plant leaves. Some plants have more sensitive tis-
sues and long exposure may cause tissue decomposition allowing sodium hypochlorite to enter the
tissue, which may inhibit the isolation of fungal endophytes. After disinfection, the plant tissue is
fragmented and deposited in culture medium for the isolation of endophytes (Figures 5.1C and D).
Selection of suitable culture media is one of the prerequisites for studying microorganisms.
Different endophytic microorganisms live in different environments and have a variety of growth
requirements, such as nutrients, pH, osmotic conditions and temperature. Due to the lack of suf-
ficient variability in the composition of the culture medium, replication of the exact environmental
conditions in the laboratory is almost impossible (Basu et al., 2015). However, some media have
been considered more suitable to isolate most fungi, bacteria and actinomycetes, such as potato
dextrose agar (VanderMolen et al., 2013), Luria Bertani agar (Ramalashmi et al., 2018) and AAC
(Akemi et al., 2013), respectively. These media have proven effective in providing the nutrients
necessary for the growth of the microorganism, such as vitamins, amino acids and sugar sources.
The addition of antibiotics (Figure 5.1C), such as 50 mg/L chloramphenicol or ampicillin, may
be used to suppress bacterial growth (Savi et al., 2015) and cycloheximide (50 µg/mL) may be used
FIGURE 5.1 Work-up scheme for isolation of endophytes: (A) plant species; (B) disinfection process;
(C) selection of culture conditions; (D) Petri dish with fragments of leaves; (E) isolated microorganism.
to inhibit the development of fungi (Gos et al., 2017). To stimulate the isolation of actinomycetes,
nalidixic acid (50 µg/mL) and cycloheximide (50 µg/mL) were used to inhibit bacterial and fungal
development (Savi et al., 2016).
A temperature range of 25‒36°C is suitable for the growth of most endophytic microorganisms.
After the incubation of the leaf fragments, the growth of endophytes needs to be verified daily,
for about 30 days, since different microorganisms or species have different growth rates. As an
example, some fungal species grow in only 2 days, while others require more than 10 days (Romão-
Dumaresq et al., 2016). The emerging mycelia are transferred to a new plate (Figure 5.1E) and
stored at 4°C in a suitable medium for further identification (Noriler et al., 2018).
Using these criteria, our group has isolated fungi and actinomycetes from the above-
mentioned medicinal plants (M. ilicifolia, S. terebinthifolius, V. divergens and S. adstringens)
to assess the diversity of endophytes. Interestingly, leaves and stems of the studied plants
were colonized by fungi with higher frequency than by actinomycetes (Gomes-Figueiredo
et al., 2007; Gomes et al., 2013; Gos et al., 2017; Hokama et al., 2017; Noriler et al., 2018; Savi
et al., 2015; Savi et al., 2016; Tonial et al., 2016). This is probably related to the chemical and
biological composition of the host leaves. Thus, the selection of plant tissues has a considerable
effect on the observed biodiversity.
5.4 DIVERSITY OF ENDOPHYTES

Once the endophytes have been isolated, the first question is how to exploit such diversity? Second,
how to work with such a large number of isolates? The first step is to group the microorganisms
based on their macro- and micromorphology, considering the growth rate, colony color, hyphae
aspects, presence/absence and spores’ morphology. Based on morphological characteristics, one
isolate from each morphotype is randomly selected for identification and bioprospecting.
The pure culture needs to be obtained through a single spore culture approach (Gilchrist-Saavedra
et al., 2006). The single spore culture ensures that the isolates are pure and not contaminated with
a close morphological species. The technique consists in making a spore solution 102 spores/mL,
which is spread on a plate containing the appropriate culture medium, and after 2 or 3 days a colony
of a single spore is transferred to a new plate and used for the next steps. In our group, the single
spore colonies are deposited in the Culture Collection “Centro de Coleções de Culturas Biológicas
do Estado do Paraná” of the Taxonline (http://taxonline.bio.br/index.php), at the Federal University
of Paraná, Brazil (http://taxonline.bio.br/colecoes/index.php?id=2-coleções-microbiológicas). The
extraction of DNA is performed using standard techniques (Noriler et al., 2018), as described by
Raeder and Broda (1985), or using a commercial kit (Savi et al., 2016).
The identification of microorganisms is based on morphological, phylogenetic and ecological
aspects, and the correct identification is a critical step to ensure biotechnological reproducibility
(Raja et al., 2017). For many years, morphological characters were used as the single criterion for
species identification (Militão et al., 2014). However, the classification of microorganisms isolated
from the environment based on morphological analysis is complicated, since it is a highly variable
group, which does not always produce spores under laboratory conditions (Rodriguez et al., 2009).
In addition, morphological analyses are time-consuming and not compatible with the identification
of several isolates in a short time. In view of these limitations, we first identify isolates based on
phylogenetic analyses and use the complete morphological analysis only to describe new species
(Noriler et al., 2018; Savi et al., 2015; Savi et al., 2016; Savi et al., 2018).
The internally transcribed spacer (ITS) and 16S rRNA regions remain the first choice for identi-
fying fungi and bacteria, respectively, at a lower level, such as genus or species. In an analysis per-
formed to select the barcode sequence for fungal identification the ITS region was selected in view
of its easy amplification in different groups, and among the ribosomal regions analyzed, ITS region
presents the highest probability of successful identification, with the most clearly defined barcode
gap between inter- and intraspecific variation (Schoch et al., 2012). However, in some cryptic gen-
era, such as Diaporthe (Gomes et al., 2013) and Fusarium (Chitrampalam at al., 2016), a multigene
sequence analysis using protein-coding genes is required for species identification.
While ITS sequences are fungal barcodes, the 18S nuclear ribosomal small subunit rRNA gene
(SSU) is commonly used in phylogenetic analysis at the family level, because it has fewer hypervari-
able domains. The 28S nuclear ribosomal large subunit rRNA gene (LSU) sometimes discriminates
species on its own or combined with ITS. As an example, LSU can be used to confirm that an isolate
can represent a new genus or new family, or even to point out an inconsistency observed in the ITS
sequence to classify species in close related genera. According to Schoch et al. (2012) for yeasts, the
D1/D2 region of LSU is useful for a long time for species identification.
Thus, we used the strategy of initially identifying the endophytes based on ITS or 16S rRNA
sequence analysis. The amplification of the ITS region can be performed using different primers,
the most used being ITS1 and ITS4, but if there is a problem in the amplification or sequencing,
other primers may be used (Table 5.2). For 16S rRNA, several primers are described in the litera-
ture, and the most used are listed in Table 5.3. Normally, the names of primers used for 16S rRNA
are numbers that designate their position in that gene in Escherichia coli.
The ribosomal sequence is compared based on the similarity to the available sequences in the
GenBank database, using the BLAST tool (Figure 5.2). The GenBank was selected to compare the
sequence since it is the largest sequence database with approximately 210 million sequences (www.ncbi.
nlm.nih.gov/genbank/statistics/), corresponding to sequences of approximately 95,000 species (www.
nature.com/nature/debates/e-access/Articles/lipman.html). Of these sequences about 172,000 repre-
sent fungal ITS sequences, in 2500 genera and 15,500 species (Schoch et al., 2012).
However, the blast result is not a conclusive identification, since approximately 20% of the fungal
ITS sequences in this database were incorrectly annotated (Federhen, 2015; Nilson et al., 2006). An
interesting alternative is to use the filter “sequence-from-type” in the blast searches, or to perform the
searches on the RefSeq Targeted Loci project (http://www.ncbi.nlm.nih.gov/refseq/targetedloci/),
TABLE 5.2
Primers Used to Amplify ITS Region in Fungi
Primer F/R Sequence Reference
ITS5 F GGAAGTAAAAGTCGTAACAAGG White et al. (1990)
ITS1 F TCCGTAGGTGAACCTGCGG White et al. (1990)
ITS3 F GCATCGATGAAGAACGCAGC White et al. (1990)
ITS2 R GCTGCGTTCTTCATCGATGC White et al. (1990)
ITS4 R TCCTCCGCTTATTGATATGC White et al. (1990)
LR1 R GGTTGGTTTCTTTTCCT Vilgalys and Hester (1990)
Note: F. forward; R. reverse.
TABLE 5.3
Primers Used to Amplify 16 rDNA Gene in Bacteria
Primers Pair Sequence (5′-3′) References
68f TNANACATGCAAGTCGRRCG McAllister et al. (2011)
518r WTTACCGCGGCTGCTGG Lee et al. (2010)
341f CCTACGGGNGGCWGCAG Klindworth et al. (2013)
785r GACTACHVGGGTATCTAATCC Klindworth et al. (2013)
799f AACMGGATTAGATACCCKG Chelius and Triplett (2001)
1193r ACGTCATCCCCACCTTCC Bodenhausen et al. (2013)
967f CAACGCGAAGAACCTTACC Sogin et al. (2006)
1391r GACGGGCGGTGWGTRCA Walker and Pace (2007)
Note: f. forward; r. reverse.
FIGURE 5.2 Work-up scheme for identification of the endophytes.

which maintains curated sets of full-length reference sequences from type for ribosomal RNAs
(Federhen, 2015).
First, we use the blast tool to identify the possible fungal or bacterial genus to which the isolate
belongs. For a final or more precise identification, a dataset containing all the sequences of type
species of the valid fungal species belonging to the respective genus is obtained through a search on
the Mycoback database (www.mycobank.org/) and for bacteria and actinomycetes using search on
the List of Prokaryotic Names With Standing in Nomenclature (www.bacterio.net/). After select-
ing the sequences of valid species, the identification is based on an evolutionary framework using
a phylogenetic approach (Figure 5.2). Phylogeny reconstructs the tree-like pattern that describes
the evolutionary relationships between species with a predictive value (Pace et al., 2012), different
from a similarity analysis via Blast. Many approaches to phylogenetic inference have been used and
the relative merits of these methods have been an important consideration for phylogenetic analy-
sis (Holder et al., 2008). The topology of the phylogenetic tree, as well as the order of branching
events, is determined from the sequences of the analyzed region and, despite the fact some methods
use distance-matrix to perform the phylogeny analysis, the most valuable methods are based on
standard statistical techniques, such as maximum likelihood and Bayesian inference (Bogusz and
Whelan, 2017).
In some genera, such as Phaeophleospora, we were able to identify an isolate as a new spe-
cies using only the ITS sequence, because, for this genus the ITS sequence has enough infor-
mation to differentiate species (Savi et al., 2018). However, it is not true for other critical
genera, such as Diaporthe, in which five genes are required for accurate species identification
(Gomes et al., 2013). In other cases, such as the Colletotrichum genus, the ITS sequence has
enough information to identify which species’ complex the isolate belongs to (Damn et al., 2012).
Within each of the Colletotrichum species complexes, different protein-coding genes are recom-
mended for species identification, such as the GAPDH intron region for identification of species
within the Colletotrichum acutatum complex (Silva et al., 2017). Thus, identification of the isolate
should be performed carefully, using sequences of the type species, and the analysis should begin
with the ITS sequence and, if necessary, other genes need to be sequenced for identification at
the species level.
Using phylogenetic analysis, we identified 46 genera (including 6 possible new genera of
the Pleomassariaceae and Xylariaceae families) and 49 species as endophytes of M. ilicifolia,
S. terebinthifolius, V. divergens and S. adstringens (Table 5.4). Among these isolates, 9 strains were
described as new species, and the description of other 15 species is in progress (Table 5.4). This
data reinforce the biodiversity found in the Cerrado, Pantanal and Atlantic Rainforest biomes and
suggest the medicinal plants found in Brazil as a repository for fungi and actinomycetes.
5.5 EXPLORING THE BIOTECHNOLOGICAL POTENTIAL

OF THE ISOLATED ENDOPHYTES
The search for interesting biological activity from microorganisms has been the basis for the devel-
opment of several biotechnological applications, mainly in the pharmaceutical and agricultural
industries (Vitorino and Bessa, 2017). The most promising compounds in the clinic for treatment of
bacterial infections were isolated from microorganisms, such as penicillin isolated from Penicillium
digitatum (Laich et al., 2002); vancomycin produced by Streptomyces orientalis (Levine, 2006);
streptomycin isolated from Streptomyces griseus and erythromycin produced by Saccharopolyspora
erythraea (Donadio et al., 1996), among several others. Besides the high exploration of microorgan-
isms for active compounds in the past, studies have shown that unknown species and genetically
different strains are still abundant in nature, and natural products remain the most promising source
for new compounds (Monciardini et al., 2014).
One of the strategies for screening of endophyte producers of antibacterial and antifungal com-
pounds, in our laboratory, is performed by dual culture (Hokama et al., 2017). In the dual cultures,
TABLE 5.4
Endophytic Microorganisms of the Medicinal Plants Maytenus Ilicifolia, Schinus
Terebinthifolius, Vochysia Divergens and Stryphnodendron Adstringens Isolated and
Studied in the BIOGEMM Laboratory – UFPR
Genus Species Reference Medicinal Plant
Acrocalymma A. medicaginis Noriler et al., 2018 Sa
Actinomadura Actinomadura sp. Gos et al., 2017 Vd
Aeromicrobium A. ponti Gos et al., 2017 Vd
Alternaria Alternaria section alternate Tonial et al., 2016; Vd/St
Noriler et al., 2018
Annellosympodiella Annellosympodiella sp. Hokama et al., 2017 Vd
Antrodia Antrodia sp. Hokama et al., 2017 Vd
Bjerkandera Bjerkandera sp. Tonial et al., 2016; Vd/Sa/St
Cladosporium Cladosporium sp. Tonial et al., 2016; Vd/St
Hokama et al., 2017
Colletotrichum Colletotrichum sp. Hokama et al., 2017; Vd/Sa
C. boninense sensu lato Noriler et al., 2018 Vd/Sa
C. gloeosporioides sensu lato Noriler et al., 2018 Vd/Sa
C. siamense Noriler et al., 2018 Vd/Sa
C. simmondsii Lima et al., 2012 St
C. acutatum Lima et al., 2012 St
C. fioriniae Lima et al., 2012 St
Coniochaeta C. nepalica Noriler et al., 2018 Vd
Corynespora C. cambrensis Noriler et al., 2018 Vd/Sa
Curvularia Curvularia sp. Noriler et al., 2018 Vd/Sa
Daldinia Daldinia sp. Noriler et al., 2018 Vd
Diaporthe Diaporthe sp. 1, 2, 3, 4 Gomes et al., 2013; Vd/Sa/Mi/St
Diaporthe cf. heveae 1, Noriler et al., 2018 Vd/Sa
D. endophytica Gomes et al., 2013 Mi/St
D. inconspícua Gomes et al., 2013 Mi
D. infecunda Gomes et al., 2013 Mi/St
D. mayteni Gomes et al., 2013 Mi
D. novem Gomes et al., 2013 Mi
D. oxe Gomes et al., 2013 Mi/St
D. paranensis Gomes et al., 2013 Mi
D. phaseolorum Gomes et al., 2013 Mi
D. schini Gomes et al., 2013 Vd/Sa/St
D. terebinthifolii Gomes et al., 2013 St
Hypoxylon Hypoxylon sp.1 Noriler et al., 2018 Vd/Sa
Irpex I. lacteus Hokama et al., 2017 Vd
Lanceispora Lanceispora sp. Hokama et al., 2017 Vd
Lasiodiplodia Lasiodiplodia sp. Noriler et al., 2018 Vd/Sa
Microbacterium Microbacterium sp. Gos et al., 2017 Vd
Microbispora Microbispora sp. 1, 2, 3 Savi et al., 2016 Vd
Micrococcus Micrococcus sp. Gos et al., 2017 Vd
Micromonospora Micromonospora sp. Savi et al., 2015 Vd
Neofusicoccum N. brasiliense Noriler et al., 2018 Vd/Sa
N. grevilleae Hokama et al., 2017 Vd
(Continued)

Endophytic Microorganisms of the Medicinal Plants Maytenus Ilicifolia, Schinus
Terebinthifolius, Vochysia Divergens and Stryphnodendron Adstringens Isolated and
Studied in the BIOGEMM Laboratory – UFPR
Genus Species Reference Medicinal Plant
Neopestalotiopsis Neopestalotiopsis sp. Noriler et al., 2018 Vd
Nigrospora Nigrospora sp. Noriler et al., 2018 Vd/Sa
N. hainanensis Hokama et al., 2017 Vd
Penicillium Penicillium sp. Tonial et al., 2016 St
Phaeosphaeria Phaeosphaeria sp. Hokama et al., 2017 Vd
Paraphaeosphaeria Paraphaeosphaeria sp. Noriler et al., 2018 Vd/Sa
Peniophora Peniophora laxitexta Hokama et al., 2017 Vd
Pestalotiopsis Pestalotiopsis sp. Gomes-Figueiredo et al., 2007; Vd/Sa/Mi
P. microspora Gomes-Figueiredo et al., 2007 Mi
Phaeophleospora Phaeophleospora sp.1, 2 Noriler et al., 2018 Vd/Sa
P. vochysiae Savi et al., 2018 Vd
Phyllosticta Phyllosticta sp. Noriler et al., 2018 Vd/Sa
Phyllosticta capitalensis Hokama et al., 2017 Vd
Pleomassariaceae Pleomassariaceae sp. Noriler et al., 2018 Vd/Sa
Polyporus Polyporus sp. Hokama et al., 2017 Vd
Pseudofusicoccum Pseudofusicoccum sp. Noriler et al., 2018 Vd/Sa
P. stromaticcum Noriler et al., 2018 Vd/Sa
Roussoella Roussoella sp. Noriler et al., 2018 Vd
Sphaerisporangium Sphaerisporangium sp. Gos et al., 2017 Vd
S. thermocarboxydus Gos et al., 2017 Vd
Streptomyces S. sampsonii Savi et al., 2015a, 2015b Vd
Xylaria Xylaria sp. Tonial et al., 2016 St
X. cubensis Figueiredo et al., 2018 Mi
Xylariaceae Xylariaceae sp.1, 2, 3, 4, 5 Hokama et al., 2017; Vd/Sa
Williamsia Williamsia sp. Gos et al., 2017 Vd
Note: Mi: M. ilicifolia, St: S. terebinthifolius, Vd: V. divergens, Sa: S. adstringens.

The names in bold represent new species or genera.
the ability of the endophyte to produce active compounds in the presence of a pathogenic strain is
assessed (Fierro-Cruz et al., 2017). First, the endophytic and pathogenic strains are cultured on a specific
medium for the growth of fungi or actinomycetes, during the time necessary for the development of the
microorganism (Savi et al., 2015a, 2015b). One disc (6 mm) of the endophyte and one of the phytopatho-
gen are inoculated on opposite sides of the Petri dish and incubated under favorable conditions for the
development of endophytes and pathogens. The inhibition percentage is calculated by comparing the
diameter of the mycelial growth of the pathogen in dual culture with the diameter of the mycelial growth
of the pathogen in the individual inoculation. The antimicrobial activity is classified as low (50–59%),
moderate (60–69%) and high (≥70%) according to the percentage of inhibition (Noriler et al., 2018).
Once any promising isolates have been selected, a small-scale culture is performed using culture
media containing different concentrations containing nitrogen and carbon sources at different tem-
peratures to select the best condition to produce active secondary metabolites (Gos et al., 2017). The
most commonly used media to produce metabolites of actinomycetes are M2, R5A and SG (Savi
et al., 2015b), and for fungi the most used are PD (potato dextrose), Czapeck and ME (malt extract)
(Savi et al., 2018). Generally, the culture is filtered over celite, the biomass is extracted with MeOH
and the supernatant is mixed with the XAD-16 resin and the metabolites which are retained on
the resin are extracted with MeOH. The recovered organics are evaporated under vacuum at 40°C
to yield the crude extracts (Savi et al., 2015a). The crude extracts produced in different culture
media are evaluated for their antibacterial or antifungal activities, using the disc diffusion evalua-
tion following the instructions of the Clinical and Laboratory Standards Institute (CLSI, 2015). The
culture condition giving the most active extract is used for any subsequent large-scale cultivation
of 8-10 liters and the metabolites are purified using various chromatographic techniques such as
HPLC, Sephadex LH-20 and TLC (Savi et al., 2018). Pure compounds are then evaluated for their
antimicrobial activity, and the compound identification is performed generally by mass spectra and
NMR analysis (Savi et al., 2015b). The Work-up scheme for production, purification and identifica-
tion of secondary metabolites produced by endophytes is shown in Figure 5.3.
1.50
1.00
0.50
0.00
0.00 2.00 4.00 6.00 8.00 10.00 12.00 14.00 16.00 18.00 20.00 22.00 24.00 26.00 28.00
Minutes
Microbacterium sp. LGMB471

8 L (shaker), using SG-Medium
10 days (36 °C, 210 rpm)
Yellow-culture broth
Filtration
Filtrate Mycelium
XAD-16 resin (4%), mixing overnight MeOH (3 × 500 mL),
Filtration evap. in vac.
Water
XAD-16, washed with water (3 × 500 mL)
HPLC-analysis
XAD-16, extracted with MeOH (3 × 800 mL)
evap. in vac. 2.7 g (yellowish-brown oily crude extract)
discarded
722 mg (yellowish-brown crude extract)
Sephadex LH-20 column (2.5 × 50 cm; MeOH),

TLC and HPLC analysis
FI (182.5 mg) FII (296.3 mg) FIII (84.0 mg) FIV (10.5 mg)
HPLC HPLC HPLC HPLC
Cyclo-(L-Pro-L-Phe) (6; 3.0 mg) 7-O-β-D-Glucosyl-genistein (1; 2.5 mg),

Cyclo-(L-Val-L-Phe) (7; 0.7 mg) 7-O-β-D-Glucosyl-daidzein (2; 0.5 mg),
Cyclo-(L-Leu-L-Phe) (8; 0.8 mg) 4′,7-Dihydroxyisoflavanone (3; 0.8 mg)
Cyclo-(L-Pro-L-Val) (4; 1.2 mg) 7-O-β-D-Glucosyl-genistein (1; 1.3 mg),

Cyclo-(L-Pro-L-Leu) (5; 2.0 mg) 7-O-β-D-Glucosyl-daidzein (2; 6.8 mg)
FIGURE 5.3 HPLC analysis of crude extract and work-up scheme for production, purification and identifica-
tion of secondary metabolites produced by endophytes, using the data produced by Savi et al. (2015b).
In our screening program for metabolites produced by endophytes of different biomes in Brazil,
we identified β-carbolines and indoles produced by a strain of actinomycetes, Microbispora sp.
LGMB259, isolated from the medicinal plant V. divergens in the Pantanal – Brazil (Savi
et al., 2015b). Among the isolated metabolites, the compound 1-vinil-β-carboline-3-carboxylic acid
showed high antibacterial and cytotoxic activities. In addition to the identification of metabolites,
the manuscript also highlighted the importance of the chemical group bound to carbon 1 in the
biological activity of β-carbolines.
We also described a new species, Phaeophleospora vochysiae (LGMF1215b), isolated
from V. divergens that produced secondary metabolites with considerable antifungal activity.
Although the strain LGMF1215 was isolated as endophyte, it produced phytotoxic perylen-
equinones as major compounds, cercosporin and isocercosporin, two toxic metabolites com-
monly produced by Cercospora species (Savi et al., 2018a). The resistance to cercosporins by
P. vochysiae and by the host V. divergens may be due to cercosporin being produced associ-
ated with fungal hyphae, in this way the compound is present in reduced form, which makes
the compound nontoxic or photoactive. In addition, strain LGMF1215 produced a new
compound having antibacterial activity, 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-
methylcyclohex-2-enone and absence of cytotoxic activity for human cell lines (Savi
et al., 2018), suggesting the possibility to using this compound to treat clinical infections
caused by bacteria.
In addition to the previously reported compounds, the endophytes isolated by our group also
produced alkaloids (Gos et al., 2017; Tonial et al., 2016), diketopiperazines (Gos et al., 2017; Savi
et al., 2018b), isocoumarins (Medeiros et al., 2018; Savi et al., 2019), perylenequinones (Savi
et al., 2018a), dioxolanones (Savi et al., 2019), isoflavones (Savi et al., 2018b), tyrosols, phenolic
acids (Savi et al., 2019) and two new compounds: 5-hydroxy-orthosporin and phenguignardic acid
butyl ester. These new compounds exhibited antibacterial and cytotoxic activity against tumor cells
(Savi et al., 2019). The structure of secondary metabolites belonging to different chemical classes
are represented in Figure 5.4.
DIAPORTHE TEREBINTHIFOLII: A PROMISING SPECIES TO CONTROL

5.6
THE CITRUS PHYTOPATHOGEN PHYLLOSTICTA CITRICARPA
In 2013, we described the new species Diaporthe terebinthifolii (Gomes et al., 2013), and since
then, we have focused on exploring its biotechnological potential to act against the phytopathogen
Phyllosticta citricarpa (Medeiros et al., 2018; Santos et al., 2016; Tonial et al., 2016). We demon-
strated that the strain LGMF907 produces secondary metabolites in ME medium that completely
inhibit the development of P. citricarpa (Figure 5.5) (Santos et al., 2016). Endophytes can be used
to produce secondary metabolites used in the pharmaceutical or agricultural industries, or they can
be used as a biological controller (Kandel et al., 2017). In order to study the possibility of using
D. terebinthifolii in the biological control of P. citricarpa, we evaluated the ability of this endo-
phyte to colonize citrus plants using a strain of D. terebinthifolii expressing the DsRed fluorescent
protein (Santos et al., 2016). Microscopic analysis demonstrated that D. terebinthifolii colonizes the
intercellular region and oil glands of citrus plants without causing any negative damage (Figure 5.5),
suggesting the potential to be used in the biological control of Citrus Black Spot disease caused by
P. citricarpa.
In a chemical analysis of D. terebinthifolii extracts, two major compounds were identified: ortho-
sporin and diaporthin (Medeiros et al., 2018). However, neither of these compounds has activity
against P. citricarpa, which suggests that the compounds responsible for the biological activity are
produced in small amounts.
An alternative way to explore the metabolic potential of endophytes is to assess the long evolu-
tionary coexistence of endophytes, epiphytes and phytopathogens with the host, resulting in differ-
ent scenarios of interactions that need to be better understood and explored (Liu et al., 2017). The
FIGURE 5.4 Representative chemical diversity of secondary metabolites produced by endophytic isolates
of different Brazilian biomes: (A) phenguignardic acid butyl ester; (B) phenguignardic acid methyl ester;
(C) 5-hydroxy-orthosporin; (D) orthosporin; (E) diaporthin; (F) kitasetaline; (G) methyl 1-(propionicacid)-
β-carboline-3-carboxylic acid; (H) 1-vinil-β-carboline-3caboxylic acid; (I) cercosporin; (J) brevianamide;
(K) 4′,7-dihydroxyisoflavanone; (L) tyrosol; (M) 3-(sec-butyl)-6-ethyl-4,5-dihydroxy-2-methoxy-6-
methylcyclohex-2-enone; (N) Cyclo-(L-Pro-L-Leu). The chemical structures were obtained using the
Chemdraw software (https://chemistry.com.pk/software/chemdraw-free/).
FIGURE 5.5 Potential of Diaporthe terebinthifolii to be used in the biological control of Phyllosticta
citricarpa: (A) D. terebinthifolii; (B) inoculation of D. terebinthifolii in citrus plant; (C) D. terebinthi-
folii colonizing citrus leaves without causing any damage; (D) extract of D. terebinthifolii; (E) extract
of D. terebinthifolii (10 µL) inhibiting citrus black spot lesions in citrus fruits; (F) induced citrus black
spot lesion.
classical approach used to select endophytes with biotechnological potential is based on the screen-
ing of a single strain in culture, as presented above (Tonial et al., 2016). However, this excludes
the interaction between different microorganisms that occupy the same environment. Thus, co-
cultivation of different microorganisms has been used to understand the chemical ecological inter-
action between different organisms and to induce the expression of inactive metabolic pathways,
or even increase the production of active compounds (Reen et al., 2015). As an example, we have
the HPLC profile of an endophytic strain, D. terebinthifolii LGMF907, cultivated in the presence
and absence of the P. citricarpa, a pathogen of citrus plants. The co-culture of both endophytic
and phytopathogenic microorganisms was performed under the same condition as the single cul-
ture. Preliminary HPLC analysis of the extract obtained from the co-cultivation showed a 10-fold
increase of a compound eluting after 9:0 min, which is produced by D. terebinthifolii in the presence
of P. citricarpa. This result suggests that the eluting compound at 9 min may be responsible for the
biological activity of D. terebinthifolii against P. citricarpa. The chemical characterization of this
compound is in progress.
5.7 CONCLUSION
Brazil has a high biodiversity and herein the diversity of endophytic microorganisms is highlighted.
The endophytes that we have isolated from medicinal plants found in the Brazilian biomes of the
Pantanal and Cerrado (Table 5.1) have represented a great source of bioactive molecules and repre-
sented in Figure 5.4 with some of them possessing a new molecular framework. The biotechnologi-
cal advances contribute to increasing the importance of Brazilian diversity, and new species and
bioactive compounds are waiting to be reported.
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6 Environmental Factors
Impacting Bioactive
Metabolite Accumulation
in Brazilian Medicinal Plants
Camila Fernanda de Oliveira Junkes,
Franciele Antonia Neis, Fernanda de Costa,
Anna Carolina Alves Yendo,
and Arthur Germano Fett-Neto
Plant Physiology Laboratory, Center for Biotechnology
and Department of Botany, Federal University of Rio Grande
do Sul (UFRGS), Campus do Vale, Porto Alegre, Brazil
CONTENTS
6.1 Introduction............................................................................................................................ 109
6.2 Irradiance............................................................................................................................... 110
6.3 Temperature........................................................................................................................... 116
6.4 Water and Salinity.................................................................................................................. 116
6.5 Mineral Nutrition and Heavy Metals..................................................................................... 118
6.6 Biotic Elicitation of Secondary Metabolites.......................................................................... 121
6.7 Seasonal Variation.................................................................................................................. 124
6.8 Final Remarks........................................................................................................................ 127
References....................................................................................................................................... 127
6.1 INTRODUCTION
Secondary metabolites help protect plants against attacks by insects, herbivores and pathogens,
improve survival under abiotic stresses, besides participating in allelopathy and interactions with
pollinators, dispersers and symbionts. Strategies for production of metabolites in culture have
been developed based on these properties to increase the yield of metabolites of interest. It is well-
known that plant treatments with elicitors, or exposure to pathogens and herbivores, cause a series
of defense reactions, including the accumulation of a variety of metabolites such as phytoalexins in
intact plants or in plant cell cultures (Matsuura et al., 2018). Elicitors can be abiotic, such as metal
ions and inorganic compounds, and biotic, such as fungi, bacteria, viruses or herbivores, plant cell
wall components, as well as chemicals derived from pathogens or herbivores and plant defense
signaling molecules.
The richness of the Brazilian flora is remarkable. Knowledge on the complexity and medici-
nal potential of its chemical components remains only partial, despite massive research efforts.
The eco-chemical roles of secondary metabolites, which are often the pharmacologically active
109
ü Release of reactive oxygen species

ü Changes in photosynthetic potential/allocation
Temperature Irradiance
of energy reserves
ü Destabilization of cell membranes
Flooding üActivation/repression of transcription factors
ü Interference on three-dimensional structure
of polypeptides
ü Interaction with enzymatic cofactors
üActivation of osmoprotectants
Biotic
agents
Change in the balance of defense compounds:

Ø flavonoids
Drought Ø terpenes
Soil chemical
Pollutants and composition Ø anthocyanins
heavy metals Ø alkaloids
Ø tannins
S
Ø glucosinolates
Salinity
FIGURE 6.1 Scheme of some key steps and players involved in environmental or external modulation of
medicinal plant secondary metabolite profile.
molecules in plants, provide a working platform for improving yields of target products. Plant
metabolic profile responses to environmental stimuli can vary with taxon, genetic makeup
and chemical nature of metabolites, and, as such, should be addressed on a case-by-case basis
whenever possible. Nonetheless, some general trends are identifiable and can serve as useful
guidelines to modulate metabolic pathways toward compounds of interest through environ-
mental variables. First, some of the key players and steps involved in environmental or external
modulation of medicinal plant secondary metabolite profile (summarized in Figure 6.1) are
explored.
Second, the Brazilian territory encompasses a wide variety of climates and regional characteris-
tics, such as precipitation and temperature regimes. The seasonality in various regions is explored
in depth in a section on the topic and a list of all plant species exemplified in this chapter is shown
in Table 6.1.
6.2 IRRADIANCE
Both irradiance intensity and quality are important environmental factors for plant growth and
development, affecting plant’s morphological, biochemical and physiological parameters (Gobbo-
Neto and Lopes, 2007). Compounds such as flavonoids, anthocyanins, tannins and carotenes are
known to respond to irradiance stress because of their capacity of absorbing UV radiation and
antioxidant activity (Bian et al., 2015). The molecular structure of phenolic compounds contributes
to convert short-wave, high-energy-destructive radiation into longer wavelengths, which are less
destructive to the photosynthetic apparatus and other cellular structures (Teixeira et al., 2013). The
effects of irradiance intensity and quality on the production of secondary metabolites in plants are
often species-specific, but in general increasing their concentration can protect plants’ structures
Bioactive Metabolite Accumulation 111
TABLE 6.1
Names Discussed in This Chapter
Aloe arborescens (sin. Aloe principis) Asphodelaceae Babosa, babosa-de-arbusto (aloe)
Aloysia citriodora (sin. Aloysia triphylla) Verbenaceae Limonete, cedrina, cidró (lemon
beebrush)
Alternanthera philoxeroides (sin. Alternanthera Amaranthaceae Mata-bicho, perna-de-saracura
philoxerina) (alligator weed)
Amburana cearensis (sin. Amburana claudii) Fabaceae Amburana, imburana, cumaru, Cerejeira
Anadenanthera colubrina (sin. Acacia colubrina) Fabaceae Angico-branco
Araucaria angustifolia (sin. Araucaria dioica) Araucariaceae Araucaria (Brazilian pine)
Baccharis dentata (sin. Baccharis macrodonta) Asteraceae Vassourinha
Baccharis dracunculifolia (sin. Baccharis Asteraceae Vassourinha, alecrim-do-campo
pulverulenta)
Baccharis trimera (sin. Baccharis genistelloides Asteraceae Carqueja
var. trimera)
Bauhinia cheilantha (sin. Pauletia cheilantha) Fabaceae Pata-de-vaca, mororó
Cordia curassavica (sin. Cordia verbenacea) Boraginaceae Erva baleeira, Maria milagrosa
Cunila galioides (sin. Hedeoma glaziovii) Lamiaceae Poejo
Dimorphandra mollis Fabaceae Fava-de-anta
Elionurus muticus (sin. Elionurus marunguensis) Poaceae Capim cheiroso (lemon grass)
Eugenia uniflora (sin. Stenocalyx uniflorus) Myrtaceae Pitanga (Surinam cherry)
Hevea brasiliensis (sin. Hevea camargoana) Euphorbiaceae Seringueira (rubber tree)
Hydrocotyle umbellata (sin. Hydrocotyle caffra) Araliaceae Acariçoba
Hypericum brasiliense (sin. Sarothra brasiliensis) Hypericaceae Milfacadas, alecrim-bravo, erva-da-vida
Hypericum polyanthemum (sin. Hypericum Hypericaceae Hipérico
rivulare)
Hyptis carpinifolia (sin. Mesosphaerum Lamiaceae Rosmaninho, mata-pasto
carpinifolium)
Ilex paraguariensis (sin. Ilex curitibensis) Aquifoliaceae Mate, erva mate
Bryophyllum pinnatum (sin. Kalanchoe pinnata) Crassulaceae Folha-da-fortuna
Lafoensia pacari Lythraceae Dedaleiro, mangaba brava, pacari
Lippia origanoides (sin. Lippia schomburgkiana) Verbenaceae Alecrim-do-campo
Lychnophora ericoides (sin. Lychnophora Asteraceae Arnica-do-campo, arnica-falsa, candeia
rosmarinus)
Martianthus leucocephalus (sin. Hyptis Lamiaceae Bamburral
leucocephala)
Maytenus ilicifolia (sin. Celastrus spinifolius) Celastraceae Espinheira santa
Mikania glomerata (sin. Mikania glomerata var. Asteraceae Guaco
montana)
Mikania laevigata Asteraceae Guaco
Myrcia tomentosa (sin. Aguava tomentosa) Myrtaceae Araçazinho
Ocimum gratissimum (sin. Ocimum dalabaense) Myrtaceae Goiaba brava
Ocimum carnosum (sin. Ocimum selloi) Lamiaceae Alfavaca, manjericão
Achetaria azurea (sin. Otacanthus azureus) Lamiaceae Aniseto, alfavaca anis, anis-do-campo
Palicourea rigida (sin. Uragoga rigida) Scrophulariaceae Erva-copaíba
Passiflora alata (sin. Passiflora phoenicia) Rubiaceae Chapéu-de-couro, bate-caixa
Passiflora edulis (sin. Passiflora vernicosa) Passifloraceae Maracujá (passion fruit)
Passiflora incarnata (sin. Passiflora edulis var. kerii) Passifloraceae Maracujá azedo
Passiflora ligularis (sin. Passiflora lowei) Passifloraceae Maracujá-guaçu (purple passion flower)
(Continued)

Paullinia cupana var. sorbilis (sin. Paullinia Passifloraceae Granadilla
sorbilis)
Pfaffia glomerata (sin. Pfaffia divergens) Sapindaceae Guaraná
Physalis angulata (sin. Physalis esquirolii) Amaranthaceae Ginseng brasileiro (Brazilian ginseng)
Pilocarpus jaborandi (sin. Pilocarpus cearensis) Solanaceae Camapu, joá-de-capote, buch-de-rã
Pilocarpus microphyllus Rutaceae Jaborandi
Piper aduncum (sin. Piper reciprocum) Rutaceae Jaborandi
Pisum sativum (sin. Pisum vulgare) Piperaceae Pimenta-de-macaco
Psidium guajava (sin. Psidium igatemyense) Fabaceae Ervilha (pea)
Psychotria brachyceras (sin. Uragoga Myrtaceae Goiaba (guava)
brachyceras)
Psychotria leiocarpa (sin. Psychotria tenella) Rubiaceae Cafezinho-do-mato
Quillaja brasiliensis (sin. Fontenellea brasiliensis) Rubiaceae Cafeeiro-do-mato
Quillaja saponaria Quillajaceae Saboneteira, pau-sabão
Schinus terebinthifolia (sin. Schinus mellisii) Quillajaceae Soapbark
Theobroma cacao (sin. Theobroma sapidum) Anacardiaceae Aroeira vermelha, roeira mansa
(Brazilian pepper tree)
Tithonia diversifolia (sin. Helianthus Malvaceae Cacau (cocoa tree)
quinquelobus)
Valeriana glechomifolia Asteraceae Margaridão, girassol mexicano
(Mexican sunflower)
Cordia curassavica (sin. Varronia curassavica) Caprifoliaceae Valeriana serrana
Aloe arborescens (sin. Aloe principis) Boraginaceae Erva-baleeira, Maria-preta
from photodamage. Some examples of impacts of irradiance quality and intensity on secondary
metabolite profiles are listed in Table 6.2.
UV radiation is an effective elicitor for boosting the biosynthesis of various plant secondary
metabolites. UV is divided into three classes: UV-C, UV-B and UV-A. Although UV-C is com-
pletely absorbed by atmospheric gases, this highly energetic radiation (200-280 nm) can severely
affect metabolism, cell structure and DNA, depending on its intensity. For example, the concen-
tration of the monoterpene indole alkaloid brachycerine, (Figure 6.2), which has antioxidant and
antimutagenic properties, can be increased by approximately an order of magnitude upon UV-C
exposure of Psychotria brachyceras Müll Arg. leaves (Matsuura et al., 2013). UV-C treatment
provides a means for obtaining plant biomass with higher yields for pharmacological applica-
tions. Lewinski et al. (2015) used UV light to accelerate maturation of processed mate (Ilex
paraguariensis A. St.-Hil.), resulting in increase of some dicaffeoylquinic acid isomers and decrease
of methylxanthines, rutin and isomers of chlorogenic acids.
Quillaja brasiliensis Mart., a native species from Southern Brazil, is known as soldier’s soap due
to the fact that its leaves and bark yield persistent foam in water (Yendo et al., 2010). Red light or
UV-C treatment of young plants and detached leaves of Q. brasiliensis promoted QB-90 accumu-
lation, an immunoadjuvant triterpene saponin fraction with strong capacity to induce cellular and
humoral immune response pathways with low toxicity to mammals (Yendo et al., 2015). These sapo-
nins showed remarkable structural similarities with saponins from Quillaja saponaria Molina bark, a
related Chilean species and one of the major sources of industrial saponins that are used as adjuvants
in vaccine formulations (Kauffmann et al., 2004). Irradiation-based treatments may be important in
improving yields of immunoadjuvant saponins of soap tree, both before and after leaf harvest.
Bioactive Metabolite Accumulation
TABLE 6.2
Examples of Abiotic Stress and Its Effect on Production and/or Accumulation of Secondary Metabolites
Species Affected Secondary Metabolite(s) Tissue/Organ Condition Effect References
Irradiance
Quillaja brasiliensis Triterpene saponin QB-90 Leaves (in stem Higher irradiance, red light Increase De Costa et al. (2013),
and post-harvest) and UV-C Yendo et al. (2015)
Kalanchoe pinnata Quercetin 3-rhamnopyranoside Leaves UV-B Higher diversity of phenolic Nascimento et al.
compounds and increased (2015)
quantity of quercitrin
Piper aduncum e-Nerolidol, linalol, α-humulene, Leaves Blue light Increase Pacheco et al. (2016)
cis-cadin-4-en-7-ol and caryophyllene
Psychotria leiocarpa GPV (N, β-d- Whole seedlings Far red and blue light enrichment Increase Matsuura et al. (2016)
glucopyranosylvincosamide)
Otacanthus azureus Essential oil Shoots Higher irradiance Increase Silva et al. (2006)
Temperature
Psychotria brachyceras Brachycerine Leaves Increased temperature Brachycerine accumulation and Magedans et al. (2017)
protection of heat-sensitive
species under severe heat stress
Ocimum selloi Elimicin, trans-caryophyllene, Leaf essential oil Temperature above 40°C Reduction David et al. (2006)
germacrene D and bicyclogermacrene
Lafoensia pacari Phenolic metabolites Leaves Warmer months Reduction Sampaio et al. (2011)
Ilex paraguariensis Total polyphenol content Leaves Drying at 45°C versus natural aging Increase Holowaty et al. (2015)
Water Availability and Osmotic Stress
Bauhinia cheilantha Flavonoid content Leaves Elevated rainfall Mild reduction Sobrinho et al. (2009)
Hypericum brasiliense Betulinic acid; isouliginosin B; Shoots and roots 15 days of drought or waterlogging Increase Abreu and Mazzafera
1,5-dihydroxyxanthone and rutin (2005)
Amburana cearenses Phenolic compounds Cotyledons Water restriction Reduction Pereira et al. (2014)
Dimorphandra mollis Quercetin, isoquercitrin and rutin Plants and Drought, flooding or 75 mM NaCl Increase Lucci and Mazzafera
seedlings (2009a, 2009b)
Physalis angulata Physalin Leaves 13 days exposure to saline Increase de Souza et al. (2013)
solution 0.9%
113
(Continued)
114
Examples of Abiotic Stress and Its Effect on Production and/or Accumulation of Secondary Metabolites
Species Affected Secondary Metabolite(s) Tissue/Organ Condition Effect References
Quillaja brasiliensis Triterpene saponin (QB-90) Leaves 150 mM NaCl or isosmotic Increase de Costa et al. (2013)
concentrations of sorbitol and
polyethylene glycol
Pilocarpus jaborandi Pilocarpine Leaves Salt stress and hypoxia Reduction Avancini et al. (2003)
Alternanthera philoxeroides Betacyanins In vitro plants 400 mM salt stress Increase Ribeiro et al. (2014)
Mineral Composition and Fertilization
Lafoensia pacari Phenolic metabolites Leaves Foliar micronutrients Cu, Fe, Mn Positive correlation Sampaio et al. (2011)
and Zn
Eugenia uniflora Spathulenol and caryophyllene oxide Leaves S, Ca and Fe balance Positive correlation Costa et al. (2009)
Selina-1,3,7 (11)-trien-8-one epoxide Leaves K, Cu and Mn Positive correlation
Flavonoids and tannins Leaves Mn and Cu Negative correlation
Myrcia tomentosa Oxygenated sesquiterpenes Flowers and Foliar concentration of Cu and P Positive correlation Sá et al. (2012)
leaves
Sesquiterpene hydrocarbons Flowers and leaves Foliar concentration of Cu and P Negative correlation
Palicourea rígida Iridoids Aerial parts Low fertility and acidic soil Increase Morel et al. (2011)
Loganin Aerial parts All the soil macro and micronutrients Negative correlation
except for Mg
Tithonia diversifolia Esters of trans-cinnamic acid Root Soil nutrients Ca, Mg, P, K and Cu Negative correlation Sampaio et al. (2016)
Lychnophora ericoides Monoterpenes and sesquiterpene Leaf essential oil Organic matter and P in soils Positive correlation Curado et al. (2006)
Passiflora incarnata Total flavonoids Leaves Soil elements Fe, B and Cu Negative correlation Reimberg et al. (2009)
Passiflora alata and Total phenols Primary branch Substrate with organic N (relatively Increase Sousa et al. (2013)

Passiflora ligularis leaves low-leaf N), rich in P and K and pH 6
Baccharisdracunculifolia γ-Muroleno, valenceno, δ-cadineno, Shoot essential oil Organic compost fertilization at Increase Santos et al. (2012)
E-nerolidol and espatulenol 30-40 t.ha−1
Schinus terebinthifolius Flavonoid and phenol Leaf essential oil Growth on poultry litter Increase Tabaldi et al. (2016)
Cunila galioides Flavonoids Leaves Soil with high level of aluminum Increase Mossi et al. (2011)
Baccharis trimera Phenolic acids and flavonoids Shoots Coal burning area Reduction Menezes et al. (2016)
Alternanthera philoxeroides Betacyanin Shoots 175 μM CuSO4 in vitro 60% increase Perotti et al. (2010)
FIGURE 6.2 Structure of brachycerine.
UV-B radiation is also an important abiotic factor to promote accumulation of secondary

metabolites such as polyphenolic compounds. Kalanchoe pinnata Pers. is a widespread species
of Crassulaceae used in popular medicine in Brazil and around the world to treat several diseases,
mainly inflammatory processes. Flavonoids isolated from the leaves are mainly quercetins, such
as quercetin 3-rhamnopyranoside. Analyses of the effects of supplemental UV-B radiation on phe-
nolic profile, antioxidant activity and total flavonoid content of leaves of K. pinnata showed that
extracts had higher diversity of phenolic compounds and increased quantity of quercitrin after
UV-B irradiation (Nascimento et al., 2015).
Light conditions can contribute to increase the production of essential oil of commercial medici-
nal and aromatic species. Ocimum spp. are generally characterized as rich in essential oils used in
pharmaceuticals, fragrances and cosmetics. The major component in oil of Ocimum gratissimum
L. is eugenol. In O. gratissimum, the essential oil yield per plant increased with light intensity as
a function of increased leaf biomass, although this was not related to variation in trichome mor-
phology, density or oil content (Fernandes et al., 2013). In contrast, Varronia curassavica Jacq.,
known as erva-baleeira, had increased essential oil content under higher irradiance in associa-
tion with higher frequency of glandular trichomes (Feijó et al., 2014). Oil terpenes were differen-
tially affected; although the concentration of caryophyllene derivatives increased with irradiance,
α-humulene amount was unchanged.
Psychotria leiocarpa Cham. and Schltdl., a highly abundant bush in the understory of the Atlantic
Forest biome, produces GPV, an antioxidant glycosylated alkaloid that accounts for up to 2.5% of
dry leaves and accumulates mainly in reproductive structures. Accumulation of GPV is stimulated
by light, particularly under far red or blue wavelength enrichment (Matsuura and Fett-Neto, 2013;
Matsuura et al., 2016).
Piper aduncum L., the monkey-pepper, is an essential oil source, with biological activities, which
include insecticidal, antimicrobial and larvicidal, among others. Pacheco et al. (2016) evaluated the
effect of different light conditions on production and profile of essential oil constituents of leaves
and roots of the species. Major compounds in the oil increased under an environment rich in blue
light. Leaves were rich in E-nerolidol, linalol, α-humulene, cis-cadin-4-en-7-ol and caryophyllene,
whereas roots had mostly apiol. While oil concentration of the former organs was stimulated by
exposure to 50% reduction in sunlight, the latter were unaffected.
Increasing irradiance promoted the total yield of essential oil per plant of Baccharis trimera
(Less.) DC., popularly known as carqueja (Silva et al., 2006). The highest total essential oil yield of
Otacanthus azureus (Linden) Ronse was also observed in treatments with 100% irradiance, with
decreasing production as light levels decreased (Serudo et al., 2013).
6.3 TEMPERATURE
Daily temperature changes have considerable influence on cell physiology. Both cold and heat can cause
severe impacts on vegetative and reproductive tissues. Chilling causes rigidification of cell membranes,
that may lead to release of cell content, accumulation of reactive oxygen species, stabilization of RNA
secondary structure, destabilization of protein complexes, reductions in enzymatic activity and impair-
ment of photosynthesis. Freezing can kill cells due to the formation of ice crystals, which lead to the
rupture of organelles and cell compartments. Heat causes unfolding of proteins, affects membrane flu-
idity, metabolism, enzymatic activity and cytoskeleton rearrangement. On the other hand, temperature
changes can regulate movements, like the opening/closing of flower corolla, and may reset the internal
clocks and diurnal synchronization. Some species require exposure to low temperature to trigger devel-
opmental processes, such as flowering or germination (Ruelland and Zachowski, 2010).
In order to tolerate temperature variations, plants have developed adaptive mechanisms. These
organisms can coordinate specific responses to the different components of abiotic stress, that
include accumulation of sugar or compatible solutes, changes in membrane composition, synthesis
of dehydrin-like proteins, synthesis of chaperones and increase of antioxidant capacity (Bita and
Gerats, 2013). The response of the different classes of secondary metabolites is variable, depending
on the magnitude of temperature variation, degree of exposure and species analyzed. In general,
the production of volatile oils seems to increase at higher temperatures, although very hot days can
lead to an excessive loss of these metabolites (Gobbo-Neto and Lopes, 2007). Total phenolic content
in leaves of Lafoensia pacari A. St.-Hil. diminished in warmer months of the cerrado (Brazilian
savannah), in part possibly due to photosynthetic limitations (Sampaio et al., 2011).
P. brachyceras is a woody understory plant with bushy habit, reaching up to 3 m in height,
and distributed between the states of Rio de Janeiro and Rio Grande do Sul (Porto et al., 2009).
This species is characterized by the production of brachycerine, an antioxidant alkaloid, which
occurs mostly in leaves and whose accumulation can be stimulated by several stressful environ-
mental conditions (Gregianini et al., 2004; Nascimento et al., 2013). Magedans et al. (2017) showed
high temperature increased brachycerine accumulation, suggesting that heat treatment represents
a viable means to improve yields of brachycerine for use as an antioxidant. Brachycerine was able
to protect heat-sensitive species against severe heat stress, suggesting its involvement in mitigating
heat-promoted oxidative imbalance (Table 6.2).
I. paraguariensis originates from the subtropical region of South America and grows in Northeastern
Argentina, Southern Brazil and Eastern Paraguay. Holowaty et al. (2015) studied the variation of dif-
ferent parameters related to mate quality, specifically levels of caffeine, total polyphenol content, color
parameters and antioxidant activity in samples obtained by three different aging methods: natural aging,
temperature-controlled aging, temperature- and humidity-controlled aging. It was noted that the differ-
ent aging methods of mate-yielded products with slight differences in their physico-chemical properties.
Considering the main interest on mate, that is the content of polyphenols and its potential benefits to
human health, the temperature- and humidity-controlled aging method proved to be the best procedure.
Negri et al., (2009) investigated the influence of drying temperature on the content of secondary metab-
olites of Maytenus ilicifolia Mart. ex Reissek, the espinheira santa, which is widely used for treating gastri-
tis and stomach ulcers, finding that their values decreased as temperature increased. Temperatures at 40°C
to 50°C were more adequate for leaf drying, preserving higher levels of active principles. Similarly, David
et al. (2006) observed a drop in main components of essential oil of Ocimum selloi Benth. (elimicin, trans-
caryophyllene, germacrene D and bicyclogermacrene), with temperature above 40°C.
6.4 WATER AND SALINITY

Changes in water availability can severely interfere with a variety of biochemical processes, such
as mobilization of reserves, photosynthetic and respiratory capacity, cellular turgor, opening and
closing of stomata, structures of membranes and organelles, osmotic stress, enzyme activity,
ATP synthesis, hormonal balance, etc. Water stress can be caused both by drought or flooding,
whose effects, although distinct, are detrimental to plants often resulting in compounded stresses.
Prolonged periods of exposure to drought, for example, may cause stomata closure, decreased CO2
uptake, reduced photosynthesis, increased photooxidative damage, decreased protein synthesis,
inactivation of chloroplast enzymes, disturbance of electron transport chain, as well as perturbation
of membrane integrity and function. All these factors directly or indirectly result in increased activ-
ity of enzymes involved in the quenching of reactive oxygen species, ROS, in order to mitigate the
harmful effects of their excess in cells. Drought stress often also provokes salinity stress, since the
lack of water increases the concentration of salts in soil and cells, which causes a difference in water
potential favoring loss of water by the plant (Selmar and Kleinwächter, 2013). Flooding conditions,
in turn, lead to a reduction in transpiration rates, difficulties in mineral nutrient uptake and accumu-
lation of the gaseous hormone ethylene, which can trigger tissue senescence, foliar abscission and
synthesis of defense compounds.
Secondary plant metabolites may respond positively or negatively to water stress (see Table 6.2).
Some species increase the synthesis of metabolites that act in osmotic adjustment, whereas accu-
mulation of other metabolites can significantly decrease as a result of nutrient deficiency, affecting
enzymatic cofactors, photosynthetic pattern and energy metabolism (Verma and Shukla, 2015).
There are several reports describing how water deficiency may lead to increased production of
several types of secondary metabolites, such as cyanogenic glycosides, glucosinolates, terpenes,
anthocyanins and alkaloids. This effect, however, depends on the degree of severity and the period
of exposure to the drought conditions, which, in the short term, seem to lead to an increased produc-
tion, whereas an opposite effect is observed in longer times of exposure. Excess water, on the other
hand, can result in the loss of water-soluble substances from leaves and roots by leaching, which
can lead to the loss of some alkaloids, glycosides and volatile oils (Gobbo-Neto and Lopes, 2007).
Depending on the degree of flooding and time of exposure, ROS generation and ethylene production
may trigger secondary metabolite accumulation.
Plant growth under drought conditions is reduced, which often leads to an increase in the concen-
tration of secondary metabolites in relation to the dry mass, but not in their total content. However,
the effect may be the reverse in some cases. In Pisum sativum L., in spite of the total biomass
of plants grown under drought stress being about a third of that of plants cultivated under stan-
dard conditions, the concentration of anthocyanins practically doubled in stressed plants (Nogués
et al., 1998). The active constituents of Hypericum brasiliense Choisy, a herb found in Southern
and Southeastern Brazil, which include betulinic acid, isouliginosin B, 1,5-dihydroxyxanthone and
rutin, increased in plants under water stress, both in hypoxic and dry conditions, although the fresh
mass of the plants decreased in both cases (Abreu and Mazzafera, 2005). Drought also increased the
amount of uliginosin B and total phenolic compounds in acclimatized Hypericum polyanthemum
Klotzsch ex H. Reich. plants (Nunes et al., 2014). Likewise, higher essential oil content of Aloysia
triphylla Britton., native from South America, was observed in the seasons of lower biomass pro-
duction (Schwerz et al., 2015).
In Bauhinia cheilantha (Bong.) D. Dietr., a native species of the caatinga, leaf flavonoid contents
responded negatively to higher rainfall rates, but without significant effects (Sobrinho et al., 2009).
Water restriction resulted in decreased total phenolic compounds in Amburana cearensis (Allemão)
A.C.Sm. seeds, an endemic tree from semiarid region of Northeastern Brazil (Pereira et al., 2014).
Hydrocotyle umbellata L., found in all Brazilian states, on the other hand, did not suffer decreases
either in biomass production or in flavonoid content when cultivated under two intensities of irriga-
tion for three months (Alves et al., 2015). Rainfall did not correlate with phenolic compounds con-
centration in bark of Anadenanthera colubrine (Vell.) Brenan, a tree of the Brazilian caatinga and
semideciduous forest (Araújo et al., 2015).
Salt stress negatively affects plant growth and influences development, impacting on the water
potential difference between the plant and exterior environment. Water deficit as a result of salt
stress affects the availability, competitive uptake and translocation of nutrients to aboveground
plant parts (Park et al., 2016). Besides under salt stress, the excessive concentrations of Na+ and
Cl− hinder the absorption and/or assimilation of other elements, including boron, zinc, calcium,
copper, magnesium, iron, nitrogen, phosphorus and potassium (Farooq et al., 2017). The imbal-
ance in nutrient uptake and assimilation and ROS generation during salt stress interfere in pho-
tosynthetic reactions, cause stomatal limitation, reduction in intercellular CO2 concentration and
damage to photosystems (Zhu, 2016). Plants show several adaptations to deal with saline stress,
such as production of osmoprotectant molecules (cell compatible solutes), hormonal regulation,
activation of antioxidant defense systems and mechanisms of ion exclusion or compartmentaliza-
tion (Acosta-Motos et al., 2017).
The effect of salinity on secondary metabolism is dual depending on severity and has been
relatively less described for medicinal plants. Among Brazilian medicinal plants, there are several
examples of the positive effect of moderate salinity on the accumulation of secondary metabo-
lites. In plants and seedlings of Dimorphandra mollis Benth, the fava-de-anta, present in the cer-
rado vegetation, quercetin, isoquercitrin and rutin amounts generally increased under water stress
by drought, flooding and salinity (NaCl 75 mM) (Lucci and Mazzafera, 2009a, 2009b). Physalis
angulata L., common throughout Brazil, also accumulated more physalin in leaves after 13 days
exposure to saline solution 0.9%, in spite of showing biomass reduction (de Souza et al., 2013).
Saponins from leaf disks of Q. brasiliensis were significantly increased by application of osmotic
stress agents, such as sodium chloride 150 mM or isosmotic concentrations of sorbitol and polyeth-
ylene glycol (de Costa et al., 2013).
Plants of Alternanthera philoxeroides Mart. (Griseb.), a native species from the temperate
regions of South America, showed increased concentration of betacyanins in stems when exposed
to 200 mM or 400 mM of salt (Ribeiro et al., 2014). Pilocarpus microphyllus Stapf. ex Wardlew.
and Pilocarpus jaborandi Holmes, commonly known as jaborandi, are distributed exclusively in
South America, mainly in Northern and Northeast Brazil (Abreu et al., 2005). The leaves of jabo-
randi are a source of the imidazolic alkaloid pilocarpine, used mainly in the first stages of glaucoma
and xerostomia treatment and for stimulation of lacrimal and salivary glands (Caldeira et al., 2017).
In contrast to A. philoxeroides, salt stress and hypoxia resulted in decreased pilocarpine amount
in P. jaborandi leaves; reduction of alkaloid amounts in salt-treated plants was concentration and
time of exposure dependent (Avancini et al., 2003). However, in callus cultures of jaborandi, salt
and osmotic stress promoted pilocarpine release in medium (Abreu et al., 2005). Accumulation of
brachycerine in P. brachyceras leaf disks was promoted by osmotic stress and abscisic acid, a key
signaling hormone in drought stress (Nascimento et al., 2013).
6.5 MINERAL NUTRITION AND HEAVY METALS

Soil composition and mineral nutrient availability can greatly influence the synthesis of second-
ary metabolites. Changes in mineral nutrition impact on the availability of metallic ions that act as
enzymatic cofactors for defense metabolites synthesis. Nitrogen and sulfur can be components of
certain molecules, such as alkaloids, cyanogenic glycosides, glucosinolates, thiophenes and non-
protein amino acids. Nutrient-deficient soils often result in lower growth rate and higher secondary
metabolite accumulation. High nitrogen supply frequently results in lower production of phenolics
(Gobbo-Neto and Lopes, 2007). Phosphorus deficiency is known to promote anthocyanin accumu-
lation (Treutter, 2010).
The metabolic effects of nutritional imbalance are not entirely predictable. Although patterns
can be recognized, it is not possible to establish a consensus on the classes of metabolites that will
be benefited or impaired in each case (see Table 6.2). This is due to the complexity of the biochemi-
cal pathways and factors that are involved in mineral nutrition. Soil pH, for example, alters the avail-
ability of some nutrients, so that uptake by plants can be disrupted and their metabolism affected.
Likewise, the presence of microorganisms in the rhizosphere may interfere with the absorption and
availability of nutrients. The positive effect of mycorrhizal fungi on acquisition of phosphorus by the
roots is well established. However, despite the recognized influence on plant development, few stud-
ies show relationships between pH or soil microorganisms in secondary metabolism. Furthermore,
environmental factors do not seem to exert a homogeneous effect on the metabolism of different
parts of the plant, which may respond differently to such external signals (Sampaio et al., 2016).
Plants of Palicourea rigida Kunth, a Brazilian cerrado bush, grown on low-fertility and acidic
soil displayed higher concentration of iridoids. Except for Mg, all soil macro- and micronutrients
showed a negative correlation with loganin concentration, particularly Ca and K (Morel et al., 2011).
Accumulation of the alkaloid pilocarpine in P. microphyllus seedlings was negatively affected by
N and K omission, but not influenced by P removal from nutrient solution (Avancini et al., 2003).
In partially immersed callus cultures, however, the omission or excess of N or P improved alkaloid
accumulation (Abreu et al., 2005). This difference in metabolic response may be related to the pres-
ence of sucrose and partial submersion of the tissues in the axenic cultures. In Tithonia diversifolia
(Hemsl.) A. Gray, found in tropical regions, the presence of sesquiterpene lactones, flavonoids and
trans-cinnamic acid derivatives in inflorescences and roots was affected by the soil nutrients Ca,
Mg, P, K and Cu (Sampaio et al., 2016). Monoterpenes and sesquiterpene hydrocarbons of the essen-
tial oil of Lychnophora ericoides Mart., a native plant from the cerrado, were strongly correlated
with organic matter, P and base saturation in soils (Curado et al., 2006). Phenolic metabolites in
the leaves of the tree L. pacari A. St.-Hil. are positively influenced by foliar micronutrients Cu, Fe,
Mn and Zn, whereas macronutrients appear to have no effect on the production of these compounds
(Sampaio et al., 2011).
In leaves of Eugenia uniflora L., widely distributed in Brazil, the accumulation of spathulenol
and caryophyllene oxide was strongly correlated with phenolic content and S, Ca and Fe balance,
whereas selina-1,3,7(11)-trien-8-one epoxide was affected by K, Cu, Mn and water availability dur-
ing the wet season (Costa et al., 2009). Negative correlation of leaf phenolic concentration with Mn
and Cu was reported for the same species in the cerrado, indicating that metabolism of flavonoids
and tannins depends on leaf mineral nutrition (Santos et al., 2011). The same authors proposed that
both Mn and Cu could affect phenolic metabolism by their action as cofactors of enzymes dedicated
to phenolic catabolism and lignin synthesis. Data indicated a potential modulation of the amount of
these metabolites by foliar or soil micronutrient application. Oxygenated sesquiterpenes in Myrcia
tomentosa DC. flowers and leaves were positively correlated with foliar concentration of Cu and P,
whereas sesquiterpene hydrocarbons showed the opposite behavior (Sá et al., 2012). M. tomentosa
leaf oil composition was shown to be influenced by minerals in leaves (N, Fe, P, K, Ca, Mg, Mn, Al)
and soil (Fe, Al, Cu), as well as rainfall (Borges et al., 2013a). Concentration of total phenolics in
leaves of M. tomentosa was affected by foliar nutrients, such as Ca, Mg (both negatively) and Mn
(positively). Rainfall and soil K were negatively correlated with the concentration of hydrolysable
tannins (Borges et al., 2013b).
The genus Passiflora originates from South America and its main distribution center is in
Northern Central Brazil. Passiflora alata Curtis cultivated in low-nutrient soil inoculated with the
arbuscular mycorrhizal fungi Gigaspora albida yielded amounts of phenolics equivalent to those of
nutrient-sufficient soil. This result indicated that mycorrhizae association can help overcome poor
soil fertility, particularly phosphate deficiency (Riter Netto et al., 2014). Similar positive effects of
G. albida inoculation on P. alata were reported depending on the level of soil fertilization, with
the fungus generally promoting flavonoid and total phenolic yield (Oliveira et al., 2015). P. alata
and Passiflora ligularis Juss. showed higher concentrations of total phenols under conditions of
low leaf N (Sousa et al., 2013). Passiflora edulis Sims. displayed higher content of tocopherols and
ascorbic acid in organic system cultivation, whereas conventionally grown passion fruit displayed
more carotenoids (Pertuzatti et al., 2015). It has been shown that the soil elements Fe, B and Cu
showed an inverse correlation with the concentration of total flavonoids in Passiflora incarnata L.
(Reimberg et al., 2009).
Baccharis dracunculifolia DC. displayed higher yields of γ-muuroleno, valenceno, δ-cadineno,
E-nerolidol and espatulenol in essential oil when submitted to organic compost fertilization at
30 t.ha−1 versus unfertilized soil (Santos et al., 2012). Yields of essential oil of O. selloi, a native plant
of South and Southeast regions of Brazil, were increased in treatments of fertilization with bovine
and avian manure (Costa et al., 2008), which also led to an increase in leaf content of the major
component, methyl chavicol. However, unfertilized plants showed a higher diversity of compounds
in essential oil. Phosphorus and nitrogen influenced the essential oil content of Schinus terebinthifo-
lius Raddi (Pinto et al., 2016), known as Brazilian pepper tree and found in South America. Overall
growth, flavonoid and total phenolics concentrations of the same species increased by application
of poultry litter at 20 t.ha−1 (Tabaldi et al., 2016). Roots of Pfaffia glomerata (Spreng.) Pedersen,
the Brazilian ginseng, showed higher total amounts of β-ecdysone at 360 days after seedling emer-
gence, but was little affected by different levels of organic fertilization (Guerreiro et al., 2009)
Many anthropogenic activities can release heavy metals in the environment. Soil and sediments
tend to accumulate these elements, becoming a large reservoir to which plants are exposed. Plants
are generally sensitive and vulnerable to varying concentrations of these elements. Although some
metals are essential for plants, involved in synthesis, structure or activity of enzymes and proteins,
these same elements can become toxic when in high concentrations. Other metals, however, have no
known function in plant metabolism, so that they induce toxicity symptoms at minimum concentra-
tions due to a variety of interactions at the molecular level that may disrupt cellular homeostasis.
The plasma membrane is one of the first structures whose function is affected by heavy metals,
due to rupture or interference in protein sulfhydryl groups, increased lipid peroxidation, inacti-
vation of proteins, decrease in phosphorylation of target molecules and consequent changes in
composition and fluidity of lipids, frequently leading to structural damage and leakage of cellular
contents (Lara Lanza de Sá e Melo Marques et al., 2011). Inside the cells, one of the first physi-
ological damages is the inactivation of several cytoplasmic enzymes, due to the modification of
their three-dimensional structure, subunit binding or displacement of essential elements through
chemical competition, leading to the interruption of normal functions and the establishment of
deficiency symptoms. In addition, heavy metals can cause oxidative stress, which results in changes
in nuclear proteins and DNA, degradation of biological macromolecules and lipid membrane per-
oxidation (Rodrigues et al., 2016). Photosynthesis is also compromised in plants exposed to heavy
metal contamination, since these elements can reduce the levels of chlorophyll and carotenoids,
via inactivation of enzymes of pigment biosynthesis, damage to electron transport chain, inactiva-
tion of enzymes of the Calvin cycle and reduction of stomatal conductance. Some elements may
interfere with the absorption and metabolism of nutrients, which compromises the development of
plants in a global way (Küpper et al., 2007).
Plants can show tolerance to heavy metals based on mechanisms modulating regulation of their
absorption in the rhizosphere and accumulation in roots. These strategies may preserve plant cell
integrity and primary functions, which, in association with the low translocation to the aerial part,
may avoid overload on the photosynthetic apparatus and damage to the vascular bundles. The
concentration of free heavy metals in the cytosol is reduced especially via compartmentalization
in subcellular structures, exclusion and/or decrease in membrane transport. In addition, produc-
tion and formation of cysteine rich peptides, known as phytochelatins and metallothioneins, can
complex several metals. In conjunction, the actions of antioxidant defense systems, both enzymatic
and non-enzymatic, are capable of removing, neutralizing or cleaning free radicals (Emamverdian
et al., 2015). All these mechanisms can also impact the synthesis and extrusion of several plant
metabolites, both from primary and secondary metabolism.
The oil of Cunila galioides Benth plants grown on soil with a high level of aluminum showed
no difference in the content of oil major components, although the concentration of flavonoids
increased significantly under the same condition (Mossi et al., 2011). In contrast, extracts of
B. trimera (Less.) DC. grown in coal-burning area had lower levels of phenolic acids and flavonoids
compared with plants grown in nearby regions without contamination by these pollutants (Menezes
et al., 2016). In vitro culture of A. philoxeroides plants supplemented with 175 μM CuSO4 had
approximately 60% increase in betacyanin yield in relation to control, although growth reduction
was also observed (Perotti et al., 2010). Aluminum or silver exposure caused a two- to threefold
increase in brachycerine accumulation in leaf disks of P. brachyceras (Nascimento et al., 2013).
6.6 BIOTIC ELICITATION OF SECONDARY METABOLITES

Biotic responses are normally mediated by signaling compounds, such as jasmonic acid (JA), jas-
monoyl isoleucine (Ile-JA) and methyl jasmonate (MeJA), synthesized from linolenic or hexadeca-
trienoic acids starting with lipoxygenase (LOX) activity. JA and related compounds have a key role
in regulation of herbivory and wounding responses by modulating global changes in gene expres-
sion. Another example of a major biotic signaling compound is salicylic acid (SA) and its methyl
analog, methyl salicylate (MeSA), which have been shown to take part in defense signaling against
pathogens, leading to systemic acquired resistance (SAR) and providing long-term defense (Heil
and Ton, 2008). Both JA and SA may co-participate and cross talk in herbivory and pathogen
responses. Ethylene (ET) also has an important role in plant protection, acting as virulence factor
of pathogens and signaling compound in disease resistance, depending on the situation (van Loon
et al., 2006). The simulation of herbivory by applying mechanical damage can induce the formation
of JA and ET (Bailey et al., 2005). Besides, SA or JA exposure triggers events such as production
of ROS and increased cytoplasmic Ca2+, which can stimulate certain biochemical reactions and
production of secondary metabolites (Lin et al., 2001) (see Table 6.3).
Q. brasiliensis leaf saponins show structural and functional similarities to those of Q. saponaria
barks, which are currently used as adjuvants in vaccine formulations (Fleck et al., 2006, 2012;
Kauffmann et al., 2004). Previous studies with saponins of Q. brasiliensis showed a pronounced
immunoadjuvant activity in experimental vaccines against bovine herpesvirus type 1 and 5, polio-
virus, rabies and bovine viral diarrhea virus in mice (Cibulski et al., 2016; Fleck et al., 2006; Yendo
et al., 2016). The accumulation patterns of the immunoadjuvant fraction of leaf triterpene sapo-
nins QB-90 in response to stress factors were examined. Higher yields of bioactive saponins were
observed upon exposure to SA, JA and by mechanical damage, as well as by applying ultrasound to
leaves (de Costa et al., 2013). A significant increase in QB-90 content was observed when leaf disks
were submitted to 1 mM SA or 40 μM JA, supporting a general defense role for these metabolites
in plants.
The shoots of A. philoxeroides (alligator weed) contain betacyanins. Addition of 100 μM MeJA
to standard in vitro culture medium resulted in a fourfold increase in the pigment amaranthine after
35 days, in spite of having a negative impact on development (Perotti et al., 2016).
Wounding seedling leaves of P. microphyllus with a hemostat resulted in a negative effect on
pilocarpine content, changing from 0.7 to 0.1 μg.mg of leaf dry weight, after 1 week of treatment
(Avancini et al., 2003). Nevertheless, exposure of seedlings to MeJa and SA led to a significant
increase in the alkaloid concentration in leaves after 5 and 9 days of treatment, respectively. In a
follow-up study performed with aseptic cultures of P. microphyllus, the amount of quantified pilo-
carpine was similar between MeJa-treated and control calluses kept for 4 and 8 days under agitation
in the dark. However, control calluses released most of the alkaloid to the medium, while MeJa-
treated calluses retained most of the metabolite (Abreu et al., 2005).
Psidium guajava L., commonly known as guava, is an evergreen tree native to South America,
including Brazil. In a study of González-Aguilar et al. (2004), the application of MeJa to mature
green guava fruits stored at 5°C for 10 days reduced deterioration and the development of chill-
ing injury symptoms. MeJa treatment resulted in an increased activity of LOX and phenylalanine-
ammonia lyase, enzymes, which have an important role in the activation of mechanisms against
different stresses, including the biosynthesis of defense secondary metabolites.
The phloroglucinol derivative uliginosin B is found in the herbaceous plant H. polyanthemum native
to South Brazil. This molecule is a promising antidepressant, which activates the monoaminergic neu-
rotransmitter system (Nunes et al., 2014; Stein et al., 2012). Furthermore, H. polyanthemum accumu-
lates benzopyrans, chlorogenic acid and flavonoids (Nunes et al., 2010). A weekly application of 2 mM
122
TABLE 6.3
Examples of Biotic Stress Effectors to Induce Production and/or Accumulation of Secondary Metabolites
Induced
Secondary Tissue/ Time for Maximum
Species Treatment Metabolite(s) Organ Dose Enhancement Maximum Increase References
A. philoxeroides Methyl Amaranthine Shoots 100 μM 35 days Fourfold Perotti et al.
Jasmonate (2016)
H. polyanthemum Salicylic acid Uliginosin B and Vegetative 2 mM (weekly 18 weeks Twofold Nunes et al.
TPC tissues application) (2014)
Salicylic acid or Uliginosin B Vegetative 10 mM/wounded 2 days for mechanical 2 days (fivefold)
mechanical tissues with fine sterile damage and SA alone and and 7 days
damage or both needle 7 days for both (threefold)
H. polyanthemum Fungal elicitation Benzopyrans Whole plants 10 ml of solution of 48 h and 72 h Benzopyrans Meirelles
with Nomuraea (HP1, HP2, dried autoclaved (benzopyrans) and 24 h (approx. 1.7-fold), et al. (2013)
rileyi HP3) and total cell powder (total phenolic compound) TPC (twofold)
phenolic (containing the
compounds equivalent of 1.5
9 × 106 spores ml−1)
P. microphyllus Salicylic acid Pilocarpine Leaves 5 mM 9 days Fourfold Avancini et al.
Methyl Pilocarpine Leaves Plant incubation in 5 days Fourfold (2003)
Jasmonate plastic bag with
cotton piece
impregnated with
50 μL of pure

MeJA
P. brachyceras Mechanical Brachycerine Leaves Wounded 75% of 2 days Twofold Gregianini
damage the total leaf area et al. (2004)
of cuttings with
scissor
Jasmonic acid Brachycerine Leaves 40 µM and 400 µM 6 days (40 µM), 4 days 2.7-fold (40 µM)
(400 µM) and 3.3-fold
(400 µM)
(Continued)
Bioactive Metabolite Accumulation
Examples of Biotic Stress Effectors to Induce Production and/or Accumulation of Secondary Metabolites
Induced
Secondary Tissue/ Time for Maximum
Species Treatment Metabolite(s) Organ Dose Enhancement Maximum Increase References
Q. brasiliensis Jasmonic acid QB-90 (triterpenic Leaf disks 40 μM 2 days 2.6-fold de Costa et al.
saponin fraction) (2013)
Salicylic acid QB-90 Leaf disks 1 mM 2 days Threefold
Ultrasound QB-90 Leaf disks 1 and 2 min 2 days after initial 1 min Twofold
treatment (40 SA exposure
kHz and 135 W)
Mechanical QB-90 Seedlings 50% of leaves were 2 days 1.5-fold
damage wounded with
scissors
V. glechomifolia Salicylic acid Valepotriates Whole-plants 0.1 mM SA and 1 72 and 96 h (0.1 mM SA), 1.7- to 1.8-fold Russowski
cultured in mM SA 48 and 72 h (1 mM SA) (0.1 mM SA), et al. (2013)
MS liquid approx. 1.5-fold
media (1 mM SA)
Ultrasound (40 Valepotriates Whole-plants 2.5 and 5 min 2.5 min for cultures in the 1.4- to 1.8-fold
kHz and 135 W) cultured in 7th day and/or 14th day of
MS liquid growth cycle – harvested
media in the 21st day of growth
cycle
123
SA increased the amount of uliginosin B and total phenolic compounds in acclimatized H. polyanthe-
num plants after 18 weeks (Nunes et al., 2014). Mechanical damage, 10 mM SA and the combination
of both treatments were able to induce higher levels of uliginosin B in leaves after 1 or 2 days. The
entomopathogenic fungus Nomuraea rileyi induced production of three benzopyrans and total phenolic
compounds in H. polyanthemum plantlets (Meirelles et al., 2013). The former compounds have been
studied for their antinociceptive and antitumoral activities (Ferraz et al., 2005; Haas et al., 2010). After
48 and 72 h of exposure of acclimatized plants to a dried autoclaved cell powder of N. rileyi, increased
levels of the three benzopyrans were observed in vegetative parts. In addition, a twofold increase in total
phenolic compounds was promoted after 24 h of fungal exposure (Meirelles et al., 2013).
P. edulis is cultivated commercially in tropical and subtropical areas of Southern Brazil. Jardim
et al. (2010) observed an increase in LOX transcripts and higher enzyme activity in response to a
specialist (Agraulis vanillae vanilla) and a generalist (Spodoptera frugiperda) caterpillar attack,
suggesting that the herbivore response in passion fruit is mediated by JA signaling pathway, which
may also impact on its secondary metabolite profile. LOX activity was previously shown to be mod-
ulated in P. edulis in response to wounding and exogenous MeJa application (Rangel et al., 2002).
The seeded-fruit transcriptome of guarana, Paullinia cupana var. sorbilis (Mart.) Ducke,
a stimulant plant native to the central Amazon basin, was performed with a focus in finding
Expressed Sequence Tags (ESTs) related to secondary metabolism. Several key genes related to
flavonoid biosynthesis, plant biotic defense pathways and purine alkaloid metabolism were well
represented in the analyzes (Angelo et al., 2008).
The effects of mechanical wounding, ethylene and 0.2 mM MeJa application on the expression of
genes associated with stress responses on leaves of Theobroma cacao L. (cocoa tree) were analyzed.
A higher expression of the transcripts of type III peroxidase, a class VII chitinase, and a caffeine
synthase was found on treated-seedlings. Gene expression profile proved to be dependent on the
ontogeny of the associated tissue (Bailey et al., 2005).
Monoterpene derived valepotriates are accumulated in both shoots and roots of Valeriana
glechomifolia Meyer, a small herb which grows in rocky fields of Southern Brazil (Salles et al., 2002). A
semi-purified valepotriate fraction from shoots and roots showed sedative effects and affected behavioral
parameters related to recognition memory (Maurmann et al., 2011). A supercritical CO2 V. glechomifolia
extract displayed antidepressant potential, which is mediated by the dopaminergic and noradrenergic
neurotransmission systems (Muller et al., 2015). The exposure of whole plants cultivated in liquid medium
to SA and ultrasound increased by twofold the amount of the metabolite, best results being recorded with
the latter treatment, which has not diminished biomass accumulation (Russowski et al., 2013).
Biomembranes derived from Hevea brasiliensis (Willd. ex A.Juss.) Müll.Arg. latex (rubber tree)
have shown wound healing properties (Frade et al., 2012). The effect of ET, JA and wounding was
analyzed in the barks of 3-month-old shoots of H. brasiliensis. Several defense and latex exudation-
related genes had increased expression upon treatment application (Duan et al., 2010). A member
of AP2/ERF transcription factors from laticifers induced by JA was fully characterized, further
supporting the involvement of this hormone in latex-based defense responses (Chen et al., 2011).
Araucaria angustifolia (Bertol.) Kuntze, the Brazilian pine, is well-known for its medicinal
properties, which include antioxidant, antibacterial, antiviral, anti-inflammatory and antiprolifera-
tive (Branco et al., 2016). Several of its active pharmacological constituents are present in the bark
resin, including various polyphenolics and terpenes. A study examining the regulation of bark resin
exudation in young Araucaria plants showed that ET, SA and JA stimulated resin yield and, in some
cases, modified monoterpene relative concentration (Perotti et al., 2015).
6.7 SEASONAL VARIATION

The climate of the Northern and Northeastern regions is characterized by small annual variation
of elevated temperatures and long photoperiod, with different average rainfall. Whereas the North
presents a rainy equatorial climate, the Northeastern is characterized by a semiarid climate. On the
other hand, the Southeast and Center-West regions are influenced by a dry season in winter and a
rainy season in the summer. In the south of Brazil, due to its latitudinal location, cold air masses
contribute to the predominance of lower winter temperatures and shorter day length, with generally
well-defined four seasons.
Several environmental factors, such as seasonality, photoperiod, circadian rhythm, temperature,
irradiance, altitude, humidity and water availability may affect plant secondary metabolism in an
integrated way (Yao et al., 2004). These factors can influence secondary compound production
throughout the year, highlighting the importance of harvest time for optimizing yield. In fact, there
are several studies describing seasonal influence on the content of diverse classes of secondary
metabolites, such as terpenes and phenolic compounds (Gobbo-Neto and Lopes, 2007).
M. tomentosa (Aubl.) DC., commonly known as guava brava, is used in folk medicine against
gastrointestinal disorders, infectious diseases and hemorrhagic conditions. Sá et al. (2012) showed
that its terpene content may vary according to the time of year. In general, the major component in
samples collected in August, October, December and February was (2E,6E)-methyl farnesoate; epi-
α-bisabolol was the main compound in April, whereas germacrene D, (2E,6E)-methyl farnesoate
and bicyclogermacrene were the main components in June. The component γ-muurolene was absent
in August and December but present in the total essential oil in February and April. Regarding the
variation of percentage in the total content of essential oil, the sesquiterpene (E)-β-farnesene had
a small variation only in August and October, but bicyclogermacrene amounts varied from 4.73%
of the total essential oil content in October to 14.71% in June. Content of (2E,6E)-methyl farneso-
ate ranged from only 5.33% of the total essential oil content in April to more than 47% in October
(Sá et al., 2012).
Elionurus muticus (Spreng.) Kuntze leaves essential oil was shown to vary throughout the year.
Also known as lemon grass, it is one of the most abundant grass species in the mid-southern por-
tion of the Pantanal biome, characterized by subtropical climate, with a short cold season and low
occurrence of frost. The period of plant harvest affected the percentage of the sesquiterpenoids (E)-
caryophyllene, bicyclogermacrene, spathulenol and caryophyllene oxide in essential oils. In winter
and spring, (E)-caryophyllene was the main component in the oils, whereas in summer and autumn
bicyclogermacrene became the main component. In spring, the antibacterial caryophyllene oxide
and spathulenol displayed higher yields than in the other seasons (Hess et al., 2007).
Martianthus leucocephalus (Mart. ex Benth.) J.F.B.Pastore, formerly known as Hyptis leuco-
cephala, is an aromatic herb whose leaves, flowers and branches produce antimicrobial essential
oils which showed activity against Bacillus cereus, Staphylococcus aureus and Candida albicans.
The chemical profile of this species was strongly affected by climatic factors during the year, and
its oil content varied from 0.1% to 0.31%. Essential oil content was positively correlated with irra-
diance but showed a negative correlation with precipitation and relative humidity. Its highest pro-
duction was between September and March, months of low rainfall and elevated level of solar
radiation. However, in May and June the essential oil production was low, but vegetative growth was
higher, suggesting that an optimized cultivation protocol can be established, so that planting could
take place in May and June whereas harvesting would be done between September and March for
improved yields (Azevedo et al., 2015). In contrast, in the related species Hyptis carpinifolia Benth.,
commonly known as rosmaninho and mata-pasto, the higher the humidity the greater the amount of
α-copaene and pinonic acid (Sá et al., 2012).
The antileishmanial K. pinnata (Lam.) Pers., leaf extract traditionally used in Brazil to treat
skin diseases and wounds, has active flavonoids that accumulate at higher levels in summer. This
seasonal peak of accumulation was coincident with higher solar irradiances. A relationship between
higher accumulation of flavonoids and irradiance availability was corroborated by comparing plant
cultivation under direct sunlight or shade. Leaves under higher irradiance had an increment of sev-
enfold in the quercetin yield (Muzitano et al., 2011).
Plants of Baccharis sp. are widely used in Brazilian folk medicine, mainly to treat gastrointes-
tinal disorders. Phenolic acids such as flavones, methylated-flavones and some flavanols, mainly
aglycones, are the major compounds described for this genus, and have several biological activi-
ties, including antimicrobial and anti-inflammatory properties (Martinez et al., 2005). Sartor
et al. (2013) showed that the total content of phenolic compounds and the flavonoid fraction of
Baccharis dentata (Vell.) G.M. Barroso undergoes seasonal variations. The highest concentra-
tions of total phenolics were recorded in autumn and winter. Rutin was the most abundant flavo-
noid, showing a concentration peak in winter. Phenolic compounds such as caffeic acid were also
most abundant in winter and summer, possibly by activation of the phenylpropanoid pathway at
more extreme temperatures. In B. dracunculifolia, caffeic acid contents were higher mostly in
summer, but also in some of the spring and early autumn months (approximately between 3%
and 6% of dry weight), further confirming that seasonality affects the profile of phenolics (Sousa
et al., 2009). Quercetin, kaempferol and apigenin concentrations were higher in spring and sum-
mer than in winter, perhaps as part of an adaptive response against photooxidative stress caused
by excess of light (Sartor et al., 2013).
Lima et al. (2017) observed that pilocarpine contents varied seasonally in three populations of
P. microphyllus. Overall, pilocarpine content varied throughout the year in all samples, with the
lowest levels being recorded in the rainy season and the highest in the dry season, suggesting a
negative influence of rainfall on alkaloid content (Lima et al., 2017). The higher accumulation of
alkaloid in the dry season may reflect its role in adaptive responses against drought, photooxidative
damage and temperature stresses.
Mikania laevigata Sch.Bip. ex Baker and Mikania glomerata Spreng. are medicinal plants popu-
larly known as guaco in Brazil. Both species are broadly used to treat inflammatory and aller-
gic conditions, particularly disorders of the respiratory system. The leaves of M. laevigata and
M. glomerata have been reported to have similar chemical compositions, and their major bioactive
constituents are kaurene-type diterpenes and derivatives of cinnamic acid. The content of couma-
rin in M. laevigata reached the highest yields in summer and was significantly increased in plants
cultivated under high shading levels, whereas this phenolic was absent in M. glomerata. The accu-
mulation of kaurene-type diterpenes in both species was favored by growth under full sunlight,
and M. glomerata had the highest seasonal accumulation of these metabolites in winter (Bertolucci
et al., 2013).
Known as aloe, Aloe arborescens Mill. contain approximately 2.0% of dry weight of compounds
with antimicrobial potential, such as the quinones barbaloin, aloe-emodin, aloin A and B and iso-
barbaloin. Cardoso et al. (2010) found that the quinone levels on leaves were higher in summer and
autumn, whereas the flavonoid contents were similar for all seasons. Winter, spring and summer
chloroform extracts presented higher antimicrobial activity than their autumn counterpart; winter
extract had the lowest Minimum Inhibitory Concentration (MIC, 128 μg.mL−1) on Bacillus subtilis.
Ethanolic extracts of summer and autumn showed low antimicrobial activity, while winter ethano-
lic extract had again the lowest MIC (256 μg.mL−1) on Klebsiela pneumoniae. The antimicrobial
effects may involve other metabolites and/or a combined action of active compounds in the extracts.
In fact, the correlation between seasonal variation, metabolite content and biological activity is
not always apparent. In Lippia origanoides Kunth., antibacterial activity against S. aureus and
Escherichia coli, as well as the average yield and overall oil composition, were little influenced by
seasonal variation (Sarrazin et al., 2015).
Cordia verbenacea DC. is a native Brazilian medicinal plant, widely distributed along the
Southeast coast of Brazil. This bushy plant is popularly known as cordia, blacksage or erva
baleeira and has been known for its properties as antiulcer, antimicrobial and anti-inflammatory
(Falcão et al., 2008; Michielin et al., 2009). Several compounds are found in the aerial parts of
C. verbenacea including α-pinene, trans-caryophyllene, aloaromadendrene, cordialin A, cordia-
lin B, rosmarinic acid and flavanols (Thirupathi et al., 2008). The phenolic rosmarinic acid is
regarded as a phytochemical marker of C. verbenaceae due to its abundance in the species. A
relatively strong positive correlation between the rainy season at a Central Brazil locality and ros-
marinic acid content was reported, suggesting the wet period is the best choice for harvesting this
medicinal plant (Matos et al., 2015). According to Queiroz et al. (2016), the harvest time during
the day did not influence the content of essential oil, but it could modify its chemical profile. Even
though the concentrations of the major compounds β-caryophyllene, xylene and γ-muurolene
displayed no differences among collection times, sabinene was found only during early morning
(6 am) harvest.
6.8 FINAL REMARKS

Both mild abiotic and biotic stresses often result in increased secondary metabolite accumulation,
and this response is usually associated with some degree of growth rate reduction. This is expected
since the flux of C, N and S toward secondary metabolism drains the pools of primary metabolite
precursors supporting growth. The degree of stress exposure must be such that it finds a balance
between maximum defense metabolism stimulation and minimal damage. This fine balance can
be achieved by appropriate combinations of time and intensity of exposure to stimuli. At the center
of several stresses signaling pathways is redox balance, which is a major regulator of secondary
metabolism acting as a network hub. Developmental stage is frequently a determinant of response
capacity, as is the type of nutrition, heterotrophic (most cell cultures), semi-autotrophic or fully
autotrophic. Seasonal and circadian variations of secondary metabolic profiles may also be consid-
ered as a factor for optimizing target product yields.
Signaling pathways mediating environmental stress or regular signals involve key phytohor-
mones, notably JA, SA and their derivatives. Such molecules and their precursors may be used to
trigger metabolic profiles of interest without the need of the environmental signal itself, sparing sig-
nificant energy and time for responses to arise. Similarly, adequate modulation of mineral nutrition
to sustain biochemical activity, including enzyme components and cofactors, or even essential and
nonessential metal levels that trigger ROS production, are also important tools in driving secondary
metabolism.
To sum up, environmental signals and mild stresses are useful tools to control secondary metab-
olism and the yield of target bioactive metabolites in Brazilian medicinal plants. There is an array
of relatively simple and low-cost crop management strategies to sustainably produce pharmacologi-
cally active phytomedicines and isolated compounds from this valuable and still relatively untapped
biological resource.
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7 Brazilian Bryophytes and
Pteridophytes as Rich Sources
of Medicinal Compounds
Adaíses Simone Maciel-Silva and Lucas Vieira Lima
Universidade Federal de Minas Gerais, Laboratório de Sistemática
Vegetal, Departamento de Botânica, Instituto de Ciências Biológicas,
Belo Horizonte, Brazil
CONTENTS
7.1 Introduction......................................................................................................................... 136
7.2 Morphology and Systematics of Bryophytes and Pteridophytes......................................... 138
7.2.1 Bryophytes............................................................................................................... 138
7.2.2 Pteridophytes........................................................................................................... 140
7.3 Collection Techniques and Processing................................................................................ 142
7.3.1 Bryophytes............................................................................................................... 142
7.3.2 Pteridophytes........................................................................................................... 144
7.3.2.1 How Big is the Plant?............................................................................... 144
7.3.2.2 What to Collect......................................................................................... 144
7.3.2.3 How Should the Collected Material be Processed?.................................. 144
7.3.2.4 What Information Goes on the Herbarium Label?................................... 145
7.3.2.5 How to Assign Names to Ferns and Fern Allies....................................... 145
7.4 Chemical Compounds in Bryophytes.................................................................................. 145
7.5 Chemical Compounds in Marchantiophyta......................................................................... 146
7.5.1 Terpenoids................................................................................................................ 146
7.5.2 Aromatic Compounds.............................................................................................. 148
7.5.3 Flavonoids and Anthocyanidins.............................................................................. 151
7.5.4 Acetogenins and Lipids........................................................................................... 151
7.6 Chemical Compounds in Bryophyta................................................................................... 152
7.7 Chemical Compounds in Hornworts................................................................................... 152
7.8 Chemical Compounds in Pteridophytes.............................................................................. 153
7.8.1 Terpenoids................................................................................................................ 153
7.8.2 Phenolics.................................................................................................................. 154
7.8.3 Flavonoids................................................................................................................ 156
7.8.4 Alkaloids.................................................................................................................. 156
7.9 Biologically Active Compounds and Their Potential Medicinal Uses................................ 157
7.9.1 Bryophytes............................................................................................................... 157
7.9.2 Pteridophytes........................................................................................................... 158
7.10 Final Remarks and Future Perspectives.............................................................................. 165
Acknowledgments........................................................................................................................... 166
References....................................................................................................................................... 166
135
7.1 INTRODUCTION
Bryophytes and pteridophytes are generalized names given to the five different plant lineages:
“bryophytes” – Marchantiophyta (liverworts, ca. 9,000 spp.), Bryophyta (mosses, 12,700 spp.),
and Anthocerotophyta (hornworts, 225 spp.) (Christenhusz and Byng, 2016; Crandall-Stotler
et al., 2009; Goffinet et al., 2009; Renzaglia et al., 2009); “pteridophytes” include Lycopodiopsida
(e.g. clubmosses, quillworts, and spikemosses, 1,290–1,338 spp.) and Polypodiopsida (e.g. ferns,
horsetails, and whisk ferns, 10,560 spp.) (PPG I, 2016). Scientists have estimated that liv-
erworts appeared ca. 470 million years ago, which is at least ∼330 Ma before the angiosperms
(flowering plants) (Magallón et al., 2015; Wellman et al., 2003). Because of their ancient origins,
many taxa in these groups could produce unique and rare phytochemical compounds and rich
sources of medicinal compounds. In this chapter, we shall henceforward use the term “bryophytes”
and “pteridophytes” to refer to those plant groups.
Bryophytes and pteridophytes produce a wide diversity of chemical compounds such as terpe-
noids, steroids, flavonoids, alkaloids, and aromatic and phenolic compounds (Asakawa et al., 2013a;
Chopra and Kumra 1988; Huneck 1983; Schofield 1985). Bibenzyl cannabinoids, pinguisane-
type sesquiterpenoids, and sacculatane-type diterpenoids are examples of chemicals exclusive
to bryophytes, while pteridophytes produce unique compounds such as the alkaloids huperzine,
lycopodine, and the triterpenoid lycophlegmariol, among others (Asakawa et al., 2013a, 2013b;
Cao et al., 2017).
Studies focusing on the phytochemistry of bryophytes and pteridophytes have increased in recent
years (Asakawa, 2001; Asakawa et al., 2013a; Asakawa and Ludwiczuk, 2017; Cao et al., 2017)
despite difficulties encountered in terms of the identification of plants and their collection in large
quantities as pure samples (especially bryophytes). Countries such as China and United States stand
out in terms of the numbers of papers published from 1999 to 2017 (Figure 7.1). The chemistries
of bryophyte species are not yet very well-known, largely because of problems in obtaining pure
samples of any species (Sabovljevic et al., 2009). The secondary metabolic compounds produced
by bryophytes and ferns show antimicrobial, antifungal, anti-Alzheimer, cytotoxic, antitumor,
vasopressin (VP) antagonist, cardiotonic, allergenic, irritant, tumor-affecting, insect antifeedants,
insecticide, molluscicide, and piscicide effects; and plant growth regulation, superoxide anion radi-
cal release inhibition, 5-lipoxygenase, calmodulin, hyaluronidase, cyclooxygenase, and anti-HIV
activities (Asakawa et al., 2013a; Liu et al., 1986a, 1986b; Sabovljevic et al., 2001; Sabovljevic
et al., 2009; Santos et al., 2010).
Ethnobotanical studies have focused on the use of those plants by human societies (in places such
as Africa, America, Europe, Poland, Argentina, Australia, New Zealand, Turkey, Japan, Taiwan,
Pakistan, China, Nepal, and different parts of southern, northern, and eastern India; Beaujard
(1998), Benjamin and Manickn (2007), Bonet and Valles (2007), Chandra et al. (2017), Glime
(2017), Hammond et al. (1998), Harris (2008)) and their potential applications in the pharmaceuti-
cal and medicinal industries (Frahm, 2004; Harris 2008; Ho et al., 2011).
Approximately 5,718 species of bryophytes and 1,332 of pteridophytes are currently known from
Brazil (Flora do Brasil, 2020). The Atlantic forest alone harbors more than 32% of all Brazilian
species of bryophytes, lycophytes, and ferns. Although chemical studies of bryophytes and pterido-
phytes have indicated their great medicinal potential, these species remain poorly investigated in
relation to their high taxonomic diversities (Pinheiro et al., 1989; Santos et al., 2010). That line of
research has been very slow in Brazil despite its high biodiversity, with research on pteridophytes
being slightly greater than on bryophytes (Figure 7.1).
The main aim of this review is to compile data that could help identify potential uses of the chemi-
cal compounds found in Brazilian species of bryophytes and pteridophytes. Here, we present acces-
sible information on the morphology and taxonomy of those plants, techniques for their collection in
the field and their taxonomic identification and herborization, the potential occurrence of secondary
metabolites and their biological activities, and the geographical distributions of Brazilian taxa.
Rich Sources of Medicinal Compounds
FIGURE 7.1 Studies published between 1999 and 2017, listed by country and year of publication; based on the Web of Science database (search topics: chemical
137
compounds and bryophytes; chemical compounds and pteridophytes; chemical compounds and ferns).
7.2 MORPHOLOGY AND SYSTEMATICS OF BRYOPHYTES

AND PTERIDOPHYTES
7.2.1 Bryophytes
Bryophytes are probably the closest modern relatives of the earliest land plants, comprising plants,
which have life cycles with alternating haploid and diploid generations with a dominant gameto-
phyte (Figure 7.2; Gerrienne and Gonéz, 2011; Vanderpoorten and Goffinet, 2009). Bryophytes
are the only land plants with a dominant, branched gametophyte, and they exhibit a large diver-
sity of morphologies as compared to tracheophytes (Gerrienne and Gonéz, 2011; Vanderpoorten
and Goffinet, 2009). In addition to the structural diversity of their gametophytes, bryophytes dis-
play physiological adaptations (e.g. poikilohydry, desiccation tolerance, efficient mechanisms for
water and nutrient uptake, and specialized life cycles) that enable their successful colonization of
many different biomes, from the tundra of the Northern hemisphere to Antarctica (Crandall-Stotler
et al., 2009; Goffinet et al., 2009; Ligrone et al., 2000; Proctor et al., 2007; Renzaglia et al., 2007,
2009; Vanderpoorten and Goffinet, 2009).
Recent phylogenies, especially those based on molecular data, have resulted in different
hypotheses concerning the evolution of bryophyte lineages and the relationships between them
(Crandall-Stotler et al., 2009; Goffinet et al., 2009; Renzaglia et al., 2007, 2009). Among the cur-
rent phylogenetic hypotheses of land plants or embryophytes is the proposal that Marchantiophyta
would have diverged early in the evolution of the group, while Anthocerotophyta is the sister group
to all vascular plants; Bryophyta would be an intermediate group. Many recent studies have focused
on understanding the evolution of land plants using ever broader frameworks, and bryophyte lin-
eages seem to mark the transition from the algal ancestors of land plants to vascular plants (Goffinet
and Buck, 2012; Goffinet et al., 2009).
Liverworts (Marchantiophyta) have thallose or leafy gametophytes (Figure 7.3A–C) with leaves
in two or three rows. Oil bodies (organelles rich in essential oils) are frequently found in the game-
tophytic cells of different liverwort taxa. Sporophytes produce one sporangium, elevated only at
maturity by a hyaline stalk (seta) that extends by cell elongation. Sporangium dehiscence is typi-
cally along four vertical lines, and no stomata are present in the sporangial walls. Spores and elaters
(elongated cells with spiral wall thickenings that facilitate spore dispersal) can be found inside the
sporangia. After germination, the spores develop into a single, branched gametophyte (Crandall-
Stotler et al., 2009; Vanderpoorten and Goffinet, 2009). Brazilian liverworts are represented by
135 genera and ca. 667 species (Flora do Brazil, 2020).
Mosses (Bryophyta) have leafy gametophytes with leaves arranged in spiral rows (Figure 7.3D).
In sporophyte, complete seta development is prior to sporogenesis and elevates a terminal sporan-
gium. Dehiscence occurs through an operculum in the majority of mosses. Stomata may occur on
the sporangium wall. No elaters are found inside the sporangium, and spores generally germinate
into filamentous sporelings called protonema, which can develop into several leafy gametophytes
(Goffinet et al., 2009; Vanderpoorten and Goffinet, 2009). Brazilian mosses are currently repre-
sented by 276 genera and ca. 890 species (Flora do Brazil, 2020).
Hornworts (Anthocerotophyta) consist of thalloid gametophytes and linear sporophytes com-
posed of a long sporangium with no seta (Figure 7.3E). Sporangia show nonsynchronous spore dis-
persal as the basal meristem adds new cells to its base. Dehiscence occurs by two longitudinal lines,
exposing spore mass and multicellular pseudoelaters, unlike the elaters of liverworts. Stomata are
present on the sporangia walls in some taxa. Endosymbiotic colonies of the cyanobacteria Nostoc
are common among hornworts (Renzaglia et al., 2009; Vanderpoorten and Goffinet, 2009). Seven
genera and 15 species of hornworts are currently known to Brazil (Flora do Brazil, 2020).
The life cycles of bryophytes (as in all land plants) are characterized by an alternation of two
generations: gametophyte and sporophyte. After the fusion of two gametes (fertilization), the zygote
develops into a sporophyte, which produces spores through meiosis. While the gametophyte has sex
Rich Sources of Medicinal Compounds
FIGURE 7.2 Life cycles of mosses and ferns, showing the alternation of two generations. Mosses have a free-living gametophyte as the dominant phase, whereas the
sporophyte is dominant in ferns.
139
FIGURE 7.3 Examples of different bryophyte groups. (A and B) Ventral and dorsal views of a leafy liverwort
(Frullania brasiliensis). (C) View of a thallose liverwort (Marchantia polymorpha). (D) Mosses (Polytrichum
sp.) with sporophytes. (E) A hornwort (Phaeoceros sp.) containing several sporophytes. Photographs (A), (B),
and (D) by Oliveira, M.F.; (C) by Oliveira, B.A.; and (E) by Araújo, C.A.T.
organs (female and male gametangia), the sporophyte develops a sporangium that produces only
spores. The sporophyte is physiologically dependent on, and permanently attached to, the game-
tophyte (maternal plant) during its complete life cycle. Bryophytes are the only extant land plants
with gametophyte as the dominant generation in their life cycles (Figure 7.2; Vanderpoorten and
Goffinet, 2009; Maciel-Silva and Pôrto 2014).
7.2.2 Pteridophytes
There are many botanical texts that describe the morphologies of pteridophytes. Our main goal
here is to provide a quick guide for nonspecialists who wish to become familiar with the sub-
ject. We recommend the following reports for more detailed approaches (Harris and Harris 1994;
Lawrence, 1977; Lellinger, 2002; Stearn, 1998).
Pteridophytes are seedless vascular plants with a life cycle showing an alternation of genera-
tion, in which the gametophytic phase is independent from the sporophyte (Figure 7.2). Unlike
bryophytes, the ephemeral and reduced phase of ferns is the gametophyte, while the sporophyte is
complex, branched, and long-lived (Gifford and Foster, 1987).
Pteridophytes are widely distributed and inhabit nearly all tropical habitats, occurring in rain
forests, high montane cloud forests, temperate forests, mangroves, and even floating or submerged
in lakes. They are often pioneer species and weedy colonizers of disturbed landscapes and can be
found scattered among rocks in semiarid landscapes, savannas, and coastal and high alpine moun-
tains, resisting droughts, fires, and cold temperatures (Sharpe et al., 2010).
Ferns and lycophytes are two distinct and ancient phylogenetic lineages of seedless vascular
plants traditionally addressed as pteridophytes (PPG I, 2016; Pryer et al., 2004). According to the
most recent classification (PPG I, 2016), two monophyletic classes are recognized: Lycopodiopsida
and Polypodiopsida.
Rich Sources of Medicinal Compounds 141
FIGURE 7.4 (A) The quillworts, Isoetes, Isoetaceae. (B) The spike-moss Selaginella, Selaginellaceae.
(C) The club-moss Phlegmariurus, Lycopodiaceae. (D) The horsetail Equisetum, Equisetaceae. (E) The
moonwort Botrychium, Ophioglossaceae. (F) Danea, Maratticeae.
Lycopodiopsida is divided into three orders, three families, and 18 genera (PPG I, 2016), and
is represented by spike mosses (Selaginellaceae), clubmosses (Lycopodiaceae), and quillworts
(Isoetaceae). Those plants are principally characterized by the presence of microphylls, sporan-
gia with transversal dehiscence, and by each fertile microphyll bearing only one sporangium on
the adaxial surface, usually forming a strobilus at the branch apex (Øllgaard, 1990; Tryon and
Tryon, 1982). Lycopodiopsida is represented by 11 genera and approximately 179 species in Brazil.
Selaginella (Figure 7.4B) is the only genus in the Selaginellaceae family (PPG I, 2016), and is
characterized by the presence of rhizophores, leaves, a ligule, heterospores, and adaxial and reni-
form sporangia (Webster, 1992). In Brazil, the family is represented by 89 species, of which 30 are
endemic (Flora do Brazil, 2020).
Lycopodiaceae (Figure 7.4C) is divided into three subfamilies, 16 genera, and an estimated 388
species (PPG I, 2016). Thedifference from the other lycophyte families is mainly by demonstrating
homospory and by having eligulate microphylls. In Brazil, this family is represented by nine genera
and 62 species, of which 31 are endemic (Flora do Brazil, 2020).
Isoetaceae (Figure 7.4A) comprises one genus and approximately 250 species. Isoetes L. is read-
ily identified bythe species’ microphylls having four air chambers in cross section, a single sunken
sporangium at the base of the microphylls, ligulate microphylls, and heterospores (Gifford and
Foster, 1987; Pigg, 1992). Isoetaceae is represented by 27 species in Brazil, of which 22 are endemic
(Flora do Brazil, 2020).
The Polypodiopsida class is divided into four subclasses, 11 orders, 48 families, 319 genera, and
an estimated 10,578 species (PPG I, 2016).
Equisetidae consists of one extant order, one family, one genus, and 15 species (PPG I 2016).
Horsetails ferns (Equisetum) (Figure 7.4D-E) are principally characterized by the presence of a pel-
tate sporangiophore, articulated rhizomes (usually hollow), and by reduced and verticillate leaves
(Hauke, 1990). Only Equisetum giganteum L. occurs in Brazil (Nóbrega and Prado, 2018).
Ophioglossidae is divided into two orders, two families, 12 genera, and an estimated 129 species.
This subclass comprises the whisk ferns (Psilotaceae), which are mainly characterized by root-
less sporophytes, with dichotomous rhizomes, aerial branches, and scale-like or leaf-like leaves.
Only Psilotum nudum (L.) P. Beauv. occurs in Brazil. The subclass also includes moonwort ferns
(Ophiglossaceae) (Figure 7.4F). Those plants are readily recognized by having hemidimorphic
fronds with eusporangia on the erect fertile portion of the frond, usually in a fertile spike or pan-
icle-like sporangial cluster arising from the base of the sterile blades (Mickel and Smith, 2004).
Ophioglossidae is represented by four genera and six species in Brazil (Prado et al., 2015).
Marattiidae has only one order, one family, six genera, and an estimated 111 species. Marattiaceae
is mainly characterized by the presence of pairs of large, persistent photosynthetic stipules that
protect the young croziers, and having free abaxial eusporangia, or eusporangia united to form a
synangium (Camus, 1990). In Brazil, this subclass is represented by three genera and six species
(Prado et al., 2015).
Polypodiidae (Figure 7.5) comprises the vast majority of extant fern diversity, with seven orders,
44 families, 300 genera, and an estimated 10,323 species (PPG I, 2016). Film ferns, tree ferns,
maidenhair ferns, among other groups, demonstrate the wide morphological diversity of this
subclass, which is characterized by leptosporangia. Polypodiidae is represented by 31 families,
134 genera, and 1,153 species in Brazil (Flora do Brasil, 2020).
7.3 COLLECTION TECHNIQUES AND PROCESSING

Scientific plant collecting is essential for many reasons, including specimen identification, herbar-
ium collections, and the establishment of DNA banks (Vanderpoorten et al., 2010). Correct tech-
niques for collecting and processing plant material should always be employed. The necessity of
obtaining collecting permits must also be emphasized, as well as export licenses (if the material is
to be taken out of the country) (Frahm, 2003; Gradstein et al., 2001; Vanderpoorten et al., 2010).
7.3.1 Bryophytes
Collecting bryophytes is generally easier than collecting flowering plants, as they generally do not
need to be pressed and can be held in simple paper bags (together with substrate samples; 1–3 cm)
and allowed to air dry. A quick guide for collecting and processing of bryophytes is provided herein.
For more details, see Gradstein et al. (2001), Frahm (2003), Vanderpoorten et al. (2010), and Glime
and Wagner (2013).
During specimen collection, plants should be selected to include all organs needed for their iden-
tification. The sizes of plant samples will vary according the species and colony extension, although
c. 4 × 4 cm samples are very common. However, some species grow intermingled (mainly tiny liver-
worts) and can be very difficult to identify in the field using just a hand lens. Sporophytes and perianths
are useful for identifications and should be searched for and collected along with the gametophytes.
Useful tools for collecting bryophytes in the field include a 10–20× hand lens, different-sized
paper bags, plastic bags (used for transporting fresh samples to the laboratory), a penknife, a chisel,
waterproof markers, a field notebook, and a GPS for recording the geographic coordinates of the
FIGURE 7.5 Polypodiidae representatives. (A) Asplenium, Aspleniaceae. (B) Sticherus, Gleicheniaceae.
(C) The film fern Hymenophyllum, Hymenophyllaceae. (D) The deer tongue fern Elaphoglossum,
Dryopteridaceae. (E) Pleopeltis, Polypodiaceae. (F) The tree fern Cyathea, Cyatheaceae. (G) Anemia,
Anemiaceae.
collection sites. Additionally, general data about the collect site should be noted, including the
locality, collector names, the date, elevations, vegetation type, conservation status, substrate type
(microhabitat), and the traits of the specimens (color, growth form, fertility, etc.). All this informa-
tion should be carefully recorded in the field on the paper bags and/or in a field notebook.
Upon returning to the laboratory, the paper bags should be opened, and the samples air-dried
(checking them every day) as quickly as possible to avoid fungal growth. If necessary, very moist
samples can be dried using an electric (or light-bulb) oven at low temperatures (40–60°C). Fresh
material (maintained in plastic bags) should be stored at approximately 10°C until examined.
Liverworts containing oil bodies should be studied within just a few days after collection, as those
oil bodies disappear quite rapidly. The oil bodies should be measured, counted, and described
before they vanish.
Dried plants can be stored in separate paper packets (with their respective herbarium specimen
labels). Very tiny plants, sporophytes, or other fertile structures should be placed in mini-packets
together with the main envelope. An A4 sheet of paper can be folded into a standard envelope
(measuring about 11 × 15 cm) for preserving bryophyte specimens.
Dissecting and compound microscopes are useful for studying bryophytes in detail in the labo-
ratory. The dry or moist plants can first be analyzed under a dissecting microscope to determine
their growth form, leaf arrangements, reproductive structures, and all likely informative characters.
Small pieces of plants (gametophyte and sporophyte, if present) may be separated using micro-
forceps and very thin needles and placed in a drop of water on a glass slide. Still under the dissect-
ing microscope, some leaves can be detached from the stem and thalloid plants can be sectioned
(to be viewed under a coverslip). Many additional characteristics can be assessed under a compound
microscope by observing prepared slides, such as cell shapes, the numbers of cells, leaf borders,
costae, and teeth on the gametophyte leaves; sporophyte details such as the peristome and stomata
should also be observed.
Local floras and specific revisions are very informative for taxonomic determinations. Contacts
with bryophyte taxonomists specializing in specific groups will increase the reliability of speci-
men determinations. Taxonomic determinations are very important for researchers examining the
chemical compounds produced by bryophyte species and must be very precise to ensure the quality
of those studies.
7.3.2 Pteridophytes
A number of factors must be taken into consideration when collecting ferns and fern allies
and depositing their vouchers in a herbarium. A practical guide concerning that subject is pre-
sented here; for more details and information refer to Fidalgo and Bononi (1989) and Peixoto
and Maia (2013).
7.3.2.1 How Big is the Plant?

This may seem an obvious point, but if you are preparing an exsiccate, the plant must fit on an her-
barium sheet. Therefore, the size of the plant matters! There are huge differences in fern shapes and
sizes. Some film ferns may be just a few centimeters long, while some tree ferns may reach up to
3 m or more. How does one proceed in such cases?
When the plants are small, the best is to collect as many specimens as needed to fill the herbar-
ium sheet. When the plant is larger than the sheet, you may fold the species into a “N” or “V” shape
to better fit the sheet. However, be sure to leave the taxonomically important portions easily visible
(such as sori). When the plants are much too big for a single sheet (such as tree ferns), the fronds
may be cut into pieces that each fit on an herbarium sheet. In those cases, the base of the petiole,
with the indument, a pair of basal pinnae, a pair of median pinnae, and the frond apex will compose
the exsiccate. Additionally, you should note the intact frond size (length and width) and estimate
the size of the rhizome. This information will be included as additional data on the exsiccate label.
7.3.2.2 What to Collect

The more information you provide the better. Therefore, the best is to collect the whole plant – with
rhizome and fronds – providing more subsidies for accurate identifications. Since some groups
of ferns can easily be identified even when sterile, whereas others are almost impossible, the best
option is to collect fertile specimens. Among ferns they can be monomorphic (with identical sterile
and fertile fronds), or dimorphic (when only a portion of the frond is fertile), or holodimorphic (with
the fertile frond being distinct from the sterile frond). In the latter case, you should collect both
sterile and fertile fronds.
7.3.2.3 How Should the Collected Material be Processed?

Once you have properly collected the plant(s) of interest, the herborization process starts by press-
ing the plants and then drying them. The recommendation is to proceed with the herborization as
soon as possible to avoid plant dehydration and shriveling. The usual way to do that is to place the
plants between sheets of newspaper. To avoid any confusion regarding the origins of the specimens
numbering the newspaper sheets with the respective collection numbers of the plants is important.
The next step is to intercalate the newspaper sheets containing the plants with cardboard, and then
FIGURE 7.6 Example of a herbarium label properly filled out with the necessary information.
put them all in a press. The press is then tied tightly with rope and placed into a drying oven. The
ideal temperature for drying plants is approximately 60°C; 2 days are usually enough to completely
dry them (although some rhizomes may be thicker and require more time). To avoid plant carboniza-
tion, check the plants regularly to determine if they are sufficiently dry. After drying, the next step
is preparing the exsicate. Different herbaria may have different techniques for fixing the plants to
the final herbarium sheets, but the two main principles are: make sure the plant is well-attached to
the sheet, and place a herbarium label on your sample.
7.3.2.4 What Information Goes on the Herbarium Label?

The format and layout of herbarium labels (Figure 7.6) may vary from one institution to the next,
but the key data still need to be provided. Recording complete information about the plant origin
on the label is very important. Those data consist of, location, collection date, name of the collector
(followed by his/her collection number), and GPS coordinates (if available). Additional notes about
the substrate (e.g. terricolous, rupicolous, or corticolous) should be included, as well as the frond
and stipe dimensions (in the case of tree ferns). The herbarium label must contain the herbarium
number (the ID number of the plant), the name of person who determined the plant’s name, and the
date of that determination.
7.3.2.5 How to Assign Names to Ferns and Fern Allies

There are many ways to identify a plant, but one of the most used methods is the consulting experts
is strongly recommended, especially when working with potential medicinal plants. The taxonomy
of ferns and fern allies can be very intricate, and species identifications may be inaccurate when
performed by nonspecialists. Therefore the collection of good and complete specimens is very
important, to make complete field notes, correctly fill out the herbarium label – and always consult
an expert.
7.4 CHEMICAL COMPOUNDS IN BRYOPHYTES

Many different classes of chemical compounds, including terpenoids, steroids, alkaloids, flavo-
noids, acetogenins, and aromatic compounds have been described in different species of bryophytes
throughout the world (Asakawa et al., 2013a). There have only been rare investigations in Brazil
focusing on the biological activities of native bryophytes (Pinheiro et al., 1989). Herein, we discuss
the potential medicinal uses of some taxa based on previous assays with specimens of the same (or
related) taxa of Brazilian bryophytes.
Large varieties of chemical compounds are found in liverworts, mosses, and hornworts,
although liverworts stand out in terms of the numbers of new and different compounds identi-
fied. Because of the essential oils stored in the oil bodies of liverworts, the chemical nature
of that bryophyte group is very complex (Asakawa et al., 2013a). Mono- and sesquiterpenoids
are rare in mosses and hornworts, but di- and triterpenoids have been isolated from cer-
tain mosses. Only ca. 5% of the total of known bryophytes worldwide have yet been studied
chemically. Although liverworts are a less speciose group than mosses, new terpenoids and
phenolic compounds with interesting biological activities are regularly isolated from them
(Asakawa, 2007).
7.5 CHEMICAL COMPOUNDS IN MARCHANTIOPHYTA

Over 3,000 compounds have been found in Marchantiophyta, including more than 800 terpe-
noids (excluding triterpenoids and tetraterpenoids), and 300 aromatic compounds (not includ-
ing flavonoids) (Asakawa et al., 2013a). Most of the compounds isolated from, or detected
in, liverworts are lipophilic terpenoids (mono-, sesqui-, and diterpenoids) and aromatic
compounds (Asakawa, 2007; Ludwiczuk and Asakawa, 2010, 2015). Approximately 80% of
the sesqui- and diterpenoids found in liverworts, noteworthy, enantiomers of those found in
higher plants (Asakawa, 2007), but pinguisane-type sesquiterpenoid compounds that have not
been found in any other organisms have been detected in Marchantiophyta, especially in the
Lejeuneaceae, Trichocoleaceae, Ptilidiaceae, Porellaceae, and Aneuraceae families(Ludwiczuk
and Asakawa, 2008).
7.5.1 Terpenoids
Monoterpenoids, sesquiterpenoids, diterpenoids, triterpenoids, and steroids have been described
from different species of bryophytes (Asakawa et al., 2013a). Terpenoids (Figures 7.7 and 7.8) are
found in the three different groups of bryophytes, and liverworts contain the most diverse classes of
diterpenoids and sesquiterpenoids (Andersen et al., 1977; Asakawa, 2001; Asakawa et al., 2013a).
Some Brazilian species of liverworts appear as very promising sources of large diversities of ter-
penoids. Among this group in Brazil, the liverwort family Plagiochilaceae stands out with at least
seven species containing identified terpenoids.
The essential oils of Plagiochila bifaria and Plagiochila stricta are rich in different mono-
terpenoids, including terpinolene (1) and β-phellandrene (3), and allo-ocimene (4), and
neo-allo-ocimene (5) (Figueiredo et al., 2005). Extracts of Plagiochila rutilans, a species with
a peppermint-like odor, contained a variety of monoterpenoids, including α-terpinene (6),
terpinolene (1), limonene (7), p-cymene (8), β-phellandrene (3), p-cymen-8-ol (9), pulegone (10),
3,7-dimethyl-2,6-octadien-1,6-olide (11), menthone (12), isomenthone (13), sabinene (15), and
β-pinene (17) (Rycroft and Cole, 2001).
Reboulia hemisphaerica (Aytoniaceae) is very rich in sesquiterpenoids (ca. 41 known com-
pounds; Table 7.1), especially 1,(10) 8-aristoladiene (= Caespitene) (18) (Toyota et al., 1999). (−)-β-
Barbatene (19), another sesquiterpenoid, is commonly found in leafy liverworts, but is also present
in the thallose R. hemisphaerica and Dumortiera hirsuta (Bardón et al., 1999a; Warmers and,
König, 1999). The liverwort R. hemisphaerica is a rich source of many sesquiterpenoids, such as
a gymnomitrane, gymnomitr-3(15)-en-9-one (20), gymnomitrol (21), and (+)-gymnomitr-3(15)-en-
4a-ol (22) (Ludwiczuk and Asakawa, 2008; Toyota et al., 1999).
FIGURE 7.7 Terpenoids reported in bryophytes.
Diterpenoids are also common in liverworts. Odontoschisma denudatum (Cephaloziaceae) pro-

duces the dolabellanes acetoxyodontoschismenol (23) and acetoxyodontoschismenetriol (24) as
major components, along with other denudatenone diterpenoids (Asakawa et al., 2013a; Hashimoto
et al., 1998a, 1998b). Additionally, the fusicoccane diterpenoid fusicorrugatol (25) has been isolated
from Plagiochila corrugata (Tori et al., 1994).
Steroids such as stigmast-4-en-3-one (26) and sitost-4-en-3-one (27) have been isolated
from the thallose liverwort Ricciocarpos natans, representing the first isolation of a steroid
ketone from a bryophyte (Asakawa et al., 2013a; Yoshida et al., 1997). Stigmast-4-en-3-one and
stigmast-4-en-3,6-dione (28) were also isolated from the leafy liverwort Frullania brasiliensis
(Bardón et al., 2002).
Triterpenoids such as hopanoids including diploptene (= hop-22(29)-ene) (30), diplopterol
(= hopan-22-ol) (31), and α-zeorin (= hopan-6α,22-diol) (32) are very common in liverworts of
the orders Jungermanniales, Metzgeriales, and Marchantiales. The genera Asterella, Reboulia,
and Plagiochasma (Aytoniaceae) stand out as rich sources of hopanoids. α-Zeorin, for exam-
ple, has been found in Reboulia hemisphaerica, Plagiochasma rupestre, and F. brasiliensis
(Wei et al., 1995; Bardón et al., 1999; Bardón et al., 2002).
FIGURE 7.8 Terpenoids and steroids reported in bryophytes.
7.5.2 Aromatic Compounds
Marthantiophyta exhibits a wide diversity of bis-bibenzyls (Figure 7.9), mostly among
Jungermannniales, Marthantiales, and Metzgeliares. Several Marchantia species are rich
sources of bis-bibenzyls; dimeric bis-bibenzyls are significant components of Riccardia spe-
cies; and Radula species are rich in bibenzyls and prenyl bibenzyls. Corsinia coriandrina
is unique in producing nitrogen- and sulfur-containing compounds (Asakawa et al., 2013a).
Plagiochila diversifolia produces three prebibenzyls: longispinone A (36), longispinone B (37),
and longispinol (38) (Heinrichs et al., 2000). Prelunularin (39) has been recorded in the thal-
lose liverworts Marchantia polymorpha and R. natans (Kunz and Becker, 1994). Lunularin
(40) and lunularic acid (41) have been identified in different thallose liverworts, including
Lunularia cruciata, D. hirsuta, M. polymorpha, and R. natans (Asakawa et al., 1996; Kunz and
Becker, 1994; Lu et al., 2006).
TABLE 7.1
Examples of Brazilian Bryophyte Species with Chemical Compounds Recorded in the
Literature (Asakawa et al., 2013a and References Therein).
Number of Chemical Compounds

Bryophyte Steroids and
Group/Family Species Aromatic Terpenoids Flavonoids Brazilian Biomes
Marchantiophyta
Acrobolbaceae Lethocolea glossophylla 5 – – AtF
Adelanthaceae Adelanthus decipiens 10 – 1 AtF
Aneuraceae Aneura pinguis – 4 – AtF, Pan
Riccardia multifida 6 – – AtF
Aytoniaceae Asterella venosa – 7 – AtF, Sav
Corsinia coriandrina 10 4 – AtF
Isotachis aubertii 5 – 2 AtF
Plagiochasma rupestre 4 5 – AtF, Pam
Reboulia hemisphaerica 5 42 – AtF
Balantiopsidaceae Isotachis aubertii – 4 – AtF
Cephaloziaceae Odontoschisma – 23 – AmF, AtF, Sav
denudatum
Corsiniaceae Corsinia coriandrina – – – AtF
Dumortieraceae Dumortiera hirsuta 9 45 5 AmF, Sav, AtF, Pan
Frullaniaceae Frullania arecae – – 1 AtF, Sav
Frullania brasiliensis 1 10 – AtF, Sav
Frullania serrata – 7 – AtF
Jungermanniaceae Anastrophyllum auritum – 18 – AtF
Lejeuneaceae Bryopteris filicina 1 20 – AmF, Sav, AtF, Pan
Cheilolejeunea trifaria – 1 – AmF, Sav, AtF, Pan
Lejeunea flava – 4 – AtF, AmF, Caa, Sav,
Pam, Pan
Marchesinia brachiata 2 2 1 AmF, Sav, AtF
Omphalanthus filiformis 1 5 – AmF, AtF, Pan
Lepidoziaceae Bazzania nitida – 5 – AtF
Lophocoleaceae Lophocolea bidentata – 3 – AmF, AtF, Sav
Pelliaceae Noteroclada confluens – 6 – AtF, Sav
Lunulariaceae Lunularia cruciata 2 9 2 AtF
Marchantiaceae Marchantia chenopoda 2 1 – AmF, Sav, AtF, Pan
Marchantia paleacea 9 6 4 AtF
Marchantia polymorpha 29 18 14 AtF
Metzgeriaceae Metzgeria conjugata – 1 – AtF
Metzgeria furcata – 6 – AmF, Sav, AtF
Pallaviciniaceae Symphyogyna brasiliensis – 13 – AmF, AtF, Sav
Symphyogyna podophylla – 6 – AtF
Plagiochila corrugata – 1 – AmF, Sav, AtF
Plagiochila cristata 1 13 – AmF, AtF
Plagiochila diversifolia 10 7 4 AtF
Plagiochila gymnocalycina 1 – – AtF
Plagiochila rutilans 7 15 1 AmF, Sav, AtF
Plagiochila stricta 9 45 2 AtF
(Continued)

Examples of Brazilian Bryophyte Species with Chemical Compounds Recorded in the
Literature (Asakawa et al., 2013a and References Therein).
Number of Chemical Compounds
Bryophyte Steroids and
Group/Family Species Aromatic Terpenoids Flavonoids Brazilian Biomes
Radulaceae Radula nudicaulis 4 9 4 AtF
Ricciaceae Ricciocarpos natans 3 5 8 AmF, AtF, Pan
Bryophyta
Aulacomniaceae Aulacomnium palustre – – 4 AtF
Hedwigiaceae Hedwigia ciliata – – 1 AtF
Polytrichaceae Polytrichum commune 5 1 2 AmF, Sav, AtF
Pottiaceae Eucladium verticillatum 4 – – AtF, Sav
Brazilian Biomes: AtF, Atlantic Rainforest; AmF, Amazon Rainforest; Caa, Deciduous Caatinga; Sav, Cerrado Savanna;
Pan, Pantanal Wetlands; Pam, Pampa Grasslands (Flora do Brasil, 2020)
FIGURE 7.9 Aromatic compounds reported in bryophytes.

FIGURE 7.10 Flavonoids (47–51) and anthocyanins (52 and 53) reported in bryophytes.
7.5.3 Flavonoids and Anthocyanidins

All main orders of Marthantiophyta (Jungermanniales, Metzgeriales, and Marchantiales) contain
flavonoids (Figure 7.10), although they are generally underrepresented in liverworts. Flavones are
more common than flavanones, and luteolin (47) and apigenin (48) are the most abundant in the
Marchantiophyta, including M. polymorpha (Adam and Becker, 1994). Luteolin-7-O-glucoside and
quercetin (49) were found in L. cruciata (Jackovic et al., 2008). A new compound, an anthocyani-
din named riccionidin A (52) and the dimer riccionidin B (53) were identified in the cell walls of
R. natans grown under axenic conditions and these were also detected in the liverwort M. polymorpha
(Kunz et al., 1994).
7.5.4 Acetogenins and Lipids

Several liverworts, such as P. rutilans, produce the acetogenins 1-octen-3-ol (54) and/or
1-octen-3-yl acetate (55) (Asakawa et al., 2013a; Rycroft and Cole, 2001). In terms of the
fatty acids found in liverworts, M. polymorpha cells grown under controlled conditions were
found to contain linolenic (56), arachidonic (57), and eicosapentaenoic acids (58) (Saruwatari
et al., 1999). Example of acetogenins and fatty acids reported in bryophytes is shown in
Figure 7.11.
FIGURE 7.11 Acetogenins and fatty acids present in bryophytes.
7.6 CHEMICAL COMPOUNDS IN BRYOPHYTA

Although the many known species of mosses other than liverworts, only a small number of them
have been chemically analyzed (Asakawa et al., 2013a). Only four monoterpene hydrocarbons
(α-phellandrene (2), β-phellandrene (3), Δ3-carene (14), and α-pinene (16)) have been detected
in Sphagnum species; and only four diterpenoids, ent-16β-hydroxykaurane (33), momilactones
A (34) and B (35), and 18 monoterpenoids, five trinorsesquiterpenoids, 72 sesquiterpenoids,
ten diterpenoids, and nine triterpenoids have been isolated from, or detected in, the entire Bryophyta
group. Moss species such as Polytrichum commune may contain several compounds, includ-
ing the steroid sitosterol (29), the aromatic compounds 4-hydroxybenzoic acid (42), 3-methoxy-
4-hydroxybenzoic acid (43), 5-hydroxy-6-methoxycoumarin-7-O-β-glucopyranoside (44), methyl
indoline-6-carboxylate (45), 5-hydroxy-7-methoxychromone (46), and the flavonoids communin
A (50) and communin B (51) (Asakawa et al., 2013a).
7.7 CHEMICAL COMPOUNDS IN HORNWORTS

Although only few species of hornworts have been chemically analyzed, the chemical constitu-
ents of Anthocerotophyta are apparently very distinct from Marchantiophyta and Bryophyta.
Several monoterpenoids, sesquiterpenes, sterols, aromatic compounds, and alkaloids have been
detected in, or isolated from, certain hornworts. However, none of those species seem to occur
in Brazilian ecosystems. For instance, Anthoceros agrestis contains glutamic acid amides,
4-hydroxybenzoic, protocatechuic, vanillic, isoferulic, and coumaric acids, and the new alkaloid
anthocerodiazonin (59; Figure 7.12) (which contains a nine-membered ring system) (Asakawa
et al., 2013a; Trennheuser et al., 1994).
It is important to stress that although the majority of the above cited species occur in Brazilian
ecosystems (Table 7.1), many of the studies reported here were carried out using specimens from
other localities around the world. Since the chemical compounds produced by plants can be influ-
enced by environmental conditions, the sites where the plants were collected and the time of year
when they were harvested will be important variables to be considered in biochemical studies. That
observation reinforces the importance of collecting detailed field data and making precise speci-
men determinations. However, the fact that many plants are cultured in vitro before analyzing their
extracts may mitigate field effects, and result in similar determinations of the classes of chemical
compounds among the taxa studied.
Brazilian liverwort species such as R. hemisphaerica (Aytoniaceae), D. hirsuta
(Dumortieraceae), M. polymorpha (Marchantiaceae), P. bifaria, and P. stricta (Plagiochilaceae)
FIGURE 7.12 A new alkaloid present in the hornwort Anthoceros agrestis.
stand out in terms of the numbers of different chemicals they produce, including aromatic com-
pounds, flavonoids, and terpenoids (Table 7.1). Those species are mostly present in the Atlantic
rain forest, although several also occur in Amazon rain forest, Cerrado (Brazilian savanna),
and Pantanal sites.
Since less than 1% of the total bryophyte diversity found in Brazil is represented in
Table 7.1, the urgency of studies examining bryophyte taxa chemical content throughout Brazil
is highlighted. Compared to other areas around the world, especially the temperate zones,
Brazil has a huge potential for harboring species containing new and interesting medicinal
compounds. Many of those bryophyte species should be found in the Atlantic rain forest –
the Brazilian biome that offers the most diverse combination of microclimatic conditions and
habitat heterogeneity.
7.8 CHEMICAL COMPOUNDS IN PTERIDOPHYTES

Despite the wide array of secondary metabolites present in ferns and fern allies, little information
is actually available concerning those phytochemicals and their potential pharmacological appli-
cations (Cao et al., 2017). Even with our current limited knowledge regarding the subject, a wide
range of alkaloids, flavonoids, polyphenols, terpenoids, and steroids has already been reported in
ferns and fern allies (Cao et al., 2017; Dong et al., 2012; Ho et al., 2011; Socolsky et al., 2007, 2012;
Xia et al., 2014). Additionally, differences in the structures of those compounds from those found in
angiosperms indicate a rich, unexplored, neglected, and potential field for pharmaceutical investiga-
tions (Cao et al., 2017).
7.8.1 Terpenoids
Terpenoids (Figures 7.13–7.16) are the largest chemical group found in ferns and fern allies. This
class includes triterpenoids, diterpenoids, and sesquiterpenoids (Ho et al., 2011). Triterpenoids such
as the filicenes are typical secondary metabolic constituents of those plants (Ho et al., 2011; Nakane
et al., 2002; Reddy et al., 2001). Many compounds have been isolated from club-moss species (Zhou
et al., 2003a, 2003b, 2004), especially the Serratenes group of naturally occurring pentacyclic trit-
erpenoids with seven tertiary methyl groups (Ho et al., 2011); many of those same compounds have
been recorded in conifers (Tanaka et al., 2004; Wittayalai et al., 2012). Diterpenoids have mainly
been found in Pteridaceae, including ent-kaurane, ent-atisane, and ent-pimarane types (Alonso-
Amelot, 2002; Cao et al., 2017; Ho et al., 2011). Sesquiterpenoids are mainly represented in ferns
and fern allies by the indane and cadinane groups (Ho et al., 2011). Pterosines are a large group
of sesquiterpenes with an indane skeleton (Cao et al., 2017), many are characteristic constituents
FIGURE 7.13 Basic structures of some serratene-type triterpenoids described in pteridophytes from the
family Lycopodiaceae.
of Bracken ferns (including peterosine B) and are well-represented in that group (Pteridium spp.)
(Hikino et al., 1970, 1971, 1972).
7.8.2 Phenolics
Phenolic compounds (Figure 7.17) are represented in ferns and fern allies mainly by chloro-
genic, caffeic, ferulic, hydroxybenzoic, hydroxycinnamic, and vanillic acids. Extracts of
Pteris (Pteridaceae) showed the presence of kaempferol 3-O-l-rhamnopyranoside-7-O-[-d-apio-
FIGURE 7.14 Basic structures of some ent-kaurane-type diterpenoids from the genus Pteris.
FIGURE 7.15 Basic structures of some atisane-type diterpenoids from the genus Pteris.
FIGURE 7.16 Examples of pterosins found in the genus Pteris (adapted from Cao et al., 2017).
FIGURE 7.17 Examples of phenolic compounds in Pteris.

furanosyl-(1-2)-O-d-glucopyranoside], 7-O-caffeoylhydroxymaltol 3-O-d-glucopyranoside, his-

pidin 4-O-d-glucopyranoside, kaempferol 3-O-l-rhamnopyranoside-7-O-d-glucopyranoside,
caffeic acid, 5-caffeoylquinic acid, 3,5-dicaffeoylquinic acid, and 4,5-di-caffeoylquinic acid
(Chen et al., 2007).
7.8.3 Flavonoids
Numerous flavonoid compounds (Figure 7.18) with medicinal properties have been
identified in ferns and fern allies (Ho et al., 2011). Flavonoids can be divided into several
classes, including anthocyanins, flavones, flavonols, flavanones, dihydroflavonols, chalcones,
aurones, flavonons, flavan, proanthocyanidins, isoflavonoids, and bioflavonoids (Iwashina,
2000). Some Selaginella-derived flavonoids stand out in terms of their potential pharmaco-
logical uses, such as amentoflavone, hinokiflavone, heveaflavone, neocryptomerin, pulvi-
natabiflavone, and 7″-O-methylamentoflavone (Cheng et al., 2008; Zhang et al., 2012a, 2012b,
2012c, 2012d).
7.8.4 Alkaloids
Alkaloids (Figure 7.19) are well-represented in fern and fern allies, especially in club-mosses
(Lycopodiaceae). The groups lycopodine, lycodine, and fawcettimine standout, especially
huperzine, a lycodine with a quinolizidine skeleton (Ho et al., 2011). Other alkaloid compounds
have been isolated from club-mosses, including clavolonine, flabelliformine, gnidioidine,
lycocarinatine, lycodoline, miyoshianine, and phlegmariurine (Thorroad et al., 2014;
Tong et al., 2003).
FIGURE 7.18 Examples of involvenflavones from Selaginella (adapted from Cao et al., 2017).
FIGURE 7.19 Examples of alkaloids from club-mosses (adapted from Cao et al., 2017).
7.9 BIOLOGICALLY ACTIVE COMPOUNDS AND

THEIR POTENTIAL MEDICINAL USES
7.9.1 Bryophytes
Chemical compounds isolated from bryophytes, especially from liverworts (Marchantiophyta),
have unique characteristics. Liverworts produce a great variety of lipophilic terpenoids, aromatic
compounds, and acetogenins that are responsible for fragrances, bitterness, pungency, and sweet-
ness, as well as allergenic responses, contact dermatitis, cytotoxic effects, antimicrobial, anti-
fungal, calmodulin inhibitory, cardiotonic, larvicidal, 5-lipoxygenase inhibitory, molluscicidal,
muscle relaxant, neurotrophic, plant growth regulatory, superoxide release inhibitory, throm-
boxane synthase inhibitory, and vasopressin antagonist activities (Asakawa, 2007; Asakawa
et al., 2013a).
When crushed, liverworts emit a very strong odor. Lipophilic terpenoids (such as
monoterpenoids) and aromatic compounds held in oil bodies are responsible for the intense sweet-
woody, turpentine, sweet-mossy, fungal-like, carrot-like, mushroom-like, or seaweed-like odors of
liverworts. Almost all liverworts that smell of mushrooms contain 1-octen-3-ol and the correspond-
ing acetate (Asakawa et al., 2013a). Allergenic contact dermatitis caused by some Frullania species
is associated with sesquiterpenes with α-methylene-γ-lactone functionality (Asakawa et al., 2013a).
The essential oils of several liverworts exhibit anti-bacterial, fungal, and viral activities
(Asakawa et al., 2013), including against Escherichia coli, Staphylococcus aureus (Lorimer
and Perry, 1994), Cladosporium cucumerinum (Scher et al., 2004), Candida albicans
(Wu et al., 2008, 2009, 2010), and he H1N1 and H5N1 influenza A virus (Iwai et al., 2011). A
Brazilian study (Pinheiro et al., 1989) with ten liverworts and 15 moss species found that 40%
of them produced chemical compounds that inhibited the growth of several bacterial strains.
Extracts of Calymperes lonchophyllum moss and an unidentified Bazzania species significantly
inhibited E. coli growth; and Leucomium lignicola extracts were active against S. aureus, Proteus
vulgaris, Klebsiella pneumoniae, and Edwardsiela tarda. Dehydrocostus lactone (from the thal-
lose liverwort Targionia lorbeeriana) likewise showed antifungal activity against C. albicans,
and insecticidal activity against Aedes aegypti (Neves et al., 1999).
Several eudesmanolides, germacranolides, and guaianolides isolated from liverworts exhibit
cytotoxic activity against KB nasopharyngeal and P-388 lymphocytic leukemia cells. Some
Marchantia species produce marchantins A, B, D, perrottetin F, and paleatin B, which show DNA
polymerase β inhibitory activity (IC50 range 14.4–97.5 µM), cytotoxicity against KB cells (IC50 range
3.7–20 µM), and anti-HIV-1 activity (IC50 range 5.3–23.7 mg/cm3) (Asakawa et al., 2008, 2009).
Lunularic acid appears to have ABA-like activity in vascular plants, inhibiting the germination
and growth of plants such as Lepidium sativum and Lactuca sativa. Yoshikawa et al. (2002) hypoth-
esized that vascular plants altered their endogenous growth regulator from lunularic acid to abscisic
acid during their evolution.
The in vitro cultivation of bryophytes appears to be the most appropriate route for large biomass
productions and the isolation of compounds showing interesting biological activities (Sabovljevic
et al., 2009).
7.9.2 Pteridophytes
The importance of ferns and fern allies to pharmacological and medical applications was noted
by the Greek botanist Theophrastus (ca. 372–287 B.C.) and by the father of pharmacognosy
Dioscorides (ca. 50 A.D.). Both the bracken fern (Pteridium spp.) and the male fern (Dryopteris
filix-mas) were mentioned in Dioscorides’ magna opus “De materia medica” (Banerjee and
Sen, 1980).
In spite of the historical importance and wide use of plants in traditional medicines (Maciel
et al., 2002), the numbers of medicinal ferns and lycophytes encountered in ethnobotanical
studies are not consistent with the therapeutic potentials often attributed to them (Reinaldo
et al., 2015).
The need for new drugs and reports of therapeutic effectiveness of compounds derived from
ferns and ferns allies present open opportunities for pharmacological research (Cao et al., 2017).
Many studies have demonstrated the pharmacological potentials of ferns and lycophytes due to the
presence of metabolites with antioxidant, anti-inflammatory, analgesic, antimutagenic, immuno-
modulatory, and neuromodulatory activities (Goldberg et al., 1975; Keller and Prance, 2015; Lee
and Lin, 1988; Nonato et al., 2009; Tomšík 2014; Wu et al., 2005).
Investigation of the potential pharmaceutical uses of Brazilian ferns and fern allies is still incipi-
ent. Santos et al. (2010) estimated that only approximately 4.7% of the Brazilian pteridoflora has
been examined in that light, even though many experiments with pteridophytes have shown their
efficacy in treating human ailments.
Antioxidant activities have been reported in many genera that (also) occur in Brazil, such as
Hypolepis, Pteridium, Dryopteris, Polystichum, Dicranopteris, Lycopodium l.s., Osmunda,
Adiantum, Pteris, Lygodium, Selaginella, and Thelypteris l.s. (Baskaran et al., 2018; Shin, 2010),
due to the presence of substances such as 2,2-diphenyl-1-picrylhydrazyl activity and 2,2′-azino-
bis-(3-ethyl benzthiazoline-6-sulphonie acid diammonium salt.
Anticancer activities have been reported in extracts of the genera Selaginella due to the actions
of lycopodine and biflavones such as amentoflavone and isocryptomerin (Silva et al., 1995); other
compounds with anticancer activities have been found in Acrostichum (Uddin et al., 1998) and the
naturalized genera Macrothelypteris (Liu et al., 2012). Antihyperglycemic and analgesic properties
were likewise observed in plants of the genera Christella and Adiantum (Paul et al., 2012; Sultana
et al., 2014; Tanzin et al., 2013).
Anti-inflammatory properties were reported in Selaginella (Dhiman, 1998), Ophioglossum,
Lygodium (Vasudeva, 1999), Christella (Gogoi, 2002), Pteris (Lee and Lin, 1988; Wu et al., 2005),
Dryopteris (Otsuka et al., 1972), Cyathea (Benjamin and Manickam, 2007; Madhukiran and Ganga,
2011), Blechnum (Nonato et al., 2009), and others. Antimicrobial activities were likewise reported
in Nephrolepis (Jimenez et al., 1979), Lygodium (Cambie and Ash, 1994), Marattia (de Boer et al.,
2005), Adiantum (Reddy et al., 2001), Equisetum (Joksic et al., 2003; Radulovic et al., 2006), and
others.
Therefore, that ferns and fern allies represent a huge unexplored group of plants with poten-
tial pharmaceutical and medical uses is undeniable, and even though very few studies have
examined the Brazilian pteridoflora, a number of bioactive compounds have already been
identified (Table 7.2). Additional studies have pointed out that extracts of Brazilian ferns and
fern allies contain antioxidant, anticancer, antiviral, anti-inflammatory, and other activities
(Table 7.3).
Table 7.4 summarizes some information about the Brazilian bryophytes and pteridophytes dis-
cussed in the chapter.
TABLE 7.2
Brazilian Species of Pteridophytes Containing Isolated Bioactive Compounds
Taxa Family Bioactive Compounds Reference
Adiantopsis flexuosa Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
aglycone,
7-O-glycosides of luteolin,
7-O-glycosides of chrysoeriol,
3-O-glycosides of kaempferol,
3-O-glycosides of quercetin
Adiantum Pteridaceae Adiantone, adiantoxide, Taylor (2003)
capillus-veneris* astragalin, β-sitosterol,
caffeic acids, caffeylgalactose,
caffeylglucose, campesterol,
carotenes, coumaric acids,
coumarylglucoses, diplopterol,
epoxyfilicane, fernadiene,
fernene, filicanes,
hopanone, hydroxyl-adiantone,
hydroxyl-cinnamic acid,
isoadiantone, isoquercetin,
kaempferols, lutein,
mutatoxanthin, naringin,
neoxanthin, nicotiflorin,
oleananes, populnin,
procyanidin, prodelphinidin,
quercetin, querciturone,
quinic acid, rhodoxanthin,
rutin, shikimic
acid,
violaxanthin, and zeaxanthin
Cyathea phalerata Cyatheaceae Kaempferol-3-neohesperidoside, Hort et al. (2008)
4-O-β-d-glucopyranosyl
caffeic acid,
4-O-β-d-glucopyranosyl
p-coumaric acid,
3,4-spyroglucopyranosyl
protocatechuic acid,
sitosterol
β- d-glucoside, β-sitosterol,
kaempferol, and vitexin
Doryopteris Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
Concolor aglycone, 7-O-glycosides of
luteolin,
7-O-glycosides of
chrysoeriol,
3-O-glycosides
of kaempferol,
and
(Continued)

Equisetum arvense* Equisetaceae Isoquercetin, Radulovic et al. (2006),
quercetin 3-O-glucoside, Mimica et al. (2008),
quercetin Milovanovic et al. (2007)
3-O-(6″-O-malonylglucoside),
5-O-caffeoyl mesotartaric
acid, monocaffeoyl meso-tartaris acid,
monocaffeoyl
meso-tartaris acid,
di-E-caffeoyl-meso-tartaric
acid, hexahydrofarnesyl acetone,
cis-geranyl scetone,
thymol, and trans-phytol
Lytoneuron Pteridaceae 7-O-Glycosides of apigenin, aglycone, Salatino and Prado (1998)
ornithopus 7-O-glycosides of luteolin,
3-O-glycosides
of kaempferol,
3-O-glycosides of kaempferol, and
Ormopteris Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
cymbiformis aglycone, 7-O-glycosides of
luteolin,
Ormopteris Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
gleichenioides aglycone, 7-O-glycosides
of luteolin,
Ormopteris pinnata Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
aglycone, 7-O-glycosides
of luteolin,
3–O-glycosides of kaempferol, and
Ormopteris riedelii Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
aglycone, 7-O-glycosides of luteolin,
Pityrogramma Pteridaceae 2′6′-Dihydroxy-4,4′- Star and Mabry (1971)
calomelanos dimethoxydihydrochalcone,
kaempferol 7-methyl ether,
apigenin 7-methyl ether
(Continued)

Psilotum nudum Psilotaceae Quercetin, kaempferol, Cambie and Ash (1994)
amentoflavone, hinokiflavone,
vicenin-2 psilotin,
3′-hydroxypsilotin
Pteridium aquilinum Desdendticiaceae p-Coumaric acid, Michael and Gillian (1984)
p-hydroxybenzoic acid,
caffeic acid, ferulic acid, vanillic acid,
kaempferol, quercetin, apigenin
Pteris altissima Pteridaceae 7-O-Glycosides of apigenin, aglycone, Salatino and Prado (1998)
Pteris angustata Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
aglycone,
Pteris decurrens Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
aglycone,
Pteris deflexa Pteridaceae 7-O-Glycosides of apigenin, aglycone, Salatino and Prado (1998)
Pteris denticulata Pteridaceae 7-O-Glycosides of apigenin, aglycone, Salatino and Prado (1998)
Pteris multifidi Pteridaceae Luteolin-7-O-glucoside, Hoang and Tran (2014), Lu et al.
16-hydroxy-kaurane-2-β-d-glucoside, (1999), Liu and Qin (2002),
luteolin, palmitic acid, Murakami and Machashi
apigenin4-O-α-l-rhamnoside, (1985), Shu et al. (2012),
quercetin, hyperin, Wang et al. (2010)
isoquercitrin, kaempferol, rutin,
apigenin-7-O-β-d-glucoside, and
pterosin
(Continued)

Pteris podophylla Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
aglycone,
Pteris propinqua Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
aglycone,
Pteris quadriaurita Pteridaceae 7-O-Gycosides of apigenin, aglycone, Salatino and Prado (1998)
7-O-glycosides of
luteolin, 7-O-glycosides of
chrysoeriol, 3-O-glycosides
of kaempferol, 3-O-glycosides of
kaempferol, and
Pteris splendens Pteridaceae 7-O-Glycosides of apigenin, Salatino and Prado (1998)
aglycone,
Pteris tripatita Pteridaceae α-Caryophyllene and octadecanoic Baskaran and Jeyachandran
acid (2010)
Pteris vitata* Pteridaceae Rutin, kaempferol monoglycoside, Salatino and Prado (1998),
kaempferol diglycoside, Sigh et al. (2008)
quercetin monoglycoside,
quercetin diglycoside
Salvinia molesta Salviniaceae Salviniside I, montbretol, Li et al. (2013)
5,6-dehydrosugiol,
7-methoxyrosmanol,
montbretyl 12-methyl ether,
11-hydroxysugiol,
sugiol, ferruginol,
7-hydroxyferruginol,
6,7-dehydroferruginol,
12-hydroxy simonellite, simonellite
Adiantum cuneatum Pteridaceae Filicene, filicenal Bresciani et al. (2003)
* Cultivated plants.
TABLE 7.3
Brazilian Species Pteridophytes with Potential Pharmacological Utility
Taxon Family Activity Reference
Adiantum radianum Pteridaceae Antioxidant Lai and Lim (2011)
Nephrolepis biserrata Nephrolepidaceae Antioxidant Lai and Lim (2011)
Microgramma vacciniifolia Polypodiaceae Antioxidant Peres et al. (2009)
Phlebodium decumanum Polypodiaceae Anticancer Chang et al. (2007)
Selaginella willdenowii* Selaginellaceae Anticancer Lee and Lin (1988)
Marsilea minuta Marsileaceae Anticancer Sarker et al. (2011)
Christella dentata Thelypteridaceae Antiviral Paul et al. (2012)
Asplenium nidus* Aspleniaceae Antiviral Chand et al. (2013), Singh (1999),
Santhosh et al. (2014)
Blechnum occidentale Blechnaceae Anti-inflamatory Nonato et al. (2009)
Adiantum caudatum Pteridaceae Antibiotic Banerjee and Sen (1980), Lakshmi and
Pullaiah (2006), Lakshmi et al. (2006),
Singh et al. (2008a)
Selaginella pallescens Selaginellaceae Antibiotic Haripriya et al. (2010), Rojas et al.
(1999)
Lygodium venustum Lygodiaceae Antibiotic Moraes-Braga et al. (2012)
* Cultivated plants.
TABLE 7.4
The Names of Plant Species Including Family and Common Names from Which Some
Natural Products Originated and Are Presented in This Chapter
Adelanthus decipiens (sin. Marsupidium brevifolium) Icacinaceae
Adiantopsis flexuosa Pteridaceae
Adiantum capillus-veneris (sin. Adiantum remyanum) Pteridaceae Common maidenhair fern, avenca
Adiantum caudatum (sin. Adiantum lyratum) Pteridaceae Common maidenhair fern, avenca
Adiantum cuneatum Pteridaceae Common maidenhair fern, avenca
Adiantum radianum Pteridaceae Common maidenhair fern, avenca
Anastrophyllum auritum (sin. Anastrophyllum Anastrophyllaceae
hintzeanum)
Aneura pinguis Aneuraceae
Anthoceros agrestis Anthocerotaceae
Asplenium nidus (sin. Neottopteris musaefolia) Aspleniaceae
Asterella venosa (sin. Fimbraria venosa) Solanaceae
Aulacomnium palustre (sin. Aulacomnium pygmaeum) Aulacomninaceae
Bazzania nitida (sin. Mastigobryum stephanii) Lepidoziaceae
Blechnum occidentale (sin. Blechnum rugosum) Blechnaceae
Bryopteris filicina (sin. Bryopteris brevis) Lejeuneaceae
Cheilolejeunea trifaria (sin. Euosmolejeunea robillardii) Lejeuneaceae
Christella dentata (sin. Cyclosorus dentatus var. Thelypteridaceae
violascens)
Corsinia coriandrina (sin. Riccia coriandrina) Corsiniaceae
Cyathea Cyatheaceae Tree fern, samambaiaçu
Cyathea phalerata (sin. Trichipteris phalerata) Cyatheaceae Tree fern, samambaiaçu
(Continued)

Dicranopteris Gleicheniaceae
Dicranopteris Pteridaceae
Doryopteris concolor (sin. Cheilanthes concolor) Pteridaceae
Dryopteris filix-mas (sin. Aspidium veselskii) Pteridaceae
Dumortiera hirsuta (sin. Marchantia hirsuta) Dumortieraceae
Elaphoglossum Dryopteridaceae Deer tongue fern
Equisetum Equisetaceae Horsetail, cavalinha
Equisetum arvense (sin. Equisetum calderi) Equisetaceae Horsetail, cavalinha
Equisetum giganteum (sin. Equisetum bolivianum) Equisetaceae Horsetail, cavalinha
Eucladium verticillatum (sin. Eucladium angustifolium) Pottiaceae
Frullania arecae (sin. Frullania crispistipula) Jubulaceae
Frullania brasiliensis Jubulaceae
Frullania serrata (sin. Frullania lacerata) Jubulaceae
Hedwigia ciliata (sin. Hedwigia macowaniana) Burseraceae
Hymenophyllum Hymenophyllaceae Film fern
Isotachis aubertii (sin. Isotachis rutenbergii) Balantiopsaceae
Lejeunea flava Lejeuneaceae
Lethocolea glossophylla Acrobolbaceae
Lophocolea bidentata (sin. Lophocolea setacea) Lophocoleaceae
Lunularia cruciata Lunulariaceae
Lygodium venustum (sin. Lygodium commutatum) Lygodiaceae
Macrothelypteris Thelypteridaceae
Macrothelypteris Thelypteridaceae
Marchantia chenopoda Marchantiaceae
Marchantia paleacea Marchantiaceae
Marchantia polymorpha Marchantiaceae
Marchesinia brachiata (sin. Marchesinia aquatica) Lejeuneaceae
Marsilea minuta (sin. Marsilea perrieriana) Marsileaceae Trevo de quatro folhas
Metzgeria conjugata Metzgeriaceae
Metzgeria furcata Metzgeriaceae
Microgramma vacciniifolia (sin. Lepicystis vacciniifolia) Microgramma
Nephrolepis biserrata (sin. Nephrolepis mollis) Nephrolepidaceae
Noteroclada confluens Pelliaceae
Odontoschisma denudatum (sin. Jungermannia denudata) Cephaloziaceae
Omphalanthus filiformis (sin. Jungermannia filiformis) Lejeuneaceae
Ormopteris gleichenioides (sin. Pellaea gleichenioides) Pteridaceae
Ormopteris cymbiformis (sin. Pellaea cymbiformis) Pteridaceae
Ormopteris pinnata (sin. Cassebeera pinnata) Pteridaceae
Ormopteris riedelii (sin. Pellaea riedelii) Pteridaceae
Pellaea mucronata (sin. Lytoneuron ornithopus) Pteridaceae
Phlebodium decumanum (sin. Phlebodium multiseriale) Polypodiaceae
Plagiochasma rupestre (sin. Aytonia rupestris) Aytoniaceae
Plagiochila bifaria (sin. Jungermannia bifaria) Plagiochilaceae
Plagiochila corrugata (sin. Jungermannia corrugata) Plagiochilaceae
Plagiochila cristata (sin. Plagiochila secundifolia) Plagiochilaceae
Plagiochila diversifolia Plagiochilaceae
Plagiochila gymnocalycina Plagiochilaceae
(Continued)

Plagiochila rutilans Plagiochilaceae
Plagiochila stricta Plagiochilaceae
Polytrichum commune (sin. Polytrichum leonii) Dryopteridaceae
Psilotum nudum (sin. Psilotum domingense) Psilotaceae
Pteridium aquilinum (sin. Pteris lanuginosa) Dennstaedtiaceae Samambaia-das-taperas
Pteris altissima (sin. Litobrochia grandis) Pteridaceae
Pteris angustata (sin. Litobrochia angustata) Pteridaceae
Pteris decurrens Pteridaceae
Pteris deflexa (sin. Pteris gaudichaudii) Pteridaceae
Pteris denticulata Pteridaceae
Pteris multifida (sin. Pycnodoria multifida) Pteridaceae
Pteris podophylla (sin. Pteris inflexa) Pteridaceae
Pteris propinqua (sin. Pteris hostmanniana) Pteridaceae
Pteris quadriaurita (sin. Pteris prolifera) Pteridaceae
Pteris splendens Pteridaceae
Pteris tripartita (sin. Litobrochia tripartita) Pteridaceae
Pteris vittata (sin. Pteris vittata f. cristata) Pteridaceae
Radula nudicaulis Radulaceae
Reboulia hemisphaerica Aytoniaceae
Riccardia multifida Aneuraceae
Ricciocarpos natans (sin. Riccia natans) Ricciaceae
Salvinia molesta (sin. Salvinia adnata) Salviniaceae
Selaginella pallescens (sin. Selaginella cuspidata) Selaginellaceae
Selaginella willdenowii (sin. Lycopodium willdenowii) Selaginellaceae
Symphyogyna brasiliensis (sin. Symphyogyna Pallaviciniaceae
lehmanniana)
Symphyogyna podophylla (sin. Jungermannia Pallaviciniaceae
podophylla)
Thelypteris Thelypteridaceae
7.10 FINAL REMARKS AND FUTURE PERSPECTIVES

That bryophytes and pteridophytes show great (but currently neglected) potential for pharmaceuti-
cal and medicinal uses is quite clear, especially Brazilian species. The taxa that have been sampled
in phytochemical studies have usually been harvested near research centers, so that our knowledge
of phytochemical diversity in different Brazilian biomes is still very limited. The native Brazilian
flora is also under constant threat, with biomes such as the Atlantic forest having less than 8% of its
original cover still intact.
The importance of floristic and ethnobotanical surveys cannot be over emphasized. Efforts must
be applied to the mapping and cataloging of mosses and their allies – an essential step in their con-
servation and for a better understanding of their diversity and distribution patterns. Investigations
must also focus on species used in traditional medicine, as well as other species within genera
known to contain bioactive compounds.
Modern tools should also be tested and employed in bioprospecting bryophytes and pterido-
phytes, especially with the onset of the genomic era – which permits to investigate gene clusters that
govern the biosynthesis of bioactive compounds (Zotchev et al., 2012) – to accelerate the processes
of discovery and cataloging chemical compounds within the Brazilian flora.
ACKNOWLEDGMENTS
ASMS thanks Fundação de Amparo à Pesquisa do Estado de Minas Gerais (FAPEMIG, APQ-
00395-14) and the Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq,
459764/2014-4). This study was also sponsored by the Coordenação de Aperfeiçoamento de Pessoal
de Nível Superior – Brasil (CAPES) – Finance Code 001 (88887.19244/2018-00).
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8 Chemical and Functional
Properties of Amazonian Fruits
Elaine Pessoa, Josilene Lima Serra,

Hervé Rogez, and Sylvain Darnet
Centre for Valorization of Amazonian Bioactive Compounds
& Federal University of Pará, Belém, Pará, Brazil
CONTENTS
8.1 Introduction............................................................................................................................ 174
8.2 Bertholletia Excelsa............................................................................................................... 187
8.2.1 Botanical Description................................................................................................ 187
8.2.2 Phytochemicals.......................................................................................................... 188
8.2.3 Mineral Content......................................................................................................... 188
8.2.4 Biological and Pharmacological Activities................................................................ 188
8.3 Genipa Americana.................................................................................................................. 189
8.3.2 Phytochemicals.......................................................................................................... 190
8.4 Myrciaria Dubia (Kunth) McVaugh...................................................................................... 192
8.4.2 Phytochemistry.......................................................................................................... 192
8.5 Spondias Mombin................................................................................................................... 194
8.5.2 Phytochemicals.......................................................................................................... 194
8.6 Astrocaryum Vulgare Mart. (Arecaceae)................................................................................ 195
8.6.1 Botanical Aspects and Occurrence............................................................................ 195
8.6.2 Phytochemistry of Tucumã Pulp and Seeds............................................................... 196
8.6.3 Biological and Pharmacological Activities of
Tucumã Pulp Oil........................................................................................................ 196
8.7 Bactris Gasipaes Kunth. (Arecaceae).................................................................................... 197
8.7.1 Botanic Aspects and Occurrence............................................................................... 197
8.7.2 Phytochemistry of Peach Palm Fruit and Seed Oil.................................................... 197
8.7.3 Biological and Pharmacological Activities of
Peach Palm Pulp Oil.................................................................................................. 197
8.8 Euterpe Oleracea Mart. (Arecaceae)..................................................................................... 198
8.8.1 Botanic Aspects and Occurrence............................................................................... 198
8.8.2 Phytochemistry of Açaí Fruits................................................................................... 198
173
8.8.3Biological and Pharmacological Activities of Açaí Fruits....................................... 199

8.8.3.1 Cardiovascular Effects.............................................................................. 199
8.8.3.2 Renal Failure Effects.................................................................................200
8.8.3.3 Effects on Lipids and Diabetes Metabolism.............................................200
8.8.3.4 Antitumor Effects......................................................................................200
8.8.3.5 Nontoxic Effects........................................................................................ 201
8.8.3.6 Other Effects............................................................................................. 201
8.9 Mauritia Flexuosa L.F. (Arecaceae).................................................................................... 201
8.9.1 Botanic Aspects and Occurrence.............................................................................. 201
8.9.2 Phytochemistry.........................................................................................................202
8.9.3 Biological and Pharmacological Activities..............................................................202
8.10 Final Remarks...................................................................................................................... 203
Acknowledgments�� 203
References�� 203
8.1 INTRODUCTION
As suggested by the last Food and Agriculture Organization (FAO) of the United Nations the
projections for the next 20 years will be the key to food security and nutrition on a worldwide
scale and a better use of biodiversity (FAO, 2017). The challenge is to develop more sustainable
cultures in the context of global climate change for a growing urban population with a changing
diet (Choudhury and Headey, 2017; FAO, 2018). Approximately 50% of all the calories consumed
are from only three cereal crops, and the genetic erosion of crops and the loss of livestock, forest
and aquatic sources are decreasing rapidly (FAO, 2018). The value of plant diversity is crucial.
The list of edible plants provides approximately 30,000 species, but only 30 of these plants form
the basis of human nutrition. The neglected and underutilized species should help to diversify
nutritional sources (FAO, 2017). The new sources should supply food or calories, but a significant
expectation is to increase the nutritional values in the diet, including a high content of vitamins,
minerals and other micronutrients (FAO, 2018). The food diversification should include sustain-
ability to preserve the biodiversity and the ecological foundations necessary to sustain life and
rural livelihoods (FAO, 2018). One agricultural system that is environmentally friendly is agro-
forestry (FAO, 2018). Agroforestry has many advantages, such as the maintenance of water and
soil quality, carbon sequestration, habitat provision for wild species and the facilitation of biologi-
cal pest control and pollination (FAO, 2018). Another crucial aspect is to increase food security
and to mitigate the over representation and use of annual plants in actual agricultural systems
(Meldrum et al., 2018). Perennial crops should represent a good alternative for human nutritional
diversification. Some neglected and underutilized types of plants with high nutritional value are
trees bearing fruit from tropical regions in Africa, Asia, Central and South America (Meldrum
et al., 2018).
The Amazonia rainforest, particularly the eastern region, is highly enriched in edible plants,
such as fruits, and the legacy of 4500 years of polyculture agroforestry by the pre-Columbian
population and biodiversity exploration (Maezumi et al., 2018). With the exception of two
fruits, Brazilian nuts and açaí, the Amazon fruits are neglected and underutilized; however,
over the last 30 years, scientific studies have highlighted the high nutritional value and medici-
nal properties of this legacy (Dutra et al., 2016; Neri-Numa et al., 2018; Oliveira et al., 2012).
This chapter presents the functional studies from a list of valuable fruits of the Amazonian
region. This list of valuable and underused fruit is very long, yet only few species have already
been functionally explored. For example, Theobroma grandiflorum (cupuaçu), Platonia insignis
(bacuri) and Endopleura uchi (uxi) are not included due to the lack of characterization and
functional studies. Theobroma cacao is excluded from this chapter, although it originated in
Properties of Amazonian Fruits 175
FIGURE 8.1 Fruit of four Amazonian trees: (A) Brazil nut (B. excelsa), (B) genipap (G. americana),
(C) camu-camu (M. dubia) and (D) yellow mombin (S. mombin).
the Amazon, because the functional studies focus on the byproduct, cocoa and chocolate, and the
research was performed outside of the native South American region. In the first part of this
chapter, we describe the functional research of the fruit of four trees, Spondias mombin,
Myrciaria dubia, Genipa americana and the well-known Brazilian nut (Bertholletia excelsa)
(Figure 8.1) (Table 8.1). In addition, palm trees are a primary element of the Amazonian land-
scape and an essential plant for the local population, and studies on four palm tree fruits are
described, Astrocaryum vulgare, Mauritia flexuosa, Bactris gasipaes and the well-known açaí
(Euterpe oleracea) (Figure 8.2) (Table 8.1) (Brokamp et al., 2011; Paniagua-Zambrana et al.,
2015; Sosnowska and Balslev, 2009).
The phytochemistry of each fruit is described with the emphasis on bioactive compounds
(Table 8.2), and an updated review shows the functional and medicinal properties using an
TABLE 8.1
Fruits from Amazonian Biome with Functional and Chemical Properties
Studied in This Chapter
Astrocaryum vulgare (sin. Astrocaryum tucumoides) Arecaceae Tucumã, awarra palm
Bactris gasipaes (sin. Bactris dahlgreniana) Arecaceae Pupunha, peach palm
Bertholletia excelsa (sin. Bertholletia nobilis) Lecythidaceae Castanha-do-Pará, Brazil nut
Euterpe oleracea (sin. Euterpe cuatrecasana) Arecaceae Açaí, Assai palm
Genipa americana (sin. Genipa venosa) Rubiaceae Jenipapo, genipap
Mauritia flexuosa (sin. Mauritia minor) Arecaceae Buriti, moriche palm
Myrciaria dubia (sin. Myrciaria paraensis) Myrtaceae Camu-camu
Spondias mombin (sin. Myrobalanus lutea) Anacardiaceae Cajá/Taperebá, yellow mombin
FIGURE 8.2 Fruit of four Amazonian palm trees: (A) açaí (E. oleracea), (B) tucumã (A. vulgare), (C) buriti
(M. flexuosa) and (D) peach palm (B. gasipaes).
TABLE 8.2
Bioactive Compounds in Amazonian Fruit
Fruit Main Bioactive Compound Bioactive Compounds
Bertholletia excelsa Selenium Nut (1-3)
Phenolic compounds: Gallic acid, gallocatechin protocatechuic acid,
catechin vanillic acid,
taxifolin, myricetin, ellagic acid, quercetin, tannins
Fatty acids: (C12:0), (C14:0), (C16:0), (C16:1), (C18:0), (C18:1),
(C18:2) and (C20:4)
Sterols: Campesterol, stigmasterol, sitosterol
Tocopherols: α-tocopherol, β-tocopherol, γ-tocopherol
Minerals: Se, Mg and P
Genipa americana Genipin (Blue pigment) Unripe fruit (4, 5)
Iridoids: Genipin, geniposide
Ripe fruit (4, 6, 7)
Iridoids: Gardoside, geniposidic acid, genipin-1-β-D-gentiobioside,
caffeoyl geniposidic acid, p-coumaroyl geniposidic acid,
feruloylgardoside, feruloylgenipin gentiobioside, genipacetal,
genipamide, genipaol, genameside
Phenolic compounds: Dicaffeoylquinic acids, 3,5-dicaffeoylquinic
acid, 4,5-dicaffeoylquinic
acid and 5-caffeoylquinic acid, quercetin, leucoanthocyanidins,
catechins, flavanones
Others: Anthraquinones, anthrone, coumarins, triterpenoids,
steroids
(Continued)

Myrciaria dubia Vitamin C Leaves (8)
Phenolic compounds: Flavonoids, tannins
Whole fruit (9-12)
Phenolic compounds: Gallic acid, ferulic acid, p-coumaric,
protocatechuic acid, catechin,
Myricetin, Rutin, narigerin
Carotenoids: β-carotene, all-trans-lutein, zeaxanthin. luteoxanthin,
violaxanthin, neoxanthin
Pulp (11-15)
Phenolic compounds: Ellagitannin B, kaempferol, quercetin
Organic acids: Ascorbic acid
Seed (16)
Betulinic acid
Seed coat (17)
Resveratrol
Spondias mombin Carotenoids Leaves (18)
Phenolic compounds: Ellagic acid and chlorogenic acid
Sterol: Sitosterol
Pulp (18-20)
Carotenoids: β-carotene, α-carotene, lutein, zeinoxanthin,
β-criptoxanthin
Phenolic compounds: Total phenolics, flavonoids
Astrocaryum Carotenoids Pulp (21, 22, 42, 43)
vulgare
Fatty acids: (C18:1), (C16:0), (C18:0), (C18:2)
Tocopherols: α-tocopherol
Carotenoids: β-carotene, α-carotene, γ-carotene, ζ-carotene,
δ-carotene, lutein, phytoene
Phytosterols: β-sitosterol, cycloartenol, arundoin
Kernel (40)
Fatty acids: (C12:0), (C14:0), (C18:1), (C18:2)
Phytosterols: β-sitosterol, Δ5-avenasterol, campesterol
Bactris gasipaes Carotenoids Mesocarp (23, 24, 27, 42-44)
Fatty acids: (C18:1), (C16:0)
Carotenoids: β-carotene, α-carotene, δ-carotene, γ-carotene,
lycopene,
Phytosterols: β-sitosterol, fucosterol
Oil kernel (41)
Fatty acids: (C12:0), (C14:0)
Phytosterols: β-sitosterol, Δ5-avenasterol, campesterol
Euterpe oleracea Anthocyanins Pulp (45-55)
Fatty acids: (C18:1), (C16:0), (C18:2)
Carotenoids: Lutein, β-carotene, α-carotene
(Continued)

Phytosterols: β-sitosterol
Phenolic compounds: Cyanidin 3-O-glucoside, cyanidin
3-O-rutinoside, homoorientin, orientin, isovitexin, (−)-epicatechin,
(+)-catechin, scoparin, taxifolin deoxyhexose, procyanidin,
p-hydroxybenzoic acid, ferulic acid, ferulic acid, gallic acid,
ellagic acid, vanillic acid
Seed (56-58)
Phenolic compounds: Catechin, polymeric proanthocyanidins
Mauritia flexuosa Carotenoids Pulp Oil (22, 26-39, 42-44)
Fatty acids: (C18:1), (C16:0), (C18:2)
Tocopherols: β-tocopherol, α-tocopherol, γ-tocopherol
Vitamins: A and E
Carotenoids: β-carotene, α-carotene, lutein
Phytosterols: β-sitosterol, stigmasterol, campesterol
Pulp extract (32)
Phenolic compounds: Protocatechuic, chlorogenic acid, caffeic acid,
(+)-catechin, (−)-epicatechin, apigenin, luteolin, myricetin,
kaempferol and quercetin
Leaf extract (32)
Phenolic compounds: Caffeic acid hexoside, naringenin,
(−)-epicatechin, vitexin, scoparin, cyanidin-3O-rutinoside and
cyanidin-3-O-glucoside
Trunk extract (32)
Phenolic compounds: Caffeic acid hexoside, naringenin,
(−)-epicatechin and kaempferol
Source: (1) Jonh and Sahidi (2010); (2) Chunhieng et al. (2008); (3) Yang (2009); (4) Bentes and Mercadante (2014);
(5) Náthia-Neves et al. (2017); (6) Ono et al. (2005); (7) Ono et al. (2007); (8) Alves et al. (2017); (9) Akter et al.
(2011); (10) Zanatta and Mercadante (2007); (11) Rodrigues-Amaya et al. (2008); (12) Anhê et al. (2018);
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in vitro model (Table 8.3) and an in vivo model with animals (Table 8.4) and humans (Table 8.5).
Many beneficial effects have already been clearly demonstrated, including in humans, and
the value of these fruits for the diversification of human nutrition. It is time to (re)discover
Amazonian hidden treasures!
TABLE 8.3
In Vitro Studies Performed with Amazonian Fruit or Fraction
Scientific Name Source Observations References
Bertholletia
excelsa H.B.K.
Nuts Allergic symptoms (vomiting, diarrhea and loss of Bartolomé et al.
consciousness) (1997)
↑ Level of specific IgE
Nuts Allergic symptoms after ingestion Brazil nut Pastorello et al.
(anaphylactic shock and laryngeal edema) (1998)
Nuts Incidence of specific IgE to Brazil nut in patients of Pumphrey et al.
different ages and sex (1999)
Nuts ↑ Reception of cholesteryl esters by the HDL Strunz et al. (2008)
Nuts ↑ Plasma selenium level ↑ Selenium levels Maranhão et al.
(2011)
Nuts ↓ Total cholesterol and LDL-c Stockler-Pinto et al.
↑ Plasma selenium levels after diet supplementation (2012)
Nuts ↑ Increase in HLD concentrations Cominetti et al.
Improvement of the Castelli I and II indexes (2012)
Nuts ↑ Plasma selenium levels and HDL-c Colpo et al. (2013)
Nuts ↑ Plasma Se and GPx activity Stockler-Pinto et al.
↑ HDL-c levels (2014)
↓ Cytokines, 8-OHdG and 8-isoprostane plasma
↓ LDL-c levels
Partially defatted nut ↓ In serum total cholesterol and non-HDL-c levels Carvalho et al. (2015)
flour
Nuts ↑ Plasma Se levels, rectal selenoprotein P (SePP) Hu et al. (2016)
and β-catenin mRNA
Nuts ↑ GPX1 mRNA expression only in subjects with CC Donadio et al. (2017)
Genipa americana L.
Fruit None cytotoxicity or interference in cell Da Conceição et al.
differentiation (2011)
↑ Antitumor effect on choriocarcinoma-derived cells
↑ Effects on trophoblast metabolism through the
MAPK pathway
Leaf, fruit and peel ↑ Tyrosinase inhibitory activity Souza et al. (2012)
Peel, pulp and seed ↑ Antioxidant activity of extracts Omena et al. (2012)
↑ Acetylcholinesterase inhibition by thin layer
chromatography
↑ Lipid peroxidation in membrane
Seed coat ↑ Antihypertensive activity Fidelis et al. (2018)
Fruit ↑ Stability at 12–20 °C and low pH (3.0–4.0) Neri-Numa et al.
↑Antioxidant capacity on digestion (2018)
Leaf Cell death of epimastigote, trypomastigote and Souza et al. (2018)
amastigote forms of Trypanosoma cruzi
Myrciaria dubia
(Kunth) McVaugh.
Pulp ↑ Levels of phenolics, ascorbic acid, Fujita et al. (2015)
proanthocyanidins, antioxidant and antimicrobial
activity
(Continued)

Pulp ↑ Properties antihyperglycemia and antimicrobial Fujita et al. (2015)
↑ Cellular rejuvenation
Peel and seeds ↑ Antimicrobial activities of the extracts Kaneshima et al.
(Acylphloroglucinol and rhodomyrtone and (2017)
acylphloroglucinols)
Seed coat ↑ Total phenolic content, antioxidant activity and Fidelis et al. (2018)
lipid oxidation
Camu-camu extracts ↑ Antihypertensive activity and angiotensin- Azevedo et al. (2018)
converting enzyme inhibitory activity
Spondias mombin L.
Leaf ↓ Larval development on nematode eggs from sheep Ademola et al. (2005)
Leaf Not activity against promastigotes of Leishmania Accioly et al. (2012)
chagasi
↑ Activity against amastigotes
Leaf ↑ Inhibition of leukocyte migration on inflammation Cabral et al. (2016)
site
Astrocaryum
vulgare Mart.
Unsaponifiable Anti-inflammatory properties with inhibition Bony et al. (2012)
fraction of pulp oil expression of COX-2, decreased NO, prostaglandin
E2, TNF-α, and IL-6 and -10 production
Bactris gasipaes
Kunth.
Mesocarp Protective effects of lipid peroxidation on liver Quesada et al. (2011)
homogenates of rats with
TBHP
Peel, pulp and seeds Antimicrobial against Staphylococcus aureus Araújo et al. (2012);
oil Araújo et al. (2013)
Euterpe oleracea
Mart.
Freeze-dried açaí ↑ Serum sulfhydryl groups and ↓ ROS formation in Kang et al. (2010)
pulp powder PMN cell
Concentrate açaí Anti-lipidemic and anti-inflammatory effects on Martino et al. (2016)
juice 3T3-L1 mouse adipocytes
Velutin isolated from LPS-induced pro-inflammatory and IL-6 production Xie et al. (2012)
açaí pulp in macrophages
Fruit pulp fractions Attenuate inflammatory stress signaling on BV-2 Poulose et al. (2012)
mouse microglial cells
Antochyanin-rich Antioxidant properties, antiproliferative properties Hogan et al. (2010)
açaí extract on C-6 rat cell and no effect on the growth of
MDA-468 human breast cancer cells
Bark, seed and total Cytotoxic effects in malignant cells lines Silva et al. (2014)
açaí fruit Barros et al. (2015)
Extract of açaí seed ↓ Cell viability and necroptosis in the MCF-7 cell Freitas et al. (2017)
Açaí oil Without genetic toxicity in rat cells Marques et al. (2016)
Polysaccharides Induction of innate immune response Holderness et al.
isolated from açaí (2011)
fruit
(Continued)

Frozen fruit pulp Antioxidant activity of H2O2 in the cerebral cortex, Spada et al. (2009)
hippocampus and cerebellum of rats
Açaí extract Antioxidant potential and protected human Torma et al. (2017)
neuron-like cells (SH-SY5Y)
Açaí extract Attenuates Mn-induced oxidative stress on rat da Silva Santos et al.
astrocytes (2014)
Açaí stone extract Vasodilator effect dependent on activation of Rocha et al. (2007)
NO-cGMP pathway in rat
Mesenteric vascular
Açaí extract Protection in human vascular endothelial cells Noratto et al. (2011)
against glucose-induced oxidative stress and
inflammation
Açaí extract Neuroprotective effects against β-amyloid exposure Wong et al. (2013)
on CP12 rat cell
Açaí extract added ↑ Transcript level of l(2)ef l(2)ef, GstD1 and MtnA Sun et al. (2010)
into the sugar–yeast ↓ Transcript level Pepck
medium ↑ Lifespan of oxidative-stressed females caused by
sod1 RNAi
Açaí extract added Dampening stress-induced expression of the GSTD1 Vrailas-Mortimer
into yeast medium and eliminates paraquat induced circadian rhythm et al. (2012)
deficits
Açaí pulp Suppressed IgE-mediated degranulation and Horiguchi et al.
transcription of the cytokine genes (2011)
Açaí pulp ↓ Bacteroides–Prevotella spp. and the Clostridium Alqurashi et al.
histolyticum groups (2017)
↑ Short-chain fatty acids
Aqueous açaí extract ↓ ROS; ↑ Polyglutamine protein aggregation; Bonomo et al. (2014)
Prevention sulfhydryl level;
Activation of gcs-1; ↓ Proteasome activity
Anthocyanin-rich ↓ ROS via DAF-16/FOXO translocation Peixoto et al. (2016)
extract
Açaí berry extract ↓ Osteoclastogenesis and osteoclast activity; ↓ Brito et al. (2016)
(IL)-1α, -6 and TNF-α; ↑ IL-3, -4, -13 and IFN-γ
Methanol/water açaí Malvidin and cyanidin: Protects BALB/3T3 cells Petruk et al. (2017)
extract against UVA irradiation;
Interferes with ROS generation; Keeps intracellular
GSH; Lipid peroxidation close to normal cellular
levels
Açaí berry water ↑ Migration of HS68 fibroblast cells; ↑ mRNA Kang et al. (2017)
extracts fibronectin expression.; ↑ mRNA fibronectin
expression; ↓ mRNA MMP-1 expression
Mauritia flexuosa L.f.
Buriti oil Low cytotoxicity on HaCat and 3T3 cell lines Zanatta et al. (2008)
humans and mice respectively
Buriti oil Photoprotective potential in after sun formulations Zanatta et al. (2010)
Buriti peel oil extract Antiplatelet/Antithrombotic activities Fuentes et al. (2013)
Peel, pulp and Chemopreventive potentialities of fruits and their Pereira-Freire et al.
endocarp by-products; Peels with higher quantities of (2018)
bioactive
(Continued)

Leaves, trunk and Moderate antimicrobial activity from leaf extract Koolen et al. (2013)
green fruits extracts against Staphylococcus aureus and Pseudomonas
aeruginosa
Extracts of stems, Stems and leaf extract inhibition the growth of S. aureus Siqueira et al. (2014)
leaves and fruits and antiproliferative activity on human tumor cell lines
IgE: immunoglobulin E, HDL-c: high-density lipoprotein cholesterol, LDL-c: low-density lipoprotein cholesterol, GPx: glutathi-
one peroxidase, mRNA: messenger ribonucleic acid, COX-2: cyclooxygenase-2, NO: nitric oxide, TNF-α: tumor necrosis
factor-α, IL: interleukin, TBHP: tert-butyl hydroperoxide, PMN: neutrophil numbers, BALB/3T3: mouse embryonic fibro-
blast cell line, BV-2: microglial cells, LPS: lipopolysaccharide, MDA: malondialdehyde, embryonic fibroblast cell line,
MCF-7: human breast adenocarcinoma cell line, SH-SY5Y: neuron-like cells, l(2)ef lethal (2): essential for life, MtnA:
metallothionein A, Pepck: phosphoenolpyruvate carboxykinase, SOD: superoxide dismutase, RNAi: RNA de interferência,
GstD1: glutathione S transferase D1, ROS: reactive oxygen species, GCS: glutamylcysteine synthetase, DAF-16: transcrip-
tion factor required in lifespan extension in mutation of the insulin-like receptor daf-2, FOXO: Forkhead box protein O,
HS68: human foreskin fibroblast, GSH: reduced glutathione, MMP: metalloproteinase.
TABLE 8.4
In Vivo Studies Performed with Amazonian Fruit or Fraction
Scientific Name Source Observation References
Bertholletia
excelsa H.B.K.
Stem Trypanocidal activity and antioxidant activity Campos et al. (2005)
Genipa americana L.
Leaf, Fruit and Peel Tyrosinase inhibitory activity Souza et al. (2012)
Peel, pulp and seed Lipid peroxidation membrane model in rats liver Omena et al. (2012)
Leaf Neuroprotective effect in the brain morphology and Nonato et al. (2018)
oxidative markers mice behavioral models
Myrciaria dubia
(Kunth) McVaugh.
Seed ↑ Suppression of paw edema formation Yazada et al. (2011)
Pulp Assessment the antioxidant, genotoxic and da Silva et al. (2012)
antigenotoxic potential on blood cells of mice after
acute, subacute and chronic treatments
Pulp ↓ White adipose tissues, glucose, total cholesterol, Nascimento et al.
triglycerides, LDL-c and insulin blood levels; ↑ (2013)
HDL-c levels
Not inflammatory markers and liver enzymes
Pulp ↑ Plasma antioxidant activity; ↓ triacylglycerol and Gonçalves et al. (2014)
total cholesterol
Fruit Improves the immune response and growth in Nile Yunis-Aguinaga et al.
tilapia (Oreochromis niloticus) 2016
Pulp Physiological parameters of tambaqui fed with Aride et al. (2018)
proportion difference of camu-camu
↑ Cortisol, glucose, proteins and triglycerides
Fruit ↑ Prevention diet-induced obesity and ameliorate Anhê et al. (2018)
Fruit ↑ Colon protective effects against DMH damage Azevedo et al. (2018)
↓ DXR mutagenicity effect
(Continued)

Fruit ↓ Induced neurodegeneration in transgenic Azevêdo et al. (2015)
Caenorhabditis elegans
Seed coat ↑ Inhibition of lipid oxidation induced by lipid Fidelis et al. (2018)
peroxidation in rats
Spondias mombin L.
Leaf ↑ Anthelmintic activity on rats infected with Ademola et al. (2005)
gastrointestinal nematode
Leaf ↑ Anti-inflammatory activity in rats using Nworu et al. (2011)
intraplantar injection of carrageenan
Leaf Gastroprotective effects against indomethacin- Sabiu et al. (2016)
induced gastric ulcer in rats
Leaf Methyl methane sulfonate (MMS) induced Oyeyemi et al. (2015)
genotoxicity in rats
Leaf Anti-inflammatory activity on model carrageenan- Cabral et al. (2016)
induced peritonitis in mice
Leaf Detoxification of hepatic and macromolecular Saheed et al. (2017)
oxidants in acetaminophen-intoxicated rats
Leaf Gastric lesion models induced by absolute ethanol Brito et al. (2018)
and indomethacin in rats
Astrocaryum
vulgare Mart.
Pulp oil Anti-inflammatory properties in a mice model of Bony et al. (2012)
endotoxic shock and a rat model of pulmonary
inflammation
↓ Pro-inflammatory cytokines
↑ Anti-inflammatory cytokines
Unsaponifiable Anti-inflammatory properties on mice model of Bony et al. (2014)
fraction of pulp oil endotoxic shock
↑ TNF-α, IL-6 and IL-10 serum concentration
Tucuma oil Hypoglycemic effect in alloxan-induced type 1 Baldissera et al. (2017)
diabetic mice
Tucuma oil Protective effect on memory, enzymatic activities Baldissera et al. (2017)
(Na+, K+-ATPase) and AChE in the brain of
alloxan-induced diabetic mice
Tucuma oil Protective action against diabetes in the alloxan- Baldissera et al. (2017)
induced diabetic mice
Improves the immune system; Changes in the
purinergic system
Tucuma oil Effective protection against lipid oxidative damage Baldissera et al. (2018)
in the liver tissue of diabetic mice
Improved the enzymatic antioxidant defense system
Bactris gasipaes
Kunth.
Ration made with Bioavailability of vitamin A in rat liver Yuyama et al. (1991)
fruit
Diet with peach ↑ Concentration of vitamin A in the liver of the rats Yuyama and Cozzolino
palm and bioavailable of zinc in the femurs (1996)
Pulp ↓ Body weight, total cholesterol in lactating rats Carvalho et al. (2013)
(Continued)

Euterpe oleracea
Mart.
ASE Protective effect against emphysema in mice de Moura et al. (2011)
ASE Potential reduction the inflammatory and oxidant Moura et al. (2012)
actions of cigarette smoke in the mouse
Diet with açaí Promotes healthy aging in SOD1-deficient female Laslo et al. (2013)
flies; ↓ 4-hydroxynonenal-protein adducts
Açaí juice Antioxidant and anti-inflammatory activities in Xie et al. (2011)
ApoE deficient mice
Seed extract Cardioprotective effects on rats subjected to Zapata-Sudo et al.
myocardial infarction (2014)
Seed extract Prevents vascular remodeling and endothelial Cordeiro et al. (2015)
dysfunction in spontaneously hypertensive rats
ASE Prevent hypertension in rats ↓ acetylcholine-induced da Costa et al. (2012)
vasodilation, ↓ MDA, carbonyl protein, SOD, CAT,
GPx, SOD1, SOD2, eNOS and TIMP-1; Prevented
vascular remodeling ↑ MMP-2
Açaí stone ↓ Growth and survival of endometriotic lesions in Machado et al. (2016)
female Sprague-Dawley rats
Açaí pulp Protection against DXR-induced DNA damage in Ribeiro et al. (2010)
liver and kidney cells on rats
Açaí pulp Rat liver: ↑ mRNA levels for γ-GCS and GPx in Guerra et al. (2011)
hepatic tissue ↓ ROS by neutrophils
ASE Improved serum levels of urea, creatinine and Na de Bem et al. (2014)
excretion
Açaí freeze-dried Protective effect on chronic alcoholic hepatic injury Qu et al. (2014)
in rats
Açaí extract Attenuates glycerol-induced acute renal failure in Unis (2015)
rats; ↓ serum urea and serum; ↓ glutathione levels
ASE Protect mice from diet-induced obesity and fatty de Oliveira et al.
liver by regulating hepatic lipogenesis and (2015)
cholesterol excretion
Açaí extract Attenuation of renal ischemia/reperfusion injury in a El Morsy et al. (2015)
rat model; ↓ MDA and IFN-γ ↑ IL-10
Frozen açaí pulp Attenuates hepatic steatosis via adiponectin- Guerra et al. (2015)
mediated effects on lipid metabolism in high-fat
diet mice
ASE ↓ Renal injury and prevented renal dysfunction in da Costa et al. (2017)
2K1C rats
ASE Protects against renal injury in diabetic and da Silva Cristino
spontaneously hypertensive rats Cordeiro et al. (2018)
Spray dried açaí Inhibits the TCC development in male Swiss mice, Fragoso et al. (2012)
powder due to its potential antioxidant action cells
Spray dried açaí Protective effect on colon carcinogenesis on rat; ↓ Fragoso et al. (2013)
powder number of invasive tumors; ↓ tumor Ki 67 cell
proliferation and net growth index
Spray dried açaí Protective effect on colon carcinogenesis in rat Romualdo et al. (2015)
powder
(Continued)

Açaí oil ↓ Tumor in comparison to rat control group Monge-Fuentes et al.
(2017)
ASE Antiangiogenic and anti-inflammatory potential; Alessandra-Perini et al.
Inhibition of DMBA carcinogenicity in breast (2018)
cancer in female Wistar
ASE ↓ Plasma malondialdehyde levels, body weight, de Oliveira et al.
plasma triglyceride, total cholesterol, glucose levels (2010)
and insulin resistance in mice fed a high-fat diet
Açaí puree Anti-atherosclerotic and anti-diabetic activity in Kim et al. (2012)
hypercholesterolemic zebrafish
Açaí pulp Hypocholesterolemic effect in rats; ↓ LDL, protein de Souza et al. (2010)
carbonylation and sulfhydryl groups; ↑ SOD ↑
paraoxonase activity
Açaí pulp Group HA of female fischer rats; ↓ serum total de Souza et al. (2012)
cholesterol, LDL and atherogenic index; ↑ HDL
Açaí extract Rabbits with diet-induced hypercholesterolemic: ↓ Feio et al. (2012)
total cholesterol, non-HDL and TG; ↓
atherosclerotic plaque area in aorta; ↓ desmosterol/
campesterol and desmosterol/β-sitosterol
Açaí fruit Prevents electrophysiological deficits and oxidative Brasil et al. (2017)
stress induced by methyl-mercury in the rat retina
Pulp extract Restoration of stressor-induced calcium Poulose et al. (2014)
dysregulation and autophagy in rat; Recovery of
depolarized brain cells from dopamine-induced
Ca2+ influx
Clarified açaí juice Prevention MDA in the cerebral cortex in mice Souza-Monteiro et al.
(2015)
ASE Prevented chronic pain in a rat spinal nerve ligation Sudo et al. (2015)
model; Prevented thermal hyperalgesia and
mechanical allodynia
Açai frozen pulp ↑ Pro-inflammatory cytokines levels in different de Souza Machado et
brain areas of Wistar rats al. (2015)
Freeze-dried açaí ↓ NOX2, NF-κB, ROS; ↑ Nrf2 hippocampus and Poulose et al. (2017)
powder frontal cortex of rats
Lyophilized açaí Improves cognition in aged rats; ↑ working memory Carey et al. (2017)
fruit pulp in the Morris water maze; ↓ NO, TNF-α
Mauritia flexuosa L.f.
Crude and refined Hypocholesterolemic effect in rats Aquino et al. (2015)
buriti oil RB: ↓ total cholesterol, LDL, triglycerides and
enzyme aspartate transaminase
RB and CB: ↑ serum and hepatic retinol and
tocopherol
Cookies made with ↑ β-carotene and monounsaturated fatty acids in the Aquino et al. (2016)
buriti oil cookies
↑ serum and hepatic retinol levels in rats fed BOC
and correlation serum retinol contents with hepatic
retinol
↓ Total and LDL-c in rats fed BOC
(Continued)

Crude and refined ↑ Serum and liver retinol deposition among neonatal Medeiros et al. (2015)
buriti oil rats fed with CB and RB
Fruit pulp Effect of MeHg on rat aversive memory acquisition Leão et al. (2017)
and panic-like behavior
HDL-c: high-density lipoprotein cholesterol, DMH: 1,2-dimethylhydrazine dihydrochloride, TNF-α: tumor necrosis
factor-α, IL: interleukin, AChE: acetylcholinesterase, ASE: açaí stone extract, SOD: superoxide dismutase, ApoE:
apolipoprotein E deficient (apoE−/−) mice, MDA: malondialdehyde, CAT: catalase, GPx: glutathione peroxidase,
eNOS: endothelial constitutive nitric oxide synthase, TIMP-1: metallopeptidase inhibitor 1, MMP: metalloprotein-
ase, DXR: doxorubicin, mRNA: messenger ribonucleic acid, γ-GCS: gamma-glutamylcysteine synthetase, ROS:
reactive oxygen species, IFN-γ: interferon-gamma, TCC: transitional cell carcinoma, DMBA: 7,12-dimethylbenz-
anthracene, LDL-c: low-density lipoprotein cholesterol, Group HA: hypercholesterolemic diet supplemented with
2% açaí pulp, TG: triacylglycerol, NOX NADPH: (nicotinamide adenine dinucleotide phosphate)-oxidoreductase,
NF-κB: nuclear factor κB, Nrf2: nuclear factor E2-related factor 2, NO: nitric oxide, RB: refined buriti oil, CB:
crude buriti oil, BOC: cookies made with buriti oil, CB: crude buriti oil.
TABLE 8.5
Functional Characterization of Amazonian Fruit Performed in Human Studies
Bertholletia
excelsa H.B.K
Nuts Allergic symptoms (vomiting, diarrhea and loss of Bartolomé et al. (1997)
consciousness)
Nuts Allergic symptoms after ingestion Brazil nut Pastorello et al. (1998)
(anaphylactic shock and laryngeal edema)
Nuts Incidence of specific IgE to Brazil nut in patients of Pumphrey et al. (1999)
different ages and sex
Nuts ↑ Reception of cholesteryl esters by the HDL Strunz et al. (2008)
Nuts ↑ Plasma selenium level ↑ Selenium levels Maranhão et al. (2011)
Nuts ↓ Total cholesterol and LDL-c Stockler-Pinto et al.
↑ Plasma selenium levels after diet (2012)
supplementation
Nuts ↑ Increase in HLD concentrations Cominetti et al. (2012)
Improvement of the Castelli I and II indexes
Nuts ↑ Plasma selenium levels and HDL-c Colpo et al. (2013)
Nuts ↑ Plasma Se and GPx activity Stockler-Pinto et al.
↑ HDL-c levels (2014)
↓ Cytokines, 8-OHdG and 8-isoprostane plasma
↓ LDL-c levels
Partially defatted ↓ In serum total cholesterol and non-HDL-c levels Carvalho et al. (2015)
nut flour
Nuts ↑ Plasma Se levels, rectal selenoprotein P (SePP) Hu et al. (2016)
and β-catenin mRNA
Nuts ↑ GPX1 mRNA expression only in subjects with CC Donadio et al. (2017)
(Continued)

Functional Characterization of Amazonian Fruit Performed in Human Studies
Myrciaria dubia
(Kunth) McVaugh.
Fruit juice Identification of seven biomarkers associated to Dickson et al. (2018)
genipapo consumption
Bactris gasipaes
Kunth.
Carotenoids extracts Promoted a low glycemic index Quesada et al. (2011)
Peach palm test ↑ β-carotene, γ-carotene, and lycopene and retinyl Hempel et al. (2014)
meal ester levels
Euterpe oleracea
Mart.
Açaí pulp ↓ Glucose and insulin levels, total cholesterol and Udani et al. (2011)
borderline significant reductions in LDL-c
Gel capsule of açaí ↓ Systolic blood pressure Gale et al. (2014)
Açaí pulp ↑ EGF and PAI-1 in overweight women; ↑ Body de Sousa Pereira et al.
weight, BMI, % truncal fat and (2015)
tríceps skinfold thickness in eutrophic women; ↓
Skinfold thickness and total body in overweight
women
Açaí juice ↑ Total antioxidant capacity of plasma; attenuation Sadowska-Krępa et al.
of muscle damage caused by exercise and (2015)
improvement of serum lipid profile
Açai functional ↑ Time for exhaustion in a shorter time with high Carvalho-Peixoto et al.
beverage intensity (2015)
Açaí pulp ↑ Catalase activity and total antioxidant capacity; Barbosa et al. (2016)
↓ Production of ROS
Açaí pulp ↓ROS, ox-LDL and malondialdehyde; ↑ Activity Pala et al. (2018)
of antioxidative paraoxonase 1
Açaí-beverage ↓ Interferon gamma and urinary level of Kim et al. (2018)
8-isoprostane
Frozen açaí pulp ↓ Incremental açaí under the curve for total Alqurashi et al. (2016)
peroxide oxidative status
IgE: immunoglobulin E, HDL-c: high-density lipoprotein cholesterol, LDL: low-density lipoprotein cholesterol, GPx:
glutathione peroxidase, mRNA: messenger ribonucleic acid, EGF: epidermal growth factor, PAI-1: plasminogen
activator inhibitor-1, BMI: body mass index, ROS: reactive oxygen species, ox-LDL: oxidized low-density
lipoprotein.
8.2 BERTHOLLETIA EXCELSA

8.2.1 Botanical Description
Bertholletia excelsa, most popularly known as the Brazil nut, belongs to the Lecythidaceae fam-
ily. This species is a native plant from South America found in various Brazilian states, such as
Maranhão, Mato Grosso, Pará, Rondônia and Amazonas. The fruit is approximately 2 kg in weight
and has a woody shell, 8 to 12 mm thick that contains 8 to 24 seeds (nuts) (Figure 8.1A). Each seed
is protected also by a woody, thick, indurate and rugose coat. An edible pale brownish-white kernel
is found inside the seed (Figure 8.1A) (Lim, 2012).
8.2.2 Phytochemicals
Brazil nut is an excellent source of fats and rich in unsaturated fats, including monounsaturated fatty
acids (MUFA) and polyunsaturated fatty acids (PUFA), phytosterols and tocopherols. However, the
phenolic content is low (Table 8.2) (Chunhieng et al., 2008; Cicero et al., 2018; John and Shahidi,
2010). The cold-pressed Brazil nut oil contains approximately 20% of saturated fatty acids (SFA),
52% MUFA and 28% PUFA. The high unsaturation level is principally due to oleic acid (C18:1,
n-9) and linoleic acid (C18:2, n-6), corresponding to 73% of the unsaturated fatty acids (UFA). The
linoleic acid content is twofold higher than that of olive oil (Cicero et al., 2018).
The unsaturated lipid fraction of the Brazil nut is also rich in β-tocopherol. However, lower lev-
els of α-tocopherol (11.3%) and γ-tocopherols (0.4%) are observed. The high β-tocopherol content,
corresponding to 88% of the total tocopherols, is a peculiar characteristic of this oil and could be
used as a marker of discrimination from other oils, such as soy oil and olive oil. The steroidal con-
tent is similar to olive oil, and β-sitosterol and squalene are the primary steroids found in the nut
(Chunhieng et al., 2008; Cicero et al., 2018).
John et al. (2010) observed that the bound phenolic compound content is 86- and 19-fold higher
in the brown skin than the kernel and whole nut, respectively. Additionally, the antioxidant activity
is higher in the skin due to the phenolic compound content. The phenolic compounds found include
gallic acid, gallocatechin, protocatechuic acid, catechin, vanillic acid, taxifolin, myricetin, ellagic
acid and quercetin. Cicero et al. (2018) reported a lower content of phenolic compounds (<0.5 μg g−1)
in the cold-pressed Brazil nut oil, including p-coumaric acid, apigenin 7-O-glucoside, luteolin and
p-hydroxybenzoic acid.
8.2.3 Mineral Content

The Brazil nut oil is rich in magnesium (1.4 g per 100 g), phosphorus (2.4 g per 100 g) and sele-
nium (0.4 to 12.7 mg per 100 g). The high selenium content present in the nut protein fraction has
an essential dietary antioxidant role. One Brazil nut of 5 g may contain up to 290 μg, which is six
times the daily selenium requirement for an adult. However, selenium toxicity occurs with the con-
sumption of more than 800 μg per day (Cardoso et al., 2017; Chunhieng et al., 2008; IOM, 2000;
Stockler-Pinto et al., 2012).
The Se present in the Brazil nut is bound to proteins, known as selenoproteins, which are used as
molecular biomarkers (Donadio et al., 2017). The Se of the protein fraction found in this nut is cova-
lently linked to two amino acids resulting in selenomethionine and selenocysteine (Chunhieng et al.,
2004). Jayasinghe and Caruso (2011) reported that the water-soluble protein fraction can be divided
into two primary sub-groups, the first with high molecular weight Se-containing proteins and the sec-
ond with low molecular weight Se-containing proteins with an abundance of methionine amino acids.
8.2.4 Biological and Pharmacological Activities

The Brazil nut is a good source of selenium, tocopherols and PUFA. In vitro (Table 8.3) and in vivo
(Table 8.4) studies revealed their respective biological properties in support of beneficial effects on
human health (Table 8.5).
In vitro assays revealed the antifungal activity of the Brazil nut oil. Five hundred microliters
of the oil nut reduced the growth of Aspergillus parasiticus colonies by approximately 38% and
inhibited aflatoxin production (Martins et al., 2014). Another study demonstrated a high trypano-
cidal activity against the trypomastigote form of the organic extracts (acetone and methanol) of
Bertholletia excelsa stem barks at concentration of 500 μg. mL−1 (Campos et al., 2005).
The high concentrations of bioactive compounds and the low toxicity risks have increased the
interests of various researches on the in vivo effects of the daily Brazil nut consumption in humans,
especially on the lipid profile and reduction of cardiovascular risks. Stockler-Pinto et al. (2012)
showed that the consumption of one nut per day (approximately 5 g containing 290 μg of selenium)
for 3 months increases the Se plasma levels in hemodialysis patients. After 12 months with the low
supplementation levels, the Se plasma levels were significantly lower (Stockler-Pinto et al., 2012).
Cominetti et al. (2012) also showed that the daily consumption of one Brazil nut per day by obese
patients for 8 weeks improved both the Se and lipid profiles, particularly high-density lipoprotein
cholesterol (HDL-c). Similar results were found by Maranhão et al. (2011) during the daily supple-
mentation of three to five Brazil nuts in the diet of obese adolescents for 16 weeks (Cominetti et al.,
2012; Maranhão et al., 2011). Additionally, the daily consumption of defatted Brazil nut flour (13 g per
day providing 227.5 μg of selenium per day) for 3 months showed the same effects on the lipoprotein
profile of dyslipidemic and hypertensive patients, such as reductions in total cholesterol, VLDL and
LDL. Therefore, the Brazilian nut is an alternative for healthy food market (Carvalho et al., 2015).
Supplementation with a higher amount of Brazil nut, 20 g(625 μg Se) and 50 g (1560 μg Se) per
day in healthy people, demonstrated that after 9 hours of ingestion, the serum low-density lipopro-
tein cholesterol (LDL-c) decreased and HDL-c increased (Colpo et al., 2013). In a study by Strunz
et al. (2008), the consumption of 11 Brazil nuts (865 μg Se) per day for 15 days increased the Se
plasma level but did not alter the serum lipid profile. The consumption of high amounts of Brazil
nut (more than two units) is not recommended due to the occurrence of Se toxicity in concentrations
higher than 800 μg. Therefore, better health effects are observed with the daily supplementation of
one Brazil nut for a period of 3 to 12 months (Stockler-Pinto et al., 2012).
Previous studies showed chemopreventive properties in colorectal cancer, as well as anti-
inflammatory activity, suggestinge that the beneficial health effects due to the consumption of the
Brazil nut are associated with the nut’s antioxidant potential and increased Se plasma levels (Hu et al.,
2016; Stockler-Pinto et al., 2012). Stockler-Pinto et al. (2014) verified that the consumption of
one Brazil nut per day for 3 months was adequate to reduce the inflammation, oxidative stress
markers and the atherogenic risk in hemodialysis patients. This result suggests an increase in the
antioxidant defenses of the patients (Stockler-Pinto et al., 2014). The supplementation of Brazil
nuts (48 μg Se per day) in the diet of people belonging to a risk group for colorectal cancer helps to
regulate colorectal cancer oncogenesis biomarkers, such as specific genes related to selenoproteins
(SePP), WNT signaling (β-catenin), inflammation (NF-κB) and methylation (DNMT1), and conse-
quently, should reduce cancer risk (Hu et al., 2016).
Furthermore, two primary Brazil nut proteins, Ber e 1 (2S albumin) and Ber e 2 (legumin),
are considered allergens for some people who have a specific immunoglobulin E to Brazil nut
(Geiselhart et al., 2018). Pastorello et al. (1998) verified the clinical symptoms of the allergens of
Brazil nut in patients after the ingestion of two nuts associated with 2S albumin. Anaphylactic shock
and laryngeal edema were the primary symptoms observed for symptomatic patients (Pastorello
et al., 1998). Many studies also reported the allergenic effect through immunochemical methods,
based on the IgE (Arshad et al., 2018; Bartolomé et al., 1997; Pumphrey et al., 1999).
The Ber e 1 allergen is a protein that possesses high stability during the pepsin digestion and is
thermostable, showing heat denaturation at approximately 110°C (Van Boxtel et al., 2008). Therefore,
the presence of Brazil nut is a required statement that must appear on food labels (FDA, 2011).
8.3 GENIPA AMERICANA

Genipa americana (Rubiaceae) is widely distributed in tropical regions and parts of subtropical
regions, such as the lowlands of the Amazon Forest and regions of Central and South America.
Genipa americana has different popular names, such as jagua, genipa, genipap and genipapo,
depending on the region. The ripe fruits (Figure 8.1B) are edible and are primarily consumed as
juice, liqueur and jelly. Fermentation of the fruit is used to produce the alcoholic beverage “cauí”
(Oliveira et al., 2012; Ono et al., 2005; UNCTAD, 2005).
Unripe genipap fruits are widely used by indigenous tribes to extract the blue pigment, exposing
the inside part of the fruit to the air. The genipap name originated from the Guarani language that
means “fruit used to paint”. The unripe fruits are lighter and shorter with a green color. The skin
of the ripe fruits is yellow-reddish, and at the final stage of maturation, the blue pigment is absent
(Bentes and Mercadante, 2014; Bentes et al., 2015).
The genipap fruits are well known for their iridoid content, primarily genipin and geniposide
(Figure 8.3) (Table 8.2). Both of these iridoids were found only in the unripe fruits. These com-
pounds decrease more than 90% during ripening, explaining the absence of the formation of the
blue pigment after the ripe fruits are opened. Geniposide is often used in Asian countries as a natural
yellow dye. This compound represents more than 70% of the total iridoid content of the unripe fruit
(Bentes and Mercadante, 2014). Genipin is a colorless iridoid from the monoterpene class, being an
excellent source of blue pigment. Genipin reacts spontaneously with primary amines and proteins
in the presence of oxygen, producing a water-soluble bluish-violet pigment. The endocarp and whole
fruit present a higher genipin content than the other parts of fruit, such as the peel, mesocarp and
seed (Náthia-Neves et al., 2017; Neri-Numa et al., 2017). In the ripe fruit, the genipin gentiobioside
is the primary compound found in the endocarp (Bentes and Mercadante, 2014).
Moreover, other iridoids are present in genipap fruits, such as gardoside, geniposidic acid,
genipin-1-β-D-gentiobioside, caffeoyl geniposidic acid, p-coumaroyl geniposidic acid, feruloylgar-
doside, feruloylgenipin gentiobioside, genipacetal, genipamide, genipaol and genamesides (Bentes
and Mercadante, 2014; Ono et al., 2005; Ono et al., 2007).
Data on the phytochemical composition of genipap reveal that the peel and fruits contain leuco-
anthocyanidins, catechins, flavanones, anthraquinones, anthrone and coumarins, triterpenoids and
steroids. The flavonoid quercetin predominates in the peel (48 μg g−1 DW) and seed (35 μg g−1 DW),
while it is found in a lower amount in the pulp (9.8 μg g−1 DW) (Omena et al., 2012).
Bentes and Mercadante (2014) detected the presence of phenolic compounds only in the unripe fruit,
including dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid and 5-caffeoylquinic
acid, while 5-caffeoylquinic acid is the primary phenolic compound found in this fruit.
The leaf extract of genipap is also a source of iridoids, and phytochemical studies confirmed
the presence of flavonoids and tannins. Two iridoids were detected only in the leaves of geni-
pap, 1-hydroxy-7-(hydroxymethyl)-1,4aH,5H,7aH-cyclopenta[c]pyran-4-carbaldehyde and iridoid
FIGURE 8.3 The chemical structure of genipin (A) and geniposide (B).
FIGURE 8.4 The chemical structure of iridoids 1-hydroxy-7-(hydroxymethyl)-1,4aH,5H,7aH-cyclopenta[c]

pyran-4-carbaldehyde (A) and 7-(hydroximethyl)-1-methoxy-1H,4aH,5H,7aH-cyclopental[c]pyran-4-
carbaldehyde (B).
7-(hydroxymethyl)-1-methoxy-1H,4aH,5H,7aH-cyclopenta[c]pyran-4-carbaldehyde (Figure 8.4)

(Alves et al., 2017; Nogueira et al., 2014).
The pulp and seed are also excellent sources of phytosterols, such as campesterol, stigmasterol,
β-sitosterol, sitostanol, Δ5-avenasterol, Δ7-stigmasterol and Δ7-avenasterol. The total phytosterol
content in the pulp and seed oil of genipap (>200 mg. 100 g−1 FW) is higher than that of the Brazil
nut (148 mg per 100 g FW) and açaí pulp (111 mg per 100 g FW) (Costa et al., 2010).

The primary compounds found in the genipap fruit are from the iridoid class. However, the bio-
activity of other compounds is better described in the literature, such as non-phenolic compounds
and steroids. The leaf extract is an excellent source of flavonoids, tannins and polysaccharides with
antioxidant activity and bioactivity (Table 8.2).
The genipap iridoids are considered as a promising source of blue pigment for food applications,
particularly acidic foods. The stability and antioxidant capacity were studied by in vitro simu-
lated digestion of the unripe genipap endocarp extract. The extract is rich in iridoids, including
genipin, genipin 1-β-gentiobioside, geniposide, gardenoside, 6′-O-p-coumaroyl geniposidic acid
and 6′-O-feruloyl-geniposidic acid. The results revealed an increase in these compounds during
the gastric phase (pH 2.0 at 37°C) and a decrease during the intestinal phase (pH 7.0 at 37°C).
Genipin is not detected after the intestinal phase, demonstrating the instability of these compounds
at neutral pH values. Interestingly, an increase of 17% to 18% was observed after in vitro digestion
(Neri-numa et al., 2018).
Genipap pulp extract showed anti-acetylcholinesterase activity. However, this effect is not
associated with the antioxidant activity and should be related to the non-phenolic compound.
Acetylcholinesterase inhibitors are essential in the symptomatic treatment of Alzheimer’s disease
to elevate the levels of endogenous acetylcholine in the brain (Omena et al., 2012).
The treatment of BeWo cells with a fruit ethanolic extract dose of 100 μg. mL−1 inhibits and
reactivates the mitogen-activated protein kinases (p38 MAPK). The consequence is an essential
effect on trophoblast metabolism and consequently may have implications for placental develop-
ment, such as the early termination of pregnancy, gestational abnormalities, or fetal growth defects.
The steroids of the extracts should be considered to be a possible cause of the effects (da Conceição
et al., 2011).
A study on the urinary metabolomics profile of volunteers who consumed 500 mL of geni-
pap juice revealed seven compounds in the metabolic pathways of the iridoids and phenolic
derivatives, including dihydroxyhydrocinnamic acid, hydroxyhydrocinnamic acid, genipic
acid, 12-demethylated- 8 -hydroxygenipinic acid, 3(7)-dehydrogenipinic acid, genipic acid

glucuronide, nonate, (1R,6R)-6-hydroxy-2-succinylcyclohexa-2,4-diene-1-carboxylate and
3,4-dihydroxyphenylacetate. These compounds were suggested to be biomarkers of genipap con-
sumption in humans (Dickson et al., 2018).
The leaf aqueous extract in concentrations equal to or higher than 30 mg. mL−1 showed anthel-
mintic activity, and above 90% of the nematode’s sheep were inhibited. The efficacy for the larval
development inhibition is higher than that for the inhibition of egg eclosion. Phytochemical analysis
of this extract using colorimetric methods identified flavonoids and tannins. However, in vivo stud-
ies are needed to assess the toxicity risks (Nogueira et al., 2014).
The antiparasitic action of the polysaccharide extracts (54% carbohydrates, with 21% being
uronic acid) from genipap leaves on the epimastigote, trypomastigote and amastigote forms of
Trypanosoma cruzi was also observed. The cell death of this protozoan may have relationship with
reactive oxygen species (ROS) molecules that cause peroxidative damage to the trypanothione
reductase and alter the redox balance (Souza et al., 2018).
Central inhibitory effect and anticonvulsant activity is another bioactivity of a heteropolysac-
charide present in the polysaccharide extract of the genipap leaf. The extract confers brain protec-
tion against oxidative stress and impairment in the number of black hippocampal neurons (Nonato
et al., 2018).
8.4 MYRCIARIA DUBIA (KUNTH) McVAUGH

Myrciaria dubia (Myrtaceae)is a tree found in the margins of rivers, streams, lakes and swamps
from the Amazon Region (Colombia, Venezuela, Peru and Brazil). The fruit is most popularly
known as camu-camu, and when ripe, the berries present a color varying from red to purple
(Figure 8.1C). The fruit is composed of 52.5% pulp, 21.2% skin and 26.3% seed. The berries are
globoid and approximately 2.5 cm in diameter, with white pulp with high ascorbic acid content that
prompts strong sour taste . Camu-camu is often consumed as juice, ice cream, cakes and liqueur
(Azevedo et al., 2018; Genovese et al., 2008; Neri-numa et al., 2018).
8.4.2 Phytochemistry
Camu-camu has a unique phytonutrient profile, with abundance of vitamin C, phenolic compounds
and carotenoids being considered a “superfruit”, with the most abundant natural source of vitamin
C in Brazil. Recently, Azevedo et al. (2018) determined the ascorbic acid content in the camu-
camu, is approximately 1100 and 946 mg per 100 g in dry weight (DW), cultivated in Amazonas
and Roraima states, respectively. Other studies have demonstrated higher contents of ascorbic
acid, approximately 1882, 2585 and 6112 mg per 100 g of the fresh pulp in Belém, Amazonas and
Roraima, respectively. Environmental factors and the difference in agroforestry systems should
explain the variation in the vitamin C content. For example, camu-camu in the Amazonas state
is cultivated in seasonally dry conditions on solid ground; in contrast, the camu-camu from the
Roraima state is from the flooded environment near the Rio Branco river (Maeda et al., 2007;
Rufino et al., 2010; Yuyama et al., 2002). Based on these data, only 10 g of fresh pulp is sufficient
to provide the daily recommended amount of vitamin C for an adult (75 to 90 mg) (IOM, 2000).
In comparison with other citrus fruits, the content of vitamin C in camu-camu nectar (340 mg per
100 g) is 3.6-fold higher than the content in orange juice (94.5 mg per 100 g) and 9.8-fold higher
than that in lemon juice (34.5 mg per 100 g) (Maeda et al., 2007; TACO, 2011).
Others bioactive compounds present in camu-camu pulp include phenolic acids (gallic acid,
ferulic acid, ellagic acid, ellagitannin B, p-coumaric and protocatechuic acid), flavonoids (myricetin,
kaempferol, quercetin, rutin, naringenin, cyanidin-3-rutinoside, and cyanidin-3-O-glucoside) and
carotenoids (β-carotene, lutein, β-cryptoxanthin, zeaxanthin, luteoxanthin, violaxanthin and neo-

xanthin). The seed has two specific compounds, betulinic acid and trans-resveratrol, in the seed
coat (Akter et al., 2011; Anhê et al., 2018; Fidelis et al., 2018; Genovese et al., 2008; Neri-numa
et al., 2018; Rodrigues-Amaya et al., 2008; Zanatta and Mercadante, 2007).
The camu-camu fruit is an excellent natural source of antioxidants. In vitro studies have confirmed
the antioxidant activity of ripe camu-camu fruit with average moisture content of 92%. Using the
2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl
(DPPH) methods, the antioxidant capacity is 1418 and 1520 μmol trolox equivalents g−1 DW, respec-
tively, and this value is lower in the unripe fruits. The average total polyphenol content found in camu-
camu fruit extract is 6550 mg per 100 g DW. The major groups found are proanthocyanidins (1854 mg
per 100 g DW), followed by ellagitannins, flavanols/flavonols and phenolic acids. Ellagic acid is found
in significant proportions in the unripe fruit, but the antioxidant activity is inferior due to the lower
anthocyanin and polyphenol concentrations (Anhê et al., 2018; Azevedo et al., 2018).

The bioactive compounds encountered in different parts of the camu-camu fruit, especially vita-
min C, phenolic acids and flavonoids, are responsible for the functional properties and related to
their high antioxidant activity. In vitro and in vivo studies using crude, aqueous or organic extracts
obtained from the pulp, leaf, skin, seed and seed coat demonstrated the reduction of lipid peroxida-
tion and positive effects on anti-inflammatory, anti-obesity, antiplasmodial and antileishmanial,
antimutagenic and antihypertensive activities (Tables 8.3 to 8.5).
The aqueous extract of the seed and seed coat were shown as excellent anti-inflammatory agent
during in vitro assays. This extract inhibited the lipid peroxidation induced by inflammatory media-
tors and suppressed the formation of carrageenan-induced paw edema in mice. Correlation analysis
by principal component analysis reveals that the aqueous extract, with higher antioxidant activity and
inhibition of lipid peroxidation, is composed of total phenolics, non-tannin phenolics, (−)-epicatechin,
chlorogenic acid, 2,4-dihydroxybenzoic acid, 2,5-dihydroxybenzoic acid and gallic acid. The propa-
none extract of the seed coat also has excellent antihypertensive activity, possibly due its ability to
chelate Cu2+, and high levels of quercetin, quercetin-3-rutinoside (rutin), t-resveratrol, ellagic, caffeic,
rosmarinic, ferulic and p-coumaric acids (Fidelis et al., 2018; Yazawa et al., 2011).
An antiobesity effect of camu-camu pulp has been observed in the treatment of diet-induced
obese mice for 8 weeks. The rats were fed a high-fat high-sucrose diet and concomitantly started
treatment with the ingestion of daily oral doses of camu-camu extract (200 mg kg−1). Another group
was treated with vitamin C (6.6 mg kg−1) for comparison. A reduction of body weight, glucose,
total cholesterol, triglycerides, LDL-c, insulin blood levels and an increase in the HDL-c levels was
observed in rats treated with camu-camu extracts, but no alterations were observed in those treated
with vitamin C. In addition, alterations in the gut microbiota were described during the treatment of
obese mice with a high abundance of Akkermansia muciniphila and a reduction of Lactobacillus.
The high incidence of A. muciniphila was positively correlated with lithocholic acid, deoxycholic
acid and ursodeoxycholic acid in plasma bile acids (BAs), and the reduction of the Lactobacillus
correlated with unconjugated BAs. A positive association between Lactobacillus and obesity was
demonstrated by the researchers. The studies indicated that the consumption of camu-camu extract
helps to prevent visceral and liver fat deposition as well as to generate alterations in the gut micro-
biota that contribute to the prevention of obesity (Anhê et al., 2018; Nascimento et al., 2013).
Whole fruit extracts of camu-camu demonstrated antimutagenic effects in mice, protecting the
colon against the damage induced by 1,2-dimethylhydrazine dihydrochloride (DMH) doxorubicin
chloridate (DXR) mutagenicity effects (Azevedo et al., 2018). In another study, the application of a
fraction with low molecular weight extracted from fresh camu-camu was able to reduce the Aβ1–42
peptide aggregation in the muscle tissues of Caenorhabditis elegans. In this Alzheimer’s disease
model, the predominance of polar basic bioactive compounds in the extract should explain the
neuroprotective effect. A similar effect was observed in the MPP+-induced oxidative dopaminergic
neurotoxicity model for Parkinson’s disease in C. elegans (Azevêdo et al., 2015).
In humans, a study on the consumption of camu-camu juice in volunteers who smoked was
conducted. One group ingested 70 mL of the juice daily (1050 mg of vitamin C), and another group
ingested a vitamin C (1050 mg) tablet for 7 days for comparative effects. The researchers observed
that camu-camu juice is an excellent antioxidant and anti-inflammatory agent (Inoue et al., 2008).
8.5 SPONDIAS MOMBIN

The yellow mombin (Spondias mombin) is a species belonging to the Anacardiaceae family, which is
distributed in tropical regions. The plant, popularly known as taperebá and cajá, is typical in the North
and Northeast regions of Brazil. The fruit is a small ovoid drupe 2.5 to 4 cm in diameter with yellow
skin (Figure 8.1D). The weight of the fruits varies from 13 to 33 g. A proportion of 70% to 82% of
the fruit is edible, and the juicy pulp has a sour-sweet taste (Tiburski et al., 2011; Vasco et al., 2008).
The primary bioactive compound class of yellow mombin fruit is carotenoids (Table 8.2). The carot-
enoids are found in the fresh pulp and are responsible for the yellow color of the ripe fruits. The
total chlorophyll content is low in the ripe fruit, and only some green spots are visible. Zielinski
et al. (2014) observed a positive correlation between the yellow color of the fruit and the carotenoid
content, particularly with β-carotene and lycopene. Hamano and Mercadante (2001) detected an
average of 2.6 mg per 100 g FW of total carotenoids in the pulp. A lower value was detected in the
juice (1.67 mg per 100 g FW). In most pulps evaluated by Silva et al. (2012), the total carotenoids
were two times higher than in this study. The fruits used were harvested from six genotypes of yel-
low mombin trees, and five showed values of the total carotenoids ranging from 3.7 to 4.1 mg per
100 g FW (Hamano and Mercadante, 2001; Silva et al., 2012; Zielinski et al., 2014).
β-Cryptoxanthin is the principal carotenoid present in the fruit, pulp and juice, and represents
more than 50% of the total vitamin A content. Other carotenoids found in the pulp are lutein,
zeaxanthin, β-carotene and α-carotene. Of these compounds, only α-carotene, β-carotene and
β-cryptoxanthin have provitamin A activity. The pulp should be considered to be a good source of
provitamin A because 100 g provides 37.2% of the RDI for adults (Hamano and Mercadante, 2001;
Tiburski et al., 2011).
Phenolic compounds are also present in the pulp with an average value of 249 mg gallic acid
equivalents (GAE) per 100 g FW (Vasco et al., 2008). The flavonoid content (range 0.14 to 0.52 mg
per 100 g FW) found by Silva et al. (2012) is relatively low in comparison to other fruits, and antho-
cyanins were not detected (Vasco et al., 2008; Zielinski et al., 2014).
The hydroethanolic extract of the yellow mombin leaf is a source of flavonoids, cinnamic deriva-
tives, triterpenoids, steroids, mono- and sesquiterpenes, alkaloids, proanthocyanidins and leucoan-
thocyanidins. Chlorogenic acid, gallic acid, ellagic acid and isoquercetin are phenolic compounds
more common in yellow mombin leaves and should be considered as excellent biomarkers for this
genus (Brito et al., 2018; Cabral et al., 2016).

The biological properties of the yellow mombin leaf have been extensively studied in the literature.
In vitro assays show a high antioxidant capacity, cytotoxicity activity against 3T3 fibroblast cells,
anthelmintic activity reducing the larval development, leishmanicidal activity on promastigotes and
antimicrobial activity (Accioly et al., 2012; Ademola et al., 2005; Cabral et al., 2016) (Table 8.3).
In vivo studies demonstrated that the biological effects are related to antioxidant activity. Brito
et al. (2018) demonstrated that ethanolic extracts of yellow mombin leaf contribute to the chronic ulcer
treatment mediated by the antioxidant activity. In particular, gallic acid and ellagic acid, isolated or
associated, stimulate the gastric mucus production, while the presence of the sulfhydryl groups and
nitric oxide consequently have antisecretory and anti-Helicobacter pylori activities (Brito et al., 2018).
Functional studies of yellow mombin leaf extract have highlighted the hepatoprotective effects in
rats, limiting the damage caused by drugs, such as indomethacin and acetaminophen. The mecha-
nism of action is associated with antioxidant systems of the extract that act as proton pump inhibi-
tors (Sabiu et al., 2016; Saheed et al., 2017).
The oral supplementation of the leaf extract of yellow mombin can also alleviate inflammatory
responses. Studies in mice demonstrated that ingestion of the extract in doses of 100 and 200 mg kg−1 per os
reduced the tumor necrosis factor (TNF-α) levels and nitric oxide production between 2 and 4 hours,
acting in the suppression of pro-inflammatory mediators (Nworu et al., 2011). Cabral et al. (2016) also
suggested that the anti-inflammatory properties of yellow mombin extract in mice is due to the antioxi-
dant properties and that chlorogenic acid and ellagic acid contribute to the pharmacological action. The
absence of cytotoxicity in cell cultures of the extract was confirmed in both studies (Cabral et al., 2016).
Anxiolytic and antidepressant effects of yellow mombin leaf extract were observed in a zebrafish
model (Danio rerio). Scototaxis and novel tank diving test (NTDT) tests revealed hypnotic and sedative
effects by immersion (25 mg L−1) and oral administration (25 mg kg−1) of extract doses. The effects should
be associated with the presence of isoquercitrin in the leaves of yellow mombin (Sampaio et al., 2018).
Oyeyemi et al. (2015) demonstrated that doses of 5000 mg kg−1 per os aqueous and hydro-
ethanolic extracts of yellow mombin leaf did not induce acute toxicity in mice. In contrast, hydro-
methanolic extracts showed genotoxicity and antigenotoxicity action. The results of the genotoxicity
tests in mice revealed genotoxic effects of hydromethanolic extracts with the potential to induce
both somatic and germline genetic damage. The extracts also showed antigenotoxicity action and
reduced genotoxicity induced by methyl methanesulfonate in bone marrow cells of the exposed
mice. Therefore, studies suggested the therapeutic effects of yellow mombin leaf extract and that
consumption for a long time should have toxic effects (Oyeyemi et al., 2015).
An in vivo study on yellow mombin juice consumption evaluated the effects of the incorporation
of daily doses of 100 and 250 mg of the dry extract (pulp and skin) per kg of body weight reconsti-
tuted in 88.2% of water in the diet of rats on the cardiac remodeling process induced by exposure to
tobacco smoke; these doses are equivalent to 329 and 610 g per day for a human of 60 kg, respec-
tively. After 2 months, the results showed attenuation of this process with a reduction of the cardiac
levels of lipid hydroperoxide, a reduction in glycolysis, and an increase in β-oxidation and oxidative
phosphorylation (Lourenço et al., 2018).
8.6 ASTROCARYUM VULGARE MART. (ARECACEAE)

8.6.1 Botanical Aspects and Occurrence
Astrocaryum vulgare, commonly known as tucumã, (Arecaceae) is primarily distributed in Brazil
(Amazonas, Amapá, Goiás, Maranhão, Pará, Piauí, Tocantins), French Guiana, Guyana and
Suriname (Henderson, 1995; Kahn, 2008). Tucumã is present in secondary vegetation, cerrado and
forests, primarily on sandy soils (Kahn 2008; Lorenzi et al., 2010). Depending on the region, the
fruit is designated tucumã, tucumã-do-Pará (Brazil); aouara (French Guiana); cumare (Colombia);
awarra (Suriname) and cumare (Venezuela) (Kahn, 2008). Tucumã is a cespitose palm that forms a
small cluster of unbranched stems reaching 4 to 10 m tall with spines in the trunks and stem parts
(Oboh, 2009), with pistillate flower with calyx urn-shaped corolla as long as the calyx. Tucumã
fruits are primarily used as sources of nutrition by populations where they occur due to the high
carotenoid content found in the pulp oil (Kahn, 2008). In Amazonia, the fruits have been providing
valuable nutrition to people for a long time (Smith, 2015).
8.6.2 Phytochemistry of Tucumã Pulp and Seeds

The tucumã palm fruit possesses a fleshy mesocarp (pulp) and a seed (Figure 8.2B), and both are
rich in oil that can represent up to 58.6% in the pulp and 37.6% in the kernel (Bora, 2001; Oboh and
Oderinde, 1989; Rodrigues et al., 2010). Pulp oil is very rich in carotenoids (Bony et al., 2012; Oboh,
2009) which imparts the characteristic orange-yellow color of this fruit.
The pulp contains 25 different fatty acids (Bora et al., 2001), and oleic acid (C18:1), a monosatu-
rated fatty acid, can represent up to 65% of the total fatty acids, followed by SFA, including palmitic
(C16:0) (25%) and stearic (C18:0) (3%) acids. Linoleic (C18:2) (2.6%) and linolenic (C:18:3) (0.2%)
acids are the most abundant PUFA found (Bony et al., 2012; Bora et al., 2001; Rodrigues et al.,
2010). The tucumã kernel oil has a high concentration of SFA in which lauric (C12:0) is prevalent
(50% to 60%), followed by myristic acid (C14:0) (20% to 29%). The monosaturated fatty acids and
PUFA present are oleic (C18:1) (13.6%) and linoleic (C18:2) (6%) acids, respectively (Bereau et al.,
2003; Bora et al., 2001; Oboh and Oderinde, 1989). The composition indicates that the proteins are
in a suitable concentration (8.44% of dry pulp) and kernels (2.06% on DW) (Bora et al., 2001).
The determination of the total carotenoid composition using High Performance Liquid
Chromatography( HPLC) has revealed different isoforms of α, β, δ, γ and ζ-carotene, phytoene and
phytofluene in tucumã pulp oil, reaching 1934 µg g−1. β-Carotene is the most prevalent carotenoid
in the pulp oil (60%), followed by phytoene (8.7%), and phytofluene isomers (4.33%) (Bony et al.,
2012; Santos et al., 2015; Silva et al., 2011) (Table 8.2).
β-Sitosterol and campesterol are the essential phytosterols in the composition of the kernel oil
(63% and 4%) and unsaponifiable extract pulp oil (32% and 8%). The presence of the Δ5-avenasterol
(or fucosterol) and stigmasterol is 30% and 1.8% in the kernel oil, respectively. In an unsaponifiable
extract of the pulp oil, arundoin and cycloartenol represent 16% and 11.4% of the total, respectively
(Bereau et al., 2003; Bony et al., 2012). The presence of α-tocopherol, β-tocopherol, γ-tocopherol
and vitamin C in pulp oil also has been shown (Bony et al., 2012; Dos Santos et al., 2015; Rodrigues
et al., 2010) (Table 8.2).
8.6.3 Biological and Pharmacological Activities of Tucumã Pulp Oil

Tucumã pulp oil is an attractive therapeutic agent due to the high amount of carotene, phytosterol, and
tocopherol and other bioactive compounds, all known for their antioxidant effects (Table 8.3). Bony et al.
(2012) demonstrated the potential of tucumã pulp oil to act as an anti-inflammatory agent in an endotoxic
shock and pulmonary inflammation model in rats (Table 8.4). In the endotoxic shock model, the cytokine
production observed has a potent effect in serum, spleen and lung by decreasing interleukin-6 (IL-6) and
TNF-α and increasing the IL-10 concentration. In the pulmonary inflammation model, a decrease in the
inflammatory cell afflux in the lung was observed, particularly eosinophils and lymphocytes. Another
study showed a similar effect of an ethanolic unsaponifiable fraction of tucumã pulp oil against the
murine J774 macrophage cell line. The extract inhibits the in vitro production of both nitrite oxide and
PGE2 by inhibiting iNOS expression and NO scavenging activity and inhibiting the activity and expres-
sion of the COX-2 enzyme, respectively. Additionally, this effect was confirmed in a model of endotoxin
shock by modulating TNF-α, IL-6 and IL-10 serum concentration in mice (Bony et al., 2014).
The beneficial effects of tucumã oil have also been assessed using several parameters in alloxan-
induced diabetic mice treated with this oil. The hypoglycemic effect improves insulin levels and anti-
oxidant/oxidant status and protects against pancreatic damage (Baldissera et al., 2017). The extract
promotes the capacity to maintain normal serum levels of ATP, ADP, AMP and adenosine, molecules
that exhibit anti-inflammatory properties. The carotenoid compounds should be related to their ben-
eficial effects (Baldissera et al., 2017) as adequate protection against neurotoxicity and consequent
memory impairment by preventing lipid peroxidation, increasing the levels of the antioxidant enzymes
catalase (CAT) and superoxide dismutase (SOD) and inhibiting Na+, K+-ATPase activity. All these
mechanisms are involved in the memory deficits of mellitus diabetes (Baldissera et al., 2017).
8.7 BACTRIS GASIPAES KUNTH. (ARECACEAE)

8.7.1 Botanic Aspects and Occurrence
Bactris gasipaes, the peach palm or pupunha (Brazil), is the only Neotropical palm with domes-
ticated populations in the Amazon region (Clement et al., 2010). Peach palm is endemic from the
tropical forest with a natural distribution that extends from Panama to Bolivia (Leterme et al.,
2005). Peach palm has a large variety of breeds and ecotypes, broadly distributed in areas, known
as landraces, located in the humid Neotropics, particularly in Amazonia (Clement et al., 2010).
Peach palm is a monoecious, allogamous (cross-pollinated), spiny, multi-stemmed palm that may
attain up to 20 m height with 15 to 25 pinnate fronds in the crown. The inflorescences appear among
the axils of the senescent fronds. The fruit bunch contains 50 to 100 single-seeded fruits. The fruit
is a drupe, which when ripe presents a fibrous red, orange or yellow epicarp, a moist starch/oily
mesocarp and a single endocarp with a fibrous and oily white kernel (Figure 8.2D) (Clement et al.,
2004; Valencia et al., 2015).
8.7.2 Phytochemistry of Peach Palm Fruit and Seed Oil

The peach palm oil has been analyzed in different populations and parts of the fruits, such as the
mesocarp and seed. In the fruit mesocarp, the lipid content demonstrates considerable variation,
ranging from 3.5% to 19%, while the kernel is more homogeneous , varying from 11.5% to 16.4%.
The monounsaturated oleic acid (C18:1) is present in large amounts in peach palm mesocarp oil
(42.8% to 60.8%), followed by saturated palmitic acid (24.1% to 42.3%) (Espinosa-Pardo et al., 2014;
Santos et al., 2013; Yuyama et al., 2003). Kernel oil analysis highlighted the high content of the SFA
(79.5%) with lauric (C12:0) and myristic (C14:0) acids as the prevalent constituents (Bereau et al.,
2003; Radice et al., 2014) (Table 8.2).
Peach palm mesocarp is an essential source of carotenoids, varying from 198 to 357.4 µg. g−1
total carotenoid content (de Rosso and Mercadante, 2007; Santos et al., 2015). Although the carot-
enoid content is different among varieties (1.1 to 7.4 mg/100 g), β-carotene (31.2% to 42.2%) and
γ-carotene (20.3% to 26.6%) are predominant (de Rosso and Mercadante 2007; Jatunov et al., 2010;
Rojas-Garbanzo et al., 2011).
Santos, et al. (2013) analyzed the minor components in oils obtained from Amazonian peach palm
fruit, and the predominant phytosterols are β-sitosterol and campesterol, present at 82.2% and 10.9%,
respectively. The α-tocopherol was the unique tocopherol detected (117 µg g−1). In the kernel oil, the
phytosterols are primarily represented by sitosterol (73.3%) and fucosterol (22%) (Bereau et al., 2003),
while the tocopherols include α-tocopherol (47 µg g−1) and β-tocopherol (7 µg g−1) (Radice et al., 2014).
8.7.3 Biological and Pharmacological Activities of Peach Palm Pulp Oil

The primary constituents of the peach palm have been investigated for their functional properties,
since they are primarily used in ethnomedicine for their effects on 11 different diseases (Sosnowska
and Balslev, 2009). Yuyama et al. (1991) showed that the vitamin A of peach palm was highly
bioavailable in the plasma and liver in male Wistar pups. The in vitro protective effects of lipid per-
oxidation on the liver homogenates of rats induced to oxidative stress with tert-butyl hydroperoxide
suggested that the carotenoids may contribute to an enhanced antioxidant defense (Quesada et al.,
2011) (Table 8.3).
A supplementation study with red peach palm fruit on Wistar rats before and post-lactation
showed an increase in HDL-c and a reduction of body weight, total cholesterol and triglycerides,
indicating that the dietary intake of the red peach palm is healthy, especially during the lactating
and post-lactating periods (Carvalho et al., 2013) (Table 8.4).
In humans, the ingestion of peach palm mesocarp promoted a lower glycemic index, similar
to those presented by legumes, and is recommended in the prevention of several chronic diseases
(Quesada et al., 2011). The bioavailability of α-carotene, β-carotene, γ-carotene, lycopene and their
isomers from peach palm fruits in humans was demonstrated by Hempel et al. (2014), who also cor-
related the significant increase in β-carotene, γ-carotene, and lycopene and retinyl ester levels with
the conversion of the ingested provitamin A carotenoids to vitamin A (Table 8.5).
Different oil extracts were tested for their antimicrobial potential on the growth of Staphylococcus
aureus, but only the bark oil inhibits S. aureus growth after 24 hours (Araújo et al., 2012). This
result was confirmed in a similar study that evaluated the antimicrobial activity of the shell, pulp
and seed oil of the peach palm on Pseudomonas aeruginosa and S. aureus. After 48 hours of oil
treatment, the antimicrobial effect of the bark of pupunha was observed in S. aureus with a 10-mm
halo of inhibition (Araújo et al., 2013).
8.8 EUTERPE OLERACEA MART. (ARECACEAE)

The Euterpe genus has approximately 28 species distributed in Central and South America
(Uhl and Dransfield, 1991). E. oleracea (açaí), a native palm of tropical South America, forms large
spontaneous populations being notable for the commercialization of the species’ fruits (Amsellem-
Laufer, 2015). The plant’s highest abundance occurs in the floodplains of the Brazilian Amazon
Basin, particularly in the states of Pará and Amazonas (Kahn and de Granville, 1992).
E. oleracea is a multicaule palm that can reach up to 25 m in height (Henderson, 1995). At
the end of each stem, there are spiral leaves pinned with pairs of leaflets. The inflorescence is
of the bunch type constituted by a central axis (rachis) and lateral branches (rachilles), in which
unisex sessile flowers are inserted, being a preferentially allogamous species performing cross-
fertilization (Bovi and Castro, 1993; Jardim and Macambira, 1996). The fruits are drupes, grouped
in clusters, in which the most popular variety is green during the initial maturation stages and
when mature assumes their black coloration (Figure 8.2A). The mature fruits exhibit a diameter of
up to 13.5 mm. The juicy exocarp is a thin layer, and the mesocarp is only 1 to 2 mm thick, while
the seed represents 85% to 95% of the total fruit volume with a solid and homogeneous endosperm
(Figure 8.2A) (Henderson, 1995; Pompeu et al., 2009; Rogez, 2000).
E. oleracea is the most valuable food resource species of wild fruit in Amazonia(Brokamp et al.,
2011). In the population of the Amazon estuary, açaí fruits are the primary staple food base and can
represent up to 42% of their diet in DW (Heinrich et al., 2011).
8.8.2 Phytochemistry of Açaí Fruits

During the past 10 years, the açaí fruit production has been scaled up from a minor local product to
an international commodity (Brokamp et al., 2011), witha the pulp with oleaginous lipids represent-
ing from 40.7% to 60.4% of dry matter (DM) and has a high content of dietary fibers (20.9% to 21.8%
of DM), proteins (6.7% to 10.5% of DM), vitamins and antioxidants (Bichara and Rogez, 2011).
The fatty acid composition of açaí revealed that MUFA compose 51.2% of all the fatty acids,
with oleic acid predominating (49.7%). The SFA and PUFA are represented by palmitic acid (25.3%)
and linoleic acid (13.5%), respectively (Rogez, 2000; Schauss et al., 2006). The predominance of
UFA, particularly oleic acid, was also reported in other studies, including extracts of the pericarp,
endocarp and fruit (Mantovani et al., 2003), enzymatic extraction process (Nascimento et al., 2008)
and analysis of the oleic content profile among different samples (Luo et al., 2012) (Table 8.2).
The phenolic content in açaí has revealed approximately 30 compounds, including flavonoids and
phenolic acids. The first studies demonstrated the presence of homoorientin, orientin, isovitexin,
(−)-epicatechin, (+)-catechin, scoparin, taxifolin deoxyhexose, procyanidin, p-hydroxybenzoic acid,
ferulic acid, ferulic acid, gallic acid, protocatechuic acid, ellagic acid and vanillic acid as non-
anthocyanin polyphenols (Del Pozo-Insfran et al., 2004; Gallori, et al., 2004; Schauss et al., 2006).
Later studies have broadened the knowledge of the non-anthocyanin polyphenols in the
açaí with the identification of additional compounds, including lignans, eriodictyol, (2S,3S)-
dihydrokaempferol 3-O-β-D-glucoside and the isomer, (2R,3R)-dihydrokaempferol 3-O-β-D-
glucoside, velutin, 5,4′-dihydroxy-7,3′,5′-trimethoxyflavone and hydroxymethylglutaryl-rhamnoside
(Dias et al., 2013; Gordon et al., 2012; Kang et al., 2011; Lichtenthäler et al. 2005; Mulabagal and
Calderon, 2012; Pacheco-Palencia et al., 2009).
Among the anthocyanins, the cyanidin 3-O-glucoside (C3G) and cyanidin 3-O-rutinoside (C3R)
are prevalent reaching 1159 to 1609 µg g−1 of the total anthocyanins in the açaí fruit (Gallori
et al., 2004; Rogez et al., 2011; Schauss et al., 2006). The non-anthocyanin phenolic content varies
depending on the fruit origin, genotypes and commercial pulps. The C3G and C3R can reach 18,942
and 34,397 μg g−1, respectively (Carvalho et al. 2017; Rogez et al., 2011).
During the fruit maturation, the profile of the anthocyanins C3G and C3R vary. In the beginning,
the two anthocyanins are present in similar proportions, but in the latter stages, C3R is more abundant
(Rogez et al., 2011). Dias et al. (2012) demonstrated that pelargonidin-3-glucoside (Pg3G), peonidin-
3-O-glucoside (Pn3G) and peonidin 3-O-rutinoside (Pn3R) are minor anthocyanins in açaí fruits.
The tocopherol composition of açaí fruits has also been demonstrated, and α-tocopherol primar-
ily predominates. Although the low content of 147.72 μg g−1 has been already reported, other studies
highlighted higher and significant contents in açaí pulp (394.3 μg g−1 DM) or oil (1101.11 mg L−1).
Açaí should be considered an excellent source of vitamin E (Bichara and Rogez, 2011; Costa et al.,
2010; Darnet et al., 2011; Ribeiro et al., 2018). The primary carotenoids are found in low concentra-
tions and include lutein (1.5 to 7.17 µg g−1), β-carotene (1.49 to 2.4 µg g−1) and α-carotene (0.03 to
0.42 µg g−1) in açaí pulp. The total phytosterol content is high (1110 µg g−1), with β-sitosterol the most
prevalent (940 µg g−1) (Costa et al., 2010; Ribeiro et al., 2010; Romualdo et al., 2015) (Table 8.2)
8.8.3 Biological and Pharmacological Activities of Açaí Fruits

The high diversity of the phenolics in açaí has led to studies that have demonstrated their significant
antioxidant properties (Table 8.3). The juice, usually called “pulp” by various authors, was charac-
terized using different in vitro methodologies, such as measuring the antioxidant capacity against
DPPH, hydroxyl, peroxyl and peroxynitrite radicals, anion superoxide, and the inhibition of lipo-
somes (Carvalho et al., 2017; Chin et al., 2008; Del Pozo-Insfran et al., 2004; Gordon et al., 2012;
Hassimotto et al., 2005; Kang et al., 2010; Lichtenthäler et al., 2005; Paz et al., 2015; Rufino et al.,
2010; Torma et al., 2017). The healthy nutrient composition and antioxidant activity of the açaí have
been supported by studies on their different physiological and beneficial effects (Tables 8.4 and 8.5).
8.8.3.1 Cardiovascular Effects

The endothelium-dependent vasodilator effects from the skin and seed extracts have been verified
in the isolated mesenteric vascular bed of the rat (Table 8.4). The vasodilator effect was detected in
both extracts, and açaí stone extract (ASE) was more potent, suggesting the possibility of using ASE
to treat cardiovascular diseases (Rocha et al., 2007). In other experiments on hypertension, ASE also
showed a significant antihypertensive effect (Cordeiro et al., 2015). In renovascular hypertensive
rats, ASE oral administration prevents the increase in blood pressure and plasma renin levels and
recovers the endothelial-dependent vasodilator effect of acetylcholine. SOD, CAT, and glutathione
peroxidase (GPx) activities are also recovered, increasing the nitrite content and protein expression
of endothelial constitutive nitric oxide synthase (eNOS) and decreasing the malondialdehyde (MDA)
and carbonyl protein levels in the mesenteric vessels (da Costa et al., 2012). The mechanism of the
antihypertensive effect of ASE is not entirely elucidated, but hemodynamic effects in normotensive
healthy individuals treated with açaí demonstrated a significant reduction in systolic pressure (Gale
et al., 2014). Similar results were previously reported (Udani et al., 2011).
Additionally, ASE has been associated with myocardial ischemia due to the induction of cardiac
dysfunction and exercise intolerance in rats (Zapata-Sudo et al., 2014). In overweight men, the
consumption of a flavonoid-rich açaí meal was associated with improvements in vascular function,
which may lower the risk of a cardiovascular event (Alqurashi et al., 2016).
8.8.3.2 Renal Failure Effects

The beneficial effect of açaí skin has been observed in renal dysfunction experiments in rats with
renal protective action by improving kidney function and decreasing the serum urea, creatinine and
blood urea nitrogen. The effects were associated with açaí’s antioxidant action, that significantly
improved renal oxidative stress markers (Unis, 2015). In a study on renal ischemia and reperfu-
sion injury (I\R), açaí skin acts to attenuate I\R induced renal damage, decreasing the blood urea
nitrogen levels, serum creatinine and renal tissue content of kidney molecule-1 (KIM-1). The reduc-
tion of MDA, myeloperoxidase (MPO), IFN-γ, caspase-3, collagen IV and endothelin-1 were also
observed (El Morsy et al., 2015).
Additionally, the oral administration of ASE during rat pregnancy prevents functional and struc-
tural changes, such as oxidative stress, increases nephron and glomerular number, glomerular volume,
and the serum levels of renin, urea, creatinine and fractional excretion of sodium (de Bem et al., 2014).
The protective effect of açaí fruit was also verified on chronic alcoholic hepatic injury in rats. A reduc-
tion was observed in serum alanine transferase (ALT) and aspartate aminotransferase (AST), MDA,
triacylglycerol (TG), and serum TNF-α and IL-6 and an increase in reduced glutathione (GSH) and
SOD (Qu et al., 2014). Based on the data in the literature, supplementation with açaí may reduce oxida-
tive stress and inflammation and consequently chronic kidney disease (Martins et al., 2018).
8.8.3.3 Effects on Lipids and Diabetes Metabolism

In mice supplemented with a high-fat diet, the oral ASE treatment increases the body weight, plasma
triglyceride, TC, glucose levels, oral glucose tolerance test and insulin resistance (HOMA index)
(de Oliveira et al., 2010). The supplementation of rat hypercholesterolemic with açaí pulp caused a
hypocholesterolemic effect by reducing the total and non-HDL-c (de Souza et al. 2010). Rats fed
with açaí pulp also exhibited a significant decrease in serum TC, LDL-c and the atherogenic index.
Açaí has a hypocholesterolemic effect in a rat model (de Souza et al., 2012).
Additionally, high-fat mice supplemented with açaí showed attenuated hepatic steatosis via adi-
ponectin-mediated effects on lipid metabolism (Guerra et al., 2015). In humans, supplementation
with açaí results in a favorable action on plasma HDL (Pala et al., 2018), a positive impact on the
reduction of LDL oxidation with a tendency to increase plasma antioxidant capacity (Sampaio et al.,
2006) and a beneficial overall role against atherosclerosis (Feio et al., 2012). In junior athletes, the
juice blend has a hypocholesterolemic activity (Sadowska-Krępa et al., 2015).
8.8.3.4 Antitumor Effects

In an experimental cancer model in the rat, different supplementations with açaí showed evidence
of both anticarcinogenic and chemopreventive effects. In the case of esophageal tumorigenesis,
a progressive inhibition was observed with a reduction in the serum levels of the cytokines IL-5
and GRO/KC. The expression of rat IL-8, a direct homolog of that of humans, is lower, and the
antioxidant capacity increases in serum (Stoner et al., 2010). Other studies reported the inhibi-
tion of urinary bladder carcinogenesis by açaí supplementation. The effects are the reduction of
DNA damage, the expression of p63 and proliferation of cell nuclear antigen (PCNA), but the diet
supplemented with açaí for 10 weeks did not significantly alter cytoplasmic and nuclear β-catenin
(Fragoso et al., 2012). Açaí should be considered to have active anticancer activity. The açaí affects
the human malignant MCF-7 cell line (Silva et al., 2014) and inhibits colon carcinogenesis induced
by 1,2-dimethylhydrazine (DMH) in Wistar rats (Fragoso et al., 2013). In an experimental model
of colon cancer induced by azoxymethane (AOM) with dextran sulfate sodium (DSS) in ICR mice,
a downregulation of myeloperoxidase (MPO) and pro-inflammatory cytokines (TNF-α, IL-1β and
IL-6) was described. The inhibition of COX-2, PCNA and Bcl-2 and an increase in Bad and cleaved
caspase-3 expression was also observed (Choi et al., 2017).
Furthermore, açaí is a useful photosensitizer to reduce melanoma carcinogenesis by increas-

ing the necrotic tissue per tumor area (Monge-Fuentes et al., 2017). Based on tumor diameter and
weight, Nascimento et al. (2016) reported an anticarcinogenic effect of açaí in anorexia-cachexia
syndrome induced by Walker-256 tumors due to antioxidant capacity.
In breast cancer induced chemically by 7,12-dimethylbenzanthracene (DMBA), experiments
in female Wistar rats demonstrated the antiangiogenic and anti-inflammatory potential of açaí. A
decrease in the number of inflammatory cells and positive macrophage cells were correlated with
the inhibition of DMBA carcinogenicity (Alessandra-Perini et al., 2018). The effects of açaí in
tumor cells have been suggested due to the anti-inflammatory, antiproliferative and proapoptotic
properties (Alessandra-Perini et al., 2018).
8.8.3.5 Nontoxic Effects

Studies in experimental models reported the absence of toxicity. Parameters, such as animal body
weight or food consumption, show no significant differences during açaí supplementation (Fragoso
et al., 2012; Fragoso et al., 2013; Schauss et al., 2010). Additionally, açaí administration by gavage
presented no genotoxic effect based on DNA damage evaluation induced by antitumor medication
(Marques et al., 2016; Ribeiro et al., 2010; Schauss et al., 2010). A micronucleus test and a comet
assay demonstrated the absence of genotoxic effects in mice supplemented with açaí. The results
were based on parameters in the bone marrow and peripheral blood cells, polychromatic erythro-
cytes and liver and kidney cells. A protective role of açaí in human health was suggested by the
reduction of DNA damage induced by doxorubicin (Ribeiro et al., 2010). Another study identified
the same effects using a bacterial reverse mutation, a chromosomal aberration, a mammalian cell
mutation assay and an in vivo micronucleus study (Schauss et al., 2010). No significant genotoxic
effects in a comet assay and micronucleus test were verified in rat cells treated with the three doses
of açaí. Similar results were reported (Marques et al., 2016).
8.8.3.6 Other Effects

Additional effects from the polysaccharide fraction of açaí were observed in the treatment of asthma
and infectious disease (Holderness et al., 2011). A protective effect was demonstrated against emphy-
sema by reducing oxidative and inflammatory reactions, and therefore, the inflammatory and oxidant
actions of cigarette smoke in mice (de Moura et al., 2011; Moura et al., 2012). The anticonvulsant prop-
erties in mice (Souza-Monteiro et al., 2015) and a significant antinociceptive effect via a multifactorial
mechanism of action suggest that açaí could be used to develop new analgesic drugs (Sudo et al., 2015).
8.9 MAURITIA FLEXUOSA L.F. (ARECACEAE)

Mauritia flexuosa is a robust, solitary-stemmed palm tree that reaches 30 m in height, with palmate
leaves that form a spherical crown, and the roots often bear a pneumatophore structure in flooded
soils (Janick and Paull, 2008). This dioecious tropical palm bears many hanging inflorescences that
are similar in both sexes. The fruit is spherical or ellipsoidal 4 to 5 cm in diameter and 5 to 7 cm
long. Under brownish-red scales, the mesocarp is pulpy and varies from yellow, orange to reddish
orange in color (Figure 8.2C). The spongy endocarp harbors the seed (Janick and Paull, 2008).
M. flexuosa is a palm tree native to South America, restricted to permanently or seasonally
flooded soils, that often forms extensive, monodominant stands designated buritizais in Brazil
(Koolen et al., 2013; Maria Pacheco Santos, 2005). The buriti is distributed in Brazil and occurs in
the states of Pará, Amazonas, Maranhão, Piauí, Bahia, Ceará and Tocantins (Pereira Freire et al.,
2016) and Colombia, Equator, Venezuela and Guyana (Delgado et al., 2007).
For the population from the Amazon region, buriti pulp is a vital part of the diet. The fruit is
used to produce juice, jam, compote, wine and ice cream (Manhães et al., 2015). Other tree palm
parts are used, including trunks as bridges, fiber and seeds as handicraft and timber or leaves in
construction (Brokamp et al., 2011).
8.9.2 Phytochemistry
Buriti fruit contains relatively high oil content (38.4% DM), similar to palm oil and other wide-
spread oleaginous crop seeds, such as canola (40% to 45%) and sunflower (35% to 45%) (Darnet
et al., 2011). Buriti oil is rich in monosaturated fatty acid (75.5% to 92.3%), in which oleic acid
(C18:1) represents approximately 75%. The SFA (18.75% to 19.6%) include palmitic acid (C16:0),
while the PUFA are approximately 2.1%, with linoleic acid (C18:2) predominating (Aquino et al.,
2012; Darnet et al., 2011; Rodrigues et al., 2010). The comparison between the crude and refined
buriti oil showed levels of PUFA and MUFA, although the refining process changes their profiles
and reduces the content of carotenoids (Aquino et al., 2012; Medeiros et al., 2015) (Table 8.2).
The quantitative composition was determined among Amazonian samples using HPLC, and
the results showed that the buriti has a high content of total carotenoids (513.87 to 1576 µg. g−1).
Twenty carotenoids were identified, with β-carotene prevalent (85.22% to 89.32%), followed
by α-carotene (3.88% to 4.75%). The primary isomers are all-trans-β-carotene, 13-cis-β-carotene,
9-cis-β-carotene (72.45%, 11.52% and 3.61%, respectively) (de Rosso and Mercadante, 2007; Santos
et al., 2015; Silva et al., 2011). The tocopherol content of the buriti fruit varies between 1129 and
1567 µg g−1, with β-tocopherol representing approximately 50% to 67%, and a contribution of
γ-tocopherol or α-tocopherol with approximately 78% and 70% of the total carotenoid content,
respectively (Costa et al., 2010; Rodrigues et al., 2010; Santos et al., 2013; Silva et al., 2011). These
results suggest that buriti can be considered an excellent source of vitamin E (Table 8.2).
β-Sitosterol, stigmasterol and campesterol are the primary phytosterols in the buriti fruit, reaching
values of 84%, 20% and 9%, respectively, from the total content (1830 µg. g−1). In the kernel, the phytos-
terol concentration is seven times smaller, with a campesterol content of 20% and β-sitosterol and stig-
masterol approximately 24% (Costa et al., 2010; Dembitsky et al., 2011; Santos et al., 2013) (Table 8.2).
The phenolic compound characterization of the fruit pulp using UHPLC–ESI(−)-MS/MS revealed
six phenolic acids, including p-coumaric acid, ferulic acid, caffeic acid, protocatechuic acid, chloro-
genic acid and quinic acid. Seven flavonoids, such as (+)-catechin, (−)-epicatechin, apigenin, luteolin,
myricetin, kaempferol and quercetin were identified. Protocatechuic and chlorogenic acids are the pri-
mary phenolic compounds (2175.93 and 11,154.15 μg g−1 DWP) (Bataglion et al., 2014). The analysis
in the leaf extracts (LE), trunk extract (TE) and fruit extract (FE) showed that myricetin, (+)-catechin,
chlorogenic acid, naringenin and rutin are present in all the extracts. Caffeic acid hexoside, narin-
genin, and (−)-epicatechin are present in both the LE and FE. Vitexin, scoparin, C3R and C3G are
only detected in the FE and kaempferol in the TE (Koolen et al., 2013) (Table 8.2).
Buriti fruit is considered a good source of provitamin A with 7280 RE/100 g and contains rela-
tively high values of total dietary fiber (22.8% FW) and protein contents (7.6% of DM) (de Rosso
and Mercadante, 2007; Rodrigues et al., 2010).

The high content of β-carotene, α-tocopherol and oleic acid in the buriti mesocarp oil have been
investigated for their effects in supplementation, protection against diseases and cosmetic formula-
tion (Tables 8.3 and 8.4).
The antioxidant capacity of methanolic extracts has been demonstrated as higher in the leaf
(iron reduction test) than in the fruit pulp (DPPH method) (Koolen et al., 2013). The same method
showed that extracts from dried fruit exhibited higher antioxidant capacity than the fresh fruit
extract (Gomes et al., 2016) (Table 8.3).
Young rats fed with crude buriti oil (CB) or refined buriti oil (RB) showed higher vitamin A con-
tent in serum and liver. The available vitamin A and E in young rats fed CB or RB is higher for all
physiological parameters. The result suggests that buriti oil is an essential source of the antioxidant
vitamins A and E and improves the lipid profile (Aquino et al., 2015). An increase in vitamin A
was demonstrated after rats consumed cookies made with buriti oil. Consequently, the lipid pro-
file and retinol content were improved, and blood glucose was not affected (Aquino et al., 2016).
Pretreatment with enriched feed also prevents the neurocytotoxic and behavioral effects caused by
MeHg. These results indicate the protective effect against cognitive deficits and the cytoplasmic
membrane damage induced by lipid peroxidation in the rat hippocampal region (Leão et al., 2017).
Buriti oil emulsion is a potential vehicle as photo blocker by decreasing the cell damage caused
by UVA and UVB radiation in X-rayed keratinocytes (Zanatta et al., 2010). The healing activity
contributes to the formation and deposition of collagen fibers and provides cellular stimulation and
proliferation (Batista et al., 2012). A protective effect is observed on platelet activation by increasing
the antiplatelet and antithrombotic activities (Fuentes et al., 2013).
Buriti oil and extracts have been tested for antimicrobial activity .Batista et al. (2012) showed
that buriti oil inhibited the bacterial growth of Enterobacter aerogenes, Bacillus subtilis, Klebsiella
pneumoniae and S. aureus. B. subtilis has greater sensitivity to buriti oil (Batista et al., 2012). The
results of the antimicrobial tests against S. aureus, P. aeruginosa, Escherichia coli, Micrococcus
luteus, and Bacillus cereus revealed a moderate effect of the methanolic extracts on the inhibition
of growth. The best results were obtained with the leaf extract against the pathogen P. aeruginosa
with a minimum inhibitory concentration (MIC) of 50 μg. mL−1 (Koolen et al., 2013).
In another study, the antimicrobial activity against different fungi and bacteria was exhibited
by triterpenes isolated from buriti roots. An MIC ranging from 50.8 to 203.5 μM was determined
(Koolen et al., 2013). Stem ethanolic extracts from buriti demonstrated the growth inhibition of
S. aureus (methicillin-susceptible S. aureus – MSSA; methicillin-resistant S. aureus – MRSA)
(31.3 μg mL−1), while the leaf extract showed activity against MRSA (62.5 μg mL−1), demonstrating
the antimicrobial potential of the extracts (Siqueira et al., 2014).
8.10 FINAL REMARKS

Amazon fruits and nuts are the most abundant sources of bioactive compounds with antioxidant action,
such as phenolic compounds, carotenoids, tocopherols, vitamin C, UFA, terpenoids and steroids.
Characteristic compounds, present in a higher amount, are a highlight for some fruits, such as vitamin
C in camu-camu fruit, carotenoids in the peach palm and tucumã fruits, iridoids in genipap and sele-
nium and UFA in Brazil nut. The synergistic effect of all these compounds showed clear evidence of
the health benefits of the consumption of these fruits associated with their high antioxidant capacity.
ACKNOWLEDGMENTS
The authors thank the Federal University of Pará (PROPESP), Conselho Nacional de Desenvolvimento
Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior
(CAPES) – Brasil (CAPES) – Finance Code 001 – for their financial support.
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9 Plant Species from the
Atlantic Forest Biome and
Their Bioactive Constituents
Rebeca Previate Medina, Carolina Rabal Biasetto,
Lidiane Gaspareto Felippe, Lilian Cherubin Correia,
Marília Valli, Afif Felix Monteiro, Alberto José Cavalheiro,
Ângela Regina Araújo, Ian Castro-Gamboa, Maysa Furlan,
Vanderlan da Silva Bolzani, and Dulce Helena Siqueira Silva
Univ. Estadual Paulista, Núcleo de Bioensaios, Biossíntese e Ecofisiologia
de Produtos Naturais – NuBBE, Araraquara, Brazil
CONTENTS
9.1 Introduction............................................................................................................................ 217
9.1.1 The Atlantic Forest Biome......................................................................................... 218
9.1.2 Natural Products in Brazil: Historical Benchmarks................................................... 219
9.2 Bioprospecting Plants From Atlantic Forest.......................................................................... 226
9.2.1 Natural Products Active on Redox Processes, Inflammation,
Chemoprevention and Related Processes.................................................................. 227
9.2.2 Cytotoxic Compounds............................................................................................... 232
9.2.3 Natural Products Active on the Central Nervous System.......................................... 236
9.2.4 Antifungal Compounds.............................................................................................. 238
9.2.5 Natural Products Active on Neglected Diseases’
Parasites.....................................................................................................................240
9.2.6 Miscellaneous Bioactive Compounds........................................................................244
9.3 Some Comments on Recent Advances in Natural Products
Research................................................................................................................................. 247
9.4 Concluding Remarks.............................................................................................................. 249
References....................................................................................................................................... 249
9.1 INTRODUCTION
Throughout history, humans have benefited from Nature’s resources and relied on natural
products (NPs) for the treatment of a wide variety of diseases. Plant-derived preparations or
mixtures constituted the primary source of biologically active NPs and served as the basis for
the foundation of medicinal systems. Even nowadays, traditional medicinal practices and eth-
nomedicinal knowledge play crucial roles in health care and are important for the discovery
of lead compounds and drug development programs (Cragg and Newman, 2013; Newman and
Cragg, 2016).
217
9.1.1 The Atlantic Forest Biome

Atlantic Forest is a biogeographic region that covers mostly Brazil and parts of Paraguay, Argentina
and Uruguay. The Brazilian Atlantic Forest was originally found along the Atlantic Ocean coast
from Rio Grande do Norte through Rio Grande do Sul states (Morellato and Haddad, 2000). This
biome is considered an important global hotspot and a priority for biodiversity conservation, that
has been severely degraded through the past five centuries, and hosts, nevertheless, ca. 15,782 plant
species, distributed in 2,257 genera and 348 families, which accounts for 5% of the world’s flora and
2% of the global endemic vascular plants (Myers et al., 2000; Stehmann et al., 2009). The unique-
ness and large proportion of endemic plant and animal species found in the Atlantic Forest biome
has been partly due to the isolation from the Amazon Rainforest by drier biomes as the Cerrado or
the even drier Caatinga (Joly et al., 2014).
Atlantic Forest is divided into two major regions, which are covered by typical vegetation types:
the Atlantic Rain Forest and the Atlantic Semi-deciduous Forest. The Atlantic Rain Forest occupies
a narrow strip of land along the coast covering mostly low to medium elevations (0–1,000 m) from
southern to northeastern Brazil and experiences a warm and wet climate without a dry season. On
the other hand, the Atlantic Semi-deciduous Forest occupies the inlands across a plateau (usually
higher than 600 m of elevation) from the country’s center to the southeast and experiences a sea-
sonal climate with a relatively severe dry season (generally from April to September). Additional
smaller ecosystems within Atlantic Forest include mangrove forests, high-altitude grasslands
(campos rupestres) and coastal dunes (restingas) with associated transitional vegetation types,
which contributes to the biome uniqueness (Morellato and Haddad, 2000).
Originally, the extension of Atlantic Forest covered around 150 million hectares. However, about
88% of the biomes’ original distribution area has been lost as a result of extensive human occupation
(Ribeiro et al., 2009). At the same time, the extraction of natural resources from Atlantic Forest dates
back to the colonization era when the Brazilian tree pau-brasil (Caesalpinia echinata, Fabaceae)
was extensively collected for extraction of red dye from the heartwood, composed of brazilin (1) and
brazileine (2) (Figure 9.1) pigments. Unfortunately, this resource was not exploited in a sustainable
way, and nowadays, the species is still classified as endangered. Subsequent deforestation mainly
FIGURE 9.1 Natural pigments and related compounds from Brazilian plants.
Plant Species from Atlantic Forest Biome 219
due to sugarcane, coffee and soybean plantations across 16th to 20th centuries have left less than
12% of the original vegetation. Geographic elements with little use to agriculture as the ridges
“Serra do Mar” and “Serra da Mantiqueira” in São Paulo, Rio de Janeiro and Minas Gerais states,
have played a key role in the vegetation conservation in these areas.
Although only small fragments of the Atlantic Forest remain, the Brazilian government and non-
governmental organizations (NGOs) have been taking actions for conservation and restoration to
mitigate this situation (Silva et al., 2010).
Bioprospecting studies have been conducted at NuBBE1 laboratories in the last 20 years aiming
at novel pharmacologically active plant-derived compounds from this beautiful biome. Important
results from Biota-FAPESP Program-funded research have largely contributed with reasonable and
science-based inputs to the establishment of efficient conservational policies, and recent data have
suggested that, over the past few decades, this ecosystem has been experiencing a positive balance
of forest change (Costa et al., 2017; Metzger and Casatti, 2006; Ribeiro et al., 2009).
The huge biodiversity of Atlantic Forest is attested by a recent research study (Flora do
Brasil, 2019), which describes 25 medicinal plant species (Table 9.1) naturally occurring in this
biome out of 71 plants still used as folk medicines in Brazil. These plants are indicated by the
RENISUS, a national list of medicinal plants to be adopted by the public healthcare system in
the country (National List of Medicinal Plants of Interest to the Unique System Health (SUS) –
Brasil, 2009).
The 25 species from the Atlantic Forest are listed as follows: Anacardium occidentale (“cajueiro”),
Ananas comosus (“abacaxi”), Apuleia ferrea (“pau-ferro”), Arrabidaea chica (“crajiru”), Baccharis
trimera (“carqueja”), Bauhinia spp. (“pata-de-vaca”), Bidens pilosa (“picão-preto”), Casearia
sylvestris (“guaçatonga”), Copaifera spp. (“Copaíba”), Cordia spp. (“erva-baleeira”), Costus spp.
(“cana-do-brejo”), Erythrina mulungu (mulungu”), Eugenia uniflora (“pitanga”), Jatropha gossy-
piifolia (“pinhão-roxo”), Lippia sidoides (“alecrim-pimenta”), Maytenus spp. (“espinheira-santa”),
Mikania spp. (“guaco”), Passiflora spp. (“maracujá”), Phyllanthus spp. (“quebra-pedra”), Portulaca
pilosa (“amor-crescido”), Schinus terebinthifolius (“aroeira-vermelha”), Solanum paniculatum
(“jurubeba”), Solidago microglossa (“arnica-brasileira”), Tabebuia avellanedae (“ipê-roxo”) and
Vernonia spp. (“assa-peixe”).
Many of these species have been the subject of multidisciplinary studies by research groups
throughout Brazil and abroad, which confirmed their remarkable chemical diversity and potential
medicinal uses. Notably, Atlantic Forest holds a fantastic biodiversity with many still-unknown spe-
cies, and thus, with great potential for chemical prospecting of active biomolecules to inspire lead
compounds for drug development and phytomedicines.
Aiming at the discovery of novel pharmacologically active plant-derived compounds from
Atlantic Forest, bioprospection studies have been conducted at NuBBE laboratories in the last
20 years. The most prominent achievements on the chemical diversity and biological investigations
from the plants collected in the Atlantic Forest obtained up to 2008 have been previously described
(Silva et al., 2010), whereas results from the past 10 years are reviewed in this chapter. All the plants
cited in this chapter are described as follows.
9.1.2 Natural Products in Brazil: Historical Benchmarks

Since the initial period right after the discovery of Brazil by Portuguese navigators in 1500,
the exploration of mineral and vegetal wealthy products from Brazilian lands was set and
focused initially on pau-brasil (Caesalpinia echinata), a plant species from Fabaceae known as
“ibirapitanga” to the native Indians, meaning red wood tree. The species trunk wood yields a
bright red pigment used since that period for tinting fabrics and as writing ink. The pigment red
1 NuBBE, Nucleus for Bioassays, Biosynthesis and Ecophysiology of Natural Products located at the Institute of Chemistry,
Sao Paulo State University (UNESP) in Araraquara, SP, Brazil.
TABLE 9.1
Anacardium occidentale (sin. Anacardium Anacardiaceae Cajueiro
microcarpum)
Ananas comosus (sin. Ananas sativa) Bromeliaceae Abacaxi
Aniba canelilla (sin. Aniba eliiptica) Lauraceae casca-preciosa
Apuleia ferrea Fabaceae pau-ferro
Baccharis trimera (sin. Baccharis genistelloides var. Asteraceae Carqueja
trimera)
Banisteriopsis caapi (sin. Banisteria caapi) Malpighiaceae caapi or yage
Bauhinia spp. Fabaceae pata-de-vaca
Bidens pilosa (sin. Bidens abadiae) Asteraceae picão-preto
Bixa orellana (sin. Bixa platycarpa) Bixaceae Urucum
Caesalpinia echinata (sin. Paubrasilia echinata) Fabaceae pau-brasil
Carapa guianensis (sin. Carapa llanocarti) Meliaceae Andiroba
Carapichea ipecacuanha (sin. Psychotria ipecacuanha) Rubiaceae ipecacuanha, cagosanga, poaia or ipeca
Casearia sylvestris (sin. Casearia subsessiliflora) Salicaceae Guaçatonga
Cinnamomum triplinerve (sin. Cinnamomum australe) Lauraceae Canela
Copaifera langsdorfii (sin. Copaiba langsdorfii) Fabaceae copaíba, copaibeira or pau-de-óleo
Copaifera spp. Fabaceae Copaiba
Cordia curassavica (sin.Cordia verbenacea) Boraginaceae erva-baleeira
Cordia spp. Boraginaceae erva-baleeira
Costus spp. Costaceae cana-do-brejo
Croton heliotropiifolius (sin. Croton salviifolius) Euphorbiaceae Velame
Cryptocarya mandioccana (sin. Oreodaphne polyantha) Lauraceae noz-moscada-do-Brasil
Cryptocarya. moschata Lauraceae Canela batalha
Dipterix odorata Fabaceae Cumaru
Erythrina verna (sin. Erythrina mulungu) Fabaceae Mulungu
Esenbeckia leiocarpa Rutaceae guarantã or guarataiá-vermelha
Eugenia uniflora Myrtaceae Pitanga
Fridericia chica (sin. Arrabidaea chica) Bignoniaceae Crajiru
Geissospermum leave (sin. Geissospermum martianum) Apocynaceae pau-pereira, quinarana or pau-forquilha
Genipa Americana (sin. Genipa venosa) Rubiaceae Jenipapo
Himatanthus lancifolius (sin. Plumeria lancifolia) Apocynaceae quina-mole or quina-branca,
Hymenaea courbaril (sin. Hymenaea multiflora) Fabaceae Its resins are known as jatobá or jutaí
Jatropha curcas (sin. Curcas lobata) Euphorbiaceae mandubiguaçu or purgueira or
pinhão-manso
Jatropha gossypiifolia (sin. Jatropha jacquinii) Euphorbiaceae pinhão-roxo
Jatropha ribifolia (sin. Adenoropium ribifolium) Euphorbiaceae Pinhão manso
Lippia sidoides Verbenaceae alecrim-pimenta
Maclura tinctoria (sin. Chlorophora tinctoria) Moraceae tatajuba, taiúva, espinheiro bravo
Maquira sclerophylla (sin. Olmedioperebea sclerophylla) Moraceae pau-tanino, rapé-de-índio or pau-de-índio
Maytenus ilicifolia (sin. Maytenus ilicifolia var. boliviana) Celastraceae espinheira santa,
Maytenus spp. Celastraceae espinheira-santa
Mikania spp. Astereaceae Guaco
Mimosa tenuiflora (sin. Mimosa hostilis) Fabaceae Vinho-de-Jurema
Moringa oleifera (sin. Hyperanthera moringa) Moringaceae drumstick tree
Ouratea multiflora Ochnaceae
Passiflora spp. Passifloraceae Maracuja
(Continued)

Peperomia obtusifolia (sin. Peperomia petenensis) Piperaceae Pepper face
Phyllanthus spp. Phyllanthaceae quebra-pedra
Pilocarpus microphyllus Rutaceae jaborandi, from the Tupi language
Ya-bor-andi
Piper crassinervium (sin. Piper novae-helvetiae) Piperaceae Pariparoba, jaguarandi or jaguarandy
Piper tuberculatum (sin. Artanthe decurrens) Piperaceae pimenta-longa
Piptadenia peregrina (sin. Anadenanthera peregrina) Fabaceae Paricá, yopo, jopo or cohoba
Plectranthus barbatus (sin. Plectranthus Lamiaceae Brazilian-boldo or false-boldo.
pseudobarbatus)
Porcelia macrocarpa (sin. Porcelia goyazensis) Annonaceae pindaíba, pindaíba-do-mato or
banana-de-macaco
Portulaca pilosa (sin. Portulaca mundula) Portulacaceae amor-crescido
Psychotria viridis (sin. Uragoga viridis) Rubiaceae chacruna or chacrona
Pterogyne nitens Fabaceae tipá, viraró, cocal or amendoinzeiro
Schinus terebinthifolia (sin. Schinus mellisii) Anacardiaceae aroeira-vermelha
Solanum grandiflorum Solanaceae Lobeira
Solanum paniculatum (sin. Solanum chloroleucum) Solanaceae Jurubeba
Solidago chilensis (sin. Solidago microglossa) Asteraceae arnica-brasileira
Spondias tuberosa Anacardiaceae Umbu
Stemodia foliosa Plantaginaceae Meladinha
Strychnos castelnaeana (sin. Strychnos castelnaei) Loganiaceae
Strychnos guianensis (sin. Toxicaria americana) Loganiaceae
Swartzia langsdorffii (sin. Swartzia brasiliensis) Fabaceae banana-de-papagaio, jacarandá-banana,
or jacarandá-de-sangue
Tabebuia avellanedae Bignoniaceae ipê-roxo
Tetrapterys mucronata (sin. Tetrapterys silvatica) Malpighiaceae
Vernonia spp. Asteraceae assa-peixe
Virola calophylla (sin. Virola lepidota) Myristicaceae Epená
Virola calophylloidea Myristicaceae
Virola theiodora (sin. Virola rufula) Myristicaceae yakee, epena and nyakwana
color is mainly associated to the presence of brazilin (1) from the crude extract, which gives bra-
zilein (2) (Figure 9.1), that is the compound’s oxidation derivative formed during the extraction
procedure (Morsingh and Robinson, 1970).
Important historical benchmarks from previous centuries contributed to the scenario that favored
the great navigations period and the discovery of America in 1492 and Brazil in 1500. Among exam-
ples is included the Crusades, which expanded the limits of known lands to the East and brought vast
information of the abundance of valuable goods as gold, pigments and spices, especially from India
and China since the 8th century. Marco Polo’s excursions and his reports on the precious products he
found during the years he lived in China gave additional impulse to the Europe interest on the explo-
ration and trade of eastern products, which brought wealth and prosperity to thousands of European
traders for a long period. Nevertheless, the taking of Constantinople by the Turkish in 1453 blocked the
path to the East and brought collapse to a relevant part of Europe’s economy at the time. Such events
gave the necessary impulse to navigators, especially from Portugal, Spain and Italy, to find a new way
to India, China and further eastern regions which had been providing Europe with goods to support its
economic activities and relative political stability (Polo, 1958/1982).
Several fleets departed from Portugal, Spain and Italy in the end of the 15th century as part of a
huge effort for the discovery of a new maritime route to the East Indies. The arrival of Columbus in
Central America in 1492 was initially thought to take place in the extreme orient, as the navigators
made their early incursions to the inlands to find cinnamon-smelling bushes and rhubarb, a precious
Chinese medicinal plant used as a cathartic. Such findings mistakenly corroborated the navigators’
expectations to have discovered the so much desired new path to the East, which was accomplished
years later by Vasco da Gama, who finally rode across the Cape of Storms, later renamed as Cape
of Good Hope in South Africa (Butler and Moffett, 1995).
The motivation to gain access to the valuable eastern spices, pigments and other valuable goods
fairly explains the avidity on the exploration of pau-brasil as soon as Portugal realized that the
discovery of Brazil and profiteering from the colony represented an additional important source
of commercial products and wealth to the reign. In addition to pau-brasil tincture, Portuguese
colonizers explored pigments from Bixa orellana (Bixaceae), known as “urucum”, which means
red in Tupi language, and Genipa americana (Rubiaceae), known as “jenipapo”, in addition to
“andiroba” oil, used for several purposes including preparation of soaps, protection of furniture
against insects attack, and as lighting fuel. Each specimen of B. orellana may bear thousands of
sea-urchin-like fruits plenty of seeds rich in bixin (3) (Figure 9.1). This norcarotenoid is still used
as a colorant for food and as a sunscreen component and was exported to Europe in huge amounts
during the 16th and 17th centuries. The extraction of bixin was often carried out using andiroba
oil, obtained from Carapa guianensis (Meliaceae) fruits, which are rich in limonoids as andirobin
(4) (Figure 9.1), a tetranortriterpene derived from euphane triterpenes. Jenipapo ripe fruits contain
genipin (5) (Figure 9.1), an iridoid from the fruit sap used in body paintings by native Indians.
Although genipin is colorless, when reacting with skin proteins produces a black color exten-
sively employed for tattoos associated to native Indians rituals and religious ceremonies (Barber
et al., 1961; Djerassi et al., 1961; Ollis et al., 1970).
Portuguese colonizers rapidly realized the potential of Brazilian flora as a source of novel inter-
esting products as well as the valuable knowledge on plant species provided by native Indians,
especially those associated to plant pigments and poisons. Chlorophora tinctoria (Moraceae) was
known in the Indian language as “tatajuba”, which means firewood or fire-colored wood. The fla-
vonol morin (6) was shown to be responsible for the color (Figure 9.1) and became soon became a
major commodity exported to Europe to be used as a fabric pigment (Pinto, 1995).
Resins, balms and spices were also the object of great interest and value to the Portuguese and
Spanish colonizers. Cinnamon was one of the most valuable spices and the discovery of cinnamon-
smelling trees by G. Pizzaro during expeditions in the Amazon basin, led him to think he would
finally break the spices monopoly by Eastern traders. The newly discovered species in fact was
not Cinnamomum australe, the original source of cinnamon found in China and India, but Aniba
canelilla (Lauraceae). Interesting comparative phytochemical studies on such species disclosed
the marked differences in their chemical constitution. Cinnamic aldehyde has been shown as the
major component of the true cinnamon bark extract, whereas the studies carried out by Gottlieb
and Magalhaes (1959) disclosed the presence of nitrophenylethane (7) as the major constituent
of A. canelilla along with eugenol (8) and methyl-eugenol (9) (Figure 9.2). Nitrophenylethane is
responsible for the cinnamon smell of A. canelilla and the first odoriferous nitro-derivative so far
described in the literature. The numerous usages of resins, essences and balms as pain relievers
and in religious ceremonies contributed to the enormous interest in the discovery of novel sources,
and Brazilian plant species played a major role in this effort. The importance of rosin in ancient
civilizations as in Greece, Macedonia and Egypt continued through the years to the navy industry
in England who were compelled to explore North America forests in the search for new sources.
Rosin or crude turpentine has been obtained as a resinous gum rich in abietic acid (10) (Figure 9.2)
among other terpenoids mainly from Pinus trees and other conifers. Copal from Brazil has been
considered as a rosin equivalent, especially in the Amazon region, where this resin has been used
as incense and in boat construction as a sealant or to improve soldering quality. Such resins were
FIGURE 9.2 Flavoring and resin-derived compounds from Brazilian plants.
known as “jatobá” or “jutaí” and were obtained mainly from Hymenea courbaril, which presents
copalic acid (11) (Figure 9.2) as amajor constituent. This compound has also been isolated from
Copaifera langsdorffii, considered the main source of copaiba oil, a balm that has been exten-
sively used to treat inflammation and as a wound-healing agent. The chemical studies on Brazilian
Copaifera specieswere prompted by continuous and current use as folk medicine through the years
which disclosed the presence of further diterpene acids such as cativic (12) and danelic acids (13)
(Figure 9.2) in their resin and gave important contribution on the detection of adulterants in copaiba
oils commercialized in Brazil. Dipterix odorata, a native tree to the Amazon basin that may reach
50 m in height, is known as cumaru and is widespread in adjacent regions to the Amazon. The fruits
are known as fava tonka and were exported to Europe for their coumarin content (14) (Figure 9.2),
and have been used as a tobacco odorant and is still widely used in the food industry as a flavoring
agent (Duke and duCellier, 1993; Gottlieb and Magalhaes, 1959; Gottlieb and Mors, 1978; Nakano
and Djerassi, 1961; Veiga et al., 1997).
Native Indians developed peculiar and efficient strategies for animal chasing, which included the
use of venom, known as “curare” on the arrow tip. Several types of curare were used by different
tribes, which were innocuous by oral administration, but paralyzed the animal within seconds when
injected into the blood circulation, as described by Gottlieb and Mors (1978). “Curare” or “urari”
was obtained from the “urariuva” tree in the region of Orinoco river. Indians from the “Ticunas”
tribe used curare from the plant Strychnos castelnaeana, whose extract was taken to Europe in
1745 for scientific investigation of the species chemical constituents. Strychnos guianensis, formerly
described as Toxicaria americana, and Strychnos toxifera were also used as sources of curare
and these plants yielded d-tubocurarine (15) and toxiferine (16) (Figure 9.3) as their active com-
pounds, respectively (Cannali and Vieira, 1967; Repke and Torres, 2006). Hallucinogenic bever-
ages and snuffs also played an important role in cultural and religious practices of South American
Indians. “Paricá” was also known as “yopo”, “jopo” or “cohoba” and was prepared from roasted
and grounded seeds of Piptadenia peregrina (synonym of Anadenanthera peregrina, Fabaceae),
which produced bufotenine (17) (Figure 9.3) as active ingredient. Whereas the analog dimethyl-
tryptamine (18) (Figure 9.3) was shown to be a major active constituent of both snuffs prepared
from Olmedioperebea sclerophylla and “Vinho-de-Jurema” (Mimosa hostilis, Fabaceae), a wide-
spread beverage used mainly by Pankararu, Xucuru and Kariri-xocó Indian tribes among others
(Ott, 2002; Pachter, et al., 1959).
Additional sources of hallucinogenic snuffs have been associated with the Myristicaceae
family, especially from the genus Virola. Although myristicaceous seeds, especially from Virola
and Iryanthera species have been extensively investigated by South American researchers, their
chemical profiles are mostly associated with lignans, neolignans and other shikimic acid-derived
FIGURE 9.3 Poisonous and hallucinogenic compounds from Apocynaceae, Fabaceae and Myristicaceae
Brazilian plant species.
biosynthetic pathways. These natural products failed to support the hallucinogenic properties of
Virola species used in snuff preparation. Indian tribes of the northwest Amazon Basin as “Puinaves”
and “Waiká” were reported to use the blood-red bark resin of Virola calophylla, V. calophylloidea
and V. theiodora in the preparation of snuffs known as “yakee”, “epena” and “nyakwana”, where
tryptamines are present in high concentrations, with 5-methoxy-N,N-dimethyltryptamine (19)
(Figure 9.3) as the major constituent. Hallucinogenic preparations still in current use in religious
rituals in Brazil as “ayhuasca” include plants containing tryptamines as Psychotria viridis and
Banisteria caapi (Barker et al., 2012; Schultes, 1969).
Important scientific expeditions took place during the 18th century aiming the discovery and
description of Brazilian geography, flora and fauna. The work “Historia Naturalis Brasiliae”
resulted from observations of the European researchers Georg Marcgrave, Johannes de Laet and the
physician Willem Piso, whose remarkable contribution to the knowledge on medicinal plants from
South America represents our first natural history compendium. Subsequent expeditions played
important roles in the gathering and systematization of accumulated information, as those carried
out by Johann B. Spix and the botanist Carl Friederich von Martius, who suggested the invita-
tion to the German pharmacist Theodore Peckolt to join the effort in the study of Brazilian flora
(Freedberg, 1999). Peckolt came to Brazil in 1847 and was a pioneer in the systematic study of
medicinal plants aimed mostly to the preparation and commercialization of remedies, which were
often carried out in the laboratories of ancient pharmacies or “boticas”. Remarkable results from
this period include the study of Plumeria lancifolia (Apocynaceae), a medicinal plant used by the
Guarani Indians to treat malaria. P. lancifolia, known as “quina-mole” or “quina-branca”, was also
used as a folk medicine to treat inflammation, gastric problems and women’s’ reproductive organs
diseases. The plant’s chemical study led to the isolation of plumeride (20, formerly named as ago-
niadin) (Figure 9.4) by Peckolt, but the structural elucidation only occurred 88 years later and is
considered the first isolated iridoid from a natural source (Santos et al., 1998; Halpen and Schmid,
1958). Additional important scientific work was carried out by the pharmacist Ezequiel Correia
dos Santos, who obtained pereirin in 1838 from Geissospermum leavis (Apocynaceae) barks, a
medicinal plant known as “pau-pereira”, “quinarana” or “pau-forquilha”, and used to treat fever
and malaria. Pereirin has been considered the first alkaloid isolated in Brazil and several studies on
pau-pereira that revealed to be a mixture of indole alkaloids with geissospermine (21) (Figure 9.4)
as the major constituent. Recent studies have demonstrated antiviral properties and potential against
AIDS and herpes infections (Pinto et al., 2002).
By the end of the 19th and beginning of the 20th centuries, phytochemical investigations on
Brazilian medicinal plants at the School of Medicine in Rio de Janeiro and School of Pharmacy in
Sao Paulo were initiated and gave important contributions as the studies on Solanum grandiflorum,
FIGURE 9.4 Bioactive compounds from Brazilian medicinal plants.
known as “lobeira”, by Domingos José Freire Junior, and coffee (Coffea arabica) by Pedro Batista
de Andrade. S. grandiflorum bears fruits rich in vitamins which constitute the basis of “guará”
wolf diet, hence the popular name is wolf fruit (“fruta do lobo”). The plant’s pulp has been used to
control diabetes and afforded the steroidal alkaloid grandiflorin, later renamed as solasonine (22)
(Figure 9.4) (Mors, 1997; Motidome et al., 1970).
Additional studies on Brazilian plants were carried out mainly due to their medicinal folk uses
and have revealed several interesting natural products and confirmed their marked chemo-diver-
sity. Ipecacuanha (Psychotria ipecacuanha, Rubiaceae) also known as “cagosanga”, “poaia”,
or “ipeca”, is a plant widespread throughout North and Northeastern regions in Brazil. Syrup
prepared from the roots presented strong emetic properties and drew attention of Europeans
for use as a purgative and antidote for poisoning. The high alkaloids content such as emetin
(23) and cephaeline (24) (Figure 9.4) was associated to the emetic activity. Further studies on
P. ipecacuanha described the antiprotozoal potential of emetin and led to the development of
dehydroemetin to treat amebiasis with less nausea side effects than emetin (Cushny, 1918; Gupta
and Siminovitch, 1977).
Cashew fruits (A. occidentale) have long been used by native Indians as an ingredient of
fermented beverages. Their nut peel is strongly allergenic due to the high content of lipo-
philic acetyl-salicylic acid derivatives in its oil, similar to urushiol. Such compounds are
known as anacardic acids (25) (Figure 9.4) and represent a mixture of organic acids with
saturated or unsaturated C15 to C17 side chains. Their strong bactericidal activity against
Streptococcus mutans and other gram-positive bacteria, including methicillin-resistant
Staphylococcus aureus (MRSA) strains, demonstrated the potential to treat dental cavity and
tuberculosis (Mathias, 1975; WHO, 2016).
Pilocarpus microphyllus (Rutaceae) is a shrub widespread in North and Northeast regions in
Brazil, known as “jaborandi”, from the Tupi language Ya-bor-andi, which means slobber-inducing
plant. The extracts have been used as a folk medicine to treat bronchitis and rheumatism and induce
FIGURE 9.5 Bioactive compounds from Brazilian medicinal plants used in pharmaceutical products.
intense salivation and sweat due to the alkaloid pilocarpine (26) (Figure 9.5), a muscarinic receptor
agonist found in the leaves. Pilocarpine has been used to treat glaucoma for over 100 years and is
on the World Health Organization’s (WHO’s) List of Essential Medicines. P. microphyllus extrac-
tivism is associated to plantations in Maranhao, to provide leaves for extraction and commercial
production of pilocarpine, especially by Merck Company, which has dominated this active principle
market for a long period (WHO, 2016).
Although Brazilian plant diversity is enormous and provides a plethora of bioactive natural
products with equally huge chemo-diversity, this biodiversityis still under-explored consider-
ing the development of phytotherapeutic products. Traditional knowledge on medicinal plants
has played an important role in the selection of promising plant species for detailed chemical-
pharmacological-toxicological aspects, essential for phytoceuticals development. Nevertheless,
very few plant species have gone through systematic investigations to afford effective and safe
commercial products. Among these, Cordia verbenaceae, popularly known as “erva-baleeira”,
constitutes an emblematic example, considering the plant’s beneficial properties to treat inflam-
mation have long been described in “De Medicina Brasiliensi” by Willem Piso in the 18th cen-
tury. Additionally, fishermen and coastal populations have also used “erva-baleeira” extract
or macerate for wound healing and treating arthritis, rheumatism and muscle pain. Integrated
multidisciplinary studies disclosed the sesquiterpenes α-humulene (27) and trans-cariophyllene
(28) (Figure 9.5) as the active constituents of C. verbenaceae essential oil, associated with the
described anti-inflammatory properties. Further investigations on the chemistry, pharmacology,
toxicology, pharmacokinetics and additional related issues completed the requirements for regis-
tering the new phytotherapy product containing the essential oil of C. verbenaceae. Joint efforts
of researchers at public universities in Sao Paulo State, public research funding agencies and Aché
Pharmaceutical Company have thus proven successful and resulted in a topical anti-inflammatory
cream to treat myofascial pain, repetitive effort lesion, arthrosis and other painful inflammatory
conditions (Basile et al., 1989; Fernandes et al., 2007).
Such findings suggest a key role played by plant extracts and their constituents in the expansion
of knowledge on the chemistry and pharmacology of Brazilian biodiversity since the early years in
Brazil colonization through the last five centuries and stimulates further bioprospection efforts by
means of integrative and collaborative research. This scenario contributes to the discovery of novel
bioactive natural products through a rational approach, which might lead to value-added bioprod-
ucts from Brazilian biodiversity along with conservational actions toward protection of biomes.
9.2 BIOPROSPECTING PLANTS FROM ATLANTIC FOREST

As part of the NuBBE research program devoted to bioprospecting bioactive metabolites of plant
species from the Brazilian Atlantic Forest, several plants have been collected, identified, extracted,
biologically screened and chemically analyzed focusing on bioactive compounds. In this chapter we
describe compounds with several activities such as antioxidant, anti-inflammatory, chemopreven-
tive, antifungal, antiprotozoal, acetylcholinesterase inhibitor, antiparasitic, cytotoxic (in vitro toxic-
ity to malignant cells), antitumor (in vivo) and anticancer.
9.2.1 Natural Products Active on Redox Processes, Inflammation,

Chemoprevention and Related Processes
Redox reactions are involved in important physiological processes associated to function and
defense of organisms and include the generation of reactive oxygen species (ROS), such as free radi-
cal superoxide anion (O2•‒), hydrogen peroxide (H2O2) and hypochlorous acid (HOCl), in addition to
reactive nitrogen species (RNS) as peroxynitrite (ONOO‒) and nitric oxide (NO•). Normal function-
ing of living systems requires a precise and delicate balance between pro- and antioxidant agents,
which is often disturbed by biotic and abiotic conditions such as irradiation, atmospheric and food
pollutants or products from metabolism. These conditions may enhance ROS and RNS generation,
and ultimately lead to oxidative stress. Such species are known to participate in degenerative pro-
cesses related to aging and in the etiology or progress of several diseases as myocardial and cerebral
ischemia, arteriosclerosis, diabetes, rheumatoid arthritis, inflammation and cancer. Therefore, the
search for natural antioxidants may represent a strategy for the discovery of new drugs, since they
could decrease the oxidative stress caused by ROS/RNS excessive production (Vellosa et al., 2011).
Several assays were developed to screen natural products as antioxidant agents exploring their
abilities to scavenge free radicals. The free radical scavenging assays using DPPH• (2,2-diphenyl-
1-picrylhydrazyl-hydrate) and ABTS•+ [2,2′-azinobis (3-ethylenebenzothiazoline-6-sulfonic acid)]
radicals have been often used to evaluate the anti-radical potential of plant extracts and isolated
compounds. The DPPH• assay is based on the transfer of a hydrogen radical to DPPH•, which reacts
as a probe, and the antiradical activity is measured through the decrease of absorbance at 517 nm.
In the ABTS•+ assay, the reaction mechanism is based only on electron transfer and the decrease
of absorbance is measured at 734 nm at several sample concentrations (Zeraik et al., 2016a,b).
Additional scavenging assays include the use of H2O2, HOCl, taurine chloramine (TauCl), O2•‒ and
NO•. H2O2 is a lesive agent due to the compound’s easy decomposition to HO •, whereas HOCl is a
strong oxidant, which could react with important constituents of biological systems such as nucleo-
tides and amino acids. The reaction between biological amines and HOCl generates chloramines,
oxidant agents whose reactivity depends on the substance’s lipophilicity. Superoxide anion (O2•‒)
is a free radical generated during cellular respiration or pathological processes as inflammatory
diseases, and may react with NO• to generate peroxinitrite (ONOO‒), which triggers oxidation of
low-density lipoprotein (LDL), a key process in atherosclerosis etiology (Vellosa et al., 2015).
In addition to the assays described above, which involve scavenging of radical species in the
evaluation of antioxidant properties, the use of electrochemical methods has been demonstrated
as a useful alternative, which could integrate the evaluation of antioxidant properties by cyclic
voltammetry and the identification and quantification of active compounds by high-performance
liquid chromatography coupled with electrochemical detection (HPLC-ED). In this case, the elec-
trochemical response is directly related to the structure of the antioxidant and the potential required
for the product’s oxidation, and establishes the chemical profile based on redox properties of the
analyzed sample (Santos et al. 2010b).
Cellular models have also been used for the evaluation of antioxidant activity, for example, the
investigation of oxidative damage in biological membranes using erythrocytes, since they present
high polyunsaturated fatty acid contents in their membranes, in addition to high cellular oxygen and
hemoglobin concentrations. Erythrocyte lipid peroxidation is associated with a variety of pathologi-
cal events; thus, this evaluation may bring useful information for diagnosis and treatments for such
conditions (Vellosa et al., 2011).
The antioxidant properties of extracts and their partially purified fractions, in addition to iso-
lated compounds from plants collected in the Atlantic Forest biome, have been evaluated by using
the assays described above, and represent an important alternative toward a rational exploration of
Brazilian biodiversity by adding value to natural products.
Pterogyne nitens (Fabaceae) is a plant popularly known in Brazil as “tipá”, “viraró”, “cocal”,
“amendoinzeiro” and “amendoim-bravo”, which can be found in Cerrado, Caatinga and Atlantic
FIGURE 9.6 Polyphenols and guanidine alkaloids isolated from stem barks and flowers of Pterogyne nitens.
Forest biomes. Twenty extracts and fractions obtained from roots, branches, unripe fruits and
stem bark of P. nitens, collected in the Botanic Garden of São Paulo (Atlantic Forest biome,
São Paulo, Brazil) were screened for free radical scavenging activity using ABTS•+ and DPPH•
radicals colorimetric assays and a TLC bleaching test nebulized with β-carotene solution.
The ethyl acetate fraction from stem bark presented the strongest activity and was selected
for further investigation. Subsequent chromatographic fractionation of this sample yielded
three flavonols, myricetin (29), mirycetrin (30) and quercitrin (31) (Figure 9.6), which showed
potent antiradical activity against DPPH• and ABTS•+, besides inhibiting β-carotene bleach-
ing, which explains the strong antioxidant activity observed for the original fraction (Regasini
et al., 2008a).
Flowers of P. nitens afforded nine phenol derivatives and two guanidine alkaloids which were
assessed for the ability to scavenge free radicals and to inhibit myeloperoxidase (MPO), an abun-
dant heme-enzyme in polymorphonuclear cells (PMNs), considered a key macromolecule in redox
processes and in the nonspecific immune response to several agents. MPO triggers the conver-
sion of H2O2 and chloride anion to water and HOCl. The excess of MPO activity and subsequent
overproduction of HOCl leads to oxidative stress to PMNs, which is associated to several inflam-
matory processes, including rheumatoid arthritis and cystic fibrosis. Therefore, MPO inhibitors
might be considered as prototypes for the development of anti-inflammatory agents. Among the
tested compounds, quercetin-3-O-sophoroside (32) and gallic acid (33) (Figure 9.6) displayed strong
MPO inhibition and antiradicalar activity against DPPH• and ABTS•+. On the other hand, the anti-
oxidant activity of guanidine alkaloids, pterogynine (34) and pterogynidine (35) (Figure 9.6) was
also evaluated, but they did not scavenge DPPH• or ABTS•+ radicals efficiently, which corroborates
the importance of structural features as phenol hydroxy groups for a strong antioxidant activity
(Regasini et al., 2008b).
The mutagenic and antimutagenic potential of ethyl acetate (EtOAc), n- butanol (BuOH) and
hydroalcoholic (HA) fractions of the ethanol extract from P. nitens leaves were evaluated using
Tradescantia pallida micronuclei assay. This is a simple and reliable assay, where T. pallida cut-
tings were treated with EtOAc, BuOH and HA fractions independently, and tetrads from the inflo-
rescences were examined for micronuclei after exposure to test samples. Fractions BuOH and HA
demonstrated mutagenic effects. Since the EtOAc fraction showed mutagenicity only at the higher
concentration tested (0.460 mg mL−1), the sample’s antimutagenic potential was investigated and
detected at lower concentrations, 0.115 and 0.230 mg mL−1. BuOH and HA samples were fraction-
ated by column chromatography resulting in the reisolation of guanidine alkaloids 34 and 35, previ-
ously obtained from P. nitens flowers (Ferreira et al., 2009).
The ethanol extract from P. nitens leaves exhibited strong antioxidant potential
against ABTS•+, DPPH• and HOCl, as indicated by low IC50 values. In the presence of
2′-azobis(2-amidinopropane) hydrochloride (AAPH) radical, a hemolysis-stimulating agent in
erythrocytes (red blood cells), the ethanol extract and the flavonol glucoside rutin, often used as
FIGURE 9.7 Flavonoids isolated from leaves and fruits of Pterogyne nitens.
positive control, exhibited anti-hemolytic activity only at low concentrations. However, in the
absence of AAPH radical, the tested sample triggered hemolysis over erythrocytes (Pasquini-
Netto et al., 2012).
Additional flavonoids isolated from leaves of P. nitens as kaempferol (36), quercetin (37) and
isoquercitrin (38) (Figure 9.7) were also assessed as ROS scavenging agents using H2O2, HOCl,
TauCl, O2•‒ and NO• assays. The evaluated samples exhibited moderate potential on the inhibition
of NO•, although none of them were able to interact with H2O2, which would be evidence of the
complexity of such interactions and corroborates the influence of structural features other than phe-
nolic hydroxy groups for effective activity toward different radical species. Quercetin (37), one of
the strongest antioxidant flavonoids, was also the most efficient agent against HOCl, TauCl and O2•‒
(Vellosa et al., 2011). Afzelin (39), kaempferitrin (40) and pterogynoside (41) (Figure 9.7), isolated
from fruits of P. nitens, were also evaluated and showed moderate scavenging abilities toward O2•‒,
HOCl and TauCl (Vellosa et al., 2015).
In addition, compounds 36–41 were evaluated in red blood cells as well, and displayed hemolytic
effects, and they inhibited hemolysis in the presence of AAPH. However, these substances intensi-
fied the hemolytic activity when tested in a mixture with HOCl. Such data could be explained by
a possible higher affinity between flavonoids and AAPH, reducing the hemolytic effects of both
agents and producing less damaging products (Vellosa et al., 2011, 2015).
Flavonoids 39–41 inhibited TauCl, produced by the stimulation of neutrophils using phorbol
12-myristate13-acetate (PMA), although these flavonoids have also promoted the death of neutro-
phils in the presence or absence of PMA. Neutrophils are important ROS-generating systems, act-
ing via oxidative burst, and constitute important components of tissue injury during inflammatory
response. Therefore, despite their well-known scavenging action toward free radicals and oxidants,
these compounds could be harmful to living organisms at the tested concentrations, through their
action over erythrocytes and neutrophils (Vellosa et al., 2015).
The flavonol isoquercitrin (38) and flavone pedalitin (42) (Figure 9.7), also isolated from
P. nitens, exhibited strong antioxidant activity by inhibiting β-carotene bleaching in a TLC assay.
In addition, a fast, low-cost and convenient cyclic voltammetry screening, and the combination of
HPLC with an electrochemical detector (HPLC-ED), confirmed the antioxidant activity of these
flavonoids. Structural features as ortho-dihydroxy groups, α-, β-unsaturated carbonyl moiety
(ring C) and β-hydroxyketone (rings A and B) are known to play a key role in antioxidant proper-
ties, since they enhance the radical stabilization after the first oxidation steps. In this regard, the use
of HPLC-ED, already established as a qualitative and quantitative technique to detect antioxidant
small molecules, is considered as a useful assay for the determination of antioxidants in complex
matrixes without previous sample preparation or pre-concentration, since this technique offers high
sensitivity and easy operation (Okumura et al., 2012).
Plectranthus barbatus Andrews (Lamiaceae) is a popular medicinal plant used to treat gastro-
intestinal and hepatic disorders and is known as “Brazilian-boldo” or “false-boldo”. The aqueous
extract from P. barbatus leaves, collected in Cajobi (São Paulo, Brazil), presented a significant
FIGURE 9.8 Clerodane diterpenes isolated from Casearia sylvestris.
free radical scavenging activity toward DPPH• and •OH, besides iron chelating mediated activity,
preventing the formation of the oxidant Fe2+-bathophenanthroline disulfonic acid (BPS) complex.
Moreover, this extract protected mitochondria against Fe2+-citrate-mediated membrane lipid peroxi-
dation, since cell swelling and malondialdehyde production was avoided with the activity persisting
even after simulation of the product’s passage through the digestive tract (Maioli et al., 2010).
C. sylvestris Swartz (Salicaceae) is a tree widely distributed in a variety of ecosystems from the
Cerrado to the tropical Atlantic Forest and the equatorial Amazon forest known as “guaçatonga”
and widely used in folk medicine to treat ulcer, inflammation and tumors. Their leaves are rich in
clerodane diterpenes as casearins and caseargrewiins, which have shown cytotoxic and antifungal
activities in several studies (Oberlies et al., 2002; Santos et al., 2010a).
Reinvestigation of C. sylvestris afforded caseargrewiin F (43) (Figure 9.8) from the leaves’ etha-
nol extract (ELCS), collected at Parque Estadual Carlos Botelho (São Miguel Arcanjo, São Paulo,
Brazil). The evaluation of chemopreventive properties suggested a protective effect of ELCS in
micronucleus (MN) test and comet assay in mice. Cyclophosphamide (CP) was used in both tests
to damage DNA and compound 43 showed a protective effect only in the comet assay. The MN
test reveals more drastic lesions in chromosome level (mutagenicity), while the comet assay detects
genomic lesions that are susceptible to DNA repair (genotoxicity). The tests were also performed
without the injection of CP, indicating that ELCS and compound 43 triggered DNA damage only at
high concentrations (Oliveira et al., 2009).
Further investigation on the mutagenic properties of C. sylvestris was carried out using
Tradescantia micronucleus assay, the MN test in mouse bone marrow cells, and the comet assay.
The same extract (ELCS) and casearin X (44) (Figure 9.8), another clerodane diterpene isolated
from this extract, were evaluated as protective agents against the harmful effects of airborne pol-
lutants from sugarcane burning. The mutagenic agent in this case was total suspended particulate
(TSP) from air, collected near Araraquara (São Paulo, Brazil) during the sugarcane-burning sea-
son. ELCS exhibited antimutagenic activity in the Tradescantia micronucleus assay and was able
to reduce DNA damage caused by TSP in the MN test and in the comet assay, while compound
44 reduced only DNA damage assessed by the comet assay. Such data suggested that C. sylvestris
extract and the isolated diterpenes might act by different mechanisms to protect DNA against dam-
age, including repairable and non-repairable damages (Prieto et al., 2012).
Casearin B (45) (Figure 9.8), also isolated from leaves of C. sylvestris, showed genotoxicity in
HepG2 cells (comet assay) at concentrations higher than 0.30 µM when incubated with the formami-
dopyrimidine-DNA glycosylase (FPG) enzyme.Whereas, DNA damage caused by H2O2 in HepG2
cells in both pre- and posttreatment experiments was reduced. Compound 45 was not mutagenic
to S. typhimurium strains TA98 and TA102, used in the Ames test, and exhibited strong inhibitory
FIGURE 9.9 Quinonemethide triterpenes isolated from Maytenus ilicifolia.
activity against aflatoxin B1 in TA 98, in addition to moderate inhibitory activity against mytomicin
C in TA 102 (antimutagenicity assays). Casearin B also displayed antioxidant activity, since the
compound was able to reduce ROS generated by H2O2 in the 2′,7′-dichlorodihydrofluorescein diace-
tate (DCFDA) assay. However, compound 45 was less effective in the inhibition of DCFH oxidation
than the positive control quercetin. In this test, DCFDA, which is a redox inactive compound, is
converted to DCFH (active ROS) by an esterase inside the cell (HepG2 cell). H2O2 and other ROS
oxidize intracellular DCFH (nonfluorescent) to DCF (fluorescent), which is then measured in the
fluorescence assay (Prieto et al., 2013).
Maytenus ilicifolia (Celastraceae), known as “espinheira santa”, is spread in tropical and sub-
tropical parts of the Atlantic Forest and is widely used in traditional medicine as anti-inflammatory,
analgesic and antiulcerogenic (Costa et al., 2008; Jorge et al., 2004). Their roots are known to accu-
mulate quinonemethide triterpenes, which constitute chemo-taxonomical markers of this genus and
present several biological activities. HPLC-DAD analyses of M. ilicifolia extracts obtained from
root barks of adult plants (E2) and roots of seedlings (E4) indicated the presence of quinonemethide
triterpenes and phenolic compounds as main chemical constituents. The DPPH• assay indicated that
rutin, a major flavonoid from the extracts under investigation, exhibited higher antioxidant activity
than the quinonemethide triterpenes maytenin (46) and pristimerin (47) (Figure 9.9), isolated from
the root barks of M. ilicifolia. Such results are probably related to different structural features, since
quinonemethide triterpenes present an α,β-unsaturated carbonyl moiety with extended conjugation
through ring B, whereas rutin bears a catechol moiety in ring B in addition to an α,β-unsaturated
carbonyl moiety conjugated to ring B. Their antioxidant properties were also monitored by voltam-
metry screening, which corroborated the results obtained by the DPPH• scavenging assay. In addi-
tion, cyclic voltammograms associated to HPLC-ED analyses suggested a synergistic interaction
between quinonemethide triterpenes and flavonols, as indicated by the mixture of rutin with com-
pound 47, or rutin with M. ilicifolia extracts, which demonstrated, in both cases, enhanced antioxi-
dant activity than the individual samples (Santos et al. 2010b).
Spondias tuberosa (Anacardiaceae) is a native plant from Northeast of Brazil spread all over arid
and semi-arid regions and is especially useful for accumulating water in the species’ tuberous roots,
which provides continuous fruit loads even in drought seasons. The fruits are popularly known as
“umbu” and may be consumed fresh, as juice, ice cream, sweet, jam or as the traditional “umbu-
zada” (fruit pulp boiled with milk and sugar). Fractionation of umbu pulp methanol extract yielded
two novel phenolic glucosides, 3,4-dihydroxyphenylethanol-5-β-d-glucose (48) and 5-hydroxyl,4-
methoxy-3-O-β-d-glucose benzoic acid (49) (Figure 9.10), along with five known compounds, 33
(Figure 9.6) and 50–53 (Figure 9.10). The isolated substances exhibited strong antioxidant proper-
ties, which were evaluated by DPPH•, ABTS•+ and ORAC assays, while compound 48 showed the
highest capacity to prevent the oxidative effects of radicals generated by AAPH on fluorescein in
the ORAC assay. The dichloromethane extract exhibited chemopreventive activity, evidenced by
strong quinone reductase (QR) enzyme induction in Hepa1c1c7 cells when compared to the positive
control 4′-bromoflavone. The fractionation of the extract provided the isolation of an anacardic acid
FIGURE 9.10 Phenolic compounds isolated from Spondias tuberosa (“umbu”).
derivative (54) (Figure 9.10), which, however, was not effective in QR induction. The elevation of
phase II enzymes such as QR could be correlated with protection against chemical-induced carcino-
genesis in animal models in the stages of initiation and promotion (Zeraik et al., 2016a,b).
9.2.2 Cytotoxic Compounds
Cancer is a disease characterized by abnormal cell proliferation that can invade nearby tissues and
spread to other parts of the body through the blood and lymph system (NCI, 2018). According to
the WHO (2018), cancer is the second leading cause of death globally and accounted for 8.8 million
deaths in 2015. Therefore, because of numerous facts, such as cancer severity, mortality, economic
issues, lack of more selective and less toxic drugs and evolving resistance to currently available
therapeutic agents, novel effective and accessible molecules are urgently required for the treatment
of this disease. New strategies are also necessary for cancer prevention, ranging from a healthier
lifestyle to prevention by chemical means, as the promotion of chemopreventive agent’s consump-
tion, either from vegetables such as broccoli, garlic and berries or nutraceuticals.
Concerning cancer treatment, plants have directly afforded natural drugs, or precursors to semi-
synthetic derivatives, in addition to prototypes which inspired purely synthetic therapeutic agents,
currently in clinical use, such as vinca alkaloids (i.e. vinblastine and vincristine), etoposide and
teniposide (semi-synthetic derivatives of the NP epi-podophyllotoxin). Probably, one of the most
recognized and noteworthy example is paclitaxel (Taxol®), an anticancer drug isolated from the
leaves of several Taxus species along with the precursor compounds(Cragg and Newman, 2013).
The studies carried out at NuBBE have initially addressed crude extracts, which have been sub-
mitted to preliminary assays for the detection of cytotoxicity against tumor cell lines and allowed the
selection of promising samples for further chemical and biological investigation to afford partially
purified fractions and isolated compounds. Their evaluation against tumor cell lines, in addition to
complementary assays in the case of promising samples, led to the discovery of active compounds
belonging to several natural products classes.
In some studies, such as that of Ouratea multiflora (Ochnaceae), the resulting compounds did
not exhibit remarkable cytotoxic activity. In other cases, potent bioactive compounds have been
discovered as those exemplified by M. ilicifolia (Celastraceae) quinonemethides.
The chromatographic separation of chemical constituents from the ethanol extract of
O. multiflora leaves, a medicinal plant used to treat inflammatory diseases such as rheumatism and
FIGURE 9.11 Structures of flavone dimers 55–58.
arthritic disorders (Carbonari et al., 2006), led to the isolation of four flavonoid dimers, namely,
heveaflavone (55), amentoflavone-7′′,4′′′-dimethyl ether (56), podocarpusflavone-A (57) and amen-
toflavone (58) (Figure 9.11). The biflavonoids were evaluated for cytotoxicity against mouse lym-
phoma (L5178) and melanoma (KB) cancer cell lines. However, none of these metabolites was
active in this assay (Carbonezi et al., 2007).
Pristimerin (47) (Figure 9.9), a quinonemethide triterpene exhibiting cytotoxic activity against
various cancer cell lines (Deeb et al., 2014), was isolated from the ethanol extract of M. ilicifolia
root barks with cytotoxic potential evaluated through MTT (3-(4,5-dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide) assay (Mosmann, 1983) in five additional human tumor
cell lines: HL-60 (promyelocytic leukemia), k-562 (chronic myelocytic leukemia), SF-295
(glioblastoma), HCT-8 (colon cancer) and MDA/MB-435 (melanoma). The selectivity of pris-
timerin (47) was also evaluated toward a normal proliferating cell line, by performing the Alamar
Blue assay with human peripheral blood mononuclear cells (PBMC), after 72 hours of drug expo-
sure. The mechanism of the action of pristimerin in leukemia cell (HL-60) cytotoxicity was also
investigated. For this purpose, the following experiments were performed: the cell viability was
determined by the trypan blue dye exclusion test, inhibition of DNA synthesis was assessed deter-
mining the amount of BrdU (5-bromo-2′-deoxyuridine) incorporated into DNA (Pera et al., 1977),
inhibitory effects of pristimerin on human topoisomerase I were measured using a Topo I Drug Kit
(TopoGEN, Inc), acridine orange/ethidium bromide (AO/EB) staining assay (McGahon et al., 1995)
was performed in order to evaluate the cell death pattern induced by increasing concentrations of
pristimerin, HL-60 cell membrane integrity was evaluated by exclusion of propidium iodide and
then cell fluorescence was measured by flow cytometry; internucleosomal DNA (lysed) was also
analyzed by flow cytometry. Pristimerin (47) displayed cytotoxic activity toward the five tumor cell
lines tested, with IC50 values ranging from 0.55 µM to 3.2 µM in MDA/MB-435 and k-562, respec-
tively. The IC50 values over PBMC from pristimerin and doxorubicin were 0.88 µM and 1.66 µM,
respectively. Subsequent experiments conducted on HL-60 cells aimed to elucidate the mechanism
of action of pristimerin in this cell line. The trypan blue test revealed that pristimerin reduced the
number of viable cells and increased the number of nonviable cells in a concentration-dependent
way, presenting morphological alterations consistent with apoptosis. However, pristimerin was
shown not to be selective to cancer cells when compared with a normal cell line, since PBMC was
inhibited at an IC50 of 0.88 µM. The assessment of DNA synthesis inhibition by BrdU incorporation
in HL-60 cells was 70% (0.4 µM) and 83% (0.8 µM), whereas pristimerin was not able to inhibit
topoisomerase I. AO/EB staining showed that all tested concentrations of pristimerin were able to
reduce the number of viable cells, with the occurrence of necrosis and apoptosis in a dependent
concentration way, which represents results quite consistent with trypan blue exclusion insights.
Furthermore, the analysis of membrane integrity and internucleosomal DNA fragmentation through
flow cytometry in the presence of pristimerin suggested that treated cells underwent apoptosis.
Therefore, these results highlight the importance of pristimerin as a representative compound of an
emerging class of cytotoxic metabolites against several cancer cell lines, displaying antiproliferative
effect by inhibiting DNA synthesis and triggering cell death likely by apoptosis (Costa et al., 2008).
A second quinonemethide triterpene, named maytenin (46) (Figure 9.9) was also isolated from
M. ilicifolia (Santos et al., 2010b). As well as 47, maytenin was assessed for cytotoxic effects by
MTT assay toward human keratinocytes (NOK cells of the oral mucosa) while they were addition-
ally assayed for antifungal activity, as antifungal agents require a broad spectrum of action and no
toxicity over the cells. Both compounds exhibited cell viability higher than 80%, suggesting that
these metabolites were not cytotoxic in the assay (Gullo et al., 2012).
Styrylpyrones isolated from Cryptocarya spp. (Lauraceae) and their derivatives have demon-
strated antiproliferative activity in a range of human cell lines, therefore, representing antitumor
potential even though the mechanism of action involved remains unknown. Cryptocaria species
may be commonly found in both Brazilian phytogeographic regions Cerrado and Atlantic Forest.
C. mandiocanna is a tree similar to C. moschata, and the latter species is popularly entitled the
“Brazilian nutmeg” (“noz-moscada-do-Brasil”) for their highly aromatic seeds. In this context,
the styrylpyrone cryptomoschatone D2 (59) (Figure 9.12) was isolated from the methylene chlo-
ride extract from leaves of Cryptocarya mandiocanna, collected in Atlantic Forest, by chromato-
graphic methods and subsequently assessed for cytotoxic activity in HPV-infected HeLa (HPV18)
and SiHa (HPV16), as well as uninfected (C33A) human cervical carcinoma cell lines and in lung
fibroblast MRC-5 cell line. The cells were submitted to a treatment with different concentrations
of compound 59 (15 µM, 30 µM, 60 µM or 90 µM) for 6, 24 hours, and for 6 hours followed by
a post-treatment recovery period of 24, 48, or 72 hours. High dose- and time-dependent cyto-
toxicity was observed for all cell lines. Furthermore, unlike the infected cells (HeLa and SiHa),
C33A cells were unable to recover their proliferative ability proportionally to the posttreatment
recovery time (Giocondo et al., 2009).
The Piperaceae family comprises groups of species, which produce amides, phenylpropanoids,
lignans, benzoic acids and chromenes, some of the main classes of natural products found in Piper
and Peperomia that represent the largest genera in Piperaceae.
Piperlongumine (60), also known as piplartine (5,6-dihydro-1-[1-oxo-3-(3,4,5-trimethoxyphenyl)-
2-propenyl]-2(1H) pyridinone), is an amide alkaloid (Figure 9.13) widespread in Piper species.
This metabolite displays strong cytotoxic activity over tumor cell lines (e.g. HL-60, k562, Jurkat
and Molt-4), in addition to other bioactivities. Further investigation on piperlongumine cyto-
toxic properties was carried out after its re-isolation from the root extract of Piper tuberculatum.
Piperlongumine was evaluated for genotoxic (mutagenic) effects and induction of apoptosis in V79
cell line (derived from Chinese hamster lung fibroblasts), as well as for mutagenic and recombinant
potential in Saccharomyces cerevisiae. The extract was found to induce dose-dependent cytotox-
icity in S. cerevisiae cultures, in addition to exhibiting weak mutagenic effect on cells during the
FIGURE 9.12 Styrylpyrone cryptomoschatone D2 isolated from Cryptocarya mandiocanna.

FIGURE 9.13 Structures of piperlongumine (60) isolated from P. tuberculatum and the synthetic
analogs (61–65).
exponential growth phase in a buffer solution, although an increase on the frequency of mutations
during growth in the medium was observed. The neutral and alkaline comet assays revealed that
piperlongumine induced G2/M cell cycle arrest, probably due to triggering DNA double strain
breaks and repair. Furthermore, treatment with this metabolite induced dose-dependent apoptosis,
which was detected by a decrease in mitochondrial membrane potential in contrast to an increase in
internucleosomal DNA fragmentation. Finally, cells surviving piperlongumine-induced DNA dam-
age could accumulate mutations as this compound proved to be mutagenic and recombinogenic in
S. cerevisiae (Bezerra et al., 2008).
The mutagenic and antimutagenic effects of piperlongumine were also evaluated in Salmonella
typhimurium strains TA97a, TA98, TA100 and TA102 by the Ames test (Morandim-Giannetti et
al., 2011). The results indicated piperlongumine efficacy for protecting genetic material against
damage caused by mutagenic agents, and therefore suggested promising antitumor uses for this
compound due to the close relationship between mutagenesis and carcinogenesis (Morandim-
Giannetti et al., 2011).
Since 2011, several studies have focused on piperlongumine due to the compound’s selective
antitumor properties, comprising about 80 articles published worldwide, including some aimed to
encourage and guide clinical trials. However, no studies addressing piperlongumine metabolism
in human organism were known, until de Lima Moreira et al. (2016) investigated the compound’s
in vitro oxidation by Cytochrome P450 (CYP450) enzyme, in addition to the enzymatic kinetic
profile catalyzed by CYP enzymes and the prediction of in vivo pharmacokinetic parameters. The
structures of four piperlongumine metabolic products were also proposed by employing liquid
chromatography coupled to high-resolution mass spectrometry (HR-LC-ESI-MS) analyses in both
negative and positive ion modes and were confirmed on the basis of their fragmentation patterns
in LC-IT-MS spectra. Subsequent isolation through LC-SPE-NMR (liquid chromatography-solid
phase extraction-nuclear magnetic resonance) allowed 1H NMR assignments for the metabolites’
characterization, which corroborated their structures. Phenotypic studies and possible piperlongu-
mine-drug interactions were reported as well. Altogether, these results propitiate a useful guide to
further clinical studies aimed at a rational exploration of piperlongumine potential in the develop-
ment of antitumor drugs.
Metastasis is the process in which cancer cells spread from the original tumor to other parts of
the body through the blood or lymph system and initiates the formation of a new tumor. The pro-
cess of cancer cell migrations to distant tissues or organs is a crucial step in metastasis (new tumor)
formation. Most of the cancer drugs currently available target inhibition of cell proliferation and
killing cancer cells, instead of cell migration. Based on piperlongumine’s (60) previously reported
activities (Bezerra et al., 2008, Morandim-Giannetti et al., 2011, de Lima Moreira et al., 2016),
and on a cell-based screening, it’s the potential to inhibit breast cancer cell line (MDA-MB-231)
migration by the Boyden chamber assay and for cytotoxic activity against normal (MCF10A) and
cancer (MDA-MB-231 and DU-145) cell lines (Valli et al., 2017) was evaluated. Furthermore, a
series of five analogs (61–65) (Figure 9.13) was designed using the concepts of molecular sim-
plification and hybridization, synthesized and evaluated in cell migration and cytotoxicity assays.
Piperlongumine (60) inhibited the migration of MDA-MB-231 cells with EC50 of 3.0 ± 1.0 µM in
the Boyden chamber assay, which is comparable to the activity of colchicine, used as positive con-
trol. Boyden chamber consists of a two-compartmental system separated by a plastic membrane
and allows the quantitative determination of a compound effect on cell migration. Piperlongumine
analog 64, which was designed by molecular simplification, was the most active from the series
(EC50 = 9.0±1.0 µM) and showed selective cytotoxicity toward normal breast cell line MCF10A,
with a selectivity index (SI) of 4.4. Finally, piperlongumine did not show interaction with micro-
tubules in the tubulin polymerization assay, which indicates that the activity must have a different
mechanism of action (Valli et al., 2017).
9.2.3 Natural Products Active on the Central Nervous System

Natural products have been used to cause effects on the central nervous system (CNS) since ancient
times. Populations all over the world have been making use of plants for rituals and medicinal
purposes and such knowledge was passed through generations, although much information may
have been lost. Ethnopharmacological research on CNS-related disorders has also been based on
traditional knowledge and is very useful for guiding natural products research on such complex
conditions.
Some plant families have been greatly reported to treat CNS diseases, such as Fabaceae,
Asteraceae, Rubiaceae and Solanaceae, and many plants were studied having ethnopharmacol-
ogy as a basis (Cooper, 1987; Ghedini et al., 2002; Mendes and Carlini, 2007; Seidler, 2001; Valli
et al., 2016). CNS disorders are neurological illnesses impairing the function or structure of the
brain, spinal cord and retina. They include Alzheimer’s disease, Parkinson’s disease, depression,
anxiety, epilepsy, multiple sclerosis (MS) and schizophrenia (Ballios et al., 2011; DiNunzio and
Williams III, 2008). The growing number of cases of these diseases in the past few years has been
often associated to the ageing of world population (Varma et al., 2016). The use of pharmaceuti-
cal drugs is still one of the most widespread strategies for treating CNS diseases. The search for
natural products for the development of novel therapeutic agents aimed at the treatment of CNS
diseases has been one of the focuses of our research group, and herein we present a few interest-
ing results.
In a screening of native Brazilian plants, the ethanol crude extract of Esenbeckia leiocarpa stems
showed acetylcholinesterase inhibition and was submitted to bioassay-guided fractionation to iso-
late the biologically active compounds. Acetylcholinesterase inhibition has been considered a major
target in the discovery of therapeutic agents to treat patients with Alzheimer’s disease as it increases
the availability of acetylcholine for synaptic transmission in the brain. The bioactivity-guided frac-
tionation of the ethanol extract from stems afforded six alkaloids: leiokinine A (66), leptomerine
(67), kokusaginine (68), skimmianine (69), maculine (70) and flindersiamine (71) (Figure 9.14). All
isolated compounds displayed in vitro acetylcholinesterase inhibition activity in the Ellman TLC
assay (Ellman et al., 1961). Leptomerine (67) showed the highest activity (IC50 = 2.5 μM), when
compared to the reference compound galanthamine (IC50 = 1.7 μM) (Cardoso-Lopes et al., 2010).
Tetrapterys mucronata Cav. (Malpighiaceae) is one of the plants used in the preparation of aya-
huasca in some regions of Brazil. Ayahuasca is a psychotropic plant decoction with a long cultural
history of uses (Carlini, 2003), especially by devotees of some religions.
FIGURE 9.14 Acetylcholinesterase inhibitory alkaloids isolated from Esenbeckia leiocarpa.
The chemical composition of T. mucronata was determined with the constituents evaluated
as acetylcholinesterase inhibitors. The ethanol extract of T. mucronata barks exhibited in vitro
acetylcholinesterase inhibition in a TLC bioautography assay (Atta-ur-Rahman et al., 2005;
Di Giovanni et al., 2008; Ellman et al., 1961). The active constituents were identified and
among the twenty-two isolated compounds, the tryptamine alkaloids, 17 (Figure 9.3) and 72–75
(Figure 9.15), inhibited acetylcholinesterase with IC50 values below 15 μM and were compared
to the positive controls galanthamine (IC50 = 2.4 μM) and tacrine (IC50 = 0.09 μM) (Queiroz
et al., 2014a).
Toxic and hallucinogenic properties were evaluated in a study performed with a water decoction,
which mimics the ayahuasca preparation, to determine the decoction chemical profile and content
of the main tryptamine alkaloids in T. mucronata stem barks. The extraction of stem barks with
ethanol afforded bufotenine (17) (Figure 9.3), 5-methoxy-N-methyltryptamine (72), 5-methoxy-
bufotenine (73) and 2-methyl-6- methoxy-1,2,3,4-tetrahydro-β-carboline (76) (Figure 9.15). A
comparison with the water decoction revealed slightly lower levels of these constituents. These four
alkaloids have been described for their toxic and hallucinogenic properties, especially bufotenine
and 5-methoxy-bufotenine. Although some previous studies have indicated that the risk of intoxica-
tion by consuming ayahuasca is minimal, lethal cases have been reported. Therefore, this study was
important to indicate that consumption of T. mucronata as an ingredient in ayahuasca preparations
may present a risk to consumers (Queiroz et al., 2015).
FIGURE 9.15 Tryptamine alkaloids from the stem bark of T. mucronata.

9.2.4 Antifungal Compounds
The increase in fungal infections represents a serious concern considering the insufficient
amounts of antifungal drugs in addition to problems with toxicity and increased resistance
to fungi. Natural compounds from the Brazilian biodiversity and their derivatives may rep-
resent an alternative in the search for new and effective antifungal therapeutic agents (Funari
et al., 2012a; Newman et al., 2000).
Phytochemical studies on Brazilian plants developed in our research group in recent years,
especially those from Atlantic Forest, have disclosed the presence of several antifungal secondary
metabolites, mainly triterpenes, diterpenes, saponins, flavonoids, alkaloids and polyketides.
The pharmacological activities already reported for M. ilicifolia extracts and pure com-
pounds instigated the study of this species against human pathogenic fungi, especially opportu-
nistic species of Aspergillus, Candida, Cryptococcus, Histoplasma, Fusarium, Trichophyton
and Paracoccidioides genera, which have been mainly associated with infection in immuno-
compromised patients (Shoham and Levitz, 2005). In this context, maytenin (46) and pristimerin
(47) (Figure 9.9), previously isolated from the root barks of adult M. ilicifolia plants (Santos
et al., 2010b), were evaluated using a qualitative analysis of a fungal viability and microdilution
method (M27-S3 – CLSI – Clinical and Laboratory Standards Institute (2008)) with modifications
to calculate their minimum inhibitory concentration (MIC) and minimum fungicide concentration
(MFC). Maytenin showed potent antifungal activity against both yeasts and filamentous fungi with
MICs ranging from 0.12 mg L−1 to 62.5 mg L−1. Compounds 46 and 47 showed same MIC values
against Histoplasma capsulatum and Paracoccidioides brasiliensis (0.48 mg L−1 and <0.12 mg L−1,
respectively), when compared to a positive control itraconazole, which exhibited MIC 0.25–2.00 mg
L−1 for H. capsulatum and MIC lower than 0.0039 mg L−1 for P. brasiliensis, and amphotericin B,
which exhibited MIC values of 0.06–0.25 and 0.015–0.25 mg L−1 for H. capsulatum and P. brasil-
iensis, respectively. Maytenin showed the best results for all tested fungal strains, while pristimerin
displayed strong activity against Candida krusei (MIC 7.81 mg L−1) and Cryptococcus neoformans
(MIC 0.97 mg L−1) and moderate activity against filamentous fungi with MICs ranging from 0.12 to
250 mg L−1. In addition, maytenin displayed a selectivity index (SI) above 1.0 for all fungal strains
tested, wherein the higher the SI, the greater the safety of the tested compound. Pristimerin exhib-
ited high SI against C. neoformans and H. capsulatum fungal strains, but maytenin displayed the
best structural features associated with a selective effect against the tested human pathogenic fungal
strains (Gullo et al., 2012).
Croton heliotropiifolius Kunth (Euphorbiaceae) is popularly known as “velame” with the
leaves and barks used in folk medicine as pills or infusions to treat gastrointestinal problems
and for weight loss (Govaerts et al., 2000). The bio-guided fractionation of ethanol extract from
C. heliotropiifolius stem barks, using chromatographic techniques to detect and isolate compounds
with antifungal activity against Candida albicans, led to the isolation of nine compounds (77–85)
(Figure 9.16). Velamone (84) and the compound’s analog, velamolone acetate (83), showed weak
antifungal activity, whereas spruceanol (85) demonstrated a relevant minimal inhibitory quantity
(MIQ), when tested against the mutant strain DSY2621 and the wild strain. Spruceanol was the
most active compound against C. albicans that was first reported on antifungal potential (Queiroz
et al., 2014b).
The antifungal activity of the Swartzia langsdorffii (Fabaceae) extract against C. albicans,
C. krusei, C. parapsilosis and C. neoformans strains was evaluated using a microdilution assay
(CLSI, 2008) leading to the isolation of bioactive constituents. S. langsdorffii is popularly known
as “banana-de-papagaio”, “jacarandá-banana” or “jacarandá-de-sangue”, and the bio-guided frac-
tionation of an ethanol extract of the leaves using chromatographic techniques afforded pentacyclic
triterpenes and saponins (Figure 9.17) with antifungal activity. The isolated substances were also
evaluated by bioautography against phytopathogenic fungal strains, and the saponins oleanolic acid
3-sophoroside (86) and 3-O-β-d-(6′-methyl)-glucopyranosyl-28-O-β-d-glucopyranosyl-oleanate
FIGURE 9.16 Compounds (77–85) isolated from stem bark of Croton heliotropiifolius.
FIGURE 9.17 Saponins and triterpenes isolated from Swartzia langsdorffii.
(87) showed moderate activity against the phytopathogens Cladosporium cladosporioides and
C. sphaerospermum (MIC 100.0 µg mL−1), whereas oleanolic acid (88) exhibited weak activity
(MIC 200.0 µg mL−1) and lupeol (89) was not active against the pathogenic strains used in this study
(Marqui et al., 2008).
The extracts and fractions obtained from P. nitens were evaluated by microdilution method
and exhibited activity against the tested strains C. albicans, C. krusei, C. parapsilosis and
C. neoformans. The n-butanol fractions from branches (MIC 15.6 µg mL−1) and roots (MIC
31.2 µg mL−1) exhibited the most potent activities against C. krusei. Such samples were selected for
chemical investigation, which led to the isolation of four guanidine alkaloids (Figure 9.18), N-1,N-
2,N-3-triisopentenylguanidine (90), and nitensidines A-C (91-93), with moderate antifungal activity
against C. krusei (MIC 5 μg mL−1) and C. parapsilosis (MIC 31.2 μg mL−1). Additionally, all extracts,
fractions and isolated compounds were evaluated against the four fungal strains and showed MFC
values greater than 1,000 μg mL−1, indicating their weak fungistatic behavior (Regasini et al., 2010).
Additional work on the antifungal properties of P. nitens flavonoids was carried out. The syner-
gistic effects of pedalitin (42) (Figure 9.7) (Regasini et al., 2008b) and amphotericin B were evalu-
ated against C. neoformans by in vitro and in vivo tests using the alternative animal model Galleria
mellonella, addressing three parameters: survival curve, fungal burden and histological analysis
FIGURE 9.18 Prenylated guanidine alkaloids isolated from Pterogyne nitens.
(Sangalli-Leite et al., 2016). In the in vitro assay amphotericin B (AmB) and 42 were tested alone
and showed MIC of 0.125 mg L−1 and 3.9 mg L−1, respectively. This assay was performed by a micro-
dilution method described by CLSI (2008) with modifications (Scorzoni et al., 2007). The combined
treatment with AmB and 42 was performed by the checkerboard broth microdilution method. The
fractional inhibitory concentration index (FICI) was calculated using the equation: ΣFIC = FICA +
FICB, where FIC is the ratio of MIC of the drug in combination with MIC alone (Odds, 2003; White
et al., 1996). The combination was considered synergistic at FICI ≤0.5, indifferent at FICI >1 and
≤4 and antagonistic at FICI >4.0. The same formula was used to calculate the fractional fungi-
cidal concentration index (FFCI), using MFC values instead of MIC (Odds, 2003). The combina-
tions tested decreased the MIC value by fourfold compared with AmB and 42 (0.03 mg L−1 and
1 mg L−1, respectively). The synergistic effect was considered promising by the results of FICI
and FFCI. In the synergistic treatment, all the combinations of AmB doses (1, 2 and 4 mg kg−1)
and pedalitin (6.25, 12.5, 25 and 40 mg kg−1) were able to increase the survival of infected larvae
(P < 0.05). Treatment with 0.3 mg kg−1 of AmB and 10 mg kg−1 of 42, alone, led to survival of larvae
up to the sixth day of the experiment by 18.7% and 0%, respectively. However, for the combined
compounds, >56% of larvae were alive at the end of the experiment. The combination of AmB at
0.3 mg kg−1 + 42 at 10 mg kg−1after 4 days resulted in almost 100% reduction of the fungal burden.
Synergism efficacy of the treatment was also observed by histopathology of untreated and treated
larvae used in the experiment. Histopathology data showed a reduction in the number of yeasts after
14 days of treatment with AmB, 42 or combination therapy.
The results for all trials were promising and the best time-kill results were obtained after 8 hours
of exposure to the tested substances. After contact with AmB and 42 either alone or in combination,
the yeast death rate was 100%. Alternative animal models such as G. mellonella were used to perform
the in vivo antifungal assay. Before infecting G. mellonella larvae, the toxicity test of compounds
and solvents was performed and compound 42 showed a toxic effect on larvae at doses ≥50 mg kg−1.
Experiments using either alone or combined compounds showed similar activities on larvae survival.
To compare the results of synergism efficacy using the alternative animal model G. mellonella, the
treatment of 42 + AmB in murine model was also performed and showed an increase in mice sur-
vival. As observed in G. mellonella, combined treatment with AmB and 42 significantly increased
the survival of mice, which suggested that this treatment was as efficient as AmB monotherapy at
higher doses, suggesting the combination of antifungal compounds as an interesting alternative that
can increase the efficiency of fungicidal treatment (Sangalli-Leite et al., 2016).
9.2.5 Natural Products Active on Neglected Diseases’ Parasites

In the last century, humanity has suffered from climate change all over the world with deterioration
of biodiversity and deforestation of biomes that harbor disease vectors as major contributors that
have significantly altered epidemic processes. This is no different in the Atlantic Forest, which is
the natural habitat of numerous vectors and transmitters of tropical diseases, especially those con-
sidered as neglected ones, such as leishmaniasis and Chagas’ disease (Wood et al., 2014). However,
nature itself can give us support to find alternatives to problems caused by these vectors.
A growing number of natural products derived from plants collected in the remaining areas
of the Brazilian Atlantic Forest are known for their bioactivities against the causative agents of
neglected diseases.
Many examples can be cited, with some related to plant species of Peperomia and Piper genera
from the Piperaceae family. Peperomia obtusifolia is a well-known ornamental plant distributed
from Mexico to South America. Despite the plant’s predominant ornamental usage, some communi-
ties in Central America use the leaves’, stems’ and fruits’ extracts to treat insect and snake bites and
as a skin cleanser (Batista et al., 2017). Previous phytochemical investigation on P. obtusifolia aerial
parts showed the presence of prenylated chromans, lignans, amides, flavonoids and other phenolic
derivatives (Batista et al., 2011; Mota et al., 2009; Tanaka et al., 1998). Crude extracts and fractions
of P. obtusifolia leaves and stems showed potent trypanocidal activity and their chemical investi-
gation afforded seven compounds (94–100), including chromanes, furofuran lignans and flavone
C-diglycosides (Figure 9.19) (Mota et al., 2009). This study revealed that the most active compounds
were the chromanes, peperobtusin A (94) and the carboxy derivative 3,4-dihydro-5-hydroxy-2,7-
dimethyl-8-(2′′-methyl-2′′-butenyl)-2-(4′-methyl1′,3′-pentadienyl)-2H-1-benzopyran-6-carboxylic
acid (95), with IC50 values of 3.1 µM (almost three times more active than the positive control ben-
znidazole, IC50 10.4 µM) and 27.0 µM, respectively. The potent trypanocidal activity observed for
these compounds seems to be related to a benzopyran nucleus substituted with isoprenyl moieties.
The assay was performed measuring the proliferation of Y strain epimastigotes growing in axenic
culture and the number of remaining viable protozoa was established by counting the parasites in
a Neubauer chamber. Cytotoxicity assays using peritoneal murine macrophages indicated that the
chromanes were not toxic at the level of the IC50 for trypanocidal activity (Mota et al., 2009), evi-
dencing a selective index compatible with their use as prototypes for the development of therapeutic
agents for Chagas disease.
Piper crassinervium was shown to accumulate antifungal, trypanocidal and antioxidant
C-geranylated metabolites derived from both benzoic acid and p-hydroquinone (López et al., 2010).
The chemical study was reported by Lopes et al. (2008) which resulted in the identification of
two prenylated benzoic acid derivatives (101 and 102), one prenylated hydroquinone (103) and two
FIGURE 9.19 Chromanes, flavonoids and lignans isolated from Peperomia obtusifolia.
FIGURE 9.20 Phenolic compounds isolated from Piper crassinervium.
flavanones (104 and 105) (Figure 9.20). In vitro trypanocidal assays showed that the most active
compound was the prenylated hydroquinone (103) with an IC50 value of 6.10 µg mL−1, comparable
to the positive control benznidazole (IC50 1.60 µg mL−1). The presence of lipophilic geranyl moiety
oxygenated in the benzyl position was regarded as a key structural moiety essential to trypanocidal
activity (Lopes et al., 2008). These results were consistent with previous data about the importance
of isoprenyl moieties for the trypanocidal activity (Batista et al., 2008; Mota et al., 2009).
Other families from Atlantic Forest flora have also been the subject of bioprospecting studies.
Porcelia macrocarpa belongs to the Annonaceae family and is widespread in the southeastern region
from Brazil (Santos et al., 2015). Nonpolar extracts of the species’ seeds displayed in vitro activ-
ity against Trypanosoma cruzi trypomastigotes. Thus, the crude bioactive extract was subjected to
chromatographic fractionation procedures to afford an acetylene fatty acid, 12,14-octadecadiynoic
acid/macrocarpic acid (106), and two acetylene di/triacylglycerol derivatives, α,α′-dimacrocarpoyl-
β-oleylglycerol (107) and α-macrocarpoyl-α′-oleylglycerol (108) (Figure 9.21), which had their
trypanocidal potential evaluated using a MTT colorimetric assay (Muelas-Serrano et al., 2000).
FIGURE 9.21 Acetylenic compounds 106–108 isolated of Porcelia macrocarpa.

Compound 106 displayed in vitro activity against T. cruzi trypomastigotes, while compounds 107
and 108 were inactive. The importance of unsaturation for the observed bioactivity was verified as
compound 106 was hydrogenated, and the resulting product was reevaluated, and shown to be inac-
tive (Santos et al., 2015).
The antiprotozoal potential of Moringa oleifera (Moringaceae) has also been investigated.
M. oleifera, also known as the “drumstick tree”, is recognized as a multipurpose and affordable
source of phytochemicals, with potential applications in medicines and functional food prepara-
tions, water purification, in addition to biodiesel production (Saini et al., 2016). The flower’s chemi-
cal studies led to the detection of flavonoids in the ethanol extract, and a trypsin inhibitor (MoFTI)
was concentrated in a fraction from the ethanol extract. The flavonoids were evaluated against
T. cruzi and for cytotoxicity to mammalian cells. Promising results were obtained both for the
extract enriched with flavonoids and MoFTI, which triggered lysis of T. cruzi trypomastigotes with
LC50/24 h of 54.2 and 41.2 μg mL−1, respectively. High selectivity indices for T. cruzi cells were
found for the extract and MoFTI evidencing this compound is a trypanocidal principle of the flower
extract from M. oleifera (Pontual et al., 2018).
Santos et al. (2012) reported the isolation of four sesquiterpene pyridine alkaloids, ilicifoliunines
A (109) and B (110), aquifoliunine E-I (111) and mayteine (112) (Figure 9.22) from the root bark
of M. ilicifolia (Celastraceae). An antileishmanial assay using promastigote forms of Leishmania
amazonensis and L. chagasi in addition to an antitrypanosomal assay employing T. cruzi epimasti-
gotes were performed using a MTT colorimetric method (Muelas-Serrano et al., 2000) with these
isolated alkaloids. Among the tested compounds, alkaloid 111 presented activity against L. chagasi
and T. cruzi, with IC50 values of 1.4 μM and 41.9 μM, respectively. Such data indicate the antipro-
tozoal high potential of 111, as compared to the positive controls pentamidine (IC50 5.1 μM) and
benznidazole (IC50 42.7 μM), drugs that are currently employed for the treatment of leishmaniasis
and trypanosomiasis, respectively. Alkaloid 109 displayed potent antitrypanosomal activity, with
an IC50 value of 27.7 μM. However, this compound was inactive against both Leishmania species.
Compounds 110 and 112 did not exhibit activity against the protozoan species tested at 100 μM.
Such results evidence the bioactivity dependence on the benzoyl substituent concomitant to the
α-carbonyl 1,2-dimethyl-ethyl moiety, present in compounds 109 and 111, but not in 110 and 112.
Interestingly, cytotoxic activity tests in mammalian normal cells, using murine peritoneal mac-
rophages, demonstrated that the two active alkaloids 109 and 111 were more selective than the
standard drug (Santos et al., 2012), which gives additional support to their use as prototypes for
antiparasitic drugs.
The antiparasitic capacity of quinonemethide triterpenes maytenin (46) and pristimerin
(47) (Figure 9.9), isolated from root barks of M. ilicifolia, were evaluated as described above
FIGURE 9.22 Sesquiterpene-pyridine alkaloids isolated from Maytenus ilicifolia.

FIGURE 9.23 Cyclic peptide ribifolin (113) and the amino acid sequence of linear ribifolin (114) from
Jatropha ribifolia.
(Santos et al., 2012). These compounds showed potent in vitro activity against L. amazonensis
and L. chagasi promastigotes as well as T.cruzi epimastigotes, with IC values in the nano-
gram range. The IC50 values obtained for compounds 46 and 47 were 0.09 nM and 0.05 nM for
L. amazonensis promastigotes and 0.46 nM and 0.41 nM for L. chagasi promastigotes, respec-
tively. The IC50 values for T. cruzi epimastigotes were 0.25 nM and 0.30 nM, respectively. These
two quinonemethide triterpenes showed stronger activity when compared to the positive controls
pentamidine for L. amazonensis (IC50 6.75 nM) and L. chagasi (IC50 4.0 nM), and benznidazole
for T. cruzi (IC50 31.20 μM). The selectivity index (SI), a relevant characteristic for defining
hit compounds, was calculated for compounds 46 and 47 by dividing their cytotoxic activity
against murine macrophages (LC50) by their leishmanicidal or trypanocidal activities. SIs for
L. amazonensis and L. chagasi were 243.65 and 46.61 for 46 and 193.63 and 23.85 for 47, whereas
for T. cruzi epimastigotes the SIs were 85.00 for 46 and 332.37 for 47. Such results indicated that
both compounds present good selectivity for trypanosomatid extracellular forms and might rep-
resent attractive prototypes for antiprotozoal drugs development (Santos et al., 2013).
Further investigation of Atlantic Forest’s plants with antiplasmodial activities concentrated on
the species Jatropha ribifolia (Euphorbiaceae) and its potential against malaria. Although malaria is
not formally considered a neglected disease, it is closely related and many challenges are still faced
when treating and controlling this disease. The latex of J. ribifolia is widely used throughout north-
eastern Brazil as a traditional herbal medicine for the antivenom activity, furthermore the seeds are
sold in markets in the region for oil production as a purgative for veterinary use (Agra et al., 1996;
Devappa et al., 2011). An orbitide, identified as ribifolin (113) and a linear ribifolin analog (114) deter-
mined in chemical studies (Figure 9.23) was prepared to be tested against malaria using the same
assay. The synthetic linear (114) and cyclic (113) peptides were evaluated toward Plasmodium falci-
parum, a protozoan that causes the most severe form of the malaria disease (Sabandar et al., 2013),
with chloroquine being used as positive control (IC50 0.3μM) for these experiments. The cyclic peptide
113 was moderately effective against the parasite, with an IC50 of 42 μM, whereas the linear analog
114 showed weak activity with an IC50 of 519 μM, which provided evidence for the importance of
cyclization to improve biological activity in this case. Their cytotoxic activity was also measured, but
none of the tested compounds exhibited any cytotoxicity against human normal cells, which gives
additional support to the use of compound 113 as a model for antiparasitic drugs (Pinto et al., 2015).
9.2.6 Miscellaneous Bioactive Compounds

Compounds isolated from plants’ crude extracts or partially purified fractions can provide an alter-
native approach to new therapies, since they may present high chemical diversity and milder or
FIGURE 9.24 Galegine (115) and additional prenylated guanidine alkaloids (116 and 117) isolated from
Pterogyne nitens.
inexistent side effects compared with conventional treatments (Jardim et al., 2015). The following
studies revealed that Brazilian flora represents a vast, largely untapped resource of potential antibi-
otic, protease inhibitors and antiviral compounds.
The flavonol glucoside kaempferitrin (40) (Figure 9.7) (Regasini et al., 2008c) and the guanidine
alkaloid galegine (115) (Figure 9.24) (Regasini et al., 2009), isolated from P. nitens, have shown
hypoglycemic effect in vivo, which might be related to a possible antidiabetic effect. Diabetes
mellitus is a metabolic disease in which the body is affected by hyperglycemia triggering changes
in carbohydrate, lipid and protein metabolism pathways. In this context, the effect of treatment with
P. nitens on diabetic rats regarding glycemic levels and physiological parameters was evaluated.
Unfortunately, the plant crude extract did not change serum glucose levels or other physiologi-
cal parameters, water or food intake and body weight, and did not improve the diabetic condition
(Souza et al., 2009). Further studies evaluated hepatobiliary toxicity and biochemical markers levels
in urine, during treatment with P. nitens extract on diabetic rats. Nevertheless, the treatment also
had no therapeutic effect on the diabetic condition (Souza et al., 2010). The effect could be further
evaluated with different doses of the extract, route of administration or severity of the induced dia-
betes to confirm the results obtained previously.
The phytochemical studies on leaves, flowers, fruits, bark and roots of P. nitens led to the iso-
lation of pterogynidine (35) (Figure 9.6) and additional unusual prenylated guanidine alkaloids
90–93 (Figure 9.18) and 115–117 (Figure 9.24), which had their antibacterial activity evaluated
against six clinically relevant multi-drug-resistant bacteria strains. Antibiotics were initially
developed for therapeutic use in the 40s and were responsible for a remarkable increase in life
expectancy. Nevertheless, their indiscriminate use has triggered the development of resistant
bacterial strains to the available antibiotics. Methicillin-resistant S. aureus (MRSA) is a resis-
tant bacteria strain to many commonly used antibiotics and is a major public health problem.
The development of new classes of antibiotics is thus of urgent and great need. Prenylated gua-
nidine alkaloids isolated from P. nitens showed strong activity against S. aureus strains, compa-
rable to that of the positive control norfloxacin, except for alchorneine (90) (Figure 9.18), which
was weakly active. The most promising compounds were galegine (115) and pterogynidine
(35; Figure 9.6), which exhibited MIC of 31.4 and 20.5 µM respectively, for all tested strains.
The side chain length and substitution pattern represented important chemical features for the
antibacterial activity. Both compounds 35 and 115 exhibited bactericidal or bacteriostatic effect
as evaluated in the minimum bactericidal concentration (MBC) assay. Their MBC values were
the same as observed in the MIC assay, and the results evidenced that both compounds killed
the bacteria, rather than just inhibiting their growth, which indicates a bactericidal activity. Such
results indicated that guanidine alkaloids may be considered promising molecular models and
could be used for further medicinal chemistry studies in the development of antibacterial thera-
peutic agents (Coqueiro et al., 2014).
Additional antibacterial compounds were isolated from Stemodia foliosa Benth. (Plantaginaceae),
popularly known as “meladinha”’ and used in Brazilian folk medicine as bioinsecticide and to
treat respiratory infections. The ethanol extract from the aerial parts of S. foliosa, afforded three
labdane diterpenoids, 6α-acetoxymanoyl oxide (118), 6α-malonyloxymanoyl oxide (119) and
FIGURE 9.25 Labdane diterpenoids isolated from the aerial parts of Stemodia foliosa.
6α-malonyloxy-n-butylestermanoyl oxide (120) (Figure 9.25), along with the triterpenes betulinic
acid and lupeol, and the steroids stigmasterol and sitosterol. The isolated diterpenes were evaluated
using a disc diffusion assay against Gram-positive bacteria S. aureus, Bacillus cereus, B. subtilis,
B. anthracis, Micrococcus luteus, Mycobacterium smegmatis and M. phlei. Compound 119 exhib-
ited moderate activity against these strains (MIC 7–20 μg mL−1) which might be associated to the
reported plant use in traditional medicine to treat respiratory infections (Silva et al., 2008).
Inappropriate treatment of hepatitis C virus (HCV) infection, a serious health problem, can
cause liver cirrhosis with risk of hepatocellular carcinoma. The high costs of the available ther-
apy as well as the potential for development of resistance evidence the need for alternative treat-
ments and stimulated the search of new and efficient antiviral compounds. M. ilicifolia afforded
flavonoids and sesquiterpene-pyridine alkaloids as major chemical constituents. Compounds 46
and 47 (Figure 9.9), 111 (Figure 9.22) and 121 (Figure 9.26) have been isolated from the root
bark of M. ilicifolia and were subjected to studies on HCV genome replication, using the lucifer-
ase assay and Huh 7.5 cells, based on HCV sub-genomic replicons (SGR) of genotypes 2a (JFH-
1), 1b and 3a. The sesquiterpene pyridine alkaloid aquifoliunine E-I (111) dramatically inhibited
HCV SGR replication, and the western blotting assay confirmed these results, as the alkaloid
also inhibited HCV protein expression. In addition, compound 111 presented activity against
a daclatasvir resistance mutant subgenomic replicon and reduced the production of infectious
JFH-1 virus (Jardim et al., 2015).
Jatropha species have shown a great potential as source of small cyclic peptides, which have
attracted much attention due to their chemo-diversity and variety of important biological activities
(Baraguey et al., 2001; Mongkolvisut et al., 2006). Chemical studies on the latex of Jatropha curcas
led to isolation, structural elucidation and conformational studies of the cyclic peptide jatrophidin
I (122) (Figure 9.27). The compound’s biological evaluation showed strong inhibitory activity in
a fluorometric protease inhibition assay using pepsin as a molecular model for aspartic protease
inhibition (Gold et al., 2007), with IC50 value of 0.88 µM, when compared to standard pepstatin A
(IC50 0.40 µM). However, the cyclic peptide did not inhibit the serine protease subtilisin, evidencing
that the observed inhibitory activity was specific for aspartic proteases (Altei et al., 2014).
FIGURE 9.26 Catechin isolated from Maytenus ilicifolia.

FIGURE 9.27 Cyclic peptide jatrophidin I (122) isolated from Jatropha curcas.
Proteases fulfill multiple roles in health and disease, and considerable interest has been expressed
in the design and development of synthetic inhibitors of disease-related proteases. Virus-encoded
proteases have been shown to be involved in the replication of many virus types. Considering pro-
teases importance, they have become important drug targets (Kang et al., 2017). Jatropha species
may thus represent an interesting alternative for bioprospecting studies aimed at the discovery of
protease inhibitory cyclic peptides.
9.3 SOME COMMENTS ON RECENT ADVANCES IN NATURAL

PRODUCTS RESEARCH
With the continued interest and reemergence of natural products in drug discovery, the development
of novel techniques for extraction, as well as for data acquisition and processing during purification
and identification procedures, is fundamental to the finding of promising lead compounds requiring
sophisticated and efficient chemical assessment of natural sources.
Traditionally, crude extracts from plants, microorganisms or animals were obtained via classical
extraction procedures, screened in bioassays, and the main compounds often passed through vari-
ous isolation steps to purity for further spectroscopic analysis and identification. Considering that
plant extracts are complex mixtures, this strategy leads unfortunately to the re-isolation and iden-
tification of many known secondary metabolites, representing an expensive and time-consuming
process (Harvey et al., 2015). However, modern techniques, database mining and bioassay guided
fractionation are being used to continue the natural products research in the Atlantic Forest and
elsewhere.
In order to arrive at the structures presented herein, dereplication (Hanka et al., 1978) pro-
cesses are used. Furthermore, for analyses and isolation the dereplication process relies on the
following hyphenated techniques: GC-MS, LC-UV, LC-MS, LC-MS/MS and LC-NMR. Data
are acquired and compared to reference compounds in commercial or in-house databases and
include the Dictionary of Natural Products (Buckingham, 1997), NuBBEDB (Pilon et al., 2017;
Valli et al., 2013), SuperNatural (Dunkel, 2006), PubChem (Bolton et al., 2008), ChEBI (Hastings
et al., 2016), NAPROC (López-Pérez et al., 2007), CAS (Chemical Abstracts Service), ChemBank,
SciFinder and ChemSpider (Hubert et al., 2017).
Strategies in bioinformatics, chemometrics and statistical tools are being refined to speed up
analyses and improve the reliability of dereplication results (Funari et al., 2013). Among the modern
dereplication tools, the Global Natural Products Social Molecular Networking (GNPS, http://gnps.
ucsd.edu) is a powerful platform for large-scale data analysis in mass spectrometry (MS). GNPS
is an open-access knowledge base, allowing the scientific community to share raw, processed or
identified MS/MS data. The platform contains more than 93 million MS/MS spectra available from
natural products and pharmacologically active substances. Nowadays, GNPS is considered the larg-
est collection of public MS/MS data and the spectral libraries are growing through user’s contribu-
tions (Wang et al., 2016).
Natural products dereplication is challenging due to the high chemo-diversity of metabolites,
especially for metabolomics studies (Schwab, 2003). Metabolomics consist of comprehensive quali-
tative and quantitative analyses of the metabolome, which represents all metabolites present in an
organism, at a specific time and under specific conditions (Yuliana et al., 2011). To achieve optimum
results, one of the most exciting techniques for separation of complex samples is multidimensional
chromatography, either gas or liquid, where multiple chromatographic separations are performed on
a given mixture (Carr and Stoll, 2015; Li et al., 2015). Unlike two-dimensional gas chromatography
(2D-GC), the utilization of 2D-LC remains relatively uncommon, nevertheless the technology’s
potential as a promising tool for metabolomics, shall probably increase use in near future(Funari
et al., 2012a, 2012b; Wolfender et al., 2015).
The inherently low sensitivity of NMR techniques has been overcome by advances in hard-
ware, software and pulse programs, and nowadays, high-quality spectra are possible to obtain
on the microgram scale (Zani and Carroll, 2017). For this reason, NMR spectroscopy has been
considered an excellent detection technique for hyphenation with liquid chromatography for sepa-
ration and identification of compounds in natural products samples. (Cieśla and Moaddel, 2016;
Kesting et al., 2011).
The advances in instrumentation also allow the coupling of complex instruments, and nowadays,
hyphenation of LC-DAD-MS-SPE-NMR as a feasible option. For dereplication, the combination of
complementary tools as DAD, MS and NMR contributes to a significant reduction of time, smaller
amounts of sample required and an increase in the identification accuracy of natural products sam-
ples (Marshall and Powers, 2017).
Additional important aspects of natural products research include extraction and fractionation
techniques. Extraction is an essential process to convert the matrix into a sample suitable for ana-
lytical studies. This procedure is the first step in the chemical investigation of natural products,
and the success of subsequent steps of any bioassay, chromatographic or spectrometric experi-
ment still depends on an efficient, reproducible and reliable extraction (Belwal et al., 2018); and in
light of green chemistry, use of alternative solvents to ensure a safe and high-quality extract are
considered (Chemat et al., 2012). For example, modern extraction techniques for natural prod-
ucts samples include supercritical fluid extraction (SFE), microwave-assisted extraction (MAE),
ultrasound-assisted extraction (UAE), accelerated solvent extraction (ASE™), pulsed electric
field (PEF)-assisted extraction, enzyme-assisted extraction (EAE), instant controlled pressure
drop (DIC) (Allaf et al., 2013; Belwal et al., 2018; Funari et al., 2014; Selvamuthukumaran and
Shi, 2017).
A recent strategy to couple the sample extraction with chromatographic systems is the online
extraction (OLE), developed at the NuBBE laboratories. OLE was invented and patented by
Ferreira et al. and consists of inserting dry or fresh plant material inside a chamber in a secu-
rity guard holder. The precolumn is then connected to a liquid chromatographic system with a
six-port valve. As the chromatographic analysis starts, the valve is switched so that the mobile
phase flows through the precolumn containing the plant material prior to entering into the chro-
matographic column; therefore, the mobile phase is used both for extraction and separation.
The new technique OLE-LC was compared with conventional sample preparations of medicinal
plants and similar peak capacity and number of peaks were found for both methods, indicating
that OLE-LC is a feasible technique encompassing sample pretreatment integrated with chro-
matographic analysis (Ferreira et al., 2016). Although this procedure is not automated yet, some
papers have been published using OLE-LC with promising results (Russo et al., 2018; Tong
et al., 2018a, 2018b).
9.4 CONCLUDING REMARKS

In conclusion, in this chapter, many examples of natural products from the Atlantic Forest are
presented. Not enough emphasize can express how critical and important this research work is to
understanding the biome, seeking answers to the challenges of biodiversity and the ever present
potential for finding promising new drugs.
Modern techniques and equipment in addition to optimized strategies to investigate chemical
composition, biological and toxicological properties of natural products, among many complemen-
tary aspects of bioprospecting and research have proven essential for unravelling the fascinating
chemo-diversity and achieving the interesting results obtained so far. Phytochemical studies on
Brazilian plants developed in our research group from Atlantic Forest, have disclosed the presence
of many diverse secondary metabolites including triterpenes, diterpenes, saponins, flavonoids, alka-
loids and polyketides presented herein and exhibiting a multitude of biological and pharmacological
activity and more remains to be discovered.
Such scenario and goals require not only state-of-the-art equipment and methodologies but well-
trained scientists who may greatly benefit from intensive collaborative work to strengthen research
networks as well as to pave the way to continue consistent and systematic phytochemical studies
for a deeper and broader knowledge on natural resources. The essential role of secondary metabo-
lites in the species survival in addition to the maintenance of environmental equilibrium reinforces
the need of urgent and thorough commitment to preservation policies, especially of biodiversity
hotspots, as is the case of Atlantic Forest. They also evidence the utmost importance of sustainable
procedures toward the exploration of this rich biome so that value-added products may outcome
alongside biodiversity protection initiatives.
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10 Plants from the Caatinga
Biome with Medicinal
Properties†
Maria da Conceição Ferreira de Oliveiraa,
Mary Anne Sousa Limaa, Francisco Geraldo Barbosaa,
Jair Mafezolia, Mary Anne Medeiros Bandeirab,
and Wellyda Rocha Aguiarb
aDepartment of Organic and Inorganic Chemistry, Federal
University of Ceará, Campus do Pici, Fortaleza, Brazil
b Medicinal Herb Garden “Francisco José de Abreu Matos”, Federal
University of Ceará, Campus do Pici, Fortaleza, Brazil
CONTENTS
10.1 Introduction.......................................................................................................................... 258
10.2 B. cheilantha (BONG.) STEUD. (FABACEAE)................................................................. 265
10.2.1 Botanical Aspects and Occurrence......................................................................... 265
10.2.2 Ethnopharmacology............................................................................................... 265
10.2.3 Chemical Studies................................................................................................... 265
10.2.4 Pharmacological Studies........................................................................................266
10.3 C. leprosum MART. (COMBRETACEAE).........................................................................266
10.3.1 Botanical Aspects and Occurrence.........................................................................266
10.3.4 Pharmacological Studies........................................................................................ 267
10.4 E. viscosa LESS. (ASTERACEAE).................................................................................... 268
10.5 Erythrina velutina WILLD. (FABACEAE)......................................................................... 271
10.5.1 Botanic Aspects and Occurrence........................................................................... 271
† Dedicated to Prof. Francisco José de Abreu Matos (In Memoriam), “father” of Farmácias Vivas (Living Pharmacies), a
scientific and social (“local plants for local people”) program with plants from Caatinga.
257
10.6 Myracrodruon urundeuva ALLEMÃO (ANACARDIACEAE).......................................... 273

10.6.2 Ethnobotany and Ethnopharmacology................................................................... 273
10.6.5 Chemical and Pharmacological Studies of Cultivated M. urundeuva................... 274
Acknowledgments�� 276
10.1 INTRODUCTION
Caatinga (“white forest” in indigenous Tupi language) is a semi-arid ecosystem found exclusively
in Brazil, which is referred to as “a mosaic of scrubs and patches of seasonally dry forest” (Santos
et al., 2011). This unique biome occupies a large geographic area (ca. 800,000 km2) of the country
that spread from the state of Ceará to the north of the state of Minas Gerais, covering about 60% of
the Northeast region (Figure 10.1). Despite being one of the largest Brazilian biomes, the scientific
knowledge of this biome’s biodiversity is still very poor (Coe and Souza, 2014; Maia, 2004; Prado
2003; Santos et al., 2011).
Two very distinct seasons are present in Caatinga: the dry and rainy seasons. During the dry
season, which occurs for most of the year (8-9 months), the vegetation has a light gray aspect after
the leaves fall (Figure 10.2b), justifying the indigenous name “Caatinga” (Coe and Souza, 2014;
Maia, 2004; Prado, 2003). Probably, because of this aspect, Martius, the famous German bota-
nist, depicted Caatinga as “silva aestu aphylla” (forest without leaves in summer) (Prado, 2003).
The rainy season is limited to few months and the total rainfall (irregular and poorly distributed)
reaches 500-1,100 mm/year. In this period, the plant leaves reappear and the landscape is com-
pletely changed (Figure 10.2a). Notwithstanding the extreme stressing conditions found in Caatinga
(high temperature, high solar radiation, low irregular rainfall and low humidity), this biome hides a
great, but highly threatened, biodiversity of animals and plants (Coe and Souza, 2014).
FIGURE 10.1 Map displaying the location of the Caatinga biome in the Northeast region of Brazil.
Plants from the Caatinga Biome 259
FIGURE 10.2 Representative pictures from the Caatinga biome during the dry (a) and rainy (b) seasons.
Because of the high-water deficit, plants from Caatinga have developed strategies to survive,
and the xeromorphic vegetation is characterized by the presence of cacti and shrubs with spines or
prickles (Figure 10.2). The diversity of plants is estimated in ca. 5,000 species that are distributed
into eight ecoregions and 12 distinct types of vegetation. A noteworthy fact is that about 800 of the
species are endemic to the biome (Coe and Souza, 2014).
Many of the plant species from Caatinga are used in folk medicine and their potential therapeutic
benefits have been reported (Albuquerque et al., 2007; Viana et al., 2013). Albuquerque et al.
reported a quantitative approach on medicinal plants from Caatinga. The authors calculated the
ethnopharmacological relative importance index (RI) of 389 medicinal species used by rural and
indigenous communities from the Northeast region of Brazil (Albuquerque et al., 2007). A similar
study investigated 119 medicinal plants used by local population from Aiuaba, in Ceará state, and
revealed some species as having great versatility of use (Cartaxo et al., 2010).
Thirteen endemic medicinal plants from Caatinga with RI ≥1 (Albuquerque et al., 2007) are
listed in Table 10.1. Among them, Maytenus rigida (RI = 1.9), Bauhinia cheilantha (RI = 1.7) and
Cereus jamacaru (RI = 1.7) are the species with the highest ethnopharmacological relative impor-
tance indexes. The broad spectrum of therapeutic properties reported for these plants, including
anti-inflammatory, antiophidic, expectorant and calmative, and for treating problems such as diges-
tive, headache, toothache, influenza and more is worth highlighting.
Table 10.2 displays 37 non-endemic medicinal plants from Caatinga with RI ≥1 (Albuquerque
et al., 2007). The highest RI values are reported for Amburana cearensis (RI = 2.0), Myracrodruon
urundeuva (RI = 2.0) and Argemone mexicana (RI = 1.8). Several uses and properties are also
reported for these non-endemic medicinal plants, which include those described for the endemic
plants.
Some of the species listed in Tables 10.1 and 10.2 are commercialized for manufacturing of
herbal medicines and had their properties scientifically proven, such as A. cearensis, Erythrina
velutina, M. urundeuva and Sideroxylon obtusifolium (Albuquerque et al., 2007).
The biological and therapeutic potential of seven medicinal plants from Caatinga (A. cearensis,
Anadenanthera colubrina, Anacardium occidentalis, Bauhinia forficata, Cissus sicyoides,
TABLE 10.1
Endemic Medicinal Plants from Caatinga with RI ≥1 (Albuquerque et al., 2007)
RI Species Family Common Names Uses and Properties
1.9 Maytenus rigida Celastraceae Bom-nome Renal problems, hepatic problems, pains in
Mart. general, rheumatism, sexual impotence,
menstrual disturbances, inflammations in
general, asthma, cough, bronchitis, blow,
injury, anemia, circulation problems, cardiac
problems, ovarian inflammation, ovarian
infection, skin ulcers and vaginal ulcers.
1.7 Bauhinia cheilantha Fabaceae Mororó, pata-de-vaca Diabetes, high cholesterol levels,
(Bong.) Steud.a and unha-de-vaca inflammations in general, spinal problems,
cough, influenza, dysphonia, asthma, blood
thinner, depurative, rheumatism, migraine,
nervous disturbances, inappetence,
helminthiasis, expectorant, calmative and
tonic.
1.7 Cereus jamacaru DC Cactaceae Mandacaru and babão Many problems, such as renal, hepatic,
respiratory, spinal, urethral, syphilis, injury,
influenza, cough, bronchitis, ulcers,
constipation, hypertension, rheumatism,
enteritis, fever, etc.
1.5 Capparis jacobinae Capparaceae Icó-preto, icó- Cough, pertussis, colds, digestive problems,
Moric. ex Eichler verdadeiro and incó skin diseases, abdominal pain, intoxication,
fever, diabetes, lung inflammation, cardiac
problems and emmenagogic.
1.5 Erythrina velutina Fabaceae Mulungu Tooth inflammation, odontalgia, headache,
Willd.a fever, maternal milk production, diabetes,
hypertension, cough, bronchitis, nervous
disturbances, insomnia, hemorrhoids,
helminthiasis and calmative.
1.4 Caesalpinia Fabaceae Catingueira and Cough, bronchitis, respiratory infection,
pyramidalis Tul. catingueira-rasteira influenza, asthma, gastritis, colic, fever,
heartburn, flatulence, diarrhea, collision,
injury, diabetes, aphrodisiac, stomachache
and expectorant.
1.4 Spondias tuberosa Anacardiaceae Umbuzeiro, imbu, Ophthalmia, venereal diseases and digestive
Arruda and umbu problems, intestinal problems, diabetes, renal
infection, menstrual disturbances, placental
delivery, throat problems, antiemetic and
tonic.
1.3 Ziziphus joazeiro Rhamnaceae Joá and joazeiro Teeth cleaning, dandruff, asthma, cough,
Mart. influenza, pneumonia, tuberculosis,
bronchitis, constipation, throat inflammation,
indigestion, scabies, seborrheic dermatitis,
itching, skin problems, head injuries, healing
and expectorant.
1.2 Commiphora Burseraceae Imburana, emburana, Renal problems, influenza, cough, bronchitis,
leptophloeos emburana-de-cambão dysphonia, inflammations in general,
(Mart.) J.B. Gillett and umburana odontalgia, colic, diarrhea, antiemetic, tonic
and healing.
(Continued)

Endemic Medicinal Plants from Caatinga with RI ≥1 (Albuquerque et al., 2007)
1.1 Croton Euphorbiaceae Angolinha, Stomach ache, diabetes, inflammations in
argyrophylloides marmeleiro-branco, general, venereal diseases, blood thinner,
Mull. Arg. and sacatinga back pain, intoxication and headache.
1.0 Capparis flexuosa Capparaceae Feijão-bravo and Cough, pneumonia, influenza, colds, digestive
(L.) L. feijão-de-boi problems, skin diseases, abdominal pain,
rheumatism and antiophidic.
1.0 Cordia leucocephala Boraginaceae Piçarra, moleque- Bleeding, throat inflammation, rheumatism,
Moric. duro and nego-duro indigestion, arthritis, rickets, calmative and
tonic.
a Although not classified as endemic by Albuquerque et al. (2007), this species is also reported as endemic (Maia, 2004;
Viana et al., 2013).
TABLE 10.2
Non-Endemic Medicinal Plants from Caatinga with RI ≥1 (Albuquerque et al., 2007)
2.0 Amburana cearensis Fabaceae Cumaru, emburana- Tooth inflammation, genital inflammation, colic,
(Freire Allemão) A. C. de-cheiro and diarrhea, intestinal problems, placental
Smith amburana. delivery, pains in general, headache, influenza,
cough, bronchitis, pertussis, sinusitis, asthma,
heartburn, skin ulcers, urinary infection,
antiophidic, antispasmodic, expectorant and
tonic.
2.0 Myracrodruon Anacardiaceae Aroeira and Inflammations, pains, infections, blow, injury,
urundeuva Allemão Aroeira-do-sertão vaginal discharge, asthma, influenza, cough,
bronchitis, tuberculosis, heartburn, gastritis,
odontalgia, placental delivery, anemia,
diphtheria, skin ulcers and uterine.
1.8 Argemone mexicana L. Papaveraceae Cardo santo and Herpes labialis, fever, influenza, asthma,
cadinho tonsillitis, bronchitis, pneumonia, uterine
inflammation, ophthalmia, thrombosis,
scrophula, physical weakness, inflammations in
general, constipation, skin ulcers,
conjunctivitis, cholagogue, laxative and
digestive.
1.6 Anadenanthera Mimosaceae Angico, angico-de- Anemia, cough, asthma, bronchitis, pertussis,
colubrina (Vell.) caroço and lung inflammation, influenza, constipation,
Brenan angico-branco inflammations in general, cancer, blood thinner,
blow, injury, scrofula, diphtheria, fissures in
foot, gastritis and expectorant.
1.6 Operculina hamiltonii Convolvulaceae Batata-de-purga Influenza, pneumonia, asthma, bronchitis,
(G. Don) D.F. Austin cough, cardiovascular problems, helminthiasis,
& Staples rheumatism, constipation, inappetence,
digestive problems, hydropsy, syphilis,
amenorrhea, teething, inflammations in general,
flatulence and laxative.
(Continued)

1.6 Rosmarinus Lamiaceae Alecrim and Menstrual disturbances, cough, influenza,
officinalis L. alecrim-de-jardim asthma, colic, fever, pains in general,
flatulence, stomachic, intestinal problems,
hepatic problems, renal problems, injury,
rheumatism, sedative, antispasmodic and
cardiotonic.
1.5 Aloe vera (L.) Burm. f. Liliaceae Babosa Hemorrhoid, heartburn, stomach problems,
cancer, dandruff, hair loss, contusion,
rheumatism, injury, helminthiasis,
inflammations in general, emollient and
emmenagogic.
1.4 Chenopodium Chenopodiaceae Mastruz Influenza, cough, bronchitis, tuberculosis,
ambrosioides L. helminthiasis, cancer, blow, digestive problems,
headache, fever, antimicrobial, expectorant,
hematoma, gastritis and stomachic.
1.4 Cymbopogon citratus Poaceae Capim-santo and Stomach ache, gastritis, ulcer, diarrhea,
(DC.) Stapf capim-caboclo indigestion, inappetence, colic, pains in
general, hypertension, insomnia, fever, cough,
calmative and diuretic.
1.4 Leonotis nepetifolia (L.) Lamiaceae Cordão-de-são- Menstrual colic, urinary retention, rheumatism,
R. Br. francisco and inflammations in general, fever, cystitis,
cravinho dysentery, calculus of kidney, childbirth pains,
paralysis, aphrodisiac, sedative, stomachic and
healing.
1.3 Cnidoscolus Euphorbiaceae Urtiga branca and Hemorrhoid, renal problems, ophthalmic
phyllacanthus (Mull. favela diseases, blow, injury, fractures, warts, skin
Arg.) Pax & L. Hoffm. problems, eye cleansing, urinary infection and
inflammations in general.
1.3 Helianthus annuus L. Asteraceae Girassol Weakness, thrombosis, fever, ulcer,
neuralgiainjury, contusion, epistaxis,
inappetence, hypercholesterolemia and
emollient.
1.3 Lippia alba (Mill.) N.E. Verbenaceae Cidreira, erva- Diabetes, hypertension, colic, diarrhea,
Br. cidreira, and melissa heartburn, inappetence, fever, headache,
anemia, cold, anticonceptive, calmative, cardiac
problems and gastritis.
1.3 Ruta graveolens L. Rutaceae Arruda and arruda Menstrual colic, headache, otalgia,
macho conjunctivitis, arthritis, nevralgy, helminthiasis,
amenorrhea, digestive problems and
antispasmodic.
1.2 Achillea millefolium L. Asteraceae Novalgina and Pains in general, fever, abscess, skin problems,
mil-folhas headache, physical weakness, intoxication,
injury, stomachic, antispasmodic and
depurative.
1.2 Anacardium Anacardiaceae Cajú, cajueiro and Diarrhea, inflammations in general, renal
occidentale L. caju-roxo infection, heartburn, tuberculosis, diabetes,
blow and antiseptic.
(Continued)

1.2 Caesalpinia ferrea Fabaceae Jucá and pau-ferro Inflammations in general, blow, throat
Mart. afflictions, bronchitis, anemia, swelling, back
pain, injury, labyrinthitis, renal problems, stress
and fatigue.
1.2 Hyptis suaveolens (L.) Lamiaceae Alfazema-brava, Digestive problems, menstrual colic,
Poit. alfavaca-caboclo amenorrhea, odontalgia, headache, fever,
and influenza, respiratory problems in general, gout
alfazema-de- and eye cleansing.
caboclo
1.2 Momordica charantia L. Cucurbitaceae Melão-de-são- Injury, STDs, lice, scabies, hemorrhoid, allergy,
caetano and diabetes, helminthiasis, rheumatism, diarrhea
melão-de-sabiá and dandruff.
1.2 Ocimum basilicum L. Lamiaceae Manjericão and Otalgia, bronchitis, cough, influenza, fever,
manjericão-roxo tonsillitis, gingivitis, diarrhea, headache,
stomachic, antispasmodic, diuretic and
antiemetic.
1.2 Plectranthus Lamiaceae Hortelã da folha Otalgia, inflammations in general, cough,
amboinicus (Lour.) grande, hortelã- bronchitis, tonsillitis, pneumonia, influenza,
Spreng. graúdo and hortelã constipation, hepatic problems, menstrual
da folha grossa disturbances, dysphonia, stomachic and
helminthiasis.
1.2 Parkinsonia aculeata L. Fabaceae Turco Migraine, thrombosis, influenza, asthma,
diabetes, hypertension, fever, epilepsy, malaria
and antiophidic
1.2 Sideroxylon Sapotaceae Quixaba, quixabeira Blow, pains in general, duodenal ulcer, gastritis,
obtusifolium (Humb. and rompe-gibão heartburn, chronic inflammation, genital injury,
ex Roem. & Schult.) ovarian inflammation, adnexitis, colic, renal
T.D. Penn. problems, cardiac problems, diabetes and healing.
1.1 Allium sativum L. Liliaceae Alho Insect bites, dysphonia, constipation, digestive
problems, odontalgia, conjunctivitis,
hypercholesterolemia, antibiotic, influenza and
throat afflictions.
1.1 Combretum leprosum Combretaceae Mofumbo Bronchitis, influenza, cough, pertussis, sweating,
Mart. diphtheria, heartburn, calmative, hemostatic
and expectorant.
1.1 Croton rhamnifolius Euphorbiaceae Pau-de-leite and Influenza, cough, stomachache, menstrual
Willd. velame disturbances, back pain, anemia, blood thinner,
hypercholesterolemia and depurative.
1.1 Hyptis pectinata (L.) Lamiaceae Alfazema-brava, Headache, odontalgia, amenorrhea, hepatalgia,
Poit. alfazema-de- hepatic problems, flatulence, rheumatism,
caboclo, and gastritis, ulcer, asthma, cough and bronchitis.
sambacaitá
1.1 Lippia sp. Verbenaceae Alecrim de caboclo Influenza, sinusitis, cough, nasal congestion,
pains in general, headache, odontalgia,
eliminate material after child delivery,
calmative and expectorant.
1.1 Mimosa tenuiflora Mimosaceae Jurema-preta and Injury, odontalgia, inflammations in general,
(Willd.) Poir. jurema fever, menstrual colic, headache, hypertension,
bronchitis and cough.
(Continued)

1.1 Ocimum gratissimum L. Lamiaceae Alfavaca, alfavaca- Digestive, flatulence, digestive problems,
branco and quioiô influenza, cough, itching, expectorant, calming,
stress, headache and fatigue.
1.1 Scoparia dulcis L. Scrophulariaceae Vassourinha and Tortion, bronchitis, influenza, cough, fever,
vassourinha-de- inflammations in general, uterine problems,
nossa-senhora helminthiasis, diabetes, amenorrhea and
emmenagogic.
1.0 Acmella uliginosa (Sw.) Asteraceae Agrião Caries, asthma, inflammations in general, injury,
Cass. hair loss, stomachic, depurative, tonic and
expectorant.
1.0 Heliotropium Boraginaceae Crista-de-galo and Foot swelling, influenza, cough, inflammations
indicum L. fedegoso in general, hepatic problems, conjunctivitis,
renal problems and diuretic.
1.0 Kalanchoe brasiliensis Crassulaceae Pratudo and coirama Pains in general, ulcer, gastritis, inflammations
Cambess. in general, injury, lung afflictions, asthma,
kidney stones and emollient.
1.0 Leucas martinicensis Lamiaceae Cordão-de-são Renal problems, rheumatism, inflammations in
(Jacq.) R. Br. francisco and general, nevralgy, sweating, flatulence, tonic,
cordão-de-frade antispasmodic and calmative.
1.0 Marsypianthes Lamiaceae Bentônica-brava and Cough, bronchitis, flatulence, fever, articular
chamaedrys (Vahl) hortelã-do-mato rheumatism, antiophidic, stimulant and
Kuntze digestive.
1.0 Tabebuia impetiginosa Bignoniaceae Pau-d'arco-roxo, Ulcer, surgical infections, inflammations in
(Mart. ex DC.) Standl. ipê-roxo general, leucorrhea, cancer, gingivitis, cardiac
problems, antimicrobial and antiseptic.
M. urundeuva and Zingiber officinalis) were reviewed by Silva et al. (2012). Additionally,
Viana et al. (2013) reviewed 16 medicinal plants from Caatinga, including A. cearensis, A. occidentalis,
M. urundeuva and Z. officinalis.
This chapter focuses on five medicinal plants from Caatinga (listed in alphabetical order), B. chei-
lantha (RI = 1.7), Combretum leprosum (RI = 1.1), Egletes viscosa (RI = 0.9), E. velutina (RI = 1.5) and
M. urundeuva (RI = 2.0), Table 10.3, which were selected based not only on their ethnopharmacological
TABLE 10.3
Summary of the Names of All Plant Species, Their Respective Family and
Common Names Discussed in This Chapter
Bauhinia cheilantha (sin. Pauletia cheilantha) Fabaceae mororó, pata-de-vaca, unha-de-vaca
Combretum leprosum (sin. Combretum Combretaceae mofumbo, mufumbo, pente de macaco
hasslerianum)
Egletes viscosa (sin. Platystephium Compositae macela, macela-da-terra
graveolens)
Erythrina velutina (sin. Erythrina splendida) Fabaceae mulungu, suinão, canivete, corticeira,
pau-de-coral, sanaduí, sananduva
Myracrodruon urundeuva Anacardiaceae aroeira, aroeira-do-sertão
RIs but also on the scientific validation of their popular uses. For each species, the botanic aspects and
occurrence, ethnopharmacology, chemical studies and pharmacological studies are presented.
10.2 B. CHEILANTHA (BONG.) STEUD. (FABACEAE)

Bauhinia is a pantropical genus that comprises about 300-350 species (Sinou et al., 2009). In Brazil,
this genus is represented by 91 species, which are commonly known as “mororó” (indigenous Tupi
language) and “pata-de-vaca” (literally translated as cow nail); the latter because of the bifoliated
leaves (Gutiérrez et al., 2011). A noteworthy detail is that the most common Bauhinia species in the
state of Ceará (Northeast region of Brazil) are Bauhinia ungulata L. (found in the litoral) and
B. cheilantha (Bong.) Steud. (found in Caatinga) (Viana et al., 2013).
B. cheilantha is a medicinal plant (Shrub above 1 m in height) with high ethnopharmacological
relative importance index (RI = 1.7). This species is an endemic plant from Caatinga, also found in
Cerrado bioma although with less frequency (Gutiérrez, 2010).
10.2.2 Ethnopharmacology
Several uses and properties are reported for B. cheilantha, such as hypoglycemia (treatment of
diabetes), calmative, blood thinner, depurative, rheumatism, migraine, nervous disturbances, inap-
petence, helminthiasis, etc. (Table 10.1). Infusion or decoction from stem-barks of the plant is used
as tonic, depurative and for treating diabetes (Agra et al., 2007). Additionally, roasted seeds from
B. cheilantha are used in a hot beverage as a substitute for coffee (Lucena et al., 2007).
10.2.3 Chemical Studies
Despite the vast popular use of B. cheilantha and the plant’s high ethnopharmacological relative
importance index, the chemical study of this species is still incipient. Phytochemical screening for
identification of secondary metabolites in organic extracts from leaves, barks and roots revealed the
presence of alkaloids, anthraquinones, steroids, flavonoids and xanthones (Luna et al., 2005).
The chemical study of the aqueous extract from dried leaves of B. cheilantha yielded the flavonoids
afzelin (1, kaempferol-3-O-rhamnoside) and quercetrin (2, quercetin 3-O-rhaminoside) (Figure 10.3).
FIGURE 10.3 Chemical structures of the secondary metabolites from B. cheilantha.

Moreover, HPLC quantitative analyses of the aqueous extract from leaves of seven B. cheilantha
specimens revealed the presence of the flavonoids rutin (3), isoquercitrin (4) and quercetin (5), shown
in Figure 10.3, besides compound 2 (Brígido, 2001). The phytochemical investigation of the ethanol
extract from the aerial parts of B. cheilantha also yielded compounds 1 and 2 (Oliveira, 2008).
The essential oils’ (extracted by hydrodistillation) composition of the leaves from three specimens
of B. cheilantha was investigated. Among the 39 compounds present in the oils, trans-caryophyllene
(6), caryophyllene oxide (7), β-elemene (8), γ-muurolene (9), spathulenol (10), α-cadinol (11) and
α-muurolol (12) were the major constituents (Brígido, 2001).
The fatty acid composition of the seeds oil of B. cheilantha revealed linoleic acid (C18:2 Δ9,12;
42.3%), palmitic acid (C16:0; 25.7%), octadec-7-enoic acid (C18:1 Δ7; 15.3%) and stearic acid (C18:0;
10.4%) as the major constituents (Brígido, 2001). In addition, high protein content (58.9%) was
found in the seeds of B. cheilantha, being even higher than those found in soybean (39.5-44.5%) and
cowpea bean (19.5-26.1%) (Teixeira et al., 2013).
10.2.4 Pharmacological Studies

The hypoglycemic property of a methanol extract (600 mg kg−1) from leaves of B. cheilantha was
confirmed in alloxan-induced diabetic rats (Silva and Cechinel-Filho, 2002). The same activity was
also observed when the ethanol extract (900 mg kg−1) from the aerial parts was tested. The lack
of acute toxicity of this extract when tested in concentrations of 1,000 and 2,000 mg kg−1 (ip), and
5,000 mg kg−1 (oral administration) in mice is worth highlighting. In this case, no mortality was
observed after 48 hours of extract administration (Oliveira, 2008).
Ethanol extracts from the aerial parts of B. cheilantha, collected in three different periods,
were assayed for their antioxidant activities (DPPH method). EC50 values of 11.67 ± 1.43 (1st col-
lection), 26.68 ± 0.23 (2nd collection) and 21.77 ± 0.13 μg mL−1 (3rd collection) were observed
(Oliveira, 2008).
The antinociceptive activity of the aqueous extract from the bark of B. cheilantha was evalu-
ated using three different tests (writhing induced by acetic acid −0.6% formalin 1% and hot
plate). The extract (administered orally: 400 mg kg−1) reduced nociception by 54.4% in the
writhing test. The analgesic effect of the extract (100, 200 and 400 mg kg−1) in the 1% formalin
test was observed (57.4%, 46.1% and 46.2% inhibition of the pain reaction, respectively, in the
2nd phase). Finally, the analgesic properties of the extract were corroborated by the hot plate
test. The extract (100, 200 and 400 mg kg−1) increased the latency time by 39.8%, 30.7% and
32.8%, respectively. Additionally, no acute toxicity was found for the extract tested at doses up
to 3 g kg−1 (Silva et al., 2005).
In summary, B. cheilantha is an endemic medicinal plant from Caatinga, which presents one
of the highest RIs. Several uses are reported for the plant including the treatment of diabetes.
Pharmacological studies support the species hypoglycemic and analgesic properties; the latter may
be associated with the calming activity reported for the species. The lack of more comprehensive
chemical and pharmacological studies that can validate the ethnopharmacological use of this spe-
cies draw attention.
10.3 C. LEPROSUM MART. (COMBRETACEAE)

C. leprosum is a member of the family Combretaceae, constituted by circa 600 species in 18 genera,
of which Terminalia and Combretum are the most important. This species is a popular medici-
nal plant, which grows wild as a shrub in the Northeast region of Brazil, mainly in the semi-arid
regions of Ceará state known as “mofumbo”, “mufumbo” or “pente de macaco” (Facundo et al.,
1993, 2005, 2008).
Combretum species are used extensively in traditional medicine against inflammation, infections,
malaria, bleeding, diarrhea, digestive disorders and diabetes, and as diuretic (Lima et al., 2012).
Infusions or decoctions from bark, bast, leaves, flowers and roots of C. leprosum are used in the folk
medicine against bronchitis, influenza, cough, pertussis, sweating, diphtheria and heartburn, and as
calmative, hemostatic and expectorant. C. leprosum showed an RI of 1.1, suggesting for this species
a very high ethnopharmacological versatility (Albuquerque et al., 2007).
The phytochemical investigation of the organic extract from leaves (hexane and ethanol) and roots
(ethanol) of C. leprosum collected in Ceará state yielded the triterpenes 3β,6β,16β-trihydroxylup-
20(29)-ene (13), arjunolic acid (14) and mollic acid (15), besides the flavonoids 5,7,3′,4′-tetrahydroxy-
3-methoxyflavone (3-O-methylquercetin, 16) and 3-O-α-l-rhamnopyranosylquercetin (quercetrin,
2), Figure 10.4 (Facundo et al., 1993). Independently, the chemical investigation of the ethanol
extract from flowers of the same plant led to the isolation of 13-16, together with the flavonoids
5,3′,4′-trihydroxy-3,7-dimethoxy-flavone (17) and 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (18),
the cycloartane triterpenes 19 and 20, and the α-d-glucopiranoside-(3β)-stigmast-5-en-3-yl (21),
Figure 10.4 (Facundo et al., 2008).

Pharmacological investigations of C. leprosum point to similar bioactivities already observed for
other species of Combretum. Oral administration of the ethanol extract (200 mg kg−1; 37.6% inhibi-
tion) from roots of C. leprosum as well as the secondary metabolite arjunolic acid (14, Figure 10.4;
100 mg kg−1; 80.8% inhibition) revealed anti-inflammatory activity for both tested samples (extract
and isolated compound) by reducing the paw edema induced by carrageenan. No activity, is worth
mentioning, was observed for the ethanol extract from leaves of C. leprosum (Facundo et al., 2005).
The gastroprotective and anti-ulcerogenic effects of the ethanol extract from the stem bark of
C. leprosum was reported (Nunes et al., 2009). The extract inhibited the gastric acid secretion and
increased the mucosal defensive factors, such as mucus and prostaglandin.
The antiproliferative and anti-inflammatory properties of the ethanol extract from the flower
of C. leprosum was demonstrated in models of skin inflammatory and hyperproliferative process
FIGURE 10.4 Chemical structures of the secondary metabolites from C. leprosum.

(Horinouchi et al., 2013). Additionally, the anti-inflammatory effect of the triterpene 3β,6β,16β-
trihydroxylup-20(29)-ene (13; Figure 10.4), isolated from leaves of C. leprosum, was also demon-
strated in mouse cutaneous wound healing model (Nascimento-Neto et al., 2015).
The ethanol extract obtained from roots of C. leprosum and its isolated compound arjunolic acid
(14; Figure 10.4) showed the capacity of neutralizing critical points in the tissue damage process
induced by Bothrops jararacussu and B. jararaca venoms. The results represented the first scien-
tific validation for the popular use of the plant against snakebites in the Northeast region of Brazil
(Fernandes et al., 2014).
The neuroprotective potential of the ethanol extract from C. leprosum to Parkinson’s disease,
a brain disorder associated with inflammatory processes, was investigated using a murine model
induced by neurotoxin MPTB. The plant extract was able to improve motor deficits, by attenuat-
ing of the hyperlocomotion and similar to the control groups (no MPTB added). According to this
study, the extract of the plant could be a new therapeutic approach for preventing Parkinson’s dis-
ease, mainly by the preservation of the dopaminergic tonus (Moraes et al., 2016).
The antioxidant, cytotoxic and genotoxic properties of the ethanol extract from flowers of
C. leprosum and is worth mentioning 5,3′,4′-trihydroxy-3,7-dimethoxyflavone (17) and 5,3′-dihydroxy-
3,7,4′-trimethoxyflavone (18) secondary metabolites, Figure 10.4, were also evaluated. In this
case, the ethanol extract was mutagenic at high concentrations (500 μg mL−1), while the flavonoid
5,3′,4′-trihydroxy-3,7-dimethoxyflavone (17) induced an increase in DNA damage. Nevertheless,
flavonoid 5,3′-dihydroxy-3,7,4′-trimethoxyflavone (18) showed a better antioxidant action with lower
toxicity and absence of genotoxicity (Viau et al., 2016).
In summary, C. leprosum is a medicinal plant that occurs in the Northeast region of Brazil,
especially in the state of Ceará, and which displays high ethnopharmacological RI of 1.1. This plant
is used in folk medicine for treating inflammations, in which the crude extracts and isolated com-
pounds were validated by pharmacological studies. Furthermore, the antiophidic potential of the
ethanol extract also corroborated the popular use for treating snakebites.
10.4 E. VISCOSA LESS. (ASTERACEAE)

E. viscosa, one of the ten reported species of Egletes, is a medicinal flowering plant (wild and annual
herb) native to the intertropical America. The species grows throughout the Northeast region of
Brazil at margins of river banks and lakes as soon as the raining season ends. E. viscosa is popularly
known as “macela” and sometimes as “macela-da-terra”, the latter contrasting from Achyrocline
satureoides (Lam.) DC. and Chrysanthemum parthenium L. (Berh.), which are native Asteraceae
plants that occur in the South and Southeast regions of Brazil (Lorenzi, 2000).
Due to stomachic, antidiarrheal, emmenagogue and diaphoretic properties, the dried flower buds of
E. viscosa are widely sold in herbal stores and supermarkets from the Northeast region of Brazil to
be used in folk medicine as a tea or decoction (Braga, 1976; Corrêa, 1984).
The major constituents of the flower buds of E. viscosa were reported (Cunha, 2003; Lima
et al., 1996). Phytochemical investigation of the hexane extract of the plant yielded centipedic
acid (22) and 12-acetoxy-hawtriwaic acid lactone (23), while the ethanol extract yielded ternatin
(24), Figure 10.5 (Lima et al., 1996). Besides compounds 22-24, barbatol (25), tarapacol (26),
FIGURE 10.5 Chemical structures of the secondary metabolites from E. viscosa.
12-epi-bacchotricuneatin (27), 15,20-epoxy-9,10-seco-4,9(10),13(20),14-labda-tetraen-18-ol (28)

and 9,10-seco-4,9(10),13(14)-labdatrien-15,20-olid-18-ol (29), Figure 10.5, were isolated from the
chloroform extract of the plant (Cunha, 2003).
The essential oils from flower buds of E. viscosa specimens from different sites in the state of
Ceará (Northeast of Brazil) were also investigated. The study revealed the existence of two chemo-
types of the plant: trans-pynocarveil acetate (30) and cis-isopynocarveil acetate (31), Figure 10.5,
based on the high content of these major volatile constituents in each essential oil (Cunha, 2003).
From these findings, the chemical composition of lyophilized teas from the flower buds (cultivated
specimens) of both chemotypes was investigated. From the trans-pynocarveil acetate chemotype
compounds 22-24 were isolated, besides the diterpene 12-acetoxy-7-hydroxy-3,13(14)-clerodandien-
18,19:15,16-diolide (32). The cis-isopinocarveyl chemotype yielded compounds 23-25, 32 and sco-
poletin (33), Figure 10.5 (Vieira et al., 2006).
The chemical investigation of the aerial parts of E. viscosa led to the isolation of 24-26, in addi-
tion to compounds 8α-hydroxylabd-14(15)-ene-13(S)-O-β-d-ribopyranoside (34), 13-epi-sclareol
(35) and spinasterol (36), Figure 10.5 (Silva-Filho et al., 2007). The phytochemical study of the
entire plant native from Peru revealed the presence of the following compounds in the plant: tarapa-
col (26), glycosides 13(R)-O-α-l-arabinopiranoside-13-hydroxy-labda-7,14-dien (37), 13R-O-α-l-
arabinopyranoside-13-hydroxy-7-oxalabda-8,14-dien (38), tarapacol 14-O-α-l-arabinose (39) and
5,7-dihydroxy-2′,4′-dimethoxyflavonol (40), Figure 10.5 (Lee et al., 2005).

The essential oil from the flower heads of E. viscosa showed a significant dose-dependent analge-
sic activity induced by acetic acid and formalin tests, besides anticonvulsant activity induced by
pentylenetetrazol in mice. The antibacterial activity of the oil was also reported against resistant
strains of Staphylococus aureus, with the MIC ranging from 0.625 to 2.5 μL mL−1 (Souza, Rao
et al., 1998).
Centipedic acid (22) and 12-acetoxy-hawtriwaic lactone (23) (Figure 10.5) were tested on
acute and chronic models of mouse ear dermatitis induced by 12-O-tetradecanoylphorbol-13-
acetate (TPA) and oxazolone. The assays indicated that these diterpenes are effective topi-
cal anti-inflammatory agents (Calou et al., 2008). Additionally, compound 23 also attenuated
capsaicin-induced ear edema and hind paw nociception in mice, possibly involving capsaicin-
receptive TRPV1 receptors, endogenous adenosine and ATP-sensitive potassium channels
(Melo et al., 2006).
Centipedic acid (22; Figure 10.5) showed significant inhibition against the gastric ulceration
induced by indomethacin, with the gastroprotective mechanism later elucidated using the mouse
model of gastric mucosal damage induced by ethanol, indicating a cytoprotective role for the diter-
pene (Guedes et al., 2002, 2008).
The flavonoid ternatin (24; Figure 10.5) showed anti-inflammatory and anti-anaphylactic proper-
ties in modulating mouse passive cutaneous anaphylaxis (PCA) and rat carrageenan-induced pleu-
risy (Souza et al., 1992). The anti-inflammatory activity of 24 was reinforced when examined for
the compound’s possible influence on thioglycolate-elicited neutrophil influx into the rat peritoneal
cavity in vivo and nitric oxide production in lipopolysaccharide (LPS)-activated mouse peritoneal
macrophages ex vivo (Rao et al., 2003).
The hepatoprotective activity of ternatin (24) was investigated using different models (Rao
et al., 1994, Rao et al., 1997; Souza, et al., 1997; Souza, et al., 1998). This flavonoid showed
marked inhibition of CCl4 serum enzymes and morbid histological changes when the model of
liver injury induced by carbon tetrachloride in rats was used (Rao et al., 1994). Hepatoprotective
activity was also confirmed through the antiperoxidative effect against lipid peroxidation. In this
model, ternatin (24) showed significant inhibition in hind paw edema induced by adriamycin in a
manner similar to vitamin E (Souza et al., 1997). Ternatin was also able to prevent significantly
acetaminophen-induced acute increase in serum enzymes, inhibition of hepatocellular necrosis
and bile duct proliferation induced by aflatoxin (Souza et al., 1998). The compound’s gastro-
protective and antidiarrheal properties were observed through significant inhibition on gastric
mucosal damage induced by hypothermic restraint stress, ethanol and indomethacin, and in both
intestinal transit and accumulation of intestinal fluids induced by castor oil, besides antagonized
the contractile response evoked by acetylcholine, histamine, serotonin and barium chloride (Rao
et al., 1997).
The antithrombotic activity of 24 was also evaluated on in vitro platelet aggregation induced
by ADP and an in vivo mouse model of tail thrombosis induced by kappa-carrageenan (KC).
Besides showing the ADP-induced platelet aggregation inhibition (concentration-dependent; IC50
of 390 μM), the flavonoid showed marked protection of mice from thrombotic challenge with
KC (Souza et al., 1994).
In summary, E. viscosa is a medicinal plant that occurs in the Northeast of Brazil (Caatinga
bioma) known for stomachic, antidiarrheal, emmenagogue and diaphoretic properties. Dried flower
buds are sold in herbal store and supermarkets, and local people use the plant as tea or decoction.
The major chemical constituents from the flower buds extract of E. viscosa, centipedic acid (22),
12-acetoxy-hawtriwaic lactone acid (23) and ternatin (24), demonstrated several pharmacological
activities related to gastrointestinal disorders, and these findings support the validation of the spe-
cies ethnobotanical use.
10.5 ERYTHRINA VELUTINA WILLD. (FABACEAE)

E. veluntina is one of the 110 species described for the Erythrina genus, which is distributed in
tropical and subtropical regions worldwide. That about 70 of the Erythrina species are native to the
Americas is noteworthy (Virtuoso et al., 2005). E. veluntina is a medium size tree plant endemic
to the plain and riverbanks of the semi-arid regions of Northeast of Brazil, popularly known as
“mulungu”, “suinão”, “canivete”, “corticeira”, “pau-de-coral”, “sanaduí”, “sananduva”, and com-
monly used in gardens and parks as an ornamental plant (Lorenzi, 1992).
The aqueous extract or tincture from harvested stem bark of E. velutina have popular use against
a number of central nervous system disorders, such as insomnia, convulsion, anxiety, nervous
cough and rheumatism (Corrêa, 1984; Lorenzi and Matos, 2002). The dried fruit of the plant
has local anesthetic action used in the form of cigarettes as a toothache remedy (Lorenzi and
Matos, 2002).
Chemical investigation of the ethanol extract from the stem bark of E. velutina yielded the fla-
vonoids 4′-O-methylsigmoidin (41), eryvellutinone (42), homohesperetin (43), phaseolin (44),
5,7,3′-trihydroxy-5′-prenyl-6-methoxyisoflavone (45), phaseollidin (46), besides erythrodiol (47)
and lupeol (48), Figure 10.6 (Cunha et al., 1996; Rabelo et al., 2001; Rodrigues, 2004).
Chemical studies of the methanol extract from seeds of E. velutina were also reported (Ozawa
et al., 2008; Ozawa et al., 2009; Ozawa et al., 2011). The alkaloids hypaphorine (49), erysodine
N-oxide (50), erythraline (51), 8-oxo-erythraline (52), erysotrine (53), erysodine (54), erysovine
(55), glycoerysodine (56), sodium erysovine 15-O-sulfate (57), erysopine 15-O-sulfate (58), 16-O-β-
glucopyranosyl coccoline (59), erymelanthine (60) and sodium erysovine N-oxy-15-O-sulfate (61),
Figure 10.6, were isolated from this part of the plant.

The tranquilizing effects of the hydro-alcohol extracts from the stem bark of E. velutina were
extensively investigated. The anxiolytic potential on the behavior of female mice submitted to the
open-field tests and to elevated plus-maze after oral or intraperitoneal administration of the extract
showed decreased locomotor activity and sedative effect (Vasconcelos et al., 2004). The effects of
acute and chronic oral administration of the extract in rats submitted to the elevated T-maze model
of anxiety suggested that the extract exerts anxiolytic-like effects on a specific subset of defensive
behaviors, which have been associated with generalized anxiety disorder (Ribeiro et al., 2006). The
extract also showed anticonvulsant effects in a strychnine-induced seizure model, with possible
action in a glycine system and a potentiation of pentobarbital sleeping time, suggesting depressant
action in the nervous central system (Vasconcelos et al., 2007). Studies with a mice model submit-
ted to elevated plus-maze, forced swim, spontaneous locomotor activity and habituation of active
chamber effects of acute and chronic treatments showed anxiolytic-like effect of the extract that
could serve as a new approach for treatment anxiety, although at low doses the extract may have an
amnesic effect (Raupp et al., 2008). Moreover, the neuroprotective property of the plant extract was
suggested, reducing the excitatory amino acid concentrations and increased the inhibitory amino
acid levels after ischemia (Rodrigues et al., 2017).
FIGURE 10.6 Chemical structures of the secondary metabolites from E. velutina.
The anti-inflammatory activity of the hydro-alcohol extract from the stem bark of E. velutina
was also demonstrated in a dextran model. The extract decreased the paw edema at 1, 2, 3, 4 and
24 hours, probably by interfering in the inflammatory processes in which mast cells have an impor-
tant role (Vasconcelos et al., 2011). Moreover, the antibacterial activity (disk diffusion method) of
both the crude ethanol extract and hexane fraction from the plant against S. aureus and Streptococcus
pyogenes was demonstrated in preliminary experiments (Virtuoso et al., 2005). A reasonable lack
of toxicity of the aqueous extract of E. velutina leaves was observed on assays of acute toxicity in
experimental animals (Craveiro et al., 2008).
An opiate-like analgesic effect of the aqueous extract from leaves in mice has been suggested,
when the antinociceptive effect was reversed by pretreatment with the opiate antagonist naloxone
(Dantas et al., 2004; Marchioro et al., 2005). However, the mechanism of action was elucidated
definitively in experiments using terminal segments of the guinea pig ileum smooth muscle, the
following was observed when contractile response involving GABAA receptor activation, acetyl-
choline release, muscarinic receptor activation, augmentation of Ca+ entry through L-type calcium
channels and calcium release from the intracellular stores (Carvalho et al., 2009). The extract also
crossed the blood-brain barrier and inhibited cholinesterase in the central nervous system of mice,
suggesting anticholinesterase activity in mouse brains (Santos et al., 2012).
The pharmacological activities of the alkaloids isolated from seeds of E. velutina were inves-
tigated (Ozawa et al., 2008; Ozawa et al., 2009). Hypaphorine (49; Figure 10.6) showed a signifi-
cant increase on non-rapid eye movement (NREM) sleep time of mice during the first hour after
treatment confirming the sleeping promoting property. The enhancing effects of the alkaloids ery-
thraline (51), erysodine (54) and erysovine (55), Figure 10.6, on the cytotoxicity of TRAIL (tumor
necrosis factor [TNF] related apoptosis-inducing ligand) against Jurkat cells were also reported.
However, these compounds showed some cytotoxicity even when their activities were moderated
in the absence of TRAIL. Compounds 8-oxo-erythraline (52), erysotrine (53) and glycoerysodine
(56), Figure 10.6, exhibited no cytotoxicity by themselves, but they acted synergistically when com-
bined by TRAIL. Erysotrine (53) showed the superior results among the alkaloids tested when
combined with TRAIL, and no cytotoxicity by itself.
In summary, the pharmacological effects of the hydro-alcohol extract from stems bark of E. velutina
in the central nervous system support the plant’s popular use as tranquilizer in the Brazilian folk
medicine. The low toxicity of the leaf extract and the antinociceptive, analgesic and anticholinester-
ase activities reported suggest new therapeutic uses for this plant.
10.6 MYRACRODRUON URUNDEUVA ALLEMÃO (ANACARDIACEAE)

Popularly known as “Aroeira-do-Sertão”, M. urundeuva is a tree found in Brazil, mainly in the
semi-arid vegetation of the Northeast region of Brazil (Cruz, 1979). The flowering period of the
plant goes from June to August, when the tree is completely leafless, and the fruiting period occurs
from August to November (Lorenzi and Matos, 2002).
10.6.2 Ethnobotany and Ethnopharmacology

Ethnobotanical records mentioned the innumerable uses of this plant. The bark is used for tanning
of animal skins due to the high content of tannins. Additionally, the bark is considered one of the
most resistant Brazilian woods, being used in civil construction girders and railway sleepers, with
noteworthy emphasize to their resistance to crushing and to physical and biological agents, even in
long-time contact with soil and water (Braga, 1976).
Ethnopharmacological studies refer to the use of the bark (deprived of inner bark), as one of the
oldest plant remedies for gynecological problems used in folk medicine of Northeastern Brazil. The
use in the oral treatment of diseases of the respiratory system, urinary tract, hemoptysis, metror-
rhagia and diarrhea, in the form of infusions or decoctions is also reported. The plant has an excel-
lent reputation in the home treatment of postpartum sequelae, and skin and mouth injury (oral and
topical concomitant uses) (Braga, 1976).
The chromatographic fractionation of the ethyl acetate extract from the inner bark of the plant yielded
several fractions with pronounced anti-inflammatory activity. One of the bioactive fractions was com-
posed by a mixture of the dimeric chalcones, urundeuvins A (62), B (63) and C (64), Figure 10.7.
Additionally, tannins were identified as the main constituents in another active fraction (Bandeira,
2002). This preliminary work on M. urundeuva demonstrated that the therapeutic activity of this plant
may be associated with a phytotherapeutic complex rather than simply a single active compound.
Liquid chromatography coupled to mass spectrometry (UPLC-ESI-QTOF MS/MS) analyses of
the inner bark extract confirmed the presence of the chalcones 62-64 (Figure 10.7), besides other
polyphenol compounds such as gallic acid derivatives tentatively identified (Galvão et al., 2018).

Studies on the medicinal properties of the inner bark of M. urundeuva were based on the vast
folk-use of this plant (Bandeira, 2002). Both hydro-alcohol and aqueous extracts were subjected
FIGURE 10.7 Chemical structures of the secondary metabolites from M. urundeuva.
to non-clinical pharmacological tests, and showed evident anti-inflammatory, analgesic, healing

and antiulcer effects, together with antihistamine and anti-bradykinin actions. Toxicological studies
have shown that these extracts are deprived practically of toxic effects orally, and point to the lack
of teratogenic effects of the plant (Viana et al., 1995). However, when tested in pregnant rats, the
extract induced bone malformations in the fetus. In this case, no anatomopathological alterations,
decreased mating capacity or infertility were observed (Carlini et al., 2013).
Preliminary clinical studies in patients with a peptic ulcer and in patients with cervicitis and ecto-
pia, using the experimental pharmaceutical preparations elixir and vaginal ointment, respectively,
supported the clinical use of the plant in these pathologies (Bandeira, 2002; Viana et al., 1995).
The antiviral potential of the leaves from M. urundeuva was investigated (Cecílio et al., 2016).
The flavonoid enriched fraction, obtained from the ethanol leaves’ extract(adult tree), showed a
pronounced action against rotavirus.
Studies revealed the pronounced anti-inflammatory, analgesic and antiulcerogenic activities of
the fraction containing the dimeric chalcones urundeuvins A-C (62-64) (Albuquerque et al., 2004;
Viana et al., 2003). This fraction was effective in reducing cell death induced by 6-hydroxydo-
pamine (6-HODA), in addition to inhibiting lipid peroxidation and preventing 6-HODA-induced
necrosis, suggesting that these compounds may be beneficial in neurodegenerative disorders, such
as Parkinson’s disease (Nobre-Júnior et al., 2009).
The anti-inflammatory and antiulcer properties of the enriched tannin fraction were evaluated
through the formalin test in mice, in rats by the model of paw edema induced by carrageenan and
in ulcer models (Souza et al., 2007). The results showed that this fraction significantly inhibited
intraperitoneal indomethacin-induced gastric ulceration and significantly decreased gastric ulcer-
ation induced by indomethacin. The results showed that the tannins present in this species have
anti-inflammatory and antiulcerogenic effects, characteristic and common to the tannins in general
(Monteiro et al., 2005; Simões et al., 2016).
10.6.5 Chemical and Pharmacological Studies of Cultivated M. urundeuva

Due to the predatory extraction of the inner bark of M. urundeuva for medicinal purposes and the
use of the wood in carpentry, this species is threatened with extinction (Galvão et al., 2018).
Since M. urundeuva exhibits secondary growth, the tissues of the inner bark and of the shoots
exhibit intense metabolic activity; therefore, they must produce the same chemical constituents and
show similar pharmacological actions. To prove this and to make a proposition to conserve this spe-
cies, agronomic studies have been carried out integrating the pharmacological and chemical studies.
These studies demonstrated that the cultivated species (shoots of 40 cm in height) maintains genetic
characteristics regarding the pharmacological activity and produces qualitatively the same chemical
markers of the inner bark, the dimeric chalcones, urundeuvines A (62), B (63) and C (64), Figure 10.7,
and tannins (Bandeira, 2002; Souza et al., 2007). Therefore, representing a possible preservation
and technological alternative to substitute the inner bark for shoots of the species studied.
Purification and isolation of the secondary metabolites from bioactive fractions resulted in the
identification of the active dimeric chalcones 62 and 63 (Figure 10.7) from the 40 cm-tall shoot
(Bandeira, 2002). Furthermore, the flavonoids quercetin (5), aromadendrinole (65) and agathisfla-
vone (66), Figure 10.7, were identified in the leaves of the cultivated specimen (Bandeira, 2002).
Flavonoids and chalcones have a known common biosynthetic precursor and both exhibit anti-
inflammatory activity (Cecílio et al., 2016). Therefore, the presence of flavonoids enforces the
medicinal use of the shoot leaves.
Similar to the inner bark of M. urundeuva (Bandeira, 2002), tannins were identified in all parts
of the cultivated plant (stem and leaf). The quantification of total tannin content yielded 8.2% for
the inner bark, 2.2% for shoot stems and 3.9% for shoot leaves (Aguiar, 2013). The data indicated
that, for the substitution of the inner bark by the shoots’ stem an amount of approximately four times
greater of stem in relation to the inner bark is necessary. On the other hand, the high content of tan-
nins in the shoots’ leaves points to a possible medicinal use of this plant part (Aguiar, 2013). This
result suggested that the young plant also has a phytotherapeutic complex, acting in an additive or
synergistic form in the pharmacological effect (Carmona and Pereira, 2013).
Liquid chromatography coupled to mass spectrometry (UPLC-ESI-QTOF MS/MS) analyses of
extracts from shoot stem and leaves of cultivated M. urundeuva, led to the tentative identification of
11 and 15 compounds, respectively (Aguiar Galvão et al., 2018). Noteworthy evidence demonstrated
that the chemical profiles of these extracts were similar (in terms of chemical classes) to those from
the inner bark of the non-cultivated plant. These findings reinforce the presence of a phytotherapeu-
tic complex in M. urundeuva and point to the similarity between the chemical classes present in the
different complex mixtures.
The neuroprotective activity of the fluid extracts prepared from stems and leaves of the cultivated
M. urundeuva shoots in a Parkinson’s disease model was evaluated (Calou et al., 2014). The oral
extracts reversed the behavioral changes, as well as decreased dopamine and dihydrophenylacetic
acid levels in rats, suggesting their potential in the prevention and treatment of neurodegenerative
conditions. These effects are related, possibly, to the anti-inflammatory and antioxidant properties
of the biologically active compounds presents in the plant shoots.
Studies on the thermal stability of the cultivated plant were performed aiming at the production of a
vegetable drug from shoots of M. urundeuva. The plant’s drying parameters were established to be per-
formed in an oven with air circulation at 40°C in order to maintain higher levels of the chemical markers
(dimeric chalcones and tannins). Residual moisture, total ash content and granulometry of the plantdrug’s
general quality specifications were possible to determine with the material dried at 40°C (Aguiar, 2013).
Gastroprotective and anti-inflammatory activities of the extracts (700 or 1,000 mg kg−1) were
assessed on ethanol-induced gastric lesions and croton oil-induced ear edema in rats, respectively.
The pharmacological assays revealed that the fluid extracts obtained from shoot stems and leaves have
pharmacological activities similar to that of the inner bark of the adult plant, as there was no signifi-
cant difference between the treated groups. The insertion of shoot leaves, especially in the mixture
with shoot stem (1:1) will be important not only from the pharmacochemical context, because flavo-
noids and tannins present in the extracts have relevant pharmacological actions (Hoensch and Oertel,
2015), but also from the point of view of the yield of the raw material. When deciding on the use of the
leaves, a significant amount of green is possible to be generated (Aguiar Galvão et al., 2018).
In summary, the ethnopharmacological data of M. urundeuva are useful not only to direct the
pharmacological studies on the inner bark but also as an approach to reverse the potential extinc-
tion of the plant. Chemical and pharmacological studies demonstrated that the therapeutic activity
of this plant may be associated with the presence of a phytotherapeutic complex (dimeric chalcones
and tannins) rather than simply an active compound. Recent studies suggest that the substitution of
the inner bark of the adult tree by the stems and leaves of the shoots (cultivated plant), which pre-
serve the same chemical and biological behavior of the adult specimen, might be an encouraging
approach and worthy of further research
ACKNOWLEDGMENTS
The authors thank Fundação Cearense de Apoio a Pesquisa (FUNCAP; grant # PNP-0058-
00137.01.00/11), Conselho Nacional do Desenvolvimento Científico e Tecnológico (CNPq; grant
# 405001/2013-4) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES)
– Finance Code 001 for the financial support. M. C. F. Oliveira and M. A. S. Lima also thank
CNPq for the research grants # 307667/2017-0 and 302804/2015-3, respectively. M. A. M. Bandeira
thanks CNPq for the financial sponsor of the project “Production of shoots of aroeira do sertão
(Myracrodruon urudeuva Allemão): reduction of anthropic pressure by predatory collection of
bark, contribution to the species conservation and social inclusion of adolescents and their rela-
tives” (grant # 557618/2009-6 – Call MCT/CNPq nº 29/2009 – Technological Extension for Social
Inclusion/Announcement 29/2009 – Theme 2: Social Technologies for Agroecology).
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11 Natural Products Structures
and Analysis of the
Cerrado Flora in Goiás
Lucilia Katoa, Vanessa Gisele Pasqualotto Severinoa,
Aristônio Magalhães Telesb, Aline Pereira Moraesa,
Vinicius Galvão Wakuia,
Núbia Alves Mariano Teixeira Pires Gomidesc,
Rita de Cássia Lemos Limad, and Cecilia Maria Alves de Oliveiraa
aInstituto de Química, b Instituto de Ciências Biológicas,
Universidade Federal de Goias, Goiânia, Brazil
cUnidade Acadêmica Especial de Biotecnologia,
Universidade Federal de Goias, Catalão, Brazil

dDepartment of Drug Design and Pharmacology,
University of Copenhagen, Copenhagen, Denmark
CONTENTS
11.1 The Brazilian Cerrado.......................................................................................................... 281
11.2 Ethnobotanical Studies......................................................................................................... 283
11.2.1 The Significance of Ethnobotany to the Research in Natural
Products Chemistry�� 284
11.2.2 The Context of Ethnobotany in Brazil and Goiás State......................................... 285
11.2.3 The Coqueiros Community.................................................................................... 286
11.2.4 Douradinha and Douradão Examples of Medicinal Plants Used
by the Goiás Population and Their Phytochemical Studies�� 288
11.3 Alkaloids as a Metabolic Target........................................................................................... 290
11.3.1 Alkaloids from Psychotria Genus.......................................................................... 291
11.3.2 Cyclopeptide Alkaloids from Amaioua Guianensis Aubl.
and Ixora Brevifolia Benth�� 295
11.4 Imaging Mass Spectrometry of Alkaloids from Psychotria and
Palicourea Leaves�� 297
Acknowledgments...........................................................................................................................300
References.......................................................................................................................................300
11.1 THE BRAZILIAN CERRADO

The Cerrado is the second largest biome of South America, occupying more than 20% of the
Brazilian territory (MMA, 2018). In terms of area, only the vegetation formation in Brazil of the
Amazon forest exceeds the Cerrado territory (Ratter et al., 1997). This biome naturally predomi-
nates almost exclusively in the Central Plateau of Brazil, although other biomes also are present, but
in a smaller proportion and covers about 2 million km2 (Ratter et al., 1997).
281
FIGURE 11.1 Distribution of the Cerrado biome in Brazil.
The Cerrado covers a continuous area in the states of Goiás, Tocantins, Mato Grosso, Mato
Grosso do Sul, Minas Gerais, Bahia, Maranhão, Piauí, Rondônia, Paraná, São Paulo and Federal
District (Figure 11.1) (Ribeiro and Walter, 2008). Small outlying distinct areas occur in Amapá,
Roraima, and Amazonas States (Eiten, 1972). The Cerrado is considered one of the 36 most impor-
tant terrestrial hotspots of the world due to its high diversity and endemic species, coupled with the
strong threat, especially anthropic, (Carmignotto et al., 2012; CEPF, 2018; Myers et al., 2000). This
biome is still listed as a World Natural Heritage Site by the United Nations Educational, Scientific
and Cultural Organization (UNESCO, 2018).
Although the Cerrado is considered a “vegetation complex” with a phytophysiognomic and eco-
logic relation with other American tropical savanna, and from other continents such as Africa and
Australia, the species have their own unique characteristics (Walter et al., 2008). The vegetation
presents numerous distinct phytophysiognomies (Figure 11.2), ranging from dense grassland, with
a sparse covering of shrubs and small trees, to nearly closed woodland, which may or may not be
associated with watercourses (Ratter et al., 1997).
The flora is surprisingly rich and presents characteristics, which are specific from the adja-
cent biomes, although some physiognomies share species with other biomes (Oliveira-Filho and
Analysis of the Cerrado Flora in Goiás 283
FIGURE 11.2 Main phytophysiognomies of Cerrado biome found in Goiás state, Brazil. (A) Campo Limpo
(Clean Field). (B) Campo Sujo (Dirty Field). (C) Vereda (Brazilian Palm Swamps). (D) Mata Ciliar (Riparian
Forest). (E) Campo Rupestre (Rupestrian Field). (F) Mata Seca (Dry Forest).
Ratter, 1995). In terms of biologic diversity, the Brazilian Cerrado is considered the richest
savanna in the world with 11,627 species of vascular plants (Mendonça et al., 2008). According
to Mendonça et al. (2008), the families of seed plants richest in species number in Cerrado are
Leguminosae (1,174 spp.), Asteraceae (1,074 spp.) and Orchidaceae (666 spp.).
The Cerrado biome is predominant in Goiás state (Figure 11.1). The Flora do Brasil (2020)
(in construction) reporting on flora of Cerrado of Goiás an amount of 5,766 species of seed plants
(angiosperms and gymnosperms) grouped in 1,210 genera and 165 families. These numbers cor-
respond to 51% of the species of seed plants reported in the Cerrado biome. This review presents
an outlook into future perspectives of research in natural products from the Cerrado in Goiás.
Considering how the development of state-of-the-art strategies has emerged during the past decade,
this allows us to assess and better understand the chemical biodiversity from Cerrado flora. This
research includes the identification of active metabolites targeting Rubiaceae species, which are
recognized as a source of potential active alkaloids. The research studies include DESI-MSI, a rapid
and efficient approach to obtain mass spectrometric imaging directly from samples, without extrac-
tion being employed to accelerate the chemical investigation of plant species directly from leaves.
11.2 ETHNOBOTANICAL STUDIES

The use of herbal medicine is increasing globally, since many species from Cerrado flora are used
in popular medicine for treatment of a broad set of diseases. The flora is an important part of health
care, mainly for rural areas in the country. Therefore, traditional medicine has been among the
several targeted strategies to choose a plant for study with one of the approaches being Ethnobotany.
11.2.1 The Significance of Ethnobotany to the Research

in Natural Products Chemistry
Ethnobotany can be defined as the study of interrelations between human beings and nature (Alcorn,
1995; Alexiades and Sheldon, 1996). Furthermore, this considers both how some social groups clas-
sify plants and employs them (Di Stasi, 1996). Another inclusive characteristic from this scientific
program is the concern in relation to the planning of the strategies of biological conservation, which
may also merge human development with cultural survival.
The interaction between human beings and natural resources leads to the ethnobotanical knowl-
edge, which can be noted in everyday life by means of actions that support knowhow, in several
human activities, for instance, fishing, cattle farming, agriculture, religious parties, bathing in
rivers, going to the field, calm talk and meal preparation (Guarim Neto, 2006).
The comprehension of use of specific plants is achieved when ecological, chemical and cultural
aspects are studied. They can be represented through language, human cognition, cultural history,
beliefs, religions, social networks and access to information (Maffi, 2005).
Moreover, religious and cultural manifestations help in the selection and confirmation of plant
species, which present some potential for various uses. For this reason alone, we believe many
researchers ought to respect and validate the traditional knowledge. Undoubtedly, this is a great
source for researchers and future generation (Radomski, 2003). Finally, they should focus on the
devolution of information that was delivered by members for the community itself.
However, one of the commitments of Ethnobotany should be a two way road of sharing tra-
ditional knowledge that generates scientific findings and partaking with those communities that
contribute to improve the quality of life of those populations (Lima, 1996).
Medicinal plants used by some communities can provide important information with environ-
mental and sustainability studies and biotechnological developments, and among them, there are
pharmacology, chemistry, agronomy considerations. Therefore, working together with the scientific
community much traditional knowledge will be recovered (Brasileiro et al., 2008).
Moreover, the more information recorded on vplant species which becomes available, the
more the research can deliver some findings, which are identified as a means of assessing effici-
cacy and safety in relation to their use. Considering the important impact of the pharmaceutical
and cosmetic industry, for example, traditional knowledge on the use of medicinal plants may
support the development of new products through rational research, which seeks to find new
lead substances. The cost-benefit may be improved by approximately 50 times (Di Stasi, 1996).
Thereupon, the research starts by means of a planning based on empirical knowledge that has
been established through a consistent practice; however, that should be evaluated on a scientific
basis (Brasileiro et al., 2008).
An outstanding amount of members of the community do not know the benefits and dangers of
all medicinal plants. Among species that are employed and indicated by society, there are some
that may frequently be used for more than one illness. Several herbs can also be used in an isolated
manner or in combination in order to handle a specific problem. The selection of a medicinal plant
is made by means of combination between experience and belief (Pasa, 2011).
Therefore, by considering folk therapeutic knowledge into account, many species can be identi-
fied through classes related to systems in accordance with the International Statistical Classification
of Diseases and Related Health Problems (ICD) (2018), which is recognized by the World Health
Organization (WHO, 2018).
Together with an ethnobotanical survey, quantitative data can be achieved, for example, the
relative importance (RI) of each species, the informant consensus factor (ICF) and the relative fre-
quency citation (RFC), which are presented as chief indicators in relation to the selection of promis-
ing species to be studied in the light of chemistry and biology.
The RI shows its preponderance, based on the number of medicinal properties, which are attrib-
uted to similar aspects by inquiry. The maximum value of RI is 2. The latter expresses which
vegetal species demonstrate great versatility relative to their uses by the indigenous population.
According to Bennett and Prance (2000), that number can be acquired through the formula:
IR = NSC + NP
where,
NSC: the number of body systems treated by a given species (NCSS) divided by the total number
of body systems treated by the most versatile species (NSCSV);
NP: relationship between the number of properties attributed to a species (NPS) divided by the
total number of properties attributed to the most versatile species (NPSV).
Through the ICF it is possible to identify the body systems, which present the highest local RI
by means of popular appointment in key categories (Trotter and Logan, 1986). Accordingly, the
formula for the factor is:
ICF = nur − nt/nur − 1
In this regard, nur: number of use citations made by inquirers to a category of ailments; nt: number
of species used.
The maximum value of ICF is 1, when there is consensus among inquirers about medicinal
plants within the ailment category.
The RFC indicates how a given species can be highlighted in relation to the others
(Begossi, 1996), indicating, however, its importance to the community under study. RFC may be
derived from the formula RFC = FC/N (0 < RFC < 1).
In this case, FC: the Frequency of Citation (FC) is the number of inquirers that speaks about the
use of species. Beyond this aspect, N: the total number of informants who participate in ethnobo-
tanical survey, not considering some categories of use.
In this context, the study of listed plant species can be investigated through recovering and
recording of popular knowledge, since those species have not only a great pharmacological, food,
agronomical potential, but also the presence of substances with structural variety and diversity.
11.2.2 The Context of Ethnobotany in Brazil and Goiás State

The ethnobotanical outlook in Latin America is characterized by a far-reaching quantity of research,
which are led by foreign researchers. Nevertheless, the substantial quantity of research made by
national researchers appears as a positive feature for Brazil and consolidates Ethnobotany into the
Brazilian scientific community (Oliveira et al., 2009).
Through a survey made by Oliveira et al. (2009), in a database from Lattes Platform, from the
Ministry of Science and Technology and the National Council for Scientific and Technological
Development, using the word “Etnobotânica”, studies by 469 researchers with a PhD degree and
964 researchers with masters, graduates, students and technical students were evaluated.
The authors who wrote this chapter completed a search, using the same keyword, in March 2018,
in Lattes Platform. Thereupon, a big increase over ten years of PhDs (1,692) and other research-
ers (2,284) were uncovered. Therefore, throughout the past nine years, the number of Brazilian
scientists who are involved with this subject has increased considerably, and hence a new ongoing
tendency of ethnobotanical studies was revealed.
In Brazil, biological diversity represents an aspect that explains the predominance of medicinal
plants, which can be estimated at 46,000 vegetable species that are known, including algae, bryo-
phytes, ferns, lycophytes, fungi, angiosperms and gymnosperms. The latter correspond to 72% of
the total species (Flora do Brasil, 2020 [unpublished]).
In particular, the Goiás state population could be expressed by multiple origin and culture as
people from rural communities, the “quilombola” families, Afro-Brazilians, “caiçaras”, among
others whose identities are linked to the flora of bioma Cerrado, with a wide use of medicinal plants,
which have a great level of endemism, because 4,400 vegetal species, among 10,000 known, are
endemic, representing 1,5% of plants around the world (Novaes et al., 2013). This context captures
interest from Brazilian researchers in relation to knowledge and ways of conducting research.
Thus, some examples of organizations in Goiás will be listed, such as The Moinho Community
in Alto Paraíso, Kalunga in Cavalcante, Monte Alegre and Teresina, Flores Velha in Flores de Goiás
and Rufino Francisco in Niquelândia city.
Moreover, a known fact is that the Southeastern region of Goiás state contains about 21 rural
communities, which present a form of organization based on family farming and the use of natural
available resources (Mendes, 2005). In particular, one of these rural communities is the so-called
Coqueiros, located in Catalão, where knowledge about medicinal plants is very important to this
research. This community has been a relevant contributor for the study of medicinal plants per-
formed by a specific group from the Laboratory of Natural Products and Organic Synthesis, from
the Institute of Chemistry, which is part of the Federal University of Goiás.
11.2.3 The Coqueiros Community

The existence of small agglomerations that are concentrated can be identified as a rural community,
in which tradition, moral, ethical and religious values safeguard them, constituting the local residents’
life and permeating a history that combines land, work, family and daily routine (Mendes, 2005).
Within the municipality of Catalão, in Goiás state, families who went to work in the construction
of the railroad around the end of the nineteenth century established a community region, seeking a
better quality of life.
There was the appearance of communities in the Catalão region, where the first local residents
of the Coqueiros community were migrants (Mendes, 2005). The members of Arcanjo and Tomés
families were the first local residents, aside from the Duarte family, which came from Portugal and
Minas Gerais. The members of these families married among themselves and then a new family
was constituted. As a result, almost all members have a family relationship and contribute to the
transfer of intrinsic knowledge throughout the community.
First and foremost, families from the Coqueiros community were countless, and they had an
average of nine children per couple. Considering the way of work on the farm, fathers looked after
cattle and crops, while mothers were responsible for undertaking domestic activities, educating
children and other works, such as producing flour, tapioca flour and growing vegetables.
In these communities, sons began to work by the age of eight. They worked with their parents.
Girls worked with their mothers, helping them with everyday tasks. Therefore, the community’s
formation has deep roots, the value of work, as well as the relation of love and respect relative to the
land, a tradition handed down from father to son.
The children’s dedication could be identified as a tool to be employed in the structuring of cus-
toms linked to many aspects of work in the countryside. This idea presents an argument for the
cultural formation of honest people. This social organization guaranteed that both knowledge and
the way of life were safeguarded and passed on to future generations.
In the current context, where this rural community integrates, there is now a drop in the number
of children, averaging two-three per couple. Younger children do not expect to stay in the field,
because of low income. Consequently, they move to the city, seeking some job opportunity and stu-
dent improvement (Mendes, 2005). Notwithstanding this migration, the community’s people have
aged (Mendes, 2005).
In this respect, in the Coqueiros community, former knowledge tends to get lost, because
younger generations have other interests and way of life, which are different from their parents.
In face of this, Ethnobotany is a crucial tool to recover such knowledge, highlighting the use of
medicinal plants, which can be recorded though interviews and participation in the population’s
daily activities.
FIGURE 11.3 A photo of a household from the Coqueiros community (3a). The second photo presents some
medicinal plants grown in farmyards from the Coqueiros community (3b). (Photo taken from authors who
wrote this chapter.)
The Coqueiros community is formed of 38 homes, where midwives, people who bless, and woods-
men are living. These social actors have a great medicinal knowledge and they are very important to
the Coqueiros community’s history. Recording their botanical knowledge retrieved from them to be
investigated by the scientific community and returned to the community is essential.
The Coqueiros community (Figure 11.3) is located in the southeastern of state of Goiás, between
47°17′ and 48°12′ West longitude and between 17°28′ and 18°30′ South latitude, in the Centre-north
of Catalão, far away from its municipal area 15 km (Figure 11.4).
Through an ethnobotanical survey that was undertaken in the Coqueiros community, it was
found that all interviewees know and employ medicinal herbs. A total of 109 plant species were
0 1700 km
BRAZIL
0 260 km
Goiás
Coqueiros community
N
Rural communities
Rural communities
0 100 km
0 16 32 km
Southeastern of Goiás
FIGURE 11.4 A map of the Coqueiros community, located in the southeastern of Goiás state, in the Centre-
north of Catalão city. (Adapted from Silva and Hespanhol, 2016.)
identified, which were distributed among 12 categories related to the body systems, according to
ICD, such as respiratory diseases, the nervous, the genitourinary, the digestive, the circulatory and
the musculoskeletal systems. Furthermore, there are other diseases, for instance, injury, poison-
ing and certain other consequences of external causes; certain infectious and parasitic diseases;
diseases of connective tissue, the skin and subcutaneous tissue; endocrine, nutritional and meta-
bolic diseases; diseases of the blood and blood-forming organs and certain disorders involving the
immune mechanism; and unknown affections or pains.
From this survey, 109 plant species were placed in 57 botanical families. The most represented
families are Fabaceae, Lamiaceae, Myrtaceae, Annonaceae, Anacardiaceae, Rutaceae, Asteraceae,
Apocynaceae, Solanaceae, Rubiaceae, Euphorbiaceae and Apiaceae.
Considering specialized literature, among all vegetal species identified, only nine plant species’
biological activity has not been registered. Nevertheless, by means of surveys, only few species
showed some biological potential, what underscores the importance of continuing this research.
Finally, the targeting of bioprospecting studies has been possible, to some extent, due to the eth-
nobotanical knowledge that was concluded through this research, which identified the potential plant
species linked to possible biotechnological development. Therefore, the group from the Laboratory
of Natural Products and Organic Synthesis has developed its research in light of popular knowl-
edge, as well as some plant species studied, such as, Hymenaea stigonocarpa Hayne (Fabaceae),
Kielmeyera coriacea Mart. & Zucc (Calophyllaceae) and Annona coriacea Mart. (Annonaceae).
Besides these, many other plant species have already been studied before. The main chemical and
biological data of these plant species are presented herein. All plant species focused on in this chap-
ter are listed in Table 11.1.
11.2.4 Douradinha and Douradão Examples of Medicinal Plants Used

by the Goiás Population and Their Phytochemical Studies
Douradinha or congonha do campo, a plant of the Rubiaceae family identified as Palicourea coria-
cea (Cham.) K. Shum., is the object of study of several research groups in Goiás, since Psychotria
coriacea is known as a cure for several diseases including treatment of kidney stones. Freitas et
al. (2011) was the first to show this plant as a diuretic agent. The diuretic effects presented by the
P. coriacea could be explained by the presence of several compounds, but mainly ursolic acid
(1), well known for the diuretic effects. In parallel, the toxicity was studied by Passos et al. (2010)
who evaluated the possible cytotoxic, genotoxic and antigenotoxic effects of the aqueous extract of
P. coriaceae in somatic cells of Drosophila melanogaster (Meigen, 1830). The results indicated no
cytotoxicity suggesting that the leaves are safe for tea consumption.
Douradinha is a classic example of a medicinal plant of Cerrado studied by different approaches.
Backed by ethnobotanical information, chemical and biological evaluation corroborate the popular
use by traditional communities in country Goiás, but further studies are still necessary to evaluate
the mechanisms involved in its biological activity and safety.
There is information also available about P. coriacea, a medicinal plant, from the Rubiaceae
family. The initial approach chosen by some researchers was based on traditional work using
phytochemical analyses as the main goal to identify the main compounds present in the plant.
Nascimento et al. (2006) described a tetrahydro β-carboline trisaccharide (2) isolated from the
roots of P. coriacea, and its structure was elucidated using spectral 2 D (Nuclear Magnetic
Resonance (NMR) methods: Correlation Spectroscopy (COSY), heteronuclear multiple-
quantum correlation (HMQC), heteronuclear multiple-bond correlation (HMBC) and Nuclear
Overhauser Effect Spectroscopy (NOESY). The aglycone was deduced by analysis of COSY,
HMQC and HMBC connectivities, and analysis of the cross-peak in the COSY and NOESY
NMR assignments confirmed the first example of strictosidinic acid incorporating a sucrose unit.
The known alkaloids, strictosidinic acid (3), epi-strictosidinic acid (4), ketone strictosidinic (5)
TABLE 11.1
Summary of the Names of All Plant Species, Their Respective Family and Common Names
Discussed in This Chapter
Amaioua guianensis (sin. Duhamelia glabra) Rubiaceae
Annona coriacea (sin. Annona geraensis) Annonaceae Araticum
Banisteriopsis caapi (sin. Banisteria inebrians) Malpighiaceae Ayahuasca, caapi, yagé
Coffea arabica (sin. Coffea bourbonica) Rubiaceae Coffee, mountain coffee, café arábica
Dysoxylum gotadhora (sin. Dysoxylum Meliaceae
binectariferum)
Galianthe ramosa Rubiaceae
Hymenaea stigonocarpa Fabaceae Jatobá-do-Cerrado
Ixora brevifolia (sin. Ixora glaziovii) Rubiaceae
Kielmeyera coriacea (sin. Bonnetia coriacea) Calophyllaceae Pau santo
Palicourea coriacea (sin. Uragoga xanthophylla) Rubiaceae Douradinha, congonha do campo
Palicourea rigida (sin. Uragoga rigida) Rubiaceae Bate-caixa, gritadeira, chapéu-de-couro,
douradão
Psychotria gracilenta (sin. Psychotria Rubiaceae
brachybotrya)
Psychotria capitata (sin. Palicourea capitata) Rubiaceae
Psychotria colorata (sin. Psychotria calviflora) Rubiaceae
Psychotria goyazensis (sin. Psychotria argoviensis) Rubiaceae
Psychotria henryi Rubiaceae
Psychotria hoffmannseggiana (sin. Palicourea Rubiaceae
hoffmannseggiana)
Psychotria ipecacuanha (sin. Carapichea Rubiaceae Ipecac
ipecacuanha)
Psychotria myriantha (sin. Psychotria myriantha) Rubiaceae
Psychotria pilifera Rubiaceae
Psychotria prunifolia (sin. Psychotria Rubiaceae
xanthocephala)
Psychotria umbellata (sin. Uragoga calva) Rubiaceae
Psychotria ulviformis (sin. Palicourea alba) Rubiaceae
Psychotria viridis (sin. Psychotria glomerata) Rubiaceae
Uncaria tomentosa (sin. Nauclea tomentosa) Rubiaceae Cat’s claw, unha de gato
and calycanthine (6) were isolated by Nascimento et al. (2006) using acid-base fractionation
of leaves and roots extracts (Figure 11.5). Calycanthine (6) was obtained as a monocrystal and
was elucidated by X-ray diffraction (Vencato, 2004). Silva et al. (2008) described the isolation
of 11 compounds belonging to several classes of chemicals, showing that P. coriacea is not an
exclusive source of alkaloids.
Another medicinal plant used in folk medicine is Palicourea rigida, called douradão, bate-caixa
that has been traditionally used in the treatment of urinary tract disorders. In some studies, cytotoxic
activity was observed (Rosa et al., 2012) as well as using various phytochemical screens to show the
presence of common phytosterols (stigmasterol, campesterol and sitosterol), flavonoids, coumarin,
iridoids and alkaloids like strictosidinic acid (3) and vallesiachotamine (7) (Alves et al., 2017; Bolzani
et al., 1992; Lopes et al., 2004; Rosa et al., 2010; Vencato et al., 2006).
P. rigida is not endemic to Goiás Cerrado but occurs in the entire Brazilian Cerrado region.
A peculiar fact of this species is that the isolation of the alkaloid from P. rigida was just for
the first time isolated from the species collected in Goiás Cerrado (Vencato et al., 2006).
FIGURE 11.5 Alkaloids and ursolic acid isolated from Palicourea coriacea extracts.
Vallesiachotamine (7) is an indolic monoterpenic alkaloid that exhibits inhibitory activity

against human melanoma cells SKMEL37 with promotion of G0/G1 cell cycle arrest, apoptosis
and necrosis (Soares et al., 2012).
Morel et al. (2011) showed the quantification of loganin (iridoid) in individuals plants collected
in random areas of the Brazilian Cerrado, suggesting the great variation of loganin, which was most
abundant in the plants collected in Luziania city (Goiás) whereas the lowest yields were in the plants
collected in Jaguara city (Minas Gerais). Based on these finding the authors observed the variation
on the loganin production occurred inside and among populations and they suggest that the concen-
tration of iridoids in P. rigida plants are influenced by both genetic and environmental variability,
supporting the great diversity from Cerrado plants due to environmental stress.
11.3 ALKALOIDS AS A METABOLIC TARGET

The strategy used by some researchers is based on chemotaxonomy, searching for specific metabo-
lites that characterize some family plants. The Rubiaceae family is the fourth largest flowering plant
family and is estimated to contain around 600 genera and between 6,000 and 13,143 species and is a
predominantly tropical family, with biomass and diversity concentrated in the tropics and subtropics.
The genus Psychotria L. and Palicourea Aubl. are among the ten largest (by species number) genera
in Rubiaceae, and Psychotria is still the largest genus with 1,834 species (Davis, 2009). Several phyto-
chemical studies corroborate that the Rubiaceae family is a well-known and a prolific source of alka-
loids with great structural diversity and pharmacological properties. As a known example the genus
Coffea L. is one of the most economically important, mainly the species Coffea arabica L., popularly
known as coffee, which has caffeine as one of the principal chemical components.
FIGURE 11.6 Biosynthetic approach proposed for strictosidine (β-carboline monoterpenic alkaloid,
Dewick, 2009).
The well-known phytotherapic Uncaria tomentosa (Willd.) DC. (Rubiaceae), popu-

larly called “unha de gato”, is used in folk Brazilian medicine and studies have shown that
alkaloids isolated from this plant have immunostimulant and antitumor activity (Nunez and
Martins, 2016).
In the case of alkaloids found in Psychotria, most of them belong to the indole, monoter-
pene indole (MIA-type) or pyrrolidinoindoline subclass (Moraes, 2013). These alkaloids are
derived from the amino acid tryptophan, which is decarboxylated by the enzyme tryptophan-
decarboxylase to form tryptamine. Once the indole ring of the tryptamine is a nucleophilic
system, β-carboline alkaloids are formed through a Mannich/Pictet-Spengler type reaction. This
reaction also allows the condensation of secologanin with tryptamine, producing strictosidine
which contains a β-carboline system and it is a precursor of approximately 3,000 MIA-type
alkaloids (Figure 11.6, Dewick, 2009).
11.3.1 Alkaloids from Psychotria Genus

Psychotria L. is the largest genus in the Rubiaceae family, comprising approximately 1,600 spe-
cies. This genus is well known through the species P. viridis Ruiz and Pav. or P. carthagenensis
Jacq., together with Banisteriopsis caapi (Spruce ex Griseb.) C.V. Morton (Malpighiaceae), in the
preparation of the psychoactive plant tea “ayahuasca”, which has been used since pre-Colombian
TABLE 11.2
New Alkaloids Isolated from Psychotria Species Reported Since 2013
Species Compound Subclass
P Psychotria henryi Alkaloid (8); Alkaloid (9) Indole
Psychohenin (10)
P Psychotria umbellata 3,4-Dehydro-18,19-beta-epoxy-psychollatine (11) Monoterpene indole
N4-[1-((R)-2-Hydroxypropyl)]-psychollatine (12) Monoterpene indole
N4-[1-(S)-2-Hydroxypropyl)]-psychollatine (13) Monoterpene indole
P Psychotria brachybotrya Brachybotryne (14) Simple indole
Brachybotryne bis-N-oxide (15) Simple indole
P Psychotria pilifera 16,17,19,20-Tetrahydro-2,16-dehydro-18- Indole
deoxyisostrychnine (16)
times for medical and religious purposes. More recently, in the last century this tea was used by syn-
cretic religious groups in Brazil, particularly “Santo Daime”, “União do Vegetal” and “Barquinha”
(Riba et al., 2004). Furthermore, some other species are used by different traditional communi-
ties with a variety of pharmacological purposes: in Amazonia the “caboclos” use the flowers of
P. colorata (Willd. ex Roem. and Schult.) Müll. Arg. as an analgesic and to treat earache and stomach
ache; the Wayapi Indians use P. ulviformis Steyerm. in an antipyretic bath and as an analgesic too;
P. ipecacuanha (Brot.) Stokes is used as a stimulant and to treat intoxication (Porto et al., 2009;
Santos et al., 2017).
The traditional use of Psychotria species has encouraged the phytochemical study of a great
number of species of this genus. These studies have shown that alkaloids are the main metabo-
lite identified/isolated in Psychotria. In fact, nine new alkaloids have been identified in the genus
(Table 11.2; Figure 11.7) since the publication of the most recent reviews on Psychotria metabolites
(Calixto et al., 2016; Klein-Júnior et al., 2014). Liu et al. (2013) studied P. henryi H. Lev., which is
used in the traditional Chinese medicine to relieve pain and eliminate dampness, and isolated two
novel dimeric indole alkaloids (8 and 9). Interestingly, compound (9) contains an unusual decacyclic
ring, which has not been described for any other dimeric alkaloid.
Important biological activities have been attributed to the secondary metabolites, mainly alka-
loids, found in Psychotria species, such as, analgesic, anti-inflammatory, anxiolytic, antidepressant,
antioxidant, antimutagenic and cytotoxic activity (Calixto et al, 2016; Magedans et al., 2017; Moller
and Wink, 2007). Psychotria alkaloids are also known for their effects on the central nervous sys-
tem as demonstrated by various reports of Psychotria species of Brazil.
These studies regard the inhibition of the enzymes, monoamine oxidases A and B (MAO-A
and MAO-B) and acetyl cholinesterase A and B (AchE and BchE), which are important targets
in the treatment of neurodegenerative disorders such as Parkinson’s disease and Alzheimer dis-
ease (Repsold et al., 2018). Phytochemical study of the leaves of P. prunifolia (Kunth) Steyerm.
resulted in the isolation of the MIA-type alkaloids prunifoleine (17) and 14-oxoprunifole-
ine (18) (Figure 11.8), which inhibited both cholinesterases (AChE and BChE) and MAO-A.
These compounds exhibit noncompetitive inhibition with IC50 values of 10 μM and 3.39 μM
for AChE, and 100 μM and 11 μM for BChE, respectively. Furthermore, these compounds
exhibited MAO-A selectivity with IC50 values of 7.41 μM and 6.92 μM. However, both com-
pounds showed a time-dependent MAO inhibition, indicating that they can act as irreversible
inhibitors, which can be dangerous since they may cause cardiovascular toxic effects (Passos
et al., 2013).
These results agree with the literature (Hamid et al., 2017; Rüben et al., 2015), which shows
that the quaternary β-carboline scaffold displays an important role in the selectivity for some
enzymes such as AChE and MAO-A. In fact, several quaternary β-carboline derivatives have been
FIGURE 11.7 Chemical structure of the new alkaloids isolated from Psychotria species published
since 2013.
synthetized to act as multitarget compounds aiming at monoamine and cholinesterase enzymes

(Revenga et al., 2015; Santillo et al., 2014).
Furthermore, numerous studies have shown that indole alkaloids display important antiprotozoal
activities (Bharate et al., 2013; Pereira et al., 2017). Prunifoleine (17) and 14-oxoprunifoleine (18)
were tested against Leishmania amazonensis Laison & Shaw, 1972, promastigotes with IC50 values
of 16.0 and 40.7 μg mL −1. Also, other MIA-type alkaloids like 10-hydroxyisodeppeaninol (19) and
FIGURE 11.8 Chemical structures of the alkaloids isolated from Psychotria prunifolia (Kunth)
Steyerm.
10-hydroxyantirhine N-oxide (20a), together with the known strictosamide (21) were isolated from
the roots and branches of P. prunifolia (Kunth) Steyerm. (Kato et al., 2012).
From the leaves of P. hoffmannseggiana (Schult.) Müll. Arg. several alkaloids were isolated,
such as N-methyltryptamine (22), harmane (23), N-methyl-1,2,3,4-tetrahydro-β-carboline (24),
(+) chimonantine (25) and the major alkaloid strictosidinic acid (3) (Naves, 2014). Strictosidinic
acid (3), first isolated from P. myriantha Müll. Arg., was assayed on rat hippocampus show-
ing a decrease in the serotonin (5-HT) levels, possibly indicating an inhibition of the precursor
enzymes of the 5-HT biosynthesis. Tryptamine and β-carboline type alkaloids were also isolated
from the leaves of P. capitata Ruiz & Pav., with bufotenine (26) and its N-oxide derivative (27) as
the major alkaloids (Wakui, 2015). Although early studies (Moraes et al., 2011) have shown the
presence of β-carboline alkaloids in P. capitata ethanol extract, only 6-hydroxy-2-methyl-1,2,3,4-
tetrahydro-β-carboline (28) was identified. From P. goyazensis Müll. Arg., the quinolone alkaloid
calycanthine (6) was isolated together with strictosidinic acid (3) and harmane (23), so far, the
first report of the occurrence of a monoterpene indole and a quinolinic type alkaloid in the same
species of Psychotria (Januário, 2015) (Figure 11.9).
The β-carboline alkaloids from P. prunifolia and from Rubiaceae and Apocynaceae spe-
cies have been assayed against inhibitors of malate synthase, an important enzyme from
Paracoccidioides spp. (PbMSL) (Costa et al., 2015). The β-carboline alkaloids are crucial for
stability in the binding pocket of PbMSL and were chosen as candidate molecules after vir-
tual screening and molecular docking studies were obtained through studies of receptor-ligand
interactions. In addition, the alkaloids 29, 30, 31 and 21 (Figure 11.10) were assayed and the
alkaloids 29 and 30 showed no cytotoxicity in A549 and MRC5 cells. This result is concomi-
tant with bioassays described by Costa et al. (2015) where alkaloid 29, isolated from Galianthe
ramosa E.L. Cabral (Freitas et al., 2014), another Rubiaceae species, is a good candidate for
antifungal development.
FIGURE 11.9 Chemical structures of alkaloids isolated from P. hoffmannseggiana, P. capitata and
P. goyazensis.
FIGURE 11.10 Structure of alkaloid assayed against enzyme isolated from Paracoccidioides spp
(PbMSL).
11.3.2 Cyclopeptide Alkaloids from Amaioua guianensis

Aubl. and Ixora brevifolia Benth
The cyclopeptide alkaloids are polyamide based composed of 13-, 14- or 15-membered macrocyclic
rings in which a 10- or 12-membered peptide-type bridge spans the 1,3 or 1,4 positions of a benzene
ring. Typically, the molecule contains two amino acids and one styrylamine unit. The 14-membered
FIGURE 11.11 Basic structure of 14-membered cyclopeptide: (A) basic terminal (end) amino acid;
(B) β-hydroxy amino acid; (C) ring-bound amino acid; (D) hydroxystyrylamine unit. Sometimes between the
(A) and (B) unit an additional (intermediary) amino acid is present and is designated as (E).
rings are more common kinds of cyclopeptides and contain four building blocks A (basic terminal
end amino acid), B (β-hydroxyamino acid); C (a ring bonded amino acid taking part in the macro-
cycle ring) and D (hydroxylstyrylamine) as illustrated in Figure 11.11.
The cyclopeptides showed broad biological activity as well as antibacterial effects (Giacomelli
et al., 2004; Morel et al., 2002; 2005), antifungal activity (Gournelis et al., 1997) and cytotoxicity
against leukemia cells L12100 and KB murine human cells (Liu et al., 1997) and they exert activity
on the central nervous system (Tuenter et al., 2017).
One example of Rubiaceae species as a source of cyclopeptide alkaloid is Amaioua guianensis
Aubl. (Rubiaceae). Initially, Oliveira et al. (2009) began this phytochemical study by examination
of promising antioxidant active extracts of Amaioua guianensis Aubl., which exhibited a moder-
ate antioxidant activity (IC50 70 μg mL −1). This extract was subjected to solvent partitioning and
chromatographic separation to provide a new cyclopeptide alkaloid (32), a mixture of two known
proanthocyanidins and iridoids.
The complete elucidation of the cyclopeptide alkaloid amaiouine (32) (Figure 11.12) was
achieved after crystallization in EtOAc of this compound as colorless needles. The NMR analy-
sis enabled elucidation of the amino-acid portions and some linkages among them were secured
after HMBC and NOESY experiments. Although the cyclopeptide skeleton was discovered after
X-ray diffraction analysis, the Oak Ridge Thermal Ellipsoid Plot (ORTEP) diagram of the crystal
structure confirmed that the skeleton was made up of proline bearing a cinnamoyl and styrylami-
nine moiety together with two units of phenylalanine. The X-ray analysis also confirmed the trans
FIGURE 11.12 Structure of cyclopeptide amaiouine isolated from Amaioua guianensis.

FIGURE 11.13 Structures of cyclopeptides ixorine e frangulanine.
configuration (3S*, 4S*) between H-3 and H-4 and was used to decisively assign the orientation
(7S*, 25S*) to the hydrogens at H-7 and H-25. Since this was the first occurrence of this cyclopep-
tide it was named amaiouine (32).
Other cyclopeptides reported for the first time in the literature from Rubiaceae species are
ixorine (33), isolated together with the known frangulanine (34) from Ixora brevifolia branches
(Figure 11.13). Medina et al. (2016) described the complete structural elucidation by NMR
analysis including stereochemical finding by analysis of NOESY experiments attributing the
L-erythro configuration to the β-substituted leucine moiety.
Since the cyclopeptides exhibited antibacterial activity, the alkaloidal mixture of ixorine (33) and
frangulanine (34) was assayed against the bacteria Escherichia coli Meigen, 1830, Pseudomonas
aeruginosa Schroeter, 1872, Staphylococcus aureus Rosenbach, 1884 and fungi Candida albicans
(C.-P. Robin) Berkhout, 1923, but no significant biological activity was observed. However, the anti-
protozoal activity in vitro against promastigotes of Leishmania amazonensis inhibited the parasite
growth with IC50 value of 54.16 μg mL−1. Leishmaniasis is regarded as a neglected disease, and
Medina et al. (2016) described for the first time the positive results in assays with these or related
cyclic peptides against this protozoal (Medina et al., 2016).
11.4 IMAGING MASS SPECTROMETRY OF ALKALOIDS FROM PSYCHOTRIA

AND PALICOUREA LEAVES
All studies involving phytochemical analysis of Palicourea or Psychotria are a product of extrac-
tion procedures, which result in information loss on the spatial localization of these alkaloids in
the plant. Visualizing the Also, this knowledge is desirable for understanding the physiological
functions and their biosynthesis. Also, the presence of alkaloids on the leaves’ surface have been
related to the defense system since this surface offers the opportunity to display a chemical barrier
to phytophagous insects and mites.
A review (Bjarnholt et al., 2014) showed mass spectrometry imaging (MSI) techniques as an effi-
cient tool to analyze metabolite plant distribution. The report also indicates that DESI-MSI provides
sensitivity and selectivity for this technique and shows this tool has become attractive due to the
simplicity requiring few sample preparation steps compared with vacuum imaging techniques. An
overview of research involving MSI of various types of plant material demonstrated a lack of study
involving DESI-MSI in leaves, mainly due to the analysis of the spatial distribution of compounds in
tissues using MS imaging is usually performed by tissue sections obtained by cryosectioning, and
that is a barrier when looking to analyze leaves or petals. For these kinds of tissues, DESI-MSI has
recently been performed using an indirect approach with porous Teflon imprints (Thunig et al., 2011)
or TLC blotting (Cabral et al., 2013). Direct imaging of the plant has been published and requires very
carefully optimization and depends on the nature of the leaf (Li et al., 2013). Kumara et al. (2015)
* relative configuration
298
FIGURE 11.14 ESI-(+)-HRMS/MS spectra of (a) prunifoleine (m/z 291.1942), (b) 10-hydroxyantirhine (m/z 313.1917); and DESI-(+)-MS/MS of (c) 10-hydroxy-
isodeppeaninol (m/z) 327 and (d) calycanthine (m/z 347). (From Kato et al., 2018.)
successfully described the spatial and temporal distribution of the alkaloid rohitukine in Dysoxylum
binectariferum (Roxb.) Hook. f. ex Bedd. seeds from the Meliaceae family.
The increasing number of phytochemical studies of Psychotria species has supported ethnobotanical,
pharmacological and chemotaxonomic studies. However, the spatial distribution of the alkaloids in this
genus is not known. Kato et al. (2018) explored the utilization of DESI-MS imaging as a tool for visual-
ization of this alkaloid distribution in leaves. As ambient DESI imaging requires a flat surface for a good
representation of molecular distribution and since reports have described that an imprint on Teflon results
on a stable, good and intense surface with porous Teflon (Li et al., 2013), the same methodology was fol-
lowed of just washing the porous Teflon with drops of methanol before making the imprints.
For imaging, the resolution was limited by the size of leaf (ca: area 1,500 mm2) and was per-
formed at 300 mm without implications (not to increase the rastering time series).
Teflon imprint DESI-MSI from leaf revealed the alkaloids have a heterogeneous distribution on
the leaf surface.
Among the alkaloids isolated from P. prunifolia, DESI experiments showed the presence of the
major alkaloids, prunifoleine (m/z 291, 17), together with the alkaloids 10-hydroxyisodeppeaninol
(m/z 327, 19) and 10-hydroxyantirhine (m/z 313, 20b). Since there was no report about DESI frag-
mentation for these alkaloids, the authors proposed a mass fragmentation pattern for these alkaloids
under DESI/ESI conditions (Figure 11.14).
Imaging DESI experiments for imprint leaves of P. prunifolia resulted in well-defined images
of m/z 291, 313 and 327 amu. The localization of prunifoleine (m/z 291amu) predominantly in the
midrib; 10-hydroxyisodeppeaninol (m/z 313 amu), in the midrib but concentrated close to petiole
and roughly a distribution of 10-hydroxyantirhine (m/z 327amu) in the whole leaf can be observed
(Figure 11.15). The imprint image of P. coriacea shows the homogeneous distribution of calycan-
thine (m/z 347amu) in the whole leaf (Figure 11.16).
FIGURE 11.15 Imaging mass spectrometry approach applied for Psychoria prunifolia leaves.
(a) Photography of Psychotria prunifolia leaf after imprint, (b) imprint in Teflon from P. prunifolia leaf,
(c) DESI-MS images of the ions of m/z 291 = [prunifoleine]+, (d) m/z 327 = [10-hydroxyisodeppeaninol + H]+,
(e) m/z 313 = [10-hydroxyantirhine + H]+ and (F) m/z 381 = [sucrose + K]+. (From Kato et al, 2018.)
FIGURE 11.16 (a) Photography of Palicourea coriacea leaf after imprint; (b) imprint in Teflon from
P. coriacea leaf, (c) DESI-MS image of the ion of m/z 347 = [calycanthine + H]+.
This was the first study findings of different localization of various alkaloids in plant leaves
using DESI-MSI, and the imaging findings are in complete accordance with the LC analyses and
histochemical results (Kato et al., 2018). The DESI-MSI is undoubtedly a powerful tool to give us
information about the spatial localization of the metabolites of plants, and these results consolidate
this technology for use in plants.
ACKNOWLEDGMENTS
The author thanks R. N. Ribeiro for photos in Figures 11.1 and 11.2 and A. M. Uemura for
adaptation of Figure 11.4. Some of the data presented were obtained with financial support from
Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Fundação de Amparo
à Pesquisa do Estado de Goiás (FAPEG) and the Coordenação de Aperfeiçoamento de Pessoal de
Nível Superior - Brasil (CAPES) - Finance Code 001.
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12 Total Synthesis of Some
Important Natural Products
from Brazilian Flora
Leonardo da Silva Netoa,b, Breno Germano de Freitas Oliveiraa,

Wellington Alves de Barrosa, Rosemeire Brondi Alvesa,
Adão Aparecido Sabinoa, and Ângelo de Fátimaa
aGrupo de Estudos em Química Orgânica e Biológica (GEQOB),
Departamento de Química, Instituto de Ciências Exatas, Universidade
Federal de Minas Gerais *UFMG), Belo Horizonte, Brazil
bInstituto Federal Farroupilha, Alegrete, Brazil
CONTENTS
12.1 Introduction�� 305
12.2 Total Synthesis of Brazilian Plant-Derived Natural Products..............................................306
12.2.1 Coumarins: A Privileged Pyrone-Phenyl in Natural Products�� 306
12.2.2 Flavonoids: A Dietary Natural Product with Health Benefits�� 308
12.2.3 Alkaloids: Ubiquitous Bioactive Natural Products�� 310
12.2.4 Terpenes: A Diverse Class of Natural Products with Valuable Bioactivities�� 314
12.2.5 Lignans: Phenylpropane Derivatives Widely Distributed in Higher Plants�� 317
12.2.6 Some Miscellaneous Synthetic Examples�� 318
12.3 Concluding Remarks�� 322
12.1 INTRODUCTION
Since ancient times, mankind has taken advantage of natural products to treat and/or prevent many
diseases and dysfunctions, either as original compounds or after modifications (Lachance et al.,
2012; Newman and Cragg, 2012). Indeed, nature contains a vast source of natural products that
exhibit a plethora of biological activities. The diversity of chemical structure makes natural prod-
ucts very valuable to pharmaceutical industries and agricultural segments as well (Modolo et al.,
2015a). Natural products from plants have been a great source of inspiration for improving the qual-
ity of human and animal life as disease therapeutics and for increasing food resources (Cragg and
Newman, 2013; Dayan et al., 2009; de Fátima et al., 2008; de Fátima et al., 2014; Rates, 2001; Rice
et al., 1998; Silva et al., 2014).
Brazil is one of the largest countries (8.5 million km2) in the world and the largest in all Latin
America, in addition to a marine area of more than 4.5 million km2. In terms of natural resources,
Brazil has five important continental biomes and the largest river system in the world, standing out
305
at the global level because it has the richest continental biota on the planet (Prates and Irving, 2015).
Brazil contains between 15% and 20% of all world biodiversity (Barreiro and Bolzani, 2009). In
addition, it has the largest number of endemic species, the largest tropical forest (the Amazon), and
two of the 19 hotspots worldwide (the Atlantic Forest and the Cerrado) places Brazil to the first
place in the list of megadiverse countries. Brazil is home to 13.2% of the world biota, which means
approximately 207,000 known species and 1.8 million projected species, including those yet to be
discovered (Prates and Irving, 2015). These characteristics make the Brazilian territory an impor-
tant resource of natural products that are valuable for the development of new drugs for improving
the quality of human and animal life.
However, various natural products that have therapeutic properties are not available in sufficient
amounts for sustainable use (de Fátima et al., 2014). Moreover, obtaining a renewable supply of
active compounds from biological sources may be problematic, especially with respect to perennial
plant species. The complexity of many natural products can also limit the scope of chemical modi-
fications necessary to optimize therapeutic use (de Fátima et al., 2006). Despite these barriers, the
total synthesis of various bioactive natural products and analogs has proven that organic synthesis is
a powerful tool for increasing the availability of valuable natural products of limited supply or very
complex structures (Burns et al., 2009; Mayer et al., 2010; Mendoza et al., 2012; Mickel et al., 2004;
Nicolaou and Snyder, 2003; Nicolaou and Sorensen, 1996; Shi et al., 2011; Su et al., 2011).
In this context, we present herein some total syntheses of selected Brazilian plant-derived natural
products of pharmacological and agricultural interest, including examples of coumarins, flavonoids,
alkaloids, terpenes, and lignoids, among others.
12.2 TOTAL SYNTHESIS OF BRAZILIAN PLANT-DERIVED

NATURAL PRODUCTS
12.2.1 Coumarins: A Privileged Pyrone-Phenyl in Natural Products
In the plant kingdom, coumarins are secondary metabolites responsible for a range of functions,
including controlling pathogen dissemination, free radical scavenging, and protection from abiotic
stress. The backbone structure of coumarins is constituted by a pyrone-phenyl system, and there
is evidence that their biosynthesis in plants occurs from the shikimic acid pathway or from mixed
routes (shikimic acid and acetate pathways) (Modolo et al., 2015b). The coumarin pyrone-phenyl
system can be prepared by methods such as the von Pechmann reaction (Chenera et al., 1993),
Claisen-Cope rearrangement (Cairns et al., 1994), Perkin reaction (Federsel, 2000), Knoevenagel
condensation (Shaabani et al., 2009), and Wittig reaction (Demyttenaere et al., 2004).
In 2004, Demyttenaere et al. described a total synthesis of 7-(2,3-epoxy-3-methylbutoxy)-6-
methoxycoumarin (1; Figure 12.1) from 2,4,5-trimethoxybenzaldehyde in four steps and a 30%
overall yield (Figure 12.1). Coumarin 1 is a substance that was first isolated from Conyza obscura
DC (Asteraceae) (Bohlmann and Jakupovic, 1979) and from petroleum Et2O extracts of the aerial
parts of Pterocaulon balansae Chodat (Asteraceae), a plant species from the southern and south-
eastern Brazilian states (Magalhães et al., 1981). The key feature of Demyttenaere et al. synthetic
approach to 1 is the access to the pyrone-phenyl system scopoletin, employing the Wittig reaction
followed by an intramolecular transesterification.
Robustic acid (3; Figure 12.2) is a pyranocoumarin that was first isolated from Derris robusta
(Fabaceae), an Indian tree, by Harper (1942), and only after approximately 20 years was its chemical
structure elucidated by Johnson and Pelter (1964). In 2001, researchers also isolated this coumarin
from petrol and dichloromethane extracts of Deguelia hatschbachii (Fabaceae), roots, a Brazilian
native species (Magalhães et al., 2001). Donnelly et al. (1995) reported a total synthesis of
robustic acid from methyl 2,4,6-trihydroxyphenyl ketone in nine steps and an 18% overall yield
(Figure 12.2). In this study, the authors employed the Claisen reaction followed by an intramo-
lecular transesterification to construct the pyrone-phenyl system of compound 5. A regioselective
Natural Products from Brazilian Flora 307
FIGURE 12.1 Synthesis of the 7-(2,3-epoxy-3-methylbutoxy)-6-methoxycoumarin (1) (Demyttenaere et al.,

2004).
FIGURE 12.2 Synthesis of robustic acid (3) (Donnelly et al., 1995).
arylation reaction was employed using an appropriate aryl lead triacetate, in part inspired from
earlier work (Barton et al., 1989; Donnelly et al., 1993). Using this methodology in the final step,
Donnelly et al. were able to prepare robustic acid, a 3-aryl-pyranocoumarin derivative.
(+)-Calanolide A (6; Figure 12.3) is a pyranocoumarin derivative found in two main Calophyllum
species: Calophyllum brasiliense (Calophyllaceae; guanandi, jacareúba, or landim) (Flora do
Brasil, 2020a; Huerta-Reyes et al., 2004) and Calophyllum lanigerum (Calophyllaceae) (Kashman
et al., 1992). (+)-Calanolide A and other similar naturally occurring pyranocoumarins, called cala-
nolides, are well-known to exhibit anti-tuberculotic activity and possess potent anti-HIV activity
(Brahmachari, 2015). Therefore, calanolides occupy an important position in the coumarin class
of compounds, and many researchers are interested in new methodologies of their total synthesis
(Brahmachari, 2015). (+)-Calanolide A was first isolated from C. lanigerum, a tropical rainfor-
est tree (Kashman et al., 1992), and, in the early 2000s, from hexane extracts of C. brasiliense
(Calophyllaceae; guanandi, jacareúba, and landim) leaves, a tree widely distributed in Brazilian
FIGURE 12.3 Synthesis of (+)-calanolide A (6) (Sekino et al., 2004).
territory (Huerta-Reyes et al., 2004). The first enantioselective total synthesis of (+)-calanolide A
was reported by Deshpande et al. (1995). Later, in 2004, Sekino et al. published a total synthesis of
(+)-calanolide A using phloroglucinol 7 as the starting material (Figure 12.3). To obtain the pyrone-
phenyl system of intermediate 8, the authors employed the von Pechmann reaction using the starting
material 7, ethyl 3-propyl-3-oxopropionic acid, and acidic conditions. This reaction is one of the
main synthetic strategies to obtain coumarins, first reported in 1883 by Hans von Pechmann and
Duisberg. It consists of a transesterification, an electrophilic attack and a dehydration (not neces-
sarily in this order) in the presence of protic or Lewis acid (Daru and Stirling, 2011). Sekino et al.
(2004) performed the total synthesis of (+)-calanolide A from phloroglucinol 7 in eight steps and in
a 14% overall yield (Figure 12.3).
12.2.2 Flavonoids: A Dietary Natural Product with Health Benefits

Flavonoids are polyphenol compounds and they are more commonly found in vegetal species. These
compounds possess the three-ring structures, and are often color responsive in many plants, and
participate in insect defense mechanisms (Jaisankar et al., 2014). Pharmacologically, flavonoids are
widely known for their antioxidant properties; they are biochemically synthesized from cinnamoyl-
CoA and three malonyl-CoA molecules to form a polyketide that is enzymatically converted to the
basic flavonoid building block (Dewick, 2002).
In 2006, Lee and Kim described an elegant synthesis for the pyranoflavone isolonchocarpin
(9; Figure 12.4) in five steps with an overall yield of 48%. Isolonchocarpin 9 is a pyranoflavone
isolated from several plant species (Canzi et al., 2014; Garcez et al., 1988; Huo et al., 2015; Wang
et al., 2015) and was isolated by Magalhães et al. (1996) from the petroleum extract of the roots of
Dahlstedtia floribunda (Fabaceae, basionym: Lonchocarpus subglaucescens; embira-de-sapo), a
species of plant native to Brazil, where the name of the substance is derived from the genus name
of the plant (Flora do Brasil, 2020b). One of the key steps of the strategy presented by Lee and Kim
is a one-pot Knoevenagel electrocyclic reaction of diketone 10 with 3-methyl-2-butenal, which pro-
vides a key starting material 11 for the construction of natural pyranoflavones. Various solvents and
catalysts were evaluated, obtaining the best yield in benzene in the presence of 10 mol% ethylamine
diacetate (Figure 12.4). Although the route presented by Lee and Kim shows a good yield at all
FIGURE 12.4 Synthesis of isolonchocarpin (9) (Lee and Kim, 2006).
stages (>70%), in the key stage, the best yield was in benzene, a highly toxic solvent. As presented
by the authors, this problem can be overcome by using fewer toxic solvents with still high yields,
such as CH2Cl2 and 88% yield (Lee and Kim, 2006).
In 2007, Urgaonkar and Shaw described a synthesis of the glycosylated flavonoid kaempferitrin
(12; Figure 12.5) in nine steps with an overall yield of 5% (Figure 12.5). Kaempferitrin 12 is a
glycosylated flavonoid derived from kaempferol. This compound was isolated from various plants
(Euler and Alam, 1982; Yang et al., 2010), including the ethanolic extract from Pterogyne nitens
(Fabaceae; amendoinzeiro, amendoim-bravo, cocal, tipá, viraró, madeira-nova, or vilão), a com-
mon plant species in Brazil (Flora do Brasil, 2020c; Regasini et al., 2008). The synthetic strategy
of Urgaonkar and Shaw to prepare 12 starts with a conversion of 7 into a functionalized ketone by
FIGURE 12.5 Synthesis of kaempferitrin (12) (Urgaonkar and Shaw, 2007).

FIGURE 12.6 Synthesis of (+)-medicarpin (16) (Yang et al., 2017).
a Houben-Hoesch reaction. Intermediate 14 was obtained by protection and benzoylation of 13.

Another important step, this strategy is the cyclization reaction of 14 to obtain the key intermediate
15, the core of the flavonoid skeleton. With the flavonoid core 15, product 12 can be obtained from
bisglycosylation. The authors tried two approaches to glycosylation, using both a rhamnose source
and an activator, noting that when acidic conditions were used, decomposition of intermediate 15
occurred. Urgaonkar and Shaw described a total synthesis from simple reagents; however, the strat-
egy requires many steps of protection and deprotection, reducing the overall yield of the route.
In 2017, Yang et al. presented an interesting example of an asymmetric synthesis for the ptero-
carpan (+)-medicarpin (16; Figure 12.6), a type of isoflavonoid; the synthesis was performed in
12 steps with a 9% overall yield. (+)-Medicarpin 16 has already been isolated from several species
of plants, among them some of Brazilian occurrence, and can be found in the petroleum extract of
the roots of Muellera montana (Fabaceae; basionym: Lonchocarpus montanus; cabelouro, or car-
rancudo) (Magalhães et al., 2007; Santos, Braga, et al., 2009), in the benzene extract of the core of
Dalbergia decipularis (Fabaceae; Sebastião-de-arruda) (de Alencar et al., 1972), and found in the
Brazilian red propolis (Li et al., 2008). The strategy presented by Yang et al. for the asymmetric
synthesis of 16 made use of a chiral oxazolidone auxiliary group to construct the two chiral centers
in one step; reacting 17 with (R)-4-benzyl-2-oxazolidinone using n-butyllithium as a base gave 18
in good yield, which was then converted to 20 via an Evans asymmetric aldol addition with the
aldehyde 19.
12.2.3 Alkaloids: Ubiquitous Bioactive Natural Products

Alkaloids are an important class of substances that contain at least one basic heterocyclic nitrogen
and are obtained from α-amino acids (AAs) or their immediate derivatives through related bio-
synthetic pathways (true alkaloids). Many alkaloids have elevated pharmacologic activity acting
primarily on the central nervous system, with relatively high toxicity for humans. Alkaloids are
distributed in a large number of subtypes, and the majority are derived from the following AAs:
l-ornithine, l-lysine, l-phenylalanine, l-tyrosine, anthranilic acid, l-tryptophan, l-histidine, and
nicotinic acid. Despite being found in animals, insects, and microorganisms, the main source of
alkaloids is in the plant kingdom, especially among angiosperms (Cordell, 1981; Hegnauer, 1963;
Roberts and Wink, 1998).
FIGURE 12.7 Synthesis of (−)-3-iso-ajmalicine (21) (Brown et al., 2002).
In 2002, Brown et al. described an approach to access three heteroyohimbine alkaloids from
secologanin, a natural biogenetic source of indole alkaloids (Contin et al., 1998). There are many
pharmacological properties reported for heteroyohimbine alkaloids, a group repeatedly found in the
Rubiaceae family, mainly in the Uncaria genus. In South America, Uncaria tomentosa (Rubiaceae;
unha-de-gato) and Uncaria guianensis (Rubiaceae; unha-de-gato), known as cat’s claw, are com-
monly used in local popular medicine (Sandoval et al., 2002). Working with the extracts of pow-
dered leaves of U. guianensis (Rubiaceae), collected from the Viro forest reserve in Pará state,
Brazil, Bolzani and coworkers (2004) isolated some oxindole alkaloids, including the less common
3-iso-ajmalicine 21. The synthesis of this alkaloid was reported by Brown et al. in 2002 in four
steps and a 38% overall yield (Figure 12.7). The key step of this proposal was the final step, where
the two central rings were constructed, and the five-ring system was all connected. This process
was accomplished by employing a Pictet-Spengler condensation, which occurs when compound 22
is treated in acidic media to afford the desired alkaloid 21. This reaction sequence can be inverted,
with the Pictet-Spengler step being performed in the initial steps, as previously reported by Brown
and Leonard (1979); however, the route described herein allowed better control over the C-3 stereo-
chemistry, although a minor amount (∼10%) of the C-3 epimer was observed on some occasions.
Another oxindole alkaloid is diaboline 23, which belongs to the same family as strychnine. In
1984, Nicoletti et al. identified this alkaloid from Brazilian biodiversity during their studies with the
Strychnos genus. Indeed, Strychnos pseudoquina (Longaniacea; quina-do-campo or falsa-quina),
a plant that grows in the Brazilian Cerrado, locally known as “quina do campo” or “falsa quina”
and used in regional medicine for the treatment against malaria and other diseases, afforded the
(+)-diaboline 23 alkaloid from ethanolic extracts of its ground and dried leaves. The structural com-
plexity of this molecule was evidenced by the synthetic approach undertaken by Ohshima et al. (2004)
that accessed this alkaloid in 28 steps and an approximately 1% overall yield (Figure 12.8). The key
step comprised a catalytic and enantioselective Michael reaction between malonate 24 and cyclic
enone 25, mediated by the AlLibis(binaphthoxide) complex (ALB) (Shimizu et al., 1998; Takayoshi
et al., 1996); the improved methodology in this work allowed the Michael adduct 26 to be obtained
on a multi-gram scale and in high enantiomeric excess (e.e.). Another very well-delineated strategy
was the construction of the BCDE ring system through the domino cyclization process starting
from compound 27 in three steps and 66% yield, affording compound 28, which already has the
basic core of the desired product. Finally, the (+)-diaboline 23 was achieved in an elegant synthetic
FIGURE 12.8 Synthesis of (+)-diaboline (23) (Ohshima et al., 2004).
route, likely the most efficient from the literature, and can be used in the synthesis of other advanced
Strychnos alkaloids. However, some drawbacks can be noted in this approach, such as the forma-
tion of a mixture of isomers in some steps, which decreases the overall yield, and an unexpected
epimerization of the C-16 stereocenter during the E-ring formation.
Another indole alkaloid found in South American plants was (+)-affinisine 29, which exhibits
relevant pharmacological activities and has been isolated from ethanolic extracts of ground whole
plant of Peschiera affinis (Apocynaceae; grão-de-galo) collected in northeastern Brazil (Santos,
Magalhães, et al., 2009; Weisbach et al., 1963). This compound is also found in Tabernaemontana
hystrix (Apocynaceae; esperta), a species of plant native to Southeastern Brazil, known as “esperta”
(Monnerat et al., 2005). Although the alkaloid affinisine 29 possesses important biological activi-
ties, there are no syntheses described to date. However, Liu and coworkers have shown an elegant
synthetic approach to obtain the (−)-enantiomer of affinisine in a total of nine steps and a 22%
overall yield (Figure 12.9) (Liu et al., 2000). Starting from l-tryptophan derivative 30, asymmetric
FIGURE 12.9 Synthesis of the enantiomer of (+)-affinisine (29) (Liu et al., 2000).
FIGURE 12.10 Synthesis of raputindole A (32) (Kock et al., 2017).
Pictet-Spengler cyclization was employed to afford adduct 31 with 100% diastereoselectivity. This
process is the key transformation and one of more used approaches to access other indole alkaloids
(Li et al., 1999; Wang et al., 1998). As stated before, although there is no synthesis to natural alka-
loid 29, an enantiomer from an intermediate posterior to compound 31 was already obtained in
previous works (Li et al., 1999; Wang et al., 1998; Yu and Cook, 1998), suggesting the possibility of
accessing the natural (+)-affinisine (29).
A new class of uncommon alkaloids, bisindoles, was established by Vougogiannopoulou et al.
(2010), who isolated four new structures from an Amazonian plant. From these structures, raputin-
dole A 32 was isolated from dichloromethane (DCM) extracts of dried and powdered roots of
Raputia simulans Kallunki (Rutaceae), a plant found in the extreme west of the Brazilian Amazon.
In 2017, Kock et al. reported the first total synthesis of raputindole A in nine steps and a 7% over-
all yield (Figure 12.10). In fact, starting from the advanced propargyl ester adduct 33, a cyclopen-
tannulation reaction was employed, catalyzed by Au(I), which resulted in the cyclopenta[f]indoline
34 with regioselectivity and good yield. Indeed, this key reaction had been previously reported by
Marsch et al. (2016), whose cyclization process was first described by Marion et al. in 2006. With
tricyclic core 34 constructed, the bisindole alkaloid raputindole A was accessed in an additional five
steps and 56% overall yield. However, the alkaloid was obtained in the racemic form together with
one epimer in a nearly 1:1 ratio, requiring a subsequent purification step with semipreparative chiral
HPLC to afford the natural (+)-raputindole A 32; obtaining the pure compound allowed the deter-
mination of the absolute configuration through calculation of the ECD spectrum (Kock et al., 2017).
In 2014, L’Homme et al. reported a concise approach for the total synthesis of the tetracyclic
alkaloid erysotrine 35 (Figure 12.11) in nine steps and 0.1% overall yield. Erysotrine 35 is an
alkaloid commonly isolated from plants of the Erythrina genus, a species that grows in tropical
countries such as Brazil. Indeed, Sarragiotto et al. (1981) have isolated several alkaloids represen-
tative of the Erythrina species, including erysotrine 35, from methanolic extracts of finely ground
flowers from Erythrina mulungu (Leguminosae; mulungu or mulungu-coral), a plant collected
in the Santa Elisa farm from Campinas, Brazil (de Lima et al., 2006). For the synthetic odyssey
proposed by Canesi and coworkers, a key step is the dearomatization of the phenolic A-ring of
compound 36, promoted by hypervalent iodine with the aim of improving the reactivity of the
enone 37. In sequence, the B-ring was constructed by an aza-Michael cyclization, followed by
a Pictet-Spengler cyclization to afford the tetracyclic core (ABCD ring) of the desired alkaloid
(Figure 12.11) (L’Homme et al., 2014). Despite the simple approach and short route, this strategy
provides a very low overall yield.
FIGURE 12.11 Synthesis of erysotrine (35) (L’Homme et al, 2014).
12.2.4 Terpenes: A Diverse Class of Natural Products with Valuable Bioactivities

The terpenoids are an important class of secondary metabolites produced by plants. These com-
pounds are formed by the union of isoprene units, and the terpenes are classified according to the
number of isoprene units. In this way, the hemiterpenes are formed by an isoprene unit (C5), mono-
terpenes by two (C10), sesquiterpenes by three (C15), diterpenes by four (C20), sesterterpenes by five
(C25), triterpenes by six (C30), and tetraterpenes by eight (C40). The terpenes have structural diversity
since after the union of the isoprene units and cyclization of these units, several structural modifica-
tions can occur during the biosynthetic process (Dewick, 2002).
In 1993, Wang et al. described the stereoselective total synthesis of 7β-acetoxyvouacapane
(38; Figure 12.12) in nine steps and an overall yield of approximately 18%. 7β-Acetoxyvouacapane
FIGURE 12.12 Synthesis of 7β-acetoxyvouacapane (38) (Wang et al., 1993).

FIGURE 12.13 Synthesis of (−)-seychellene (42) (Srikrishna and Ravi, 2008).
is a tetracyclic furano diterpene isolated from plant species of the genus Pterodon, such as Pterodon
apparicioi (Leguminosae; sucupira), a plant species from the banks of the Rio Cipó, state of Minas
Gerais, Brazil (Fascio et al., 1976; Hansen et al., 2010). The key feature of Wang and cowork-
ers’ synthetic approach to 38 is access to the intermediary 39 using the stereoselective reductive
hydrolysis of a tosylhydrazone 40. This reduction step does not work when traditional methods are
used (LiAlH4/THF; Li(t-BuO)3AlH/THF; NaBH4/MeOH; Raney nickel/MeOH) because of steric
hindrance of the 7-carbonyl group due to the 14α-methyl group (Figure 12.12) (Wang et al., 1993).
The main disadvantage of the synthesis route for 7β-acetoxyvouacapane proposed by Wang and
coworkers is that the furano diterpene is obtained in racemic form.
In 2008, Srikrishna and Ravi described the stereoselective total synthesis of (−)-seychellene 42
from (R)-carvone in 18 steps and a 13% overall yield (Figure 12.13). (−)-Seychellene is a tricyclic
sesquiterpene hydrocarbon isolated from Cedrela odorata (Meliaceae; cedro, cedro-branco, cedro-
rosa, or cedro-vermelho) and Toona ciliata (Meliaceae; cedro australiano), two plant species from
Viçosa, state of Minas Gerais, Brazil (Flora do Brasil, 2020d; Maia et al., 2000). The key features
of Srikrishna and Ravi’s synthetic approach to 42 are the generation of intermediate 44 via tandem
intermolecular Michael addition-intramolecular Michael addition and the access of two vicinal qua-
ternary carbon atoms in the tricyclic structure 46 (Figure 12.13). In addition, the authors obtained
optically pure (−)-seychellene 42, and they used the readily available monoterpene (R)-carvone 43
as the starting material (Figure 12.13) (Srikrishna and Ravi, 2008). The main disadvantage of the
synthetic route to (−)-seychellene 42 proposed by Srikrishna and Ravi is the large number of steps
required.
In 2009, Surendra and Corey described a short enantioselective total synthesis of the pentacyclic
triterpene lupeol (47; Figure 12.14) in eight steps and a 10% overall yield. Lupeol is a pentacyclic
triterpene isolated from several plants, including the plant species Cordiera macrophylla (Rubiaceae;
basionym: Alibertia macrophylla; marmelada-de-cachorro) herbs, shrubs, or trees from the Atlantic
Forest and Cerrado of São Paulo, state of São Paulo, Brazil (Bolzani et al., 1991; Flora do Brasil,
2020e). The key feature of Surendra and Corey’s synthetic approach to 47 is the access to epoxide 50
with correct stereochemistry, which is essential for stereocontrolled cation olefin polycyclization
(50 for 51; Figure 12.14). This synthesis was possible due to the careful choice of the starting material.
FIGURE 12.14 Synthesis of lupeol (47) (Surendra and Corey, 2009).
In 2014, Tran and Cramer described the biomimetic synthesis of (+)-viridiflorol 52 from
(+)-2-carene in seven steps and an overall yield of approximately 20% (Figure 12.15). (+)-Viridiflorol
is a sesquiterpene alcohol isolated from Cedrela odorata (Meliaceae; cedro, cedro branco, cedro
rosa, or cedro vermelho) and Toona ciliata (Meliaceae; cedro australiano), two plant species from
Viçosa, state of Minas Gerais, Brazil (Flora do Brasil, 2020d; Maia et al., 2000). One of the key
features of Tran and Cramer’s synthetic approach to 52 is access to intermediate 55 using stere-
oselective olefination from ketoaldehyde 53 (Figure 12.15). This step does not work well when a
FIGURE 12.15 Synthesis of (+)-viridiflorol (52) (Tran and Cramer, 2014).

high reaction temperature or an alteration of the base or solvent is employed, resulting in a loss of
diastereoselectivity (Figure 12.15) (Tran and Cramer, 2014). Another critical step in this synthesis to
obtain (+)-viridiflorol is the cyclization of intermediate 56. The first attempt of direct cyclization of
the olefin 56 using McMurry conditions or related couplings failed. Thus, the aldehyde was unpro-
tected, and cyclization was performed under McMurry conditions with complete diastereoselectiv-
ity (Figure 12.15) (Tran and Cramer, 2014).
12.2.5 Lignans: Phenylpropane Derivatives Widely Distributed in Higher Plants

Formed from two units of phenylpropanoid derived from cinnamic acid, in which biosynthesis is
catalyzed by different enzymes, lignans are dimeric structures with high diversity in plants; differ-
ent structural derivatives of cinnamic acid are found in this class of compounds (Dewick, 2002).
Some authors hold that the term lignans should be employed only when the dimers are coupled from
the C3 side chain, as the nomenclature used for another type of coupling is neolignans (Dewick,
2002). There are currently more than 7,000 natural varieties of lignans, and one of the pioneers
and major contributors in Brazil in the isolation and identification of this class of compounds was
Dr. Otto Gottlieb (1978) (Fazary et al., 2016). Within medicinal chemistry, lignans exhibit cardio-
vascular (Ghisalberti, 1997), antioxidant (Lee et al., 2004), anti-inflammatory (Kim et al., 2009),
anticancer (Lee et al., 2014), antibacterial (Bai et al., 2018), and antiviral (Charlton, 1998) activi-
ties. Due to the diverse pharmacological properties associated with this class, many lignan natural
products isolated from plants have become desired synthetic targets in the search for new structures
with different applications (Xu et al., 2018).
Plants of the Lauraceae family, common in Latin America and found in the Amazon region, are
sources of bioactive neolignoids, such as eusiderin A 58 and B 59 (Figure 12.16), isolated from the
species Licaria aurea (Lauraceae; folha-de-ouro or folha-dourada) and Licaria rigida (Lauraceae;
louro-fígado-de-galinha), respectively, which have antiviral activity (Braz-Filho et al., 1981; Flora
do Brasil, 2020f; Marques et al., 1992; Pilkington et al., 2018). In 2012, Pilkington and Barker
reported an enantioselective divergent synthesis of these structures in 11 steps with a yield of 0.7%
for eusiderin A and 0.5% for eusiderin B, in addition to the absolute determination of the stereo-
chemistry of eusiderins.
To synthesize different eusiderins, the authors traced a strategy involving a general route,
varying only the reagent used in the last step responsible for the side chain and the substituents
of the attached aromatic ring. The synthesis has two key steps: the first key step is a Mitsunobu
reaction between the phenolic derived from the previously synthesized o-vanillin and (S)-ethyl
lactate, thus fixing the first chiral center in the structure, followed by a reduction with DIBAL
at −78°C that results in the formation of aldehyde 62 (Figure 12.16), a common intermediate for
the other eusiderins, which undergoes an addition of an aromatic organometallic reagent that dif-
ferentiates eusiderin A 58 from eusiderin B 59; after a reduction and cyclization in Amberlyst 15,
formation of the intermediate occurs (63, 64), which undergoes the second key step of eusiderin
synthesis (Suzuki reaction). Then, the eusiderins are synthesized from the intermediates 63 and 64,
and the boronate ester side chain derived from organoboron is used, since the Suzuki reaction is a
carbon-carbon cross-coupling reaction. A disadvantage faced in the synthetic route proposed by
Pilkington and Barker (2012) is the step involving the formation of the second stereogenic center
of the 1,4-benzodioxane ring, wherein the cyclization generates the cis and trans isomers in a
ratio of 1:5, resulting in a reduction in the reaction yield due to the need to separate the products
for the last step of the synthesis.
Another bioactive lignan is fargesin (65; Figure 12.17) that has anti-inflammatory (Yue et al.,
2018) and antimycobacterial (Jiménez-Arellanes et al., 2012) activities, which can be found in
Brazilian plants of the Aristolochia labiata (Aristolochiaceae; basionym Aristolochia galeata;
angelicó, buta, crista-de-galo, milhomens, papo-de-peru, or peru-bosta) and Virola flexuosa
(Myristicaceae; ucuuba or ucuuba-folha-grande) species, which are from Brazilian Cerrado and
FIGURE 12.16 Synthesis of (−)-eusiderin A (58) and (−)-eusiderin B (59) (Pilkington and Barker, 2012).
Amazonian regions, respectively (Cavalcante et al., 1985; da Silva et al., 2011; Flora do Brasil,
2020g, 2019h; Lopes and Bolzani, 1988). In order to find methods for the synthesis of bioactive
lignans, Yoda et al. (2005) propose a synthetic route of furofuran lignans starting from the mono-
terpene lactone with eight steps and 6.4% overall yield. The synthetic strategy is based on the
opening of the lactam ring forming the first stereogenic center, which after oxidation undergoes a
stereoselective Grignard reaction. The main characteristic of the synthetic approach of Yoda et al.
is the production of tetrol 70 from the coupling reaction between 69 and the corresponding aldehyde
followed by deprotection and reduction reactions; in the hold of tetrol, the authors show the method
of forming the furofuran ring from mesylation. However, a negative point of the fargesin synthesis is
that the cyclization final step forms the methyl piperitol diastereoisomer as the major product, soon
abruptly reducing the overall yield for these syntheses.
12.2.6 Some Miscellaneous Synthetic Examples

Many other interesting examples of the total synthesis of Brazilian plant-derived natural prod-
ucts of pharmacological and agricultural interest are reported elsewhere. For instance, rotenone
FIGURE 12.17 Synthesis of fargesin (65) (Yoda et al., 2005).
(71; Figure 12.18) and tephrosin (76; Figure 12.19), members of a class of secondary metabolites of
plant origin known as rutenoids, were isolated from plants of the Fabaceae family, both exhibiting
pesticide and insecticide activities (Garcia et al., 2010). Rotenone (71; Figure 12.18) is a metabo-
lite isolated from numerous plants that is marketed as an insecticide and shows activities such as
cytotoxicity, genotoxicity, and larvicidal activity against the dengue vector Aedes aegypti (Estrella-
Parra et al., 2014; Huang et al., 2009; Vasconcelos et al., 2012). The fact that rotenone is marketed
as an insecticide generates health warnings since studies indicate that this metabolite can lead to the
manifestation of Parkinson’s disease and is even used as a Parkinson’s inducer for scientific models
FIGURE 12.18 Synthesis of rotenone (71) (Georgiou et al., 2017).

FIGURE 12.19 Synthesis of Tephrosin (76) (Garcia et al., 2010).
(Lin et al., 2018; Maturana et al., 2014). Rotenone (71) can be found in the plant Deguelia urucu
(Fabaceae; basionym: Derris urucu and Lonchocarpus urucu; timbó-urucu) of occurrence in Brazil
(Fang and Casida, 1999; Flora do Brasil, 2020i). In 2017, Georgiou et al. published the first total
stereoselective total synthesis for rotenone in 17 steps with a 4% overall yield. The presented strat-
egy involved two key transformations: The first transformation was previously reported by Pelly et
al. (2007), consisting of the Pd π-allyl-mediated cyclization of 72 to obtain the dihydrobenzofuran
skeleton 73. And, the second key transformation was a 6-end hydroarylation of intermediate 74 to
construct the chromene 75 precursor for rotenone (Georgiou et al., 2017).
Tephrosin (76; Figure 12.19) was isolated by Braz-Filho et al. (1975) from the ethanolic extract of
the aerial wood of D. urucu (Fabaceae; basionym: Derris urucu and Lonchocarpus urucu; timbó-
urucu) and by Parmar et al. (1988) from the seeds of Tephrosia candida (Fabaceae; tefrósia or
anil branco) (ANVISA, 2010; Flora do Brasil, 2020i). In 2010, Garcia et al. reported a convergent
total synthesis with seven steps starting from 3,4-dimethoxyphenol with an 8% overall yield. The
main step for the synthesis is the convergence step between the routes, a coupling between the key
intermediates 77 and 78 under Mitsunobu conditions followed by cyclization with Grubb’s second-
generation catalyst to afford intermediate 79 (Garcia et al., 2010).
One example of α-pyrone total synthesis was reported in 2018 by Vaithegi and Prasad when
they described, for the first time, the total synthesis of cryptopyranmoscatone B2 (80; Figure 12.20)
in 16 steps and a 0.9% overall yield. The cryptopyranmoscatone B2 80 is one of the six cryp-
topyranmoscatones isolated from the branch of stem bark of Cryptocarya moschata (Lauraceae;
canela-batalha or canela), a tree found in the Atlantic woods in the southeastern region of Brazil
(Cavalheiro and Yoshida, 2000). Obtaining cryptopyranmoscatone B2 80 is not a simple synthe-
sis, and the low overall yield is justified because the molecule possesses five stereogenic centers.
Among the 16 steps involved in the product preparation, the olefin metathesis and Brown’s allylation
stand out, being that the latter forms homoallylic alcohol as the unique diastereoisomer. However,
Vaithegi and Prasad highlight the formation of the pyran ring as a key synthetic step since the opti-
mized conditions by the authors favored the formation of cis tetrahydropyran as the major product
to fix the stereochemistry of the pyran ring. Some disadvantages of the total synthesis proposed by
Vaithegi and Prasad are steps that have yields of less than 50%, such as the selective protection of
FIGURE 12.20 Synthesis of cryptopyranmoscatone B2 (80) (Vaithegi and Prasad, 2018).
primary alcohol with TES and acryloylation, a key step for lactam ring formation, in addition to
performing another protection step due to the experimental conditions for 82 formation.
In 2012, Peng et al. described a short total synthesis of the pyranochalcone lonchocarpin
(84; Figure 12.21) in three steps and a 24% overall yield. Lonchocarpin is a flavonoid isolated
from Derris floribunda (Fabaceae; timbó-venenoso) collected in the vicinity of Manaus, state
of Amazonas, Brazil (Braz-Filho et al., 1975). The synthetic approach of Peng et al. to access
84 is quite short and simple. First, the key intermediate 87 was obtained by the condensation of
2,4-dihydroxylacetophenone 86 with 3-methylcrotonaldehyde 85, as described in Figure 12.21;
then, compound 86 was protected by MOM-Cl, and Claisen-Schmidt condensation between the
protected compound and benzaldehyde led to 84 in a good yield.
The name of the Brazilian plant species referred to as in this chapter and additional information
are summarized in Table 12.1.
FIGURE 12.21 Synthesis of lonchocarpin (84) (Peng et al., 2012).

TABLE 12.1
The Names of All Plant Species Including Family and Common Names from Which Some
Aristolochia labiata (basionym Aristolochia galeata) Aristolochiaceae Angelicó, buta, crista-de-galo,
milhomens, papo-de-peru or peru-bosta
Calophyllum brasiliense (sin. Calophyllum lucidum) Calophyllaceae Guanandi, jacareúba or landim
Calophyllum lanigerum (sin. Calophyllum frutescens) Calophyllaceae
Cedrela odorata (sin. Cedrela angustifolia) Meliaceae Cedro, cedro-branco, cedro-rosa or
cedro-vermelho
Conyza obscura (sin. Webbia kraussii) Asteraceae
Cordiera macrophylla (sin. Alibertia macrophylla) Rubiaceae Marmelada-de-cachorro
Cryptocarya moschata (sin. Cryptocarya moschata f. Lauraceae Canela-batalha or canela
angustifolia)
Dahlstedtia floribunda (sin. Lonchocarpus subglaucescens) Fabaceae Embira-de-sapo
Dalbergia decipularis Fabaceae Sebastião-de-arruda
Deguelia hatschbachii Fabaceae
Deguelia urucu (basionyms: Derris urucu; Lonchocarpus urucu) Fabaceae Timbó-urucu
Derris floribunda Fabaceae Timbó-venenoso
Derris robusta (sin. Brachypterum robustum) Fabaceae
Erythrina verna (sin. Erythrina mulungu) Leguminosae Mulungu or mulungu-coral
Licaria aurea (sin. Acrodiclidium aureum) Lauraceae Folha-de-ouro or folha-dourada
Licaria chrysophylla (sin. Licaria rigida) Lauraceae Louro-fígado-de-galinha
Muellera montana, (basionym: Lonchocarpus montanus) Fabaceae Cabelouro or carrancudo
Pterocaulon balansae Chodat (sin. Pterocaulon paniculatum) Asteraceae
Pterodon emarginatus (sin. Pterodon apparicioi) Leguminosae Sucupira
Pterogyne nitens (sin. Pterogyne nitens f. parvifolia) Fabaceae Amendoinzeiro, amendoim-bravo, cocal,
tipá, viraró, madeira-nova or vilão
Raputia simulans Kallunki Rutaceae
Strychnos pseudoquina Longaniacea Quina-do-campo or falsa-quina
Tabernaemontana catharinensis (sin. Peschiera affinis) Apocynaceae Grão-de-galo
Tabernaemontana hystrix (sin. Tabernaemontana gracillima) Apocynaceae Esperta
Tephrosia candida (sin. Robinia candida) Fabaceae Tefrósia or anil branco
Toona ciliata (sin. Toona microcarpa) Meliaceae Cedro australiano
Uncaria guianensis (sin. Ourouparia guianensis) Rubiaceae Unha-de-gato
Uncaria tomentosa (sin. Nauclea polycephala) Rubiaceae Unha-de-gato
Virola flexuosa Myristicaceae Ucuuba or ucuuba-folha-grande
12.3 CONCLUDING REMARKS

Brazilian flora is undoubtedly one of the most plentiful sources of inspiration for the development
of new drugs. Nevertheless, few products have emerged from this rich source of chemical diver-
sity, and few have their total synthesis described so far, especially by Brazilian organic synthetic
groups. The total synthesis of various bioactive natural products and analogs from Brazilian flora
has proven that organic synthesis is a powerful tool for increasing the availability of valuable natu-
ral products of limited supply for confirmation and/or correction of the structure of such natural
products. Moreover, the unique carbon-carbon scaffolds of such natural products represent a great
opportunity and a challenge for the synthetic organic chemist, with a push to develop new synthetic
methodologies to synthesize such interesting natural products. In general, Brazil has the potential to
be one of the most important countries in the world in the field of fine chemicals, pharmaceuticals,
and agrochemicals, and it is critical to respond to contemporary challenges by having effective and
continuous supportive policies.
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Index
Note: Italicized page numbers refer to figures, bold page Psychotria hoffmannseggiana, 295
numbers refer to tables Psychotria prunifolia, 294
pteridophytes, 156, 157
β-Sitosterol, 196 reaction with, 20
γ-aminobutyric acid (GABA), 76 Solanaceae, 74
structural diversity, 45
A structures of, 50
Abiotic stress, 109, 113–114, 116, 306 test for, 19
Açaí palm, see Euterpe oleracea tyrosine, 45
Açaí stone extract (ASE), 181, 199 Alkaloid brachycerine, 112, 113, 115
Acetogenins, 145, 151, 152, 157 Alkylating guanine, 75
Acetoxyvouacapane, 314, 314–315 Allium sativum, 10, 263
Acetylcholinesterase (AChE), 45, 49, 179, 191, 226, Almond oil, 29
236–237, 237 Aloe arborescens, 111, 112, 126
Acid-insoluble ash content, determination of, 21 Aloysia triphylla, 111, 117
Acids, 29–30, 32, 152, 159, 177–178, 181, 185, 188, 192 Alternanthera philoxeroides, 111, 114, 118, 121
Acid value, fixed oils, 30, 30 Aluminum chloride, 20
Acridones, 45, 48, 51 Alzheimer’s disease, 45, 191, 193–194, 236
Acrostichum, 158 Amaioua guianensis, 289, 295–297, 296
Active pharmaceutical ingredients (API), 8, 9, 15 Amaryllidaceae alkaloids, 45
Active principles, quantitative analysis of, 22–23, 25 Amazonian fruits, 173–203
Adenocalymma imperatoris-maximilianni, 41 Astrocaryum vulgare, 175, 177, 180, 183, 195–196
Adiantopsis flexuosa, 159, 163 Bactris gasipaes, 175, 177, 180, 183, 187, 197–198
Adiantum, 158, 159, 162–163 Bertholletia excelsa, 175, 176, 179, 182, 186, 187–189
Adiantum capillus-veneris, 159, 163 bioactive compounds in, 176–178
Adiantum cuneatum, 162, 163 Euterpe oleracea, 175, 177–178, 180–181, 184–185,
Adoxaceae, 80 187, 198–201
Advertising/advertisement functional characterization of, 186–187
medicinal plants, 4 Genipa americana, 175, 176, 179, 182, 189–192
nonprescription medicines, 9 Mauritia flexuosa, 175, 175, 178, 181–182, 185–186,
prescription medicines, 9 201–203
Aedes aegypti, 157, 319 Myrciaria dubia, 175, 177, 179–180, 182–183, 187,
Aerothionin, 38, 39 192–194
Affinisine, 312, 312–313 Spondias mombin, 175, 177, 180, 183, 194–195
Aflatoxins, determination of, 24 in vitro studies, 179–182
Agrobacterium, 72 in vivo studies, 182–186
Agroforestry, 174, 192 Amazonian rainforest, 92
Akkermansia muciniphila, 193 Amburana cearensis, 44, 111, 117, 259, 261
Alamar Blue assay, 233 American and European Pharmacopoeias, 15–16
Alcohol content, determination of, 27 American Pharmacopoeia, 24
Alibertia macrophylla, 315, 322 Anacardiaceae, 80, 92, 112, 175, 194, 220–221, 231,
Alkaloid(s), 35, 42, 45–51, 47, 48–49, 50, 290–300, 310–314 260–262, 273–276
Anthoceros agrestis, 152, 153, 163 Anacardium occidentale, 80, 83, 219, 220, 262
anthranilic acid, 45 Anadenanthera colubrine, 117
biosynthesis, 47 Anadenanthera peregrina, 221, 223
from Brazilian plants, 48–49 Analgesic, 231
classification of, 45 antiulcerogenic, 231
club-mosses, 157 Bauhinia cheilantha, 266
cyclopeptide, 295–297, 295–297 drug development, 201
imaging mass spectrometry of, 297–300 opioid, 77
as metabolic target, 290–291 pteridophytes, 158
ornithine, 45 Andiroba oil, 222
Palicourea coriacea, 288, 289, 290 Anemia, 143, 260–263
Paracoccidioides, 295 Angiosperms, 56, 136, 310
phenylalanine, 45 Angolini, C. F. F., 41
Psychotria, 290–294, 295 Aniba canelilla, 220, 222
Psychotria capitata, 289, 295 Annonaceae, 43, 48, 80, 221, 289
Psychotria goyazensis, 295 Annona coriacea, 288, 289
329
330 Index
Anthoceros agrestis, 152, 153, 163 phytochemistry, 197

Anthocerotophyta, 136, 138, 152–153, 153 in vitro studies, 180
Anthocyanidin, 151, 151 in vivo studies, 183
Anthocyanins, 110, 110, 117, 151, 199, 177 Baljet reaction, 19
Anthranilic acid, 45 Banana-de-papagaio, see Swartzia langsdorffii
Anthraquinones, 19, 176 Banisteria caapi, 220, 224
Antidepressant Banisteriopsis caapi, 220, 289, 291–292
anxiolytic and, 195 Barbatimão, 23
uliginosin, 121 Barker, D., 317
Valeriana glechomifolia, 124 Batista, L. M., 203
Antifungal compounds, 238–240 Bauhinia cheilantha, 111, 113, 117, 259, 260, 264, 265,
Antitumor effects, Euterpe oleracea, 200–201 265–266
Antiulcerogenic, 93, 231, 274 Beaujard, 136
Anxiolytic and antidepressant, 195 Bellete, B. S., 38
Aplysinamisine II, 38, 39 Benjamin A., 136
Aporphines, 45, 48 Bennett, B. C., 285
Apparent density, 26 Bentes, A. d. S., 190
Aquifoliaceae, 80, 111 Benzylisoquinoline alkaloids (BIA), 76, 77
Arabidopsis thaliana (Brassicaceae), 71, 72 Bertholletia excelsa, 175, 187–189
Araucaria angustifolia, 111, 124 bioactive compound, 176
Arenosclera brasiliensis, 38 biological and pharmacological activities, 188–189
Argan oil, 29 functional characterization of, 186
Aristolochia labiata, 317–318, 322 minerals, 188
Aroeira-do-Sertão, see Myracrodruon urundeuva phytochemicals, 188
Aromatic compounds, 148, 149–150, 150–151 in vitro studies, 179, 188
Artemisia annua, 77 in vivo studies, 182, 188
Aspergillus flavus, 24 BeWo cells, 191
Aspergillus parasiticus, 24, 188 Bignoniaceae, 44, 57, 80, 220–221, 264
Asplenium, 143, 163 Bioactive compounds, 40–41
Asteraceae, 28, 31, 80, 54, 57, 80, 111–112, 220–221, 262, Astrocaryum vulgare, 177
264, 268–270, 322 Atlantic Forest biome, 244–247
Asterella, 147 Bactris gasipaes, 177
Astrocaryum vulgare, 175, 195–196 Bertholletia excelsa, 176
bioactive compound, 177 bryophytes, 136, 137, 145–152, 157–158
biological and pharmacological activities, 196 Euterpe oleracea, 177–178
phytochemistry, 196 Genipa americana, 176
in vitro studies, 180 Marchantiophyta, 146–152
in vivo studies, 183 Mauritia flexuosa, 178
Atlantic Forest biome, 217–249 Myrciaria dubia, 177
antifungal compounds, 238–240 pteridophytes, 136, 137, 153–158, 154–157, 159–163
Atlantic Rain Forest, 218 Spondias mombin, 177
Atlantic Semi-deciduous Forest, 218 Bioactive metabolite accumulation, 109–127
bioactive compounds, 244–247 biotic elicitation of secondary metabolites, 121–124
biodiversity, 219 irradiance, 110–115, 113
bioprospecting, 219, 226–247 mineral nutrition and heavy metals, 114, 118–121
conservation and restoration, 219 overview, 109–110
cytotoxic compounds, 232–236 seasonal variation, 124–127
natural products, 219–232, 236–237, 240–244 temperature, 113, 116
overview, 218–219 water and salinity, 113–114, 116–118
plant species, 219, 220–221 Biodiversity, 37–38, 38, 46, 174
Atlantic Rain Forest, 92, 93, 218 of animals and plants, 258
Atta sexdens rubropilosa, 38 Atlantic Forest biome, 219
Azevedo, L., 192 chemistry and pharmacology, 226
deforestation of biomes and, 240
B exploration, 174
plant, 78, 91
Baccharis dentata, 111, 126 preservation, 174, 218, 249
Baccharis dracunculifolia, 111, 119–120, 126 pteridophytes and bryophytes, 136
Baccharis trimera, 80, 111, 114, 115, 220 Biomes, 78, 80–82, 92–93
Bacillus cereus, 125, 203, 246 Atlantic Forest, see Atlantic Forest biome
Bactris gasipaes, 175, 197–198 Caatinga, see Caatinga biome
bioactive compound, 177 Bioprospecting, Atlantic Forest biome, 219,
biological and pharmacological activities, 197–198 226–247
functional characterization of, 187 Biota-FAPESP Program, 219
Index 331
Biotic elicitation, secondary metabolites, 121–124, Calendula officinalis, 10

122–123 California poppy culture cells, 74
Biotic stress, 122–123, 127 Calophyllum brasiliense, 307–308, 322
Bisindoles, 313 Calophyllum lanigerum, 307, 322
Bixa orellana, 83, 220, 222 Calymperes lonchophyllum, 157
Bixin, 218, 222 Campesterol, 81, 159, 176–178, 185, 196
BLAST tool, 95 Camu-camu, see Myrciaria dubia
Blechnum, 158, 163 Cancer, 232–236
Bolzani, V. S., 311 Cancer cells, 236
Bonet, 136 Candida albicans, 125, 157, 238, 297
Bononi, V. L. R., 144 Candida krusei, 238
Bony, E., 196 Candolle, De, 35
Boraginaceae, 22, 31, 80, 111, 112, 220, 261, 264 Capillary electrophoresis (CE), 23
Boric acid complexation, 20 Capillary enzyme reactors (ICER), 49, 51
Bornträger reaction, 19 Carapa guianensis, 220, 222
Botrychium, 141 Carapichea ipecacuanha, 78, 82, 220, 289
Botrytis cinerea, 83 Cardiac glycosides, 19
Brachycerine, 112, 113, 115, 116, 118, 122 Cardiovascular effects, Euterpe oleracea, 199–200
Braz-Filho, R., 320 Cardoso, F. L., 126
Brazilian Atlantic Forest, 218, 226, 241 Carotenoids, 119–120, 177–178, 187, 194, 196, 202
Brazilian-boldo, see Plectranthus barbatus Caruso, J. A., 188
Brazilian Pharmacopoeia, 8, 17 Carvone, 315
Brazilian savanna, see Cerrado biome Casearia sylvestris, 43, 53, 82, 220, 230, 230
Brazil nut, see Bertholletia excelsa Cashew fruits, 225
Bromelia antiacantha, 80, 83 Castor oil, 29
Bromeliaceae, 80, 220 Catharanthus roseus, 75
Bromine water, 20 CE, see Capillary electrophoresis (CE)
Brown, R. T., 311 Cedrela odorata, 315–316, 322
Bryophytes , 140, 147, 149–150, 152 Celastraceae, 22, 23, 31, 42, 80, 92, 111, 220, 231, 260
aromatic compounds in, 150 Cell migration, 236
biologically active compounds, 157–158 Cell physiology, temperature, 113, 116
chemical compounds, 136, 137, 152 Cell proliferation, 236
chemistry, 136 Centipedic acid, 268, 269, 270
collecting and processing, 142–144 Central nervous system (CNS)
compared to tracheophytes, 138 diseases, 236
defined, 138 disorders, 236
flavonoids and anthocyanins in, 151 natural products (NP) active on, 236–237
life cycles of, 138–140, 139 Cercospora, 101
metabolic compounds, 136 Cerrado biome, 92, 281–300, 282
morphology and systematics of, 138–140 alkaloids, 290–300
phylogenies, 138 Coqueiros community, 286–288, 287
physiological adaptations, 138 Douradinha, 288–290
phytochemistry of, 136 ethnobotany, 283–286
species of, 136 overview, 281–283
terpenoids and steroids in, 148 plant species, 289
in vitro cultivation, 158 Chagas’ disease, 240
Buriti fruit, 201–202 Chandra, S., 136
Chemical compounds, see Bioactive compounds
C Chemical markers, quantitative analysis of, 22–23, 25
Chemical reference substance (CRS), 8
Caatinga biome, 92, 257–276, 258–259 Chemoprevention, 227–232
Bauhinia cheilantha, 265, 265–266 Chemosystematics system, 35
Combretum leprosum, 266–268, 267 Chikungunya virus, 40
Egletes viscosa, 268–270, 269 Chloral hydrate test solution, 18
endemic medicinal plants, 260–261 Chlorophora tinctoria, 220, 222
Erythrina veluntina, 271–273, 272 Chondodendron tomentosum, 35, 78
Myracrodruon urundeuva, 273–276, 274 Christella, 158, 163
non-endemic medicinal plants, 261–264 Chromatographic fingerprinting, identification
overview, 258–259 tests, 20–21
plant species, 264 Chrysoerythol, 38, 40
Caenorhabditis elegans, 183, 193–194 Cicero, N., 188
Caesalpinia echinata, 34, 34, 42, 218, 219–221, 220 Cinnamomum australe, 220, 222
Cajá, see Spondias mombin Cinnamon, 222
Calanolide, 308, 308 Citrus aurantium, 10, 43
332 Index
Citrus limonia, 43, 63–64 Database

Citrus reticulata, 41, 42 GenBank, 95
Citrus variegated chlorosis (CVC), 62 Mycoback, 96
Cladosporium cladosporioides, 239 De Andrade, Pedro Batista, 225
Cladosporium cucumerinum, 157 Decree, 2, 3, 6
Club-mosses, 156, 157 Deguelia hatschbachii, 306, 322
Codex Alimentarius, 24 Deguelia urucu, 320, 322
Coffea arabica, 225, 289, 290 De Laet, Johannes, 224
Collecting and processing De Lima Moreira, F., 235
bryophytes, 142–144 Demyttenaere, J., 306
pteridophytes, 144–145 Derris floribunda, 321, 322
Colletotrichum, 97, 98 Derris robusta, 306, 322
Colletotrichum acutatum, 97 Desdendticiaceae, 161
Columbus, 222 Deshpande, P. P., 308
Combretum leprosum, 263–264, Diabetes mellitus, 200, 245
266–268, 267 Diaboline, 311, 312
Cominetti, C., 189 Diaporthe, 95, 97, 98
Commiphora species, 33, 42 Diaporthe terebinthifolii, 101–103
Companies, HMP, 9–10 chemical analysis of, 101
Compounding potential of, 103
of herbal medicinal products (HMP), 4, 4–5 Dicranopteris, 158, 164
pharmacies, 1, 4, 5 Dimorphandra mollis, 44, 58, 78, 81, 111, 113, 118
Computational and Medicinal Chemistry of São Paulo Dipterix odorata, 220, 223
University, 37 Diterpenes, 52, 126, 230, 230, 238, 270, 314
Congonha do campo, see Douradinha Diterpenoids, 147; see also Terpenoids
Constantinople, 221 Donnelly, D. M. X., 306–307
Contamination, see Radioactivity contamination Doryopteris concolor, 159, 164
Conyza obscura, 306, 322 Dos Santos, Ezequiel Correia, 35, 224
Copaifera langsdorffii, 81, 223 Douradinha, 288, 289
Copper acetate, 20 Dragendorff’s test, 19
Coqueiros community, 286–288, 287 Drosophila melanogaster, 92, 288
Cordia verbenacea, 22, 31, 111, 126, 220, 226 Drug, defined, 2
Cordiera macrophylla, 315, 322 Drug development, 201
Corey, E. J., 315 Drugstore, defined, 3
Corsinia coriandrina, 148, 149, 163 Drumstick tree, see Moringa oleifera
Costa, F. G., 294 Dry extracts, 16, 24–27
Coumarins, 19, 56, 57, 62, 306–308 Dryopteris, 158, 164
Cramer, N., 316–317 Dryopteris filix-mas, 158, 164
CRISPR/Cas9, 75–76 Dry residue, determination of, 25
Croton heliotropiifolius, 220, 238, 239 Duisberg, C., 308
Cryptocaria, 234 Dumortiera hirsuta, 146, 148, 149, 164
Cryptocarya mandiocanna, 234, 234 Dysoxylum binectariferum, 299
Cryptocarya moschata, 220, 234, 320, 322
Cryptococcus neoformans, 238 E
Cryptopyranmoscatone, 320, 321
Cucurbitaceae, 80, 263 Ecosystems, 56, 62–65
Culture media, 93 Edwardsiela tarda, 157
Cunila galioides, 111, 114, 120 Egletes viscosa, 264, 268–270, 269
Cupressus sempervirens, 33, 42 Elaphoglossum, 143, 164
Curare, 223 Elionurus muticus, 111, 125
Cyanobacteria, 138 Emetine, 78, 79, 82
Cyanogenic glucosides, 75 Endemic medicinal plants, 260–261
Cyathea,143, 158, 159, 163 Endophytes, 92
Cyatheaceae, 143, 159, 163 biotechnological potential of, 91–93, 97–101
Cyathea phalerata, 159, 163 defined, 91
Cyclopeptide, 295–297, 295–297 diversity of, 94–97
Cysteine rich peptides, 120 HPLC analysis, 100
Cytotoxic compounds, 232–236 identification of, 95–97, 97
isolation of, 93–94, 94
D microorganisms of medicinal plants, 98–99
phylogenetic analysis, 97, 98–99
Da Gama, Vasco, 222 Enfleurage, 28
Dahlstedtia floribunda, 308, 322 Enzymatic stability, 49
Danea, 141 Equisetaceae, 81, 141, 160, 164
Index 333
Equisetidae, 142 Ferric chloride, 19, 20

Equisetum, 141, 142, 158, 160, 164 Fidalgo, O., 144
Equisetum arvense, 160, 164 Finished product, 8
Equisetum giganteum, 81, 142, 164 Fiocruz, 3
Erva-baleeira, see Cordia verbenaceae Fistularin-3, 38, 39
Ervanaria, 2–4 Fixed oils, 16, 29–30, 30
Erysotrine, 271, 272, 314, 314 Flavobacterium okeanokoites, 74
Erythrina mulungu, 43, 48, 220, 313, 322 Flavonoids, 20, 40, 56, 57–59, 62, 63, 110, 114, 149–150,
Erythrina veluntina, 271–273, 272 177, 241, 308–310
Erythromycin, 97 bryophytes, 151
Escherichia coli, 55, 77, 95, 126, 157, 203, 297 citrus fruit waste, 38, 40
Esenbeckia leiocarpa, 220, 236, 237 marchantiophyta, 151
Esperta, 312, 322; see also Tabernaemontana hystrix pteridophytes, 156, 156
Espinheira santa, see Maytenus ilicifolia Pterogyne nitens, 229
Essential oils, 16 Flavonoids, test for, 20
Bauhinia cheilantha, 266 Flavonol morin, 218, 222
content determination, 22 Flindersia (Flindersioideae), 35–36
Egletes viscosa, 269–270 Flowering plants, see Angiosperms
physico-chemical tests, 28–29 Food diversification, 174
Ester value, 16, 30 Food security, 174
Ethnobotany Foreign matter, determination of, 21–22
Cerrado biome, 283–286 Forward genetics, 72, 73
defined, 284 Freitas, P. C. M., 288
Ethylmethanesulfonate (EMS), 72, 75 Frothing test, 20
Ethylnitrosourea (ENU), 75 Fruits, see Amazonian fruits;Buriti fruit
Eucalyptus, 28, 93 Frullania, 157
Eucalyptus citriodora, 28, 31 Frullania brasiliensis, 140, 147, 149, 164
Eucalyptus globulus, 28, 31 Fusarium, 95, 238
Eugenia uniflora, 111, 114, 119, 220
Euphorbiaceae, 58–59, 81, 111, 220, 238, 244, 261–263 G
European Pharmacopoeia, 24
Eusiderin, 317, 318 Galanthamine-type, 45, 49
Eusporangia, 142 Galegine, 245, 245
Euterpe oleracea, 175, 198–201 Galleria mellonella, 239–240
antitumor effects, 200–201 Gametophytes, 138, 139, 140,
bioactive compound, 177–178 Gamma-aminobutyric acid (GABA), 76, 272
biological and pharmacological activities, 199–201 Gas chromatography (GC), 21, 25, 28
cardiovascular effects, 199–200 Gauva, see Psidium guajava
diabetes metabolism, 200 Geissoschizoline, 34, 35
effects on lipids, 200 Geissospermum laeve, 35, 42
functional characterization of, 187 Geissospermum leavis, 224
nontoxic effects, 201 Gelatin, 20
phytochemistry, 198–199 GenBank database, 95
renal failure effects, 200 Genetics, 73
in vitro studies, 180–181 forward, 72
in vivo studies, 184–185 reverse, 72–75
Execution of histological cuts, 18 Genipa americana, 175, 175, 189–192, 220, 222
Expressed Sequence Tags (ESTs), 124 bioactive compound, 176
Extractable material, 22 biological and pharmacological activities, 191–192
Extraction ratio, plant derivatives, 25 phytochemicals, 190–191
in vitro studies, 179, 191
F in vivo studies, 182, 191
Genipap fruits, see Genipa americana
Fabaceae, 81; see also Caesalpinia echinata Genipin, 82, 176, 190, 190, 218, 222
Falsa quina, 311, 322 Genome, synthetic, 77
False-boldo, see Plectranthus barbatus Genome editing, 72–75
Fargesin, 317–318, 319 Gigaspora albida, 119
Farmácia Viva, 3, 4, 4, 5 Ginkgolides, Ginkgoaceae, 74
Fatty acids Glime, J. M., 136, 142
Bauhinia cheilantha, 266 Global natural products social molecular networking
Marchantiophyta, 151, 152 (GNPS), 37, 247–248
Ferns, 140 Glycine max, 10, 29, 31, 75
life cycle, 138, 139 González-Aguilar, G. A., 121
naming, 145 Good clinical practice (GCP), 7
334 Index
Good manufacturing practices (GMP), 3, 5, 6, 8, 15 Human pathogenic fungi, 238

Gottlieb, Otto Richard, 35, 37, 56, 222, 223 Hydrocotyle umbellata, 111, 117
Gradstein, S. R., 142 Hymenaea stigonocarpa, 288, 289
Gram-positive bacteria, 225, 246 Hymenea courbaril, 81, 83, 220, 223
Granulometry, characterization test, 26 Hymenophyllum, 143, 164
Groppo, M., 35 Hyoscyamine 6β-hydroxylase (H6H), 83
Guanidine alkaloids, 228, 228, 240, 245 Hyoscyamus niger, 83
Guava (Psidium guajava), 112, 121 Hypericum brasiliense, 111, 113, 117
Hypericum polyanthemum, 111, 117, 121–124, 122
H Hypochlorite TS, 18
Hypolepis, 158
Haemagglutination test, 20 Hyptis carpinifolia, 111, 125
Hager’s test, 19
Hallucinogenic snuffs, 223–224 I
Halpern, O., 35
Hamano, P. S., 194 ICH, see International Conference on
Hammond, 136 Harmonization (ICH)
Handroanthus impetiginosus, 78, 80 Identification, physico-chemical tests, 15, 16, 18–21
Harper, S. H., 306 by chromatographic fingerprinting, 19–20
Harris, E. S. J., 136 macroscopic/microscopic botanical
Heavy metals examination, 18
determination of, 23 by phytochemical prospection/chemical
secondary metabolites, 118–121 identification, 19, 19–20
Helianthus annuus, 10, 262 Ilex paraguariensis, 80, 111, 112, 113, 116
Helicobacter pylori, 195 Imaging mass spectrometry, 297–300
Hemidimorphic fronds, 142 Immobilized enzyme reactors (IMER), 49
Hempel, J., 198 Industrialized HMP, regulations, 5–10, 6
Herbal active pharmaceutical ingredient (IFAV), 6–7, 8, 10 Inflammation, 227–232
Herbal drug, 2, 5, 8, 10 Informant consensus factor (ICF), 28
Herbal medicinal products (HMP), 2, 6–8, 15 Infrared spectroscopy (IR), 23
companies, 9–10 Internally transcribed spacer (ITS), 95
compounding of, 4, 4–5 International Conference on Harmonization (ICH), 15–16
industrialized, 5–10, 6 International Union of Pure and Applied Chemistry
licenses, 9, 10 (IUPAC), 15
manufacturers of, 8 Iodine value, fixed oils, 30
notification, 6 Ipecacuanha, 82, 220, 225
registration dossier of, 6, 7 Iridoids, 82, 114, 176, 190–191, 191
regulations, 1–10 Irradiance, 110–115, 113
subclasses, 6 Iryanthera, 223
technical evaluation, 6 ISO 2859, 17
Herbal preparations, 2 Isoetaceae, 141, 141–142
Herbal raw material, 2, 8, 9 Isoetes, 141, 141–142
Herbarium, labeling, 145, 145 Isoflavones, 10, 74, 101
Herborization, 144 Isolonchocarpin, 308, 309
Hesperidin, 38, 63–64, 64, 80 Isopentenyl diphosphate (IPP), 51
Hesperitin, 38, 40 Isoquinolines, 45, 48
Hevea brasiliensis, 111, 124 Isosakuranetin, 38, 40
High performance liquid chromatography (HPLC), 21, 25, ITS region, 95, 96
63–65, 196, 227 IUPAC, see International Union of Pure and
Histoplasma capsulatum, 238 Applied Chemistry (IUPAC)
Historia Naturalis Brasiliae, 224 Ixora brevifolia, 289, 297
Holowaty, S. A., 113, 116
Homologous recombination, 74 J
Homology-directed repair, 76
Homolycorine-type, 45, 49 Jasmonic acid, 121
Homopurpuroceratic acid B, 38, 39 Jasmonoyl isoleucine, 121
Hornworts, see Anthocerotophyta Jatobá, 223, 223
Horsetails ferns, see Equisetum Jatropha, 246–247
Hortia, 35–36, 36 Jatropha curcas, 220, 246, 247
Hortia arborea, 35 Jatropha ribifolia, 220, 244, 244
Hortia brasiliana, 35 Jayasinghe, S. B., 188
Hortia oreadica, 35 Jenipapo, 222
HPLC, see High performance liquid chromatography John, J. A., 188
(HPLC) Jungermanniales, 147, 151
Index 335
K Lycophytes, 136, 140, 158, 285

Lycopodiaceae, 141, 141, 154
Kaempferitrin, 229, 229, 245, 309, 309 Lycopodiopsida, 136, 140, 141, 141
Kalanchoe pinnata, 44, 58, 111, 113, 115 Lycopodium, 158
Karl Fischer method, see Volumetric methods Lygodium, 158, 163–164
Kato, L., 299 Lytoneuron ornithopus, 160, 164
Kedde reaction, 19
Keller-Killiani reaction, 19
Kielmeyera coriacea, 288, 289 M
Kim, D. H., 308–309 Macela, see Egletes viscosa
Klebsiella pneumoniae, 126, 157, 203 Macroscopic botanical examination, identification tests, 18
Kock, M., 313 Magalhaes, M., 222
Kumara, P. M., 297–299 Magistral preparation, compounded HMP, 5
Magnesium, 20, 65, 188
L Maia, L. C., 144
Mandelin reaction, 19
Lactobacillus, 193 Manickam, V. S., 136
Lactuca sativa, 157 Manufacturers of HMP, 8
Lafoensia pacari, 111, 113–114, 116 Maranhão, P. A., 189
Lani, R., 40 Marattia, 158
Large subunit rRNA gene (LSU), 95 Marattiaceae, 141, 142
Lauraceae, 35, 42, 44–45, 59–60, 81, 220, 222, 234, 317, Marattiidae, 142
320, 322 Marcgrave, Georg, 224
Lead acetate reaction, 20 Marchantiales, 147
Lecythidaceae, 175, 187 Marchantia polymorpha, 140, 148, 149, 164
Lee, Y. R., 308–309 Marchantiophyta, 138
Legal reaction, 19 acetogenins, 151, 152
Leishmania amazonensis, 243–244, 293, 297 anthocyanidin, 151, 151
Leishmania chagasi, 180, 243–244 aromatic compounds, 148, 149–150, 150–151
Leishmaniasis, 240 chemical compounds in, 146–152
Lemon grass, 111, 125 fatty acids, 151, 152
Lepidium sativum, 157 flavonoids, 151
Leptosporangia, 142 terpenoids, 146–147, 147–148
Lewinski, C. S., 112 Martianthus leucocephalus, 111, 125
L’Homme, C., 313 Mass spectrometry (MS), 23, 41, 247–248
Licaria aurea, 317, 322 Matricaria recutita, 10, 28, 31
Licaria rigida, 317, 322 Mauritia flexuosa, 175, 201–203
Licenses, HMP, 9, 10 bioactive compound, 178
Liebermann-Burchard reaction, 20 biological and pharmacological activities, 202–203
Life cycles phytochemistry, 202
of bryophytes, 138–140, 139 in vitro studies, 181–182
ferns, 138, 139 in vivo studies, 185–186
mosses, 138, 139 Mayer’s test, 19
pteridophytes, 139 Maytenin, 51, 231, 234, 238, 243
Lignans, 56, 317–318 Maytenus aquifoliun, 51
defined, 56 Maytenus ilicifolia, 23, 31, 42, 80, 92, 92–93, 98–99, 111,
neolignans and, 56, 61, 64 116, 220, 231, 231–234, 238, 243, 243–244, 246
Lima, D. F., 126 Medicago truncatula, 71–72
Linolenic acids, 30 Medical plants, trade of, 4
Lipids, 151, 152 Medicarpin, 310, 310
Lippia origanoides, 82, 111, 126 Medicinal plants
Liquid chromatography, 41, 275 advertising, 4
Liquid extracts, 16, 20, 24–27 defined, 2
Liu, Y., 292 endophytic microorganisms, 98–99
Liverworts, 136, 138, 157 forward genetics, 72, 73
Lonchocarpin, 321, 321 genome editing, 72–75
Lonchocarpus subglaucescens, 308, 322 molecular biology, 78–79, 83
Lonchocarpus urucu, 320, 322 reverse genetics, 72–75
Lopes, A. A., 241–242 secondary metabolites, 109–110, 110
Lunularia cruciata, 148, 149, 164 taxonomic classification of, 92
Lunularic acid, 148, 157 Medina, R. P., 297
Lunularin, 148 Meliaceae, 41–42, 44, 52, 54, 80, 81, 220, 289, 315–316, 322
Lupeol, 315, 316 Mendonça, R. C., 283
Lychnophora ericoides, 111, 114, 119 Menispermaceae, 35, 42, 81
336 Index
Mercadante, A. Z., 190, 194 activity on neglected diseases’ parasites,

Metabolic compounds, bryophytes, 136 240–244
Metastasis, 235–236 alkaloids, 45–51
Methicillin-resistant Staphylococcus aureus Atlantic Forest biome, 219–232, 236–237
(MRSA), 245 in Brazil, 33–65
Methyl jasmonate, 121, 122 Cerrado biome, see Cerrado biome
Metzgeriales, 147, 151 coumarins, 306–308
Mevalonate (MVA), 51, 52, 77 dereplication, 248
Mg-hesperidin complex, 65 development of agrochemicals, 33
Michael reaction, 311 development of pharmaceuticals, 33
Microorganisms, endophytic flavonoids, 308–310
classification of, 95 lignans, 317–318
identification of, 95 molecular biology tools of, 71–84
medicinal plants, 98–99 overview, 33–41
Microscopic botanical examination, identification tests, 18 research, 247–248
Mikania glomerata, 111, 126 secondary metabolites, 41–56
Mikania laevigata, 80, 111, 126 synthesis, see Synthesis of natural products
Mineral nutrition terpenes, 51–52, 314–317
heavy metals and, 114, 118–121 use in Mesopotamia, 33
secondary metabolites, 118–121 Neglected diseases’ parasites, natural products (NP)
Mitogen-activated protein kinases (p38 MAPK), 191 activity on, 240–244
Mitsunobu reaction, 317 Negri, M. L. S., 116
Molecular biology Nicoletti, M., 311
medicinal plants, 78–79, 83 NMR spectroscopy, 248
of natural products, 71–84 Nobiletin, 38, 40
overview, 71–72 Nomuraea rileyi, 122, 124
tools, 71–84 Non-endemic medicinal plants, 261–264
use of, 79, 83–84 Nonhomologous end-joining, 76
Monounsaturated fatty acids (MUFA), 29, 188 Nonprescription medicines, advertising, 9
Montanine-type, 45, 49 Nontoxic effects, Euterpe oleracea, 201
Moonwort ferns, see Ophiglossaceae Norberto Peporine Lopes, 37
Moraceae, 42, 56, 81, 220 Norcarotenoid, 222
Morel, L. J. F., 290 Nostoc, 138
Moringa oleifera, 220, 243 Novel tank diving test (NTDT), 195
Morphology and systematics NuBBE database, 41–42
of bryophytes, 138–140 Nuclear magnetic resonance (NMR), 23
of pteridophytes, 140–142 Nucleus of Bioassays, Biosynthesis and Ecophysiology of
Mors, Walter Baptist, 35, 223 Natural Products (NuBBE), 37, 83–84
Mosses, life cycle, 138, 139; see also Bryophytes Nucleus of Research in Natural and Synthetic
Muellera montana, 310, 322 Products, 37
Mulungu, see Erythrina veluntina Nyctaginaceae, 82
Murexide reaction, 19
Mycoback database, 96 O
Mycoplasma mycoides, 76–77
Myocardial ischemia, 199 Ocimum gratissimum, 111, 115, 264
Myracrodruon urundeuva, 80, 259, 261, 264, 273–276, 274 Ocimum selloi, 111, 113, 116, 120
Myrciaria dubia, 175, 192–194 Odontoschisma denudatum, 147, 149, 164
bioactive compound, 177 Official preparation, compounded HMP, 5
biological and pharmacological activities, 193 Ohshima, T., 311
functional characterization of, 187 Oils, 29
phytochemistry, 192–193 Oliveira, F. C., 285, 296
in vitro studies, 179–180, 193 Olive oil, 29
in vivo studies, 182–183, 193 Olmedioperebea sclerophylla, 220, 223
Myrcia tomentosa, 111, 114, 119, 125 Online extraction (OLE), 248
Myristicaceae, 35, 221, 224, 322 Ophiglossaceae, 141, 142
Ophioglossidae, 142
N Opioid, 77
Opportunistic species, 238
Narigenin, 38, 40 Optical rotatory power, determination of, 29
Nascimento, C. A., 288–289 Orange (Citrus aurantium), 10
Natural pigments, 218 Ordinance, 2, 3
Natural products (NP), 34, 217 Ormopteris cymbiformis, 160, 164
active on central nervous system (CNS), Ormopteris gleichenioides, 160, 164
236–237 Ormopteris pinnata, 160, 164
Index 337
Ormopteris riedelii, 160, 164 Phlegmariurus, 141

Ornithine, 45 Phyllosticta citricarpa, 101–103, 103
Oryza sativa, 72, 76 Phylogenies, bryophytes, 138
Osmunda, 158 Physalis angulata, 112–113, 118
Otacanthus azureus, 111, 113, 115 Physico-chemical tests, 24
Otto reaction, 19 for essential oils, 28–29
Ouratea multiflora, 220, 232–233 for fixed oils, 29–30, 30
Over the counter (OTC), 6 for phytomedicines, 30–31, 31
Oxidative stress, 120, 126, 181, 185, 189, 192, 197, 200, for plant derivatives, 24–27
227–228 for plant raw materials, 15, 16, 18–24
Oyeyemi, I. T., 195 quality, 15, 16, 18–24
Physiological adaptations, bryophytes, 138
P Phytochemical prospection, identification tests, 19, 19–20
Phytochemicals/phytochemistry
Pacheco, F. V., 115 Astrocaryum vulgare, 196
Paclitaxel, 78, 232 Bertholletia excelsa, 188
Palicourea coriacea, 288–289, 289, 290, 300 of bryophytes, 136
Palicourea rigida, 111, 114, 119, 289–290 Euterpe oleracea, 198–199
Palm tree, see Mauritia flexuosa Genipa americana, 190–191
Palythoa, 40, 41 modern, 35
Palytoxin, 40, 41 Myrciaria dubia, 192–193
Pampa, 80–82, 92, 150 of pteridophytes, 136
Pantanal (Swampland), 92 Spondias mombin, 194
Papaver somniferum, 33, 42, 74 Phytomedicines, 15, 30–31, 31
Paracoccidioides, 238, 294, 295 Phytopharmaceuticals, 78, 79
Passiflora alata, 111, 114, 119 Phytosterols, 177, 178, 188, 191, 196, 197, 202
Passiflora edulis, 111, 114, 119, 124 Pictet-Spengler cyclization, 313
Passiflora incarnata, 111, 114, 119 Pilkington, L. I., 317
Passiflora ligularis, 114, 119 Pilocarpine, 36, 37, 78, 79, 82, 114, 226
Passos, D. C., 288 Pilocarpus, 36, 37
Pathogenic fungi, 238 Pilocarpus jaborandi, 112, 114, 118
Pau-brasil, 34, 42, 218, 220 Pilocarpus microphyllus, 82, 112, 118, 221, 225–226
Paullinia cupana, 82, 112, 124 Piperaceae, 43–44, 49, 59, 112, 221, 82, 234
Peach palm, see Bactris gasipaes Piper aduncum, 112–113, 115
Peach palm pulp oil, 197–198 Piper crassinervium, 221, 241–242, 242
Peckolt, Theodoro, 34, 224 Piperidine, 45, 49
Pelly, S. C., 320 Piperlongumine, 234–236, 235
Peng, F., 321 Piper tuberculatum, 43, 49, 221, 234–235
Penicillin, 97 Piptadenia peregrina, 221, 223
Penicillium digitatum, 97 Piso, Willem, 224, 226
Peperomia obtusifolia, 221, 241, 241 Pisum sativum, 112, 117
Pereirine, 35 Pityrogramma calomelanos, 160
Perennial crops, 174 Pizzaro, G., 222
Peschiera affinis, 312, 322 Plagiochasma, 147
Pesez reaction, 19 Plagiochila bifaria, 146, 164
Pesticide residues, determination of, 23–24 Plagiochila corrugata, 147, 149, 164
Pew reaction, 20 Plagiochila diversifolia, 148, 149, 164
Pfaffia glomerata, 112, 120 Plagiochila rutilans, 146, 149, 165
pH, determination of, 27 Plagiochila stricta, 146, 149, 165
Phaeoceros, 140 Plant crude drug, 15
Phaeophleospora, 96, 99 Plant derivatives, 15
Phaeophleospora vochysiae, 101 extraction ratio, 25
Pharmaceutical industries, 3 physico-chemical tests, 15–16, 16
Pharmaceutical ingredient supplier, 3 quality, 15–16, 16
Pharmacies, 1, 3, 4, 5 receiving and sampling, 17, 17
Pharmacopoeia, 7 Plant raw materials
Phenolics; see also Bioactive compounds physico-chemical tests, 15, 16, 18–24
pteridophytes, 154–156, 155 quality, 15, 16
Pteris, 155 receiving and sampling, 17, 17
Spondias tuberosa, 232 Plant species, 9, 31, 42–45, 94, 163–165
Phenols, in Lamiaceae, 74 Atlantic Forest biome, 219, 220–221
Phenylalanine, 45 Caatinga biome, 264
Phenylpropanoids, 56, 57–60, 61 Cerrado biome, 289
Phenypropane derivatives, 317–318 secondary metabolites, 111–112
338 Index
Plasma membrane, 120 Pteris angustata, 161, 165

Plasmodium falciparum, 244 Pteris decurrens, 161, 165
Plectranthus barbatus, 221, 229–230 Pteris deflexa, 161, 165
Pleomassariaceae, 97, 99 Pteris denticulata, 161, 165
Pleopeltis, 143 Pteris multifidi, 161
Plumbagin, Droseraceae, 74 Pteris podophylla, 162, 165
Plumbaginaceae, 82 Pteris propinqua, 162, 165
Plumeria lancifolia, 34, 42, 224 Pteris quadriaurita, 162, 165
Plumierid, 34, 34 Pteris splendens, 162, 165
Polo, Marco, 221 Pteris tripatita, 162
Polyphenols, 228 Pteris vitata, 162
Polypodiidae, 142, 143 Pterocaulon balansae, 44, 57, 322, 306
Polypodiopsida, 142 Pterodon apparicioi, 315, 322
Polystichum, 140, 158 Pterogyne nitens, 221, 227–229, 228, 229, 239, 240,
Polytrichum commune, 150, 152, 152, 165 245, 245, 322
Polyunsaturated fatty acids (PUFA), Pulp oil, 178, 180, 183, 196
29–30, 188 Purealidin L, 38, 39
Porcelia macrocarpa, 221, 242, 242 Purity and integrity, physico-chemical tests,
Prance, G. T., 285 15, 16
Prasad, K. R., 320 Putrescine N-methyltransferase (PMT), 83
Prescription medicines, advertising, 9 Pyranoflavone isolonchocarpin, 308
Price regulation of HMP, 10 Pyrazines, 38, 39
Pristimerin, 54, 80, 231, 233–234, 243 Pyridine, 45, 49, 243, 243, 246
Processing, see Collecting and processing Pyrrolizidine alkaloid, 45, 49
Proteus vulgaris, 157
Protoberberine, 45, 48 Q
Protospacer Adjacent Motif (PAM), 76
Pseudomonas aeruginosa, 182, 198 Quality
Pseudovibrio denitrificans, 38, 39 HMP, 8, 10
Psidium guajava, 112, 121 medicinal plants, 4
Psilotaceae, 161, 165 methods, 8
Psilotum nudum, 142, 161, 165 physico-chemical tests, 15, 16, 18–24
Psychotria, 290–294, 297–300 phytomedicines, 15
Psychotria brachyceras, 112, 112, 113, 116 plant derivatives, 15–16, 16
Psychotria capitata, 289, 294, 295 plant raw materials, 15, 16
Psychotria coriacea, 288, 299, 300 THP, 8
Psychotria goyazensis, 289, 294, 295 Quantitative analysis, physico-chemical tests, 15, 16
Psychotria hoffmannseggiana, 289, 294, 295 Quantum chemical calculations, 40
Psychotria ipecacuanha, 82, 220, 289, 225 Quézia Cass group, 40
Psychotria leiocarpa, 112–113, 115 Quillaja brasiliensis, 112, 112–114
Psychotria prunifolia, 289, 292, 294, 294, 299, 299 Quillaja saponaria, 112, 112
Psychotria viridis,221, 224, 289 Quillworts, see Isoetaceae
Ptedoron pubescens Benth, 35 Quina-do-campo, 42, 311, 322
Pteridaceae, 159–162
Pteridium, 158 R
Pteridium aquilinum, 161, 165
Pteridophytes Radioactivity contamination, 24
alkaloids, 156, 157 Radula, 148, 150, 165
biologically active compounds, 158, 159–163 Raputia simulans, 313, 322
chemical compounds, 136, 137, 153–157, Raputindole A, 313, 313
154–157 Ravi, G. A., 315
collecting and processing, 144–145 Raw materials, see Plant raw materials
defined, 140 Raymond-Marthoud reaction, 19
flavonoids, 156, 156 Reactive nitrogen species (RNS), 227
labelling, 145 Reactive oxygen species (ROS), 186–187, 192, 227
life cycle, 139 Reboulia, 147, 149, 165
morphology and systematics of, Reboulia hemisphaerica, 146, 149, 165
140–142 Redox reactions, 227–232
phytochemistry of, 136 Refraction index, determination of, 29
size of, 144 Registration dossier of HMP, 6, 7
species of, 136 Regulations
terpenoids, 153–154, 154 addressing, 4, 6
Pteris, 155, 158, 162 compounding of HMP, 4, 4–5
Pteris altissima, 161, 165 industrialized HMP, 5–10, 6
Index 339
Relative density, determination of, 26, 29 Sesquiterpenes, 51, 114

Renal failure effects, Euterpe oleracea, 200 Seychellene, 315, 315
RENISUS, 219 Shaw, J. T., 309–310
Resolução da Diretoria Colegiada (RDC), 2–9, 4, 6, 15 Shinoda reaction, 20
Resolution, 2 Silva, C. F. C., 194
Reverse genetics, 72–75 Silva aestu aphylla, see Caatinga biome
Ribifolin, 244 Silva-Junior, E. A., 38
Ricciocarpos natans, 147, 148, 150, 165 Sinensetin, 38, 40
RNAi, 74 Single guide RNA (sgRNA), 76
Robustic acid, 306, 307 Small-scale culture, 99
Rotenone, 318–320, 319 Small subunit rRNA gene (SSU), 95
Rubiaceae, 43, 48–49, 53, 82, 238, 111–112, 175, Social thermotherapy, 3
220–221, 289, 322 Softening/re-hydrating dehydrated material, 18
Rutaceae, 36, 42, 42–44, 48, 57, 59, 82, 112, 220–221, Soft extracts, 24–27
262, 322 Solanaceae, 82, 112, 163, 221
Solanum grandiflorum, 221, 224–225
S Solanum lycopersicum, 76
Solanum tuberosum, 74
Saccharomyces cerevisiae, 77, 234–235 Solubility, determination of, 26
Saccharopolyspora erythraea, 97 Soluble solids, 25–26
Salacia campestris, 43, 51 Solvent residues, determination of, 25–26
Salicaceae, 82, 220, 230 Sorghum bicolor, 75
Salinity, secondary metabolism, 116–118 Soybean, 10, 31, 75
Salkowski reaction, 20 Soy oil, 29
Salmonella typhimurium, 235 Species of pteridophytes, 136, 159–162
Salvia miltiorrhiza, 74 Spix, Johann Baptist, 34, 224
Salviniaceae, 162, 165 Spodoptera frugiperda, 124
Salvinia molesta, 162, 165 Spondias mombin, 175, 194–195
Sanitary regulation, 2 bioactive compound, 177
Santos, M. F. G., 197 biological and pharmacological activities,
Santos, M. G., 158 194–195
Santos, W. P., 243 phytochemicals, 194
Sapindaceae, 82, 112, 162 in vitro studies, 180, 194
Sapindales, 42 in vivo studies, 183, 195
Saponification value, 16, 30 Spondias tuberosa, 221, 231–232, 232, 260
Saponins, 20, 239 Sporangia, 138
Saturated fatty acids (SFA), 29, 188 Sporangium dehiscence, 138
Schinus terebinthifolius, 92–93, 98–99, 114, 120, 219 Sporophytes, 138, 140, 140, 142
Schistosoma mansoni, 35 Srikrishna, A., 315
Schmid, H., 35 Staining, 18
Scototaxis, 195 Staphylococcus aureus, 125, 180, 182, 198, 225,
Seasonal variation, 124–127 270, 272
Secondary metabolites, 24, 100, 102, 113–114, 265, 267, Stemodia foliosa, 43, 53, 221, 245–246, 246
269, 272, 274 Steroids, 20, 148, 149–150, 176
alkaloids, 45–51 Stiasny reaction, 20
biotic elicitation of, 121–124, 122–123 Stockler-Pinto, M. B., 188–189
distribution of, 41–56 Streptococcus mutans, 225
eco-chemical roles of, 110 Streptococcus pyogenes, 76, 272
heavy metals, 118–121 Streptomyces griseus, 97
irradiance, 109–110 Streptomyces orientalis, 97
mineral nutrition, 118–121 Streptomyces wadayamensis A23, 41
modulation of medicinal plant, 110, 110 Streptomycin, 97
overview, 109–110 Strictosidine, 48, 78, 291, 291
phenylpropanoids, 56 Strychnos castelnaeana, 221, 223
plant species, 111–112 Strychnos guianensis, 221, 223
salinity, 116–118 Strychnos pseudoquina, 311, 322
structural diversity, 41–56 Strychnos toxifera, 223
terpenes, 51–55 Stryphnodendron adstringens, 31, 81, 92, 92–93, 98–99
UV radiation, 110–115 Styrylpyrones, 234
Sekino, E., 308 Subclasses, HMP, 6
Selaginella, 141, 141, 156, 157, 158, 163, 165 Sunflower, see Helianthus annuus
Selaginellaceae, 141, 141, 163, 165 Surendra, K., 315
Selenium, 176, 179, 186 Suzuki reaction, 317
Serratia marcescens 3B2, 38, 39 Swartzia langsdorffii, 221, 238, 239
340 Index
Swinglea glutinosa, 42, 48, 51 Toddalioideae, 35–36

Synthesis of natural products, 305–323 Toona ciliata, 315–316, 322
alkaloids, 310–314 Total ash content, determination of, 21
coumarins, 306–308 Toxicaria americana, 221, 223
flavonoids, 308–310 Toxic glycoalkaloids, 74
lignans, 317–318 Tradescantia micronucleus assay, 230
overview, 305–306 Tradescantia pallida, 228
rotenone, 318–320, 319 Traditional herbal product (THP), 2, 3, 6, 6–9
tephrosin, 319–320, 320 Tran, D. N., 316–317
terpenes, 314–317 Transcription activator-like effector nucleases
Synthetic biology, 76–78 (TALEN), 74
Trans-p-coumaric acid, 56
T Triterpenes, 20, 51, 231, 231, 239
Triterpenoids, 147, 154, 176
Tabernaemontana hystrix, 312, 322 see also Terpenoids
Taperebá, see Spondias mombin Triticum aestivum, 76
Tapped density, 26 Trypanosoma cruzi, 179, 192, 242–243
Tatajuba, 220, 222 Tryptamine alkaloids, Tetrapterys
Taxonline, 95 mucronata, 221, 237
Tazettine-type, 45, 49 Tubocurarine, 34, 35, 79
T-DNA vector, 74 Tucumã, see Astrocaryum vulgare
Technical evaluation, HMP, 6 Tumor cell lines, 182, 233
Technology
CRISPR/Cas9, 75–76 U
genome editing, 75
RNAi, 74 Uliginosin B, 113, 117, 121, 122, 124
Temperature, cell physiology, 113, 116 Umbu, see Spondias tuberosa
Tephrosia candida, 320, 322 Uncaria guianensis, 311, 322
Tephrosin, 319–320, 320 Uncaria tomentosa, 82, 289, 291, 311, 322
Terpenoids/terpenes, 51–55, 53–54, 149–150, Único de Saúde (SUS), 3, 4, 5, 10
314–317 University of San Diego, 37
2-C-methyl-D-erythritol 4-phosphate Unsaturated fatty acids (UFA), 188
(MEP), 52 Urgaonkar, S., 309–310
biosynthesis of, 52 Urticaceae, 82
from Brazilian plants, 52–54 Urucum, 220, 222
indole alkaloids, 45, 48–49 UV radiation, 110–115
Marchantiophyta, 146–147, 147–148 UV-Vis chromophores, 41
mevalonate pathways (MVA), 52
pteridophytes, 153–154, 154 V
structures of, 55, 147, 148
Test(s) Vaithegi, K., 320
acid-insoluble ash content, determination of, 21 Valeriana glechomifolia, 112, 124
aflatoxins, 24 Validation, 6
essential oils content, determination of, 22 of analytical procedures for
extractable material, 22 HM, 9
foreign matter, determination of, 21–22 of analytical procedures for THP, 9
heavy metals, determination of, 23 HMP methods, 8
pesticide residues, determination of, 23–24 Valles, 136
physico-chemical tests, 15, 16 Vancomycin, 97
quantitative analysis of active principles, 22–23 Vanderpoorten, A., 142
quantitative analysis of chemical markers, 22–23 Varronia curassavica, 80, 112, 115
radioactive contamination, 24 Velame, see Croton heliotropiifolius
total ash content, determination of, 21 Verbenaceae, 31, 82, 111, 220, 262–263
water and volatile material, determination Verongidoic acid, 38, 39
of, 21 Vibrational circular dichroism, 40
Tetrahydroisoquinoline, 45, 48 Vinblastine, 79
Tetrapterys mucronata, 221, 236–237, 237 Vincristine, 79
Thelypteris, 158, 164–165 Viridiflorol, 316, 317
Theobroma cacao, 112, 124 Virola, 223–224, 221, 322
Theophrastus, 158 Virola flexuosa, 317, 322
Thin-layer chromatography (TLC), 21, 25, 28, 30 Viscosity test, 27
Tithonia diversifolia, 112, 114, 119 Vitamin C, 177, 192–194
Toddaliinae, 35 Vitexin, 78, 79, 80, 159, 178, 202
Index 341
Vitis amurensis, 83 X
Vochysia divergens, 92, 92, 98–99
Volumetric methods, 21 Xerostomia (dry mouth), 36
Von Martius, Carl Friederich, 34, X-rays, 72
224, 258 Xylariaceae, 97, 99
Von Pechmann, Hans, 308 Xylella fastidiosa, 62–63, 65
Vougogiannopoulou, K., 313
Y
W Yang, X., 310
Wagner, D. H., 142 Yoda, H., 318
Wagner/Bouchardat test, 19 Yoshikawa, H., 157
Wang, F., 314–315 Yuyama, L. K. O., 197
Wasicky reaction, 19
Water Z
biochemical processes, 116–118
salinity and, 113–114, 116–118 Zea mays, 76
volatile material and, 21 Zebrafish, 185, 195
Watson–Crick base pairing, 75 Zielinski, A. A. F., 194
Winteraceae, 82 Zinc-finger nucleases (ZFN), 74

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What is the focus of the book series described?

What is the focus of the book series described?

What areas of knowledge does the series intend to cover?

What areas of knowledge does the series intend to cover?

Brazilian Medicinal Plants

Natural Products Chemistry of Global Plants

Recent Titles in this Series:

Traditional Herbal Remedies of Sri Lanka

Medicinal Plants of Bangladesh and West Bengal

Brazilian Medicinal Plants

© 2020 by Taylor & Francis Group, LLC

No claim to original U.S. Government works

Printed on acid-free paper

International Standard Book Number-13: 978-1-138-09375-1 (Hardback)

Library of Congress Cataloging-in-Publication Data

Names: Modolo, Luzia V., author.

Visit the Taylor & Francis Web site at

and the CRC Press Web site at

Chapter 1 Unraveling the Complexities of Brazilian Regulations for

Chapter 2 Physico-Chemical Methods for the Quality Control of Medicinal Plants,

Chapter 3 The Widening Panorama of Natural Products Chemistry in Brazil........................... 33

Chapter 4 Molecular Biology Tools to Boost the Production of Natural Products:

Chapter 5 Diversity of Endophytes and Biotechnological Potential............................................ 91

Chapter 6 Environmental Factors Impacting Bioactive Metabolite Accumulation

Chapter 7 Brazilian Bryophytes and Pteridophytes as Rich Sources of

Chapter 8 Chemical and Functional Properties of Amazonian Fruits...................................... 173

Chapter 9 Plant Species from the Atlantic Forest Biome and

Chapter 12 Total Synthesis of Some Important Natural Products from

R. Cooper, Ph.D., Editor-in-chief

Mary Ann Foglio

ary Ann Foglio is Senior Researcher at the Faculty of Pharmaceutical

Michelli Massaroli da Silva Lidiane Gaspareto Felippe

Sylvain Darnet Arthur Germano Fett-Neto

Fernanda de Costa Chirlei Glienke

Lucilia Kato Luzia Valentina Modolo

Maria da Conceição Ferreira de Oliveira Josilene Lima Serra

1.2 VARIOUS DEFINITIONS AND APPROACHES

1.2.2 Medicinal Plants Trade, as Established by Law 5,991/1973

2.2 IMPORTANT PUBLICATIONS

on Harmonization), and the Brazilian organizations such as INMETRO (National Institute of

2.3 RECEIVING AND SAMPLING PLANT RAW

2.4 PHYSICO-CHEMICAL TESTS FOR THE QUALITY

2.4.1.2 Identification by Phytochemical Prospection or Chemical Identification

TABLE 2.3 (Continued)

2.4.1.3 Identification by Chromatographic Fingerprinting

2.4.2 Determination of Water and Volatile Material

2.4.3 Determination of Total Ash Content

2.4.4 Determination of Acid-Insoluble Ash Content

2.4.5 Determination of Foreign Matter

2.4.6 Determination of Essential Oils Content

2.4.8 Quantitative Analysis of the Active Principles and/or Chemical Markers

2.4.9 Determination of Heavy Metals

2.4.10 Determination of Pesticides Residues

2.4.12 Determination of Aflatoxins

2.5 PHYSICO-CHEMICAL TESTS FOR THE QUALITY CONTROL OF PLANT

2.5.1 Determination of Dry Residue (or Soluble Solids)

2.5.2 Determination of Solvent Residues

2.5.4 Apparent Density (or Tapped Density)

The determination of the pH is important, not only as a characterization parameter of liquid

2.3 RECEIVING AND SAMPLING PLANT RAW

2.4 PHYSICO-CHEMICAL TESTS FOR THE QUALITY

2.5 PHYSICO-CHEMICAL TESTS FOR THE QUALITY CONTROL OF PLANT

2.6 PHYSICO-CHEMICAL TESTS FOR THE QUALITY

2.7 PHYSICO-CHEMICAL TESTS FOR THE QUALITY

2.8 PHYSICO-CHEMICAL TESTS FOR THE QUALITY

3.1 INTRODUCTION AND AN OVERVIEW OF NATURAL PRODUCTS

3.2 STRUCTURAL DIVERSITY AND DISTRIBUTION OF

4.6 MOLECULAR BIOLOGY IN THE CONTEXT

4.7 USE OF MOLECULAR BIOLOGY TO IMPROVE THE PRODUCTION

5.5 EXPLORING THE BIOTECHNOLOGICAL POTENTIAL

7.2 MORPHOLOGY AND SYSTEMATICS OF BRYOPHYTES