Synergistic Antifungal Activity of Ethanolic Extracts of Dragon Fruit (Hylocereus

polyrhizus) peels and Flamingo Lily (Anthurium andraeanum) flower against Aspergillus
fumigatus.

A Thesis
to be Presented to the Faculty of the College of Pharmacy
Mindanao Medical Foundation College

In Partial Fulfillment

Of the Requirements for the Degree of

Bachelor of Science in Pharmacy

Betonio, Cheery Lyn

Bulay, Debbie Joyce
Saldivar, Weena Lou
Veloso, Luc
2019
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Chapter 1

THE PROBLEM AND ITS SETTING

Introduction

Present antibiotics have become less and less effective due to antimicrobial resistance

that is rapidly emerging and spreading globally. Microorganisms continue to evolve and

develop new resistance mechanisms in line with the misuse and overuse of antimicrobials.

This has presented a serious threat to our capability to treat and stop the spread of common

infections with antibiotics that leads to a prolonged illness, disability, and eventually death.

And as this crisis continues to rise, scientists are forced to search for new medicinal agents

(Ventola, 2015).

Fungi of the genus Aspergillus are widely distributed in our surroundings, both

indoors and outdoors, that’s why most people are often exposed to aspergillus. This type of

mold rarely affects healthy individuals, however, for people who are immunocompromised,

Aspergillus species can produce a spectrum of diseases that affects the lungs or other organs

and tissues. There are 180 known species of Aspergillus and about 20 of them can cause

human infections. Aspergillus fumigatus is the most common causative agent of aspergillosis

of various types (Singh et al, 2015). Commonly occurring types of aspergillosis are mild,

allergic forms and generally not life-threatening but can affect immunocompetent hosts. In

contrast, invasive aspergillosis is less common and occurs mostly in immunocompromised

patients but, it can be the main cause of morbidity and mortality. However, the occurrence

has likely shifted due to the increasing number of organ transplants and the usage of

immunosuppressant agents. According to the Centers for Disease Control and Prevention

(CDC), in the United States, the cases of invasive aspergillosis has increased with an average

of 3% every year from 2000 to 2013. In 2014, about 15,000 hospitalizations that occurred in
 2

the United States are aspergillosis-associated, with an estimated cost of $1.2 billion (Benedict

& Chiller, 2018).

According to the intensive care unit (ICU) autopsy studies in a broad US healthcare

network, aspergillosis was one of the top 4 most common diagnoses that likely lead to death.

It was found out that invasive aspergillosis was the most common type of fungal infection

(25%) among stem cell transplant recipients and was the second most common type of fungal

infection (59%) among solid organ transplant recipients (Webb et al, 2018).

Many Asian countries failed to demonstrate accurate epidemiological surveys about

fungal infections of various types. However, candidiasis and aspergillosis are still the most

commonly significant opportunistic mold and yeast infection. Opportunistic invasive fungal

infections (IFIs) in this region has a significant impact on public health as the number of

immunocompromised patients expanded (Slavin, 2012).

Similarly, infectious disease is still the main cause of morbidity in the Philippines, but

fungal disease surveillance has not been done. Nowadays, serious fungal diseases have

gained recognition with the dramatic increase in the number of immunocompromised patients

related to AIDS, tuberculosis and autoimmune diseases. Estimates derived from various

programs suggests aspergillosis and candidiasis as the major causes of fungal infections in

the Philippines with an estimation of 1,852,137 in 2016 (Batac et al, 2017).

One of the major challenges in treating invasive aspergillosis is the suspected

emergence of resistant strains of Aspergillus fumigatus. This is because treatment options for

this type of infection are limited and associated with serious toxicities or drug interactions.

Antifungal resistance with a particular interest in Aspergillus species has been increasing and

isolated worldwide due to prolonged clinical exposure and agricultural use as fungicides. In

clinical isolates of A. fumigatus from Regional Mycology Laboratory Manchester, UK overall

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resistance frequency is about 17% in 2007 with a significant increase since 2004 (Howard et.

al, 2009).

In the past decades, many efforts were done to address the pan-resistance of fungi to

clinically available antifungals. Many investigational drugs that are still in preclinical and

clinical studies have the potential to overcome resistance to azole and echinocandins

(Wiederhold, 2017). Much work has also been done in an attempt to evaluate the antifungal

activities from various botanical sources. Medicinal plants and their products like combined

essential oils showed potentials to cure mycotic infections and can be used as

pharmaceuticals or preservatives (Bansod et al., 2008 and Rajes et al., 2004). Some plant

extract combinations showed even higher inhibition than traditional antibiotics against certain

species of microorganisms with the presence of phenolic compounds, flavonoids, and

anthocyanidins (Altemimi et al, 2017).

Since botanical extracts have the potentials to be considered as alternatives to

conventional antimicrobial agents, they should be evaluated especially in this era of

antimicrobial drug resistance. Plants with a particular interest in this study are red-fleshed

dragon fruit (Hylocereus polyrhizus) and flamingo lily (Anthurium andraeanum). Several

studies evaluating their antimicrobial and pharmacologic use and have concluded that these

plants have the potential to inhibit certain microbes (Abima shazhni et al, 2016 & Ismail et al,

2017). The researchers wanted to know if there is a synergistic effect when ethanolic extracts

of red-fleshed dragon fruit peels and flamingo lily flowers are combined.

Review of Related Literature and Studies

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This provides relevant and related information from reliable researchers, journals, and

websites of various authors to help the researchers in recognizing conditions that are

important in conducting the experiment.

Related Literature

A. The Plants

Figure 1. Dragon Fruit (Hylocereus polyrhizus)

Source: https://picture-graffiti.blogspot.com/2019/01/physical-dragonfuit

Taxonomic Classification

Kingdom: Plantae

Class: Mongoliopsida

Order: Caryophyllales

Family: Cactaceae

Genus: Hylocereus

Species: H. polyrhizus

Plant Description
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Native from Central and South America, dragon fruit (Hylocereussp ) or “pitaya” is

grown as a night-flowering ornamental vine and as a fruit crop across the world. Dragon fruit

stems are scandent or clambering and branch profusely, growing between 5 to 10 meters or

longer. The flowers are large and white, which are nocturnal and scented hence the name

“moonflower” or “Lady of the Night”. The fruit is propagated through seeds and cuttings.

The brightly colored oblong fruits are about ten centimeters in length and can weigh up to a

pound. They have pink to magenta-colored skin due to the betacyanins present and has the

appearance of succulent, fleshy scales overlapping, leaving small, green-tipped protrusions

along its length. The skin is thin, with an average thickness of only three millimeters, so the

flesh to rind ratio is high (Wybraniec et al., 2007).

Plant Cultivation

Dragon fruit is a tropical fruit that grows best in an area wherein rainfall is uniformly

distributed. It requires free draining soil with sandy to clay loam types and has a pH ranging

from 5.3 – 6.7 with high organic matter is most preferable. Even so, the plant can tolerate

poor soil conditions and temperatures range about 20 – 30 °C. The plant requires good

amount of sunlight, but not for a very long period of time so shading is important (Fruit

Research and Development Institute, 2015).

Plant Phytochemicals

The plant predominantly contains steroids (29.77%) and triterpenoids (16.46%).

Supercritical carbon dioxide extracts of dragon fruit were analyzed by gas chromatography-
mass spectrometry analysis. Main constituents of H. polyrhizus were identified as beta-
amyrin (15.87%), alpha- amyrin (13.90%), octacosane (12.2%), gammasitosterol (9.35%) and
others (Lou et al, 2014).
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Figure 2. Flamingo Lily (Anthurium andraeanum)

Source: hhtps://copiahomeandgarden.com/product/anthurium

Taxonomic Classification

Kingdom: Plantae

Class: Magnoliopsida

Order: Alismatales

Family: Araceae

Genus: Anthurium

Species: A. andraeanum L.

Plant Description

Anthurium andraeanum, also known as flamingo lily or tail flower, is a

monocotyledonous perennial ornamental plant. It is popular for its brightly colored, heart-

shaped leaves. And a flower with waxy spathes or flower bracts and a protruding

inflorescence also known as a spadix. Each flower will last about six weeks in plant or when

cut and placed in a vase with water. It is one of the most globally traded flowers since it is a

popular pot or cut flower (Harb et al., 2010). Flamingo lily is an herbaceous, perennial and
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rhizomatous evergreen plant of small height and slow annual growth rate. It has the

cultivation of life from five to ten years.

Plant Cultivation

Flamingo lily grows best in humid and low-light conditions. It requires soils that is

moist and high on organic matter. Humidity is a significant factor in cultivation this plant,

and should not drop below 50% for more than a few days. It prefers shady areas and may

tolerate acidic, sandy or loamy soils (Gilman, 2018).

Plant Phytochemicals

Flamingo lily contains phytochemicals such as alkaloids, flavonoids, phenols,

phlobotannins, steroids, and tannins. The presence of these phytochemicals varies with the

plant parts and type of solvent used. Reportedly, the flowers extracted with ethanol was

believed to be positive with most phytochemicals. The spadix contains calcium oxalate which

is toxic to cats and dogs (Abima shazhni et al, 2016).

Plant Phytochemical Screening

Plants produce several compounds with various chemical structures called

phytochemicals or secondary metabolites. These compounds are found in smaller quantities

but of higher value than primary plant metabolites, many act as antioxidants, sex attractants,

antibiotic agents and many other medicinal agents. These metabolites are isolated and

structurally identified using new sophisticated instrumental techniques for phytochemical

studies in the search of bioactive compounds (Aguinaldo et al, 2005).

Itraconazole

Itraconazole is a triazole antifungal agent with a broad spectrum antifungal activity.

This drug that acts by inhibiting CY450 dependent enzyme 14-alpha-sterol demethylase,
 8

thereby disrupting the ergosterol biosynthesis pathway. Itraconazole exhibits fungistatic

activity against yeasts and fungicidal activity against Aspergillus species. This is indicated for

many systemic infections caused by fungi such as Blastomyces, Sporothrix, Aspergillus, etc.

it is extensively used in the treatment of dermatophytoses, especially onychomycoses. It has

adverse effects similar to other azole derivative antifungals (Lester & Hope, 2013). In the

study, itraconazole is used as a positive control.

Normal Saline Solution

Normal saline solution (NSS) is a mixture of sodium chloride in water (0.9%) and has

several uses in medicine. Normal saline solution is used for preparing microbial suspensions

when it is necessary to deliver a set number of microbes to an identification test battery,

specifically for the growth media used for disk susceptibility testing (Villanova, PA, 2018).

This can reduce some types of bacteria and irritation since it is isotonic with body fluids. In

this study, a normal saline solution is used as a negative control.

Zone of Inhibition

The zone of inhibition visibly shows the sensitivity of inoculated microorganisms to

the agent or drug as a circular area around the spot of the antibiotic, which is measured in

millimeters. Diameters of the zone of inhibition are used in computing for MIC in disk

diffusion or agar diffusion test. The zone of inhibition diameter in mm are to be interpreted

as: <10 mm (inactive), 10 – 13 mm (partially active), 14-19 mm (active) and >19 (very

active) (Barnard, 2019).

Broth Dilution Method

Broth dilution method is an option in providing both quantitative (MIC) and

qualitative results. It can be performed by two ways: macro dilution or microdilution, they

only differ on the volume of the broth. It involves the preparation of twofold dilutions of
 9

antibiotic agents in a liquid growth medium dispensed in test tubes. The antibiotic containing

tubes are inoculated with a standardized bacterial suspension and examined for bacterial

growth as evidenced by turbidity. Minimal inhibitory concentration (MIC) will be measured

as the lowest concentration that prevented growth (Ahmed, 2019).

Minimal Inhibitory Concentration (MIC)

Minimal Inhibitory Concentration (MIC) is the lowest concentration of an

antimicrobial agent that prevents the visible growth of microorganisms. MIC can be

determined by either the broth dilution test or E- test method. It is important in determining

the effective dose of an antimicrobial

Related Studies

A study exploring the biological activities of red-fleshed dragon fruit revealed the

presence of various phytochemicals and antimicrobial activity of its peel extract. The

identified chemical constituents were oxygenated terpenes such as 5-cedranone, eucalyptol,

and terpineol. The antimicrobial activity that was evaluated against 5 microbial strains with

Aspergillus having 17.5± 0.70 inhibition zone via cup agar method. The extracts of

hylocereus polyrhizus exhibited strong antimicrobial activity which may be due to the co-

activity of its oxygenated terpenes (Ismail et al., 2017).

Flamingo lily flower was also studied by various researchers for its chemical

constituents, antimicrobial and pharmacologic activity. On a phytochemical screening, results

showed the presence of alkaloids, flavonoids, phenols, phlobatannins, steroids, and tannins

(Abima Shazhni et al, 2016). Specifically, the ethanol extract of the flower was tested

positive for alkaloids, flavonoids and steroids. In the same study, different plant parts were

extracted with different solvents (aqueous, alcohol, chloroform, dimethyl sulfoxide and
 10

ethanol). The results showed that ethanolic extract of the flower part exhibited superior zone

of inhibition about 20mm against Aspergillus fumigatus via agar well diffusion method.

A study evaluating the nutraceutical potential of and antioxidant benefits of red pitaya

made quantification of the flesh polyphenols of the peels via sub-fractionation method. The

extraction attained higher polyphenolic contents than the past studies. The extracted phenolic

compounds have shown to inhibit a wide range of bacterial and fungal food pathogens. They

concluded that red pitaya may be used for nutraceutical manufacture formulation and food

applications as the fruit has antioxidants and preservative properties (Tenore et al, 2011).

This research was designed to evaluate the efficacy of the red dragon fruit

(Hylocereus polyrhizus), as an antimicrobial agent. Extracts of the fruit peel sample were

acquired with solvent maceration pH 5. The peel extracts were noted for phytochemical

properties, complete phenols, antioxidants and antimicrobial activity. Extracts were combined

with elevated levels of phytochemical compounds and complete extract phenols with

antioxidant and antimicrobial activity. Red dragon fruit peel extracts prepared with different

percentages and evaluated their physicochemical properties, nutrients, antioxidant activity,

and microbiological profile. (Temak et al, 2018).

Standard procedures were used to screen plant chemicals and the presence of

(alkaloids, flavonoids, phenols, phlobatannins, steroids and tannins) were demonstrated in

this study. The utilization of the agar Well Diffusion method against four bacterial and two

fungal pathogens has been used to determine the anti-microbial activity of plant extracts. The

aim of this research was to examine the phytochemical and antimicrobial compounds of

various components of Anthurium andraeanum. Flower, leaf, stem and root plant materials

have been obtained using various solvents such as aqueous, acetone, dimethyl sulfoxide,

chloroform and ethanol. (Renu et al, 2016).The plant extracts have shown results showed to

have highly inhibited the samples of the grams of negative E.coli and K.pneumoniae bacterial
 11

strains and fungal pathogen Aspergillus fumigatus. The floral extracts showed superior

inhibition area for the Aspergillus fumigatus, a bacterial pathogen and E.coli, fungal

pathogen. The findings show antimicrobial values that may be helpful in assessing the

usefulness of treating microbial illnesses by the decorative plant A.andraeanum.

Theoretical Framework

Aspergillus fumigatus is a ubiquitous saprophytic fungus, with airborne conidia.

Immunocompetent hosts may normally eliminate the conidia but are still vulnerable to

allergic bronchopulmonary aspergillosis (ABPA) and aspergilloma and mild infections.

However, it may cause severe and usually fatal invasive aspergillosis to

immunocompromised patients. A. fumigatus is predominantly identified by the morphology

of their conidia and conidiophores, and, species are distinguished through biochemical and

molecular characterizations in addition to morphological data. This species is thermophilic

and can grow at 50°C and still survive maintained temperatures up to 70°C (Leonard &

Badii, 2016). 

Plants yield a wide variety of secondary metabolites also called phytochemicals that

has pharmacologic values, including antimicrobial activities. Phytochemicals are influenced

by environmental conditions and growth conditions such as lack of nutrients, pathogen attack,

competitive co-habitation plant species and insect predation. Phytochemicals found in various

plant parts may have different biological activities such as enzyme inhibitory properties,

kinase affecting properties which may prevent cancer development, and antimicrobial

properties.

Theoretically, antimicrobial agents microbial processes such as inhibiting cell wall

synthesis, inhibiting microbial nucleic acid or protein synthesis, disrupting microbial

membrane or blocking key enzymes (Willey et al, 2008). The antimicrobial activity of a
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plant extract may be associated with the presence of flavonoids, steroids, saponins, tannins,

and organosulfates such as isothiocyanates. They are widely distributed in plant kingdom but

with varying concentrations that in one genus, a certain species may have higher

concentrations than another.

Red pitaya or red-fleshed dragon fruit was found out to have high concentrations of

steroids and triterpenoids with predominant constituents identified as α- and β – amyrin,

octacosane, and γ-sitosterol (Luo, et al, 2014). On the other hand, flamingo lily’s (Anthurium

andraeanum) predominant constituents are alkaloids, flavonoids, phenolic compounds,

phlobatannin, steroids and tannins in the entire plant (Abima shazhni et al, 2016). Both plants

are studied for their antimicrobial activities, with H. polyrhizus against Aspergillus having

17.5± 0.70 inhibition zone via cup agar method and with the flower extracts of A.

andraeanum showing greater inhibitions of certain types of bacteria and fungi.

Itraconazole is used for the treatment of several fungal infections such as

aspergillosis, onychomycosis, blastomycosis, and cryptococcal meningitis in both

immunocompromised and non-immunocompromised patients. It has been said that

itraconazole works by inhibiting the cytochrome P-450- dependent enzyme which results in

impairment of ergosterol synthesis (Lester, J. M. et al, 2013)

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Conceptual framework

Independent variables Dependent Variables

Concentration of ethanolic H. Antifungal Activity

polyrhizus peel extract

- Zone of Inhibition

Concentration of ethanolic A.
- Minimal inhibitory Concentrations
andraeanum flower extract
(MICs)

Concentration of combined ethanolic

extracts of H. polyrhizus and A.

andraeanum

Positive Control (Itraconazole)

Negative Control (Normal Saline

Solution) Figure 3. Conceptual Framework of the Study

Figure 3 illustrates the conceptual framework of the study. It shows two types of

variables, the independent and dependent variables. The independent variables are the

variables that are changed or controlled in the experiment to perceive effects on the

dependent variables. The independent variables include the concentrations of the individual

and combined ethanolic extracts of red-fleshed dragon fruit (Hylocereus polyrhizus) peels

and flamingo lily (Anthurium andraeanum) flowers, the positive control (itraconazole) and

negative control (normal saline). The dependent variables of the study are the zones of

inhibition revealed by the disk diffusion method (Kirby-Bauer test), and the values of

minimal inhibitory concentrations of the individual and combined extracts of the plants used

by broth dilution test.

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Statement of the Problem

The main purpose of this study is to evaluate the synergistic antifungal activity of

ethanolic extracts extracted from dragon fruit peel (Hylocereus polyrhizus) and flamingo lily

flower (Anthurium andraeanum).

Specifically, this study aimed to answer the following questions:

1. What types of phytochemicals are present in the ethanolic extracts of red-fleshed

dragon fruit (Hylocereus polyrhizus) peels and flamingo lily flower (Anthurium

andraeanum)?

2. Does the combined extracts of red-fleshed dragon fruit (Hylocereus polyrhizus)

and flamingo lily flower (Anthurium andraeanum) have antifungal activity against

Aspergillus fumigatus?

3. What are the individual and combined minimal inhibitory concentrations (MIC) of

ethanolic extracts of red-fleshed dragon fruit (Hylocereus polyrhizus) and

flamingo lily flower (Anthurium andraeanum)?

4. Is there a significant difference among the minimal inhibitory concentrations

(MICs) of the individual plant extracts from the minimal inhibitory concentration

(MIC) of the combined plant extract?

Null Hypothesis

H 0 – There is no significant difference between the minimal inhibitory concentrations

(MICs) of the individual and combined extracts of red-fleshed dragon fruit (Hylocereus

polyrhizus) and flamingo lily flower (Anthurium andraeanum).

H A – There is a significant difference between the minimal inhibitory concentrations

(MICs) of the individual and combined extracts of red-fleshed dragon fruit (Hylocereus

polyrhizus) and flamingo lily flower (Anthurium andraeanum).

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Significance of the Study

The study intended to investigate the possible synergistic antifungal activity of the

combined ethanolic extracts of red-fleshed dragon fruit (Hylocereus polyrhizus) and flamingo

lily flower (Anthurium andraeanum). Thus, this study enabled the discovery of a possible

alternative antifungal agent. Hence, this study would benefit the following:

To the Public. This may serve as an informative text in regards to the potential use of

plants mentioned above as herbal or medicinal plant particularly in treating fungal infections

if they decide an alternative for mild aspergillosis.

To the Department of Health and Food and Drugs Administration. The data

gathered in this study may give additional information about the use of the combination of

these plants as a natural antifungal agent, may it to serve as a preservative, cure or herbicide.

To the Department of Agriculture. The result of this study should give benefits to

the department as it may serve as a reference in using these plants as an alternative fungicide

to protect crops and plants from fungal infestations.

To future researchers. The data and results of this study would provide additional

information about the individual and combined antifungal values of red-fleshed dragon fruit

(Hylocereus polyrhizus) and flamingo lily flower (Anthurium andraeanum).

Definition of Terms

Operational definition of terms provided to understand the study are the following:

Aspergillosis is a disease caused by a fungus called Aspergillus.

Aspergillus fumigatus is a fungal strain that was tested against the plant extract for minimal

inhibitory concentration (MIC) determination.

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Broth Dilution Method is the method used to determine the minimal inhibitory

concentration (MIC) of the plant extracts.

Anthurium andraeanum is the plant sample being evaluated in the study for its antifungal

and synergistic action with Hylocereus polyrhizus.

Hylocereus polyrhizus is the plant sample being evaluated in the study for its antifungal and

synergistic action with Anthurium andraeanum.

Itraconazole is an antifungal drug used as the positive control in this study.

Minimal inhibitory concentration (MIC) is the dependent variable of the study; it shows

the lowest concentration of the agent that inhibited the growth of the fungal strain.

Normal saline solution is a reagent used as the negative control in this study.

Phytochemical screening is the method used to determine the presence of secondary

metabolites from the plant samples.

Synergistic effect is a concept that describes how the interactions of two or more variables
that can produce a better result than its added individual outcome.
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Chapter II
METHODOLOGY

This chapter contains information concerning the research design of the study,

research locale, research procedures such as plant authentication, preparation of plant extract,

phytochemical screening procedures, preparation of inoculum, sensitivity testing by disc

diffusion method, determination of the minimal inhibitory concentrations (MICs) using broth

dilution method and the statistical analysis to be used by the researchers.

Research Design

This study will utilize experimental design which focuses on evaluating the antifungal

and synergistic activity of ethanolic extracts extracted from dragon fruit peel (Hylocereus

polyrhizus) and flamingo lily flower (Anthurium andraeanum). Experimental research design

is the primary approach to be used to investigate causal (cause/effect) relationships between

one variable and another (Key, 1997). The following tests are to be performed in the course

of the study: Phytochemical Screening, Sensitivity Testing by Disc Diffusion Method,

Minimal Inhibitory Concentration by broth dilution method and statistical analysis. Data to

be gathered from the laboratory procedures will be analyzed and interpreted for the

formulation of answers to the problems of the study.

Research Locale

The plant materials, red-fleshed dragon fruit, and flamingo lily flower were to be

collected from Maryland Dragonfruit Gardens, Tagum City and Baganihan, Marilog District,

Davao City, respectively. The Aspergillus fumigatus strain will be derived from ATCC®
 18

204305™ 01021P Kwik Stick pack. The experimental procedures will be conducted in the

laboratories of Mindanao Medical Foundation College, Inc., P. Villanueva St., Agdao, Davao

City. The chemicals, instruments, and equipment were to be provided by the institution for

the researchers to conduct the study.

Sampling Technique

The researchers will use a purposive sampling design for the collection of the plant

samples to be used for the extraction and evaluation of their activity against the fungal

sample. Specific plant parts will be used for the preparation of plant extracts, which are peels

of the red-fleshed dragon fruit and flowers of flamingo lily from spathes to spadix, and their

combined extracts. Positive control and negative control to be used in the study were

Itraconazole and distilled water, respectively. Aspergillus fumigatus species will be derived

from ATCC® 204305™ 01021P Kwik Stick pack to be inoculated and incubated, accessible

for sensitivity testing.

Research Instruments

The researchers will use needed laboratory apparatuses for the preparation and

phytochemical screening of plant samples, and sensitivity testing of Aspergillus fumigatus

against the plant extracts (H. polyrhizus and A. andraeanum), positive control (Itraconazole)

and negative control (Normal Saline Solution).

Plant Authentication

The Dragon fruit and Flamingo Lily plant samples will be authenticated by Mrs.

Orcheliza L. Paramo, MA, PhD, a botanist and faculty member of Davao City National High

School, F. Torres St., Davao City.

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Research Procedures:

Preparation of the Plant Material and Extract

The plant samples, the dragon fruit (Hylocereus polyrhizus) and flamingo lily flower

(Anthurium andraeanum), will be cut into small pieces and washed thoroughly with water.

Then, it will be thoroughly air dried at room temperature. The dried material will be

grounded using a Wiley mill and then treated using 80% ethyl alcohol completely

submerging the sample. The mixture will then be filtered through a cheesecloth and the

filtrate will be concentrated under a rotary evaporator. The exact volume of concentrated

extract will be measured and recorded in case the equivalent weight of the plant extract is

needed.

Phytochemical Screening

The plant extracts were to be screened for presence of alkaloids, steroids and

flavonoids responsible for its antimicrobial activity. The screening will be performed by

standard phytochemical test protocols described by Aguinaldo et al. (2005).

A. Screening for Alkaloids

Take the equivalent of 20g plant material from the stock plant extract prepared and

place in an evaporating dish; evaporate to a syrup consistency over a steam bath. Add about

0.5g sodium chloride; stir and filter. Wash the residue with enough 2M HCl to bring the

filtrate to a volume of about 5 mL;

a. Take 1 ml of the filtrate and test with 2-3 drops of Dragendorff’s reagent.

(positive result: orange precipitate)

b. Take another 1 ml of the filtrate and test with 2-3 drops of Wagner’s reagent

(positive result: red precipitate)

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c. Record the relative amount of precipitation observed as follows.

(+) slight turbidity

(++) definite turbidity

(+++) heavy precipitation

B. Screening for Steroids

Take an equivalent of 10 g plant material from the plant extract and evaporate this to

incipient dryness over a water bath. Cool to room temperature and defat with 6 mL hexane

and 3 mL water to remove the colored pigments. Heat the defatted aqueous layer over a water

bath to remove the residual hexane, then cool to room temperature. Divide the mixture into 3

portions for screening of steroids and flavonoids, and one as a control. To 1/3 portion of the

defatted aqueous layer, free from hexane, add 3 ml of ferric chloride, and stir. With the test

tube in an inclined position, add cautiously from a pipette, 1 m of concentration sulfuric acid

(CAUTION: very corrosive), letting the acid trickle down along the insides of the test tube.

Allow the mixture to stand upright and observe for any coloration at the interface of the acid

and the aqueous layers (positive result: a reddish-brown color, which may turn to blue or

purple, indicates the presence of 2 – deoxysugars).

C. Screening for Flavonoids

The test to be used detect the presence of flavonoids was Wilstatter “cyandin” test,

which determines compounds having the γ-benzopyrone nucleus. Dilute the other 1/3 portion

of the defatted aqueous layer with 3.3 ml of 80% ethyl alcohol and filter. Treat the filtrate

with 0.5 ml concentration hydrochloric acid (12M) and add 3 to 4 pieces of magnesium

turnings. Observe any color change within 10 minutes and compared with the control tube. If

definite coloration occurs dilute with an equal volume of water and add 1 ml of octyl alcohol.
 21

Shake well and allow to stand. Note the color in each layer (positive result: colors ranging

from orange to red, to crimson and magenta, and occasionally to green or blue may be

observed.

Preparation of the Inoculum

Streak the fungi test organism in Sabouraud glucose agar slant or plate culture.

Incubate it at 30 degree Celsius for 5 to 7 days until the spores and aerial hyphae have

luxuriantly developed, scrape off loopfuls of the spores and some hyphal fragments and

suspend the spores in isotonic saline containing 0.05% Tween 80 and dilute the spore

suspension with more saline to the desired concentration by monitoring with hemocytometer.

This suspension has a concentration of 10^6 spores/ml. And dilute 1.0mL of the above

suspension with a concentration of 10^6 spores/mL to a final volume of 200mL with

Sabouraud glucose broth and use this inoculum immediately in the determination of the MIC

of the plant extract against filamentous fungi.

Sensitivity Testing by Disc Diffusion Method (Singh et al, 2017)

10 microliters of the ethanolic extract of each plant extract was pipetted and

transferred to a sterile 6mm diameter Whatman filter paper. It was left to dry for 2 hours. The

solidified filter paper disc was placed on the test organism seeded plate and was incubated at

37° C for 24 hours. The zone of inhibition diameter in mm will be interpreted as: <10 mm

(inactive), 10 – 13 mm (partially active), 14-19 mm (active) and >19 (very active).

Preparation of Plant Extract for Assay

Take an equivalent of 40mg (0.40 g) of each crude ethanol plant extract in a

previously sterilized 50ml beaker and then add to the extract in the beaker 10ml of Mueller

Hinton broth. Tween 80 can be added to the broth with a concentration of 0.1% v/v (0.1ml

Tween 80 in 100ml broth added before autoclaving) to enhance solubility of the water
 22

immiscible components of the plant. Swirl the contents in the aluminum-capped beaker to

effect an even homogenization of the extract and the broth. Transfer the suspension into the

sterile regular-sized test tubes and vortex the contents for 15 seconds. In the absence of a

vortex mixer, roll the tube between both palms for 30 seconds. A 1ml potion of the

suspension/mixture contains 4mg or 4000 mcg of the plant extract. Do this with the

individual plant extracts (Hylocereus polyrhizus & (Anthurium andraeanum) and the

combined plant extracts (1:1).

Determination of Minimal Inhibitory Concentration (MIC)

Sterilize 14 screw capped test tubes (13mm x 100mm) and number each tube

accordingly. With a sterile 5ml sterile pipette, introduce 1ml of Mueller-Hinton broth (MHB)

into tubes #2 to #11. To tube #12, introduce 2ml of MHB. Pipette 1ml of the plant extract

prepared previously into tube #1 and #2 and then covers the tubes well. Shake gently or

vortex the contents of tube #2 for 5 seconds. Aseptically withdraw 1 ml from tube #2 and the

transfer this to tube #3. Shake or vortex tube #3. With another clean sterile 1ml pipette,

aseptically with 1ml from tube #3 and transfer to tube #4 then shake or vortex. Continue this

process until 1ml has been withdrawn from tube #9 and subsequently added to tube #10 and

shake or vortex the contents. Pipette off 1ml from tube #10 and discard this. Introduce 1ml

of the diluted bacterial/fungal inoculum previously prepared into tubes 1 to 11 and tube 13.

To tube #13, add 1ml of the antibiotic standard and to tube #14 add 1 ml of the distilled

water. With all the tubes tightly capped, gently shake or vortex the contents and incubate the

tubes at 35 degree Celsius for 18 to 24 hours. After incubation, examine the tubes for fungal

growth. This can be visible as turbidity in the tube or as whitish pellet at the bottom of the

tube. The tube with lowest concentration of plant extract at which NO GROWTH or turbidity

is observed is considered as the MIC of the plant extract against the test organism. (Note:

incubation period for filamentous fungi must be 30 degree Celsius or lower for 3 to 5 days).
 23

For reading and documentation, the minimal inhibitory concentration is usually

evaluated by purely visual examination of the tubes. The higher the value of the MIC for the

test organism, the more resistant is the organism to the plant extract. The lower the value of

the MIC, the more sensitive is the organism to the plant extract.
 24

Collection of Plant Material

Preparation of the Plant Material and Extract

Phytochemical Screening

Preparation of Inoculum

Sensitivity Testing by Disc Diffusion Method

Preparation of Plant Extract for Assay

Determination of Minimal Inhibitory Concentration (MIC)

H. polyrhizus H. polyrhizus and A. andraeanum A. andraeanum

Statistical Analysis

Interpretation and Conclusion

Recommendation

Figure 4. Research Flow.

 25

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