Presence of Garcinol in Garcinia binucao (Batuan) in vitro

A Research Paper
Presented to

The College of Medicine

University of St. La Salle
Bacolod City

In partial fulfilment
Of the requirements for
Physiology and Biochemistry

By:
Aguila, Juan Paolo
Ama, Andrick Cole
Balsomo, Esa Katrina
Garrucho, Geraldine
Gimony, Jn
Jochico, Jane Marie
Ladines, Gian Kevin

February 2015

ACKNOWLEDGEMENTS

The completion of this study would not have been possible without the support,
guidance and effort of those people who have contributed during the course of our work.
We would like to express our gratitude to Dr. Julian Raca, Jr. and Dr. Charibel Escandelor
for their counsel and scrutiny and especially for taking interest in our study.

We would also like to thank the Herbanext Laboratories Inc. in connection with
Donato C. Cruz Trading Corporation for lending some of their machines and equipments.
The kindly suggestions and guidance of Mr. Philip Cruz and Ms. Chris Ann Dublin has
helped us throughout our research period.

We give our deepest gratitude to Ms. Roselyn Usero and the rest of the Negros
Prawn Producers Cooperative Analytical & Diagnostic Laboratory staff for allowing us to
conduct our research in their facility. Their patience and cooperation are much appreciated.

To all our families and friends who have one way or another gave us their
encouragement and prayers and most especially to God for sustaining us throughout the
whole research period and providing the knowledge and wisdom to accomplish our
endeavours, thank you.

ABSTRACT

This is an experimental research which aimed to detect the presence of Garcinol in

Garcinia binucao fruit extract in vitro. Specifically, it aimed to identify the compound
Garcinol in G. binucao using HPLC (High Performance Liquid Chromatography) Method
using a food grade solvent as extractant, to determine the wavelength at which Garcinol of
G. binucao peaks and to determine the concentration of Garcinol in G.binucao. G. binucao
was oven-dried at 60OC, finely grinded, and 200g was weighed for soaking with 96% foodgrade ethanol, then filtered, and extracted using rotary evaporator at 60-65 OC for 2.5 hours.
10% aliquot was used to load HPLC and results revealed a peak at a retention time of 17.520.0 minutes indicating the presence of Garcinol in the extract. Since G. binucao is an
endemic fruit in Negros and Panay, not many studies have been made pertaining to it. With
the positive presence of the phenolic compound Garcinol in G. binucao, it is recommended
that further studies be made particularly on its Garcinol concentration and benefits.

TABLE OF CONTENTS

ACKNOWLEDGEMENT...i
ABSTRACT....ii

a.
b.
c.
d.
e.
f.
a.
b.
a.
b.
c.
d.
e.

I. INTRODUCTION....1-5
Background of the Study.....1
Objectives....2
Operational Framework...3
Significance of the Study.4
Scope and Limitations.4
Definition of terms......5
II. REVIEW OF RELATED LITERATURE ..6-9
Garcinol...6
Garcina sp. containing Garcinol.7
III. METHODOLOGY.10-11
Preparation of Samples .10
Acquisition of Reference Compound10
Preparation for Extraction.....10
Extraction Procedure.11
Analysis of Garcinol..11
IV. RESULTS AND DISCUSSION12-15
V. CONCLUSION AND RECOMMENDATION...16
REFERENCES.17-18
APPENDIX..19-26
CHAPTER I
INTRODUCTION
Background of the Study
Plants which include the genus Garcinia are basically identified because of their
fruit exemplified with a thick endocarp. There are about 610 scientific plant names of
species rank for the genus Garcinia and 418 of these have accepted species names[1] They
belong to the group of trees and shrubs distributed around tropical Asia, Africa and

Polynesia; thus they exist and thrive over a wide range of environments. These plants have
a rich source of bioactive molecules including xanthones, flavonoids, benzophenones,
lactones and phenolic acids.[2] Garcinia species are well documented producers of useful
organic compounds. Most notable of which is Garcinol. It has been isolated from several
Garcinia species; however, Garcinia indica is the most common source. This organic
compound is documented to possess anti-inflammatory, antioxidant, anticancer, and
antibacterial activity observed as a yellow oil or pigment [3]. It also possesses Anti-ulcer
and anti-HIV activity

[4]

. Some of its biological properties are only beginning to be

elucidated.
Garcina binucao, commonly known as batuan or binukaw, is endemic and widely
distributed in the Luzon and Visayas regions particularly in Panay and Negros. It is grown
as a home garden tree and its fruits maybe eaten raw or used for souring local dishes. Its
fruits are yellowish when mature, somewhat rounded, and 4 cm 8 or more in diameter. They
have a firm outer covering and contain a very acid pulp with several seeds.

Currently, there isnt much research on G. binucao although studies of related

Garcinia species on Garcinol and its biological implication are of particular interest due to
its biological activities. Moreover, G.binucao is native to Negros and Panay islands and
could easily be acquired for local studies.
General Objective:

To detect the presence of Garcinol in Garcinia binucao fruit extract in vitro.

Specific Objectives:
1.

To identify the compound Garcinol in G. binucao using HPLC (High Performance

Liquid Chromatography)Method using a food grade solvent as extractant.

2.

To determine the wavelength at which Garcinol of G. binucao peaks.

3.

To determine the concentration of Garcinol in G.binucao.

Operational Framework

Figure 1. Operational Framework

Figure 1 illustrates the flow of the study by stating the steps leading to the detection
of Garcinol in G. binucao.

Significance of the Study

The results of this study may be useful to the following:
1. To the promotion of Batuan not only as food, condiment or ingredient in local
dishes but also its biological effects.
2. To local researchers so they would patronize on doing research on a local fruits
and set about its medical implications.
3. To food industries and businesses that they could help encourage the use of
Batuan in local and national setting by manufacturing various products out of it.
4. To the Philippine Government so they could fund more projects or researches
regarding Batuan as a potential international export product.

Scopes and Limitations

The study only tackled on the presence of Garcinol in G. binucao whole fruit. It did
not individually identify the presence and concentration of Garcinol in the different parts of
the fruit (e.g exocarp). Also, the study only included those G. binucao which are already
available in the market to represent the usual cultivated crop in the locality.

Definition of terms:
1.

[5]

Chromatogram

a graph showing the quantity of a substance leaving a

chromatography column as a function of time

2. Endemic prevalent in or limited to a particular locality, region, or people
3. Gaussian Shape is a characteristic symmetric bell curve shape
4.[6]HPLC (High Performance Liquid Chromatography) a separation technique that
involves the injection of a small volume of liquid sample into a tube packed with tiny
particles
5. Negative Control - a specimen or sample known to lack a particular substance, which is
used as a standard for measuring a tested substances effect
6. Retention Time the time interval between sample injection and the maximum
emergence of the peak
7. Rotavap (Rotary Evaporator) - a device used in chemical laboratories for the efficient
and gentle removal of solvents from samples by evaporation
8. Sonication - the process of dispersing, disrupting, or inactivating biological materials,
such as viruses, by use of sound-wave energy
9. Food Grade Ethanol - used in food applications since FCC Grade ethanol (Food
Chemical Codex) adds heavy metal specification limits to the grades. This can be either of
fermentation or synthetic origin.
10. Exocarp - the outermost layer of the fruit wall

CHAPTER II
REVIEW OF RELATED LITERATURE

Garcinol
Garcinol is a polyisoprenylated benzophenone (PPB) derived from Garcinia sp. It is
a well-known anti-oxidant (i.e., emulsified garcinol suppressed superoxide anion
comparably to DL alpha-tocopherol), anti-carcinogen and also has anti-microbial
properties.[7] Garcinol is a yellowish pigment. [8]. In fact all Garcinia species have some
amount of garcinol.[15] There is ample data to suggest potent antioxidant properties of this
compound which have been used to explain most of its observed biological activities.
However, emerging evidence suggests that garcinol could be useful as an anti-cancer agent,
and it is increasingly being realized that garcinol is a pleiotropic agent capable of
modulating key regulatory cell flavouring. Garcinol is an example of a prenylated chalcone,
which has many beneficial effects in human health and disease.[9]
According to Padhye S, Ahmad A, Oswal N and Sarkar FH (2009) [4], results of their
research suggest that garcinol can play an important role in the treatment of gastric ulcers
caused by the hydroxyl radicals or chronic infection with Helicobacter pylori, which,
together with cells from gastric mucous membrane, produces hydroxyl radicals and
superoxide anions. Presently, treatment with Clarithromycin antibiotic is the therapy of
choice for treating H. pylori infection, which, however, suffers from side effects and
emergence of rapid resistance. They stated that Garcinol may be a viable alternative to
conventional antibiotics.

It is also mentioned in their study that Garcinol shows antibacterial activity against
Methicillin-resistant Staphylococcus aureus (MRSA) which is comparable to that of the
antibiotic Vancomycin (MIC 3-12 g/mL for garcinol Vs. 6 g/mL for Vancomycin). It
also inhibits topoisomerases I and II (IC50 = 43 and 55 g/mL respectively) at
concentrations comparable to that of Etoposide (IC50 = 70 g/mL for topoisomerases II).
However, they made clear that although this compound has been shown to exhibit
therapeutic activity against gram-positive and gram-negative cocci, mycobacteria and
fungi, it has been found to be inactive against gram-negative enteric bacilli, yeasts and
viruses.[4]

Garcinia sp containing Garcinol

Garcinia indica
Kokum, whose botanical name is Garcinia indica, is an ornamental fruit tree native
to India. Its tiny fruit turns from red to deep purple as it ripens, and is harvested and dried
in the spring. The dried rind of the fruit, used as a culinary and medicinal agent, is almost
black tinged with purple, has gnarly edges and is slightly sticky, according to Healing
Spices. It has a somewhat sour taste but a faint, slightly sweet aroma. Kokums major
active ingredient is garcinol, a substance that has antibacterial, antioxidant and antiinflammatory properties. [10]

Garcinia cambogia / Garcinia gummi-gutta

Brindle berry (scientific name Garcinia gummi-gutta) is a tropical plant belonging
to the Garcinia genus and is indigenous to Indonesia. Although called a berry, the fruit of
8

this plant resembles a small pumpkin and its color varies from green to light yellow.
Historically, Garcinia cambogia (brindle berry) has been employed for curing gastric
ulcers. A study published in 2002 suggests that this herb mainly acts by means of one of its
constituents called garcinol. It has been shown that garcinol is effective in lessening acidity
in the stomach as well as protecting the gastric mucosa.[11]

Garcinia mangostana L.
Mangosteen (Garcinia mangostana L.) is a tropical tree native to Southeast Asia and
native to the Malay Archipelago. It is believed that the Queen of England was so delighted
by the mangosteens unique taste and exquisite lavour that she named it Queen of Fruits.
The outer shell of the fruit, pericarp (rind), is firm (softens during riping), typically 4-6 cm
in diameter, and contains astringent phytochemicals which discourage infestation by
insects, fungi, plant viruses and bacteria. The same phytochemicals are pigments giving the
exocarp its characteristic purple color, including phenolics acids, also called phenols. These
pigments have antioxidant properties which afford the fruit further protection from
ultraviolet radiation and free radicals generated during photosynthesis. Isolation of exocarp
pigments has permitted their identity to be revealed as xanthones, mainly garcinol and
mangostin, which, as phenolics, make the exocarp highly astringent and inedible.[12]

Garcinia binucao
Binucao (locally known as batuan) is a tree reaching a height of about 25 m and a
diameter of 40 cm. Leaves are opposite, shiny and smooth. The newly emerged leaves often
exhibit a reddish color. The branches tend to be pendulous. The bark is black in color.
9

Flowers are small, red and borne in clusters. Fruits are yellowish when mature, somewhat
rounded, and 4 cm or more in diameter. They have a firm outer covering and contain a very
acid pulp with several seeds. Binucao is common and widely distributed throughout Luzon
and the Visayan Island.Ten hectares are planted for this crop at ECG Farm in Negros
Occidental.[13]

Identification of Garcinol
In studies conducted by Darji, Shetgiri and Dmello (2010) and Jones, Vezele,
Worthen, Isaac, and Smith (2014), they used High Power Liquid Chromatography (HPLC)
in singling out Garcinol from G. indica and G. kola respectively.
For G. indica, they studied its antioxidant and antihyperlipidemic activity. In order
to identify Garcinol using HPLC, they used a Qualisil BDS C18 Column with a flow rate of
1 ml/min. The retention time was 7.43 minutes. For the mobile phase, they used Methanol
and the detector was set at 280 nm.[16]
For G. kola where the researchers only analyzed the different extraction methods,
for HPLC, they used an Agilent Zorbax SB-C8 (4.6x250mm, 5 ) column. The mobile
phase was 80:20 Acetonitrile:Water (.1% Acetic Acid) and a flow rate of 1 mL/min.
Retention time was 20 minutes and at 200nm to 400nm.[17]

10

CHAPTER III
METHODOLOGY

Preparation of Samples
Three kilograms (3kg) of G. binucao fruits were bought at the Libertad Public
Market in Bacolod City which was harvested from Gihulngan farms in Negros Oriental.
The sample preparation was conducted in D.Z. Cruz Farms Laboratory in Barangay Taloc,
Bago City. The fresh whole fruits were washed with tap water and were cut into quarters
before it was oven dried at 60C for two days. After two days when the samples were
completely dried, these were subjected to size reduction into coarse grinds using a grinder.
After which the dried weight of the sample was obtained using a digital balance. The
coarsely grinded samples were then stored in a refrigerator at 4C.
Acquisition of Reference Compound
Ten milligrams (10mg) of pure Garcinol: sc-200891 standard was ordered from Axil
Scientific Pte Ltd in Singapore and was stored at 0OC at Negros Prawn Producers
Cooperative (NPPC) Analytical and Diagnostic Laboratory, Door 1&2, NOLKFI Bldg., 6 th
Street, Bacolod City.
Preparation for Extraction
The weighed dried sample was soaked in 96% food grade ethanol with 1:10 ratio. In
the course of the study, the researchers obtained a total of 455 grams of the dried sample.
Only 200 grams of the sample was used and was soaked in 2000 ml of the food grade

11

ethanol for 24 hours. The sample was then vacuum filtered using a #2 Whatmann filter
paper in Buchner Funnel.
Extraction Procedure
The researchers used the Rotary Evaporator (ROTAVAP) for general extraction of
organic compounds from the grinded dried fruit sample of G. binucao at NPPC. In this
procedure, the filtrate was subjected to ROTAVAP for 2.5 hours with a running temperature
of 60-64OC.
Analysis of Garcinol
After isolating the sample extract from the food grade ethanol, 40 ml of the sample
extract was obtained and 1 ml was used for analysis using High Performance Liquid
Chromatography (HPLC) Method (Shimadzu) UV-Vis. The 1 ml liquid extract was diluted
to 10 ml with 80% Acetonitrile. Due to a highly concentrated product, the dilution was
again repeated with a total proportion of the liquid extract and 80% Acetonitrile in 1:100
ratio. It was then sonicated for 10 minutes. After which, 3 mg of the Garcinol standard was
prepared with a single dilution of 10 ml using 80% Acetonitrile and was sonicated for 10
minutes. The diluted liquid extract and the Garcinol standard were then filtered using 0.45
um microfilter before it was injected in the HPLC. The column used was 150mm x 4.0mm
with a run time of 20 minutes. The flow rate was 1ml per minute and the oven temperature
was 40OC using 200 and 215 wavelengths respectively. [14]

12

CHAPTER IV
RESULTS AND DISCUSSION

GRAPH 1

Data 1 Garcinol Standard

13

Graph 1 shows the chromatogram of 3 mg Garcinol Standard in 215 nm wavelength at 20

minutes run time as represented by Data 1. The Garcinol peak was most prominent between
17.5 to 20 minutes unlike 0.0 to 2.5, 2.5 to 5.0, and 7.5 minutes retention time. Also due to
the Gaussian shape of the Garcinol peak at 17.5 to 20 minutes, this was made as the basis
for detection of Garcinol from the Sample Extract by the administering chemist.

GRAPH 2

Data 1 Garcinol Standard Data 2 Acetonitrile (Blank)

Graph 2 shows the results for reliability of the Garcinol Standard against the negative
control, Acetonitrile, which are represented by Data 1 and 2, respectively, in 215 nm
wavelengths at 20 minutes run time still. Although the chromatogram of Data 2 at 0.0 to 2.5
minutes showed small peaks, the administering chemist ruled out these as insignificant
findings due to asymmetry and absence of the Gaussian shape.

14

GRAPH 3

Data 1 Sample Extract

Data 2 Garcinol Standard

Initially done at 215 nm wavelengths at 20 minutes run time, the administering chemist was
not able to find a Garcinol peak between 17.5 to 20 minutes retention time from the Sample

15

Extract. Taking to account the concentration and viscosity of the Sample Extract, it was
diluted with a ratio of 1:100 and the wavelengths were reduced to 200 nm instead.
Graph 3 shows the Garcinol peak of the Sample Extract against the Garcinol Standard.
Regardless of the presence of peaks between 0.0 to 15.0 minutes run time from the Sample
Extract, they were ruled out as insignificant since they did not match the expected retention
time of 17.5 to 20.0 minutes. The administering chemist identified the peak between 17.5 to
20.0 minutes retention time was due to the presence of Garcinol because of its Gaussian
shape and emergence at the same time frame as the Garcinol Standard.
Results based on the chromatograms of the Sample Extract revealed positive for Garcinol
but in low concentration.

16

CHAPTER V
CONCLUSIONS AND RECOMMENDATIONS

Conclusions
Based on the results, the Garcinol of G. binucao showed its peak at the wavelength
of 200 nm through trial and error, and with the concentration of 7.5 ppm. It is therefore
concluded that there is presence of Garcinol in the endemic G. binucao of Negros and
Panay.

Recommendations
1. The researchers recommend that there would be a quantitative study on the Garcinol
content of Garcinia binucao plant parts eg. leaf, stem, roots.
2. It would be highly recommended to identify the specific parts of the fruit which contains
the highest Garcinol content.
3. Further studies on Garcinol in different maturation stages are also encouraged
4. This study can be recommended to serve as basis for future studies concerning Garcinol
in Garcinia species.
5. An in depth study on the uses and biochemical properties of Garcinol to expound on the
already known facts
6. Isolation and identification of other extractable compounds aside from Garcinol which
may also serve as breakthrough in health and medical practice.

17

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[1]

Garcinol. Retrieved from: http://www.theplantlist.org/browse/A/Clusiaceae/Garcinia/

[2]

K.N. Varalakshmi, C.G. Sangeetha, A.N. Shabeena, S.R. Sunitha and J. Vapika. 2010.

[3]

Garcinol. Retrieved from: http://www.drugs.com/npp/garcinol.html

[4]

Padhye S, Ahmad A, Oswal N and Sarkar FH. 2009. Emerging role of Garcinol, the

antioxidant chalcone from Garcinia indica Choisy and its synthetic analogs. Journal of
Hematology & Oncology 2009, 2:38 doi:10.1186/1756-8722-2-38.
[5]

R. E. Coronel, 2011. Important and Underutilized Edible Fruits of the Philippines. DA-

BAR

and

UPLBFI.

Manila,

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from:

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training.com/2013/12/27/how-to-read-a-chromatogram/
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http://polymer.ustc.edu.cn/xwxx_20/xw/201109/P020110906263097048536.pdf

[7]

Majeed, Muhammed, Hadmaev, and Vladimir. 2006. Bioavailable composition of natural

and synthetic HCA. Sabinsa Corporation. United States Patent: 7,063,861.

[8]

Atre A. 2011. Application of Garcinia indica as a Colorant and Antioxidant in Rice

Extrudates. Graduate School-New Brunswick Rutgers, The State University of New Jersey.
[9]

Shailendra R, Shailesh D, Shruti R, Rashi K, Deepti B, Ajit D., et al. 2013. Identification

of antibacterial component from extract of Garcinia indica fruit rinds using LC/MS/MS.
ASMS 2013 MP34-689. Shimadzu Corporation.
[10]

Perrin B. 2014. What Are the Health Benefits of Kokum? Retrieved from:

http://www.livestrong.com/article/448787-what-are-the-health-benefits-of-kokum/
[11]

Brindle

Berry.

http://www.herbs2000.com/herbs/herbs_brindle_berry.htm
18

Retrieved

from:

[12]

What is Mangosteen? Retrieved From: http://mangosteenhealthtree.com/

[13]

Florido HB and Cortiguerra FF. 2003. Lesser Known Edible Tree Species. Research

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[14]

S.N.P. Kumar, D.G.B. Gowda, K. Mantelingu, K.S. Rangappa, 2013. Development and

validation of a reversed-phase HPLC method for the analysis of garcinol and isogarcinol in
Garcinia indica. Journal of Pharmacy Research. Volume 7, Issue 1, Pages 103106
[15]

Ramachandran HD. 2014. Plant Profile, Phytochemistry and Pharmacology of Garcinia

indica: A Review. Int. J. Pharm. Sci. Rev.Res., 27(2), JulyAugust 2014; Article No.66,
Pages: 376-381.
[16]

Darji K.K., Shetgiri P., and Dmello P. M. 2010. Evaluation of Antioxidant and
Antihyperlipidemic Activity of Extract of Garcinia indica. IJPSR, 2010; Vol. 1 (12): 175181
[17]

Jones M.D., Vezele Y., Worthen D., Isaac G., and Smith N. 2014. A Study Comparing
UPC2-MS Analysis of Various Garcinia kola Extraction Procedures. Waters Corporation.

19