Presence of Garcinol in Garcinia binucao (Batuan) in vitro

A Research Paper Presented to

The College of MedicineUniversity of St. La SalleBacolod City

In partial fulfilmentOf the requirements forPhysiology and Biochemistry

By:Aguila, Juan PaoloAma, Andrick ColeBalsomo, Esa KatrinaGarrucho, Geraldine Gimony, JnJochico, Jane MarieLadines, 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% food-grade ethanol, then filtered, and extracted using rotary evaporator at 60-65OC for 2.5 hours. 10% aliquot was used to load HPLC and results revealed a peak at a retention time of 17.5- 20.0 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...iABSTRACT....ii

I. INTRODUCTION....1-5a. Background of the Study.....1b. Objectives....2c. Operational Framework...3d. Significance of the Study.4e. Scope and Limitations.4f. Definition of terms......5II. REVIEW OF RELATED LITERATURE ..6-9a. Garcinol...6b. Garcina sp. containing Garcinol.7III. METHODOLOGY.10-11a. Preparation of Samples .10b. Acquisition of Reference Compound10c. Preparation for Extraction.....10d. Extraction Procedure.11e. Analysis of Garcinol..11IV. RESULTS AND DISCUSSION12-15V. CONCLUSION AND RECOMMENDATION...16REFERENCES.17-18

APPENDIX..19-263

CHAPTER IINTRODUCTION

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. 1

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 StudyThe 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 LimitationsThe 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 time2. Endemic prevalent in or limited to a particular locality, region, or people3. Gaussian Shape is a characteristic symmetric bell curve shape4.[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 particles5. Negative Control - a specimen or sample known to lack a particular substance, which is used as a standard for measuring a tested substances effect6. Retention Time the time interval between sample injection and the maximum emergence of the peak7. Rotavap (Rotary Evaporator) - a device used in chemical laboratories for the efficient and gentle removal of solvents from samples by evaporation8. Sonication - the process of dispersing, disrupting, or inactivating biological materials, such as viruses, by use of sound-wave energy9. 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 wallCHAPTER IIREVIEW OF RELATED LITERATURE

GarcinolGarcinol 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 GarcinolGarcinia indicaKokum, 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 anti-inflammatory properties. [10]

Garcinia cambogia / Garcinia gummi-guttaBrindle 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 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 binucaoBinucao (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. 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 GarcinolIn 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]

CHAPTER IIIMETHODOLOGY

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 CompoundTen 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., 6th Street, Bacolod City.Preparation for ExtractionThe 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 ethanol for 24 hours. The sample was then vacuum filtered using a #2 Whatmann filter paper in Buchner Funnel. Extraction ProcedureThe 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]

CHAPTER IVRESULTS AND DISCUSSION

GRAPH 1 Data 1 Garcinol Standard 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.

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 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.

CHAPTER VCONCLUSIONS AND RECOMMENDATIONS

ConclusionsBased 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.

Recommendations1. 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.

REFERENCES

[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, Philippines. Retrieved from: http://lab-training.com/2013/12/27/how-to-read-a-chromatogram/[6] 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. Retrieved from: http://www.herbs2000.com/herbs/herbs_brindle_berry.htm[12] What is Mangosteen? Retrieved From: http://mangosteenhealthtree.com/[13] Florido HB and Cortiguerra FF. 2003. Lesser Known Edible Tree Species. Research Information Series on Ecosystems Volume 15 No. 3. Retrieved from: http://erdb.denr.gov.ph/publications/rise/r_v15n3.pdf[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): 175-181

[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.

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