ajbb54180-183

  American Journal of Biochemistry and Biotechnology 5 (4): 180-183, 2009ISSN 1553-3468© 2009 Science Publications   Corresponding Author: Syamsudin, Faculty of Pharmacy, Pancasila University, Jl., Srengseng Sawah, Jagakarsa,South Jakarta, Indonesia 180 Chemical Composition of Propolis from Different Regions inJava and their Cytotoxic Activity 1 Syamsudin, 2 Sudjaswadi Wiryowidagdo, 3 Partomuan Simanjuntak and 4 Wan Lelly Heffen 1 Department of Pharmacology, 2 Department of Biology of Pharmacy, Faculty of Pharmacy,Pancasila University, Jl., Srengseng Sawah, Jagakarsa, South Jakarta, Indonesia 3 Department of Natural Product, Research Center for Biotechnology, Indonesian Institute of Sciences,Jl. Raya Bogor km 46, West Java, Indonesia 4 Department of Research and Development, Dharmais Cancer Hospital, Jl. Letjen. S. Parman Kav 84-86,West- Jakarta, Indonesia Abstract:   Problem statement: Propolis samples from tropical zones, such as Java (Indonesia) with its   vast biodiversity, have become a subject of increasing scientific   and economic attention. Theassociation of the chemical composition of propolis from   different geographic regions with cytotoxicactivities lead   to the identification of active principles, a fundamental tool   to achieve standardization of this bee product. Approach: The purpose of this research was evaluate the quality of propoliscollected at different places in Java (Indonesia) based on cytotoxic activity. The ethanolic extracts of propolis from different areas in Java were tested for cytotoxicity against tumor cell lines (T47D, MCF-7, Hela, Myeloma and Vero) using MTT assay. Propolis samples were collected from Batang (CentralJava), Lawang (East Java) and Sukabumi (West Java). Results: The extract of propolis from Batangshowed the most potent activity of T47D and MCF-7 with IC 50 34.67±8.3 and 37.8±2.5 µg mL − 1 . Theextract of propolis from Sukabumi showed the most potent activity of Hela cell with IC 50 147.34±8.9.However, all propolis extract did not show activity of myeloma and Vero cells. Conclusion: Ethanolicsextract of three propolis samples from Batang (Central Java), Lawang (East Java) and Sukabumi (WestJava) regions in Java were investigated using GC-MS. From 37 compounds identified, 7 among of them were found for the first time in propolis. This indicated that the secondary metabolite extract of propolis from Batang (Central Java) obtained in the study has antiproliferative activity of breastcarcinoma cells (T47D and MCF-7). Key words: Propolis, Java, cytotoxicity activity, GC-MS, MTT assay INTRODUCTION Propolis is a resinous hive product collected byhoneybees (  Apis mellifera , L.) from various plantsources. Bees use it as a construction material, tosmooth internal walls of the hive and preserve it formextreme moisture and drought condition. They also useit to embalm dead invaders and in such way, preventthe development and spread of microbial diseases.Propolis is widely used in traditional medicine and isreported to have of pharmacological activities. Besidesits traditional uses, it has recently gained popularity as afood supplement in numerous countries, claimed toimprove health and prevent diseases [2,3] . Variousbiological activities, such as anticancer, antioxidant,antibiotic effects have been reported for propolis and itscomponents [10] .The composition of the propolis depends on theplace and time of collection. As a consequence, morethan 160 components have been identified so far,among which phenolic compounds, includingflavonoids, are major components [4] . Due to theincreasing interest in the characteristics of Indonesianpropolis, we undertook a study using samples collectedin different regions. We evaluated the quality of propolis collected at different places in Java (Indonesia)based on cytotoxic activity. MATERIALS AND METHODSPropolis: Propolis samples were collected from   different regions in Indonesia in April 2007 Sukabumi(West Java), Batang (Central Java) and Lawang (East   Am. J. Biochem. & Biotech., 5 (4): 180-183, 2009 181Java). Hand-collected propolis samples were keptdesiccated in the dark up to their processing. Extraction: One gram of each sample was cut intosmall pieces and extracted at room temperature with50 mL of 70% ethanol (twice after 24 h). The alcoholicextract was evaporated under vacuum at 50°C untildryness. The percentage of extracted matter was asfollows: Batang propolis 0.56% w/w, Lawang propolis0.43% w/w and Sukabumi propolis 0.67% w/w. GC-MS-MS analysis: The GC-MS-MS analysis wasperformed using temperature programming. Thecolumn oven temperature was initially held at 100°Cfor 2 min, then programmed to rise to 280°C at a rate of 20°C min − 1 and held for 5 min. The total run-time was16 min. The temperatures of the injector port and theinterface were set at 250 and 280°C, respectively. Thecarrier gas (helium) flow rate was 1.0 mL min − 1 . Theionization energy was set at 70 eV. The mass spectrawere collected by scanning from m/z 50-550 at 2 secintervals. Identification of compounds: Peaks were identifiedusing computer searches in commercial referencelibraries. Reference compounds were co-chromatographed when possible to confirm GCretention times. Cell culture and assay for cytotoxic activity: Thehuman cervix carcinoma (HeLa), Myeloma, MCF-7,T47D and Vero cell lines were supplied by CancerHospital Center Dharmais, Jakarta, Indonesia. Thehuman tumor cytotoxicities were determined followingprotocols established by the NCI [11] . Cellular viabilityin the presence and absence of experimental agents wasdetermined using the standard 3-(4,5-dimethylthiazol-2-yl)-2,5-dimethyltetrazolium bromide (MTT, Sigma, St.Louis, MO) assays as described previously. In brief,exponentially growing cells were harvested and a 50 íLsuspension containing 2500 cells was plated in 96-wellmicrotiter plates (Falcon, Becton Dickinson, NJ). After24 h of incubation at 37°C under 5% CO 2 to allow cellattachment, the cells were treated with varyingconcentrations of test specimens in their respectivemedium (100 íL) and incubated for 4 days under thesame conditions as above. After adding a solution of MTT for 4 h, the amount of formazan formed wasmeasured spectrophotometrically at 590 nm usingImmuno Mini NJ-2300 plate reader. The inhibitionConcentration, IC 50 which is the drug concentration thatinhibits 50% of cell lines growth, was determined fromthe graph. The experiment was conducted in triplicates. Data analysis: The absorbance of the formazansolution at 590 nm was determined on thespectrophotometer. IC 50 values (dose of drug thatproduces a 50% reduction in the absorbance comparedto the control) were determined from the dose-responsecytotoxic curves. Data obtained were analyzed usingthe 2-way Analysis Of Variance (ANOVA) andDuncan ′ s Mean Separation Test using the percentage of cell viability and IC 50 values as parameters. RESULTS Propolis is a resinous product collected by bees(  Apis mellifera ) from tree exudates, mainly resins of leaf buds mixed with beeswax to form a sealingmaterial in their honeycombs, smooth out the internalwalls and protect the entrance from intruders [5,7] .Propolis samples from tropical zones, such as Indonesiawith its vast biodiversity, have become a subject of increasing scientific and economic attention. Theassociation of the chemical composition of propolisfrom different geographic regions with biologicalactivities lead to the identification of active principles, afundamental tool to achieve standardization of this beeproduct [14] . Propolis cytotoxicity on cultures of humanand animal tumor cells, including breast carcinoma,melanoma, colon and renal carcinoma cell lines [6] .Table 2 lists cytotoxicity activity of propolis of cancer cell lines, i.e., T47D, MCF-7, Hela, Myelomaand Vero. The results indicated that the extract of propolis from Batang showed the most potent activityof T47D and MCF-7 with IC 50 34.67±8.3 and37.8±2.45 µg mL − 1 . The extract of propolis fromSukabumi showed the most potent activity of Hela cellwith IC 50 147.34±8.9. However, all propolis extract didnot show activity of Myeloma and Vero cells. Thisindicated that the secondary metabolite extract obtainedin the study has antiproliferative activity of breastcarcinoma cells (T47D and MCF-7). T47D and MCF-7cell containing Estrogen Receptor (ER) were used as amodel for studying the events associated with responseto chemotherapy of breast cancer cell [12] . DISCUSSION Although the components of propolis responsiblefor its cytotoxic   action were not investigated in thisresearch, Present study opens   a new perspective forfurther investigation. A large amount   of work dealingwith antitumor action of propolis and its components   indicates their promising usefulness and claims for newPropolis samples were collected from three regions inJava area (Indonesia), Batang (Central Java), Lawang   Am. J. Biochem. & Biotech., 5 (4): 180-183, 2009 182(East Java) and Sukabumi (West Java), each themcharacterized by some types of pre dominant trees orshrubs. The chemical composition of the three Javasamples was investigated using GC-MS after silylation.Each individual substance is presented in Table 1.The peak numbers in the Table 1 are givenaccording to the retention time only to the major peaks.The following compounds were identified for the firsttime in propolis: 1,3-bis(trimethylsilylloxy)-5,5-proylbenzene, 3,4-dimethylthioquinoline, 4-oxo-2-thioxo-3-thiazolidinepropionic acid, D-glucofuranuronic acid, dofuranuronic acid, patchouleneand 3-quinolinecarboxamine. Four new sugar and sugarderivates have been identified: arabinofuranose, D-ribose, threitol and arabinitol. Silanol is also newcompound which is found in propolis.   The variabilityof constituents of propolis in three samples showed thatthey were collected by the honeybee from differentplants depending on the geographic location [9] . Thespecific composition of plant population varies as afunction of elevation, soil type and moisture. The plantpopulation include many native and introduced speciesof flowering trees, shrubs and herbs [13] . The plant srcinof propolis determines its chemical diversity. Bee glue ′ schemical composition depends on the uniqueness of thelocal flora at the site of collection and thus on thegeographic and climatic characteristics of this site.This fact results in the strikling diversity of propolischemical composition, especially of propolissrcinating from tropical regions [1] .The three samples of propolis in the research haveone thing in common. All of them contain phenolicacids. Phenolics acids are considered to play a positiverole in the prevention of human diseases. As a populartraditional medicine, propolis is a rich source of caffeicacid. The active components of the propolis responsiblefor its clinical usages were extensively studied [8] . TheBatang sample showed the presence of groups of aromatics acids, terpenes and quinoline. The 3,4-dimethylthioquinoline and 3-quinolinecarboxamine wereidentified in the ethanolic extracts and also new inpropolis. The existence of quinoline as 3,4-dimethylthioquinoline and 3-quinolinecarboxamine inBatang Propolis indicated that there could be anotherplant source for propolis which need more investigation.In contrary to Batang sample, The Lawang samplecontains very high amounts of aromatic acids, wherethe dominant vegetation was native plant and C. petandra and  E. longam trees. The 4-oxo-2-thioxo-3-thiazolidinepropionic acid and glucofuranuronic acidwere found for the first time in propolis. It alsocontained terpene which is also new to propolis:patchoulene. Table 1: Chemical composition of ethanolic extracts of Batang,Lawang and Sukabumi propolis samples (percentage of total ion current, GC-MS)Compound Batang Lawang Sukabumi Aliphatic acids Hexadecanoic acid - - 0.72 Aromatic acids Benzoic acid - 0.41 - Phenylic acid 94.22 95.62 94.51Dofuranuronic acid - - 0.32 D-glucofuranuronic acid - 0.56 -1,3-bis(trimethylsilyloxy)-5,5- 2.40 - -propilbenzene 4-oxo-2-thioxo-3- - 0.79 - thiozolidinepropionic acidTerpenes Abietic acid1-Naphtalenemethanol 3.76 95.62 -Patchoulene - 0.27 -- 0.27 - Quinoline 3,4-dimethylthioquinoline3-quinolinecarboxamine 0.53 - - Sugars and alcoholic sugars D-mannopyranoseD-xylose 0.31 - -Arabinofuranose 0.24 - -D-ribose 0.23 - -D-galactose 0.15 - -D-mannitol - 0.51 -D-glucitol - - 1.62Erythritol - - 1.62Threitol 0.81 0.86 0.88Arabinitol - - 0.86- - 0.81 Others Glycerol 0.81 0.86 0.88Table 2: IC 50 values (µg mL − 1 ) of ethanolic extract of propolis fromdifferent area in Java in Hela, myeloma, Vero, MCF-7 andT47D cell lines. Values are mean of 3 separatedeterminations and errors represent the SD valuesIC 50 of ethanolic extract of propolis-------------------------------------------------------------------Batang Lawang SukabumiCell lines (Central Java) (East Java) (West Java)T47D 34.670±8.3 267.67±9.3 367.81±8.7Hela 589.31±4.1 674.35±3.8 147.34±8.9Myeloma >1000 >1000 >1000Vero >1000 >1000 >1000MCF-7 37.8±2.5 178.45±6.3 276.45±9.8 The Sukabumi propolis sample was gathered froma bee hive situated near the mountains at about 700 maltitude, temperature 20-26 ° C and the humidity in theranges of 85-96%. In this area there are relatively highnumber C. petandra and  H. brasiliensis trees. Itcontained very low amounts of aromatic acids. Fromthose samples, there is also silanol compound which isfound for the first time in propolis.   Silanol ispolysiloxanes which are the most common and one of the most important polymer chemistry. Probably this is   Am. J. Biochem. & Biotech., 5 (4): 180-183, 2009 183because the propolis sample was taken from the areawhere rubber plants grow.   The extract latex productfrom Hevea rubber plants is polydimathylsiloxaneelastomer. CONCLUSION From the results of the GC-MS-MS analysis weconclude that Ethanolics extract of three propolissamples from Batang (Central Java), Lawang (EastJava) and Sukabumi (West Java) regions in Java wereinvestigated using GC-MS. From 37 compoundsidentified, 7 among of them were found for the firsttime in propolis. A large amount of work dealing withcytotoxic activity of propolis and its claims for newinvestigations.   In order to explore propolis's potential asa cancer chemopreventive   and chemotherapeutic agent,our laboratory is investigating propolis   action in breastcarcinoma cells.   REFERENCES 1. Bankova, V., 2005. Recent trends and importantdevelopments in propolis research. Evid. BasedComplement. Alternat. Med., 2: 29-32. DOI:10:1093/ecam/nch059   2. Bankova, V.S., S.L. 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