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Plants have being used worldwide for the treatment of diseases. These are a source of bioactive compounds which may have healing properties. Medicinal plants may provide new therapeutic solutions in the form of extracts or compounds which may be active against pathogens (Kaikabo, 2009) and cancers.
Many anti-cancer agents have been isolated from various plant. Scientists are still attempting to explore the bioavailability of anti-cancerous compounds in unexplored plant species. Medicinal plants have proved to successfully aid in various ailments leading to mass screening for their therapeutic components. As at currently, the search for plants derived compounds rich in anticancer and antimicrobial properties has been high because of their medicinal importance and application in treatments of cancer and infections. Cancer has been adjudged as the current leading cause of death. It has been showed that approximately two-thirds of anticancer drugs approved worldwide up to 1994 were of plant origin .
Due to rapid occurrences of multiple drug resistant strains of pathogens to existing antimicrobial. There is the need to take bold approach and urgent search for new antimicrobials from medicinal plants. Many medicinal plants have been screened extensively for their antimicrobial potential worldwide [4-6]. Any substance that can either inhibit the growth or kill pathogenic organisms with relatively low toxic effects to host cells are considered good candidates as potential antimicrobial agents .
However, majority of chemically derived compounds used as antimicrobial and anticancer are not without toxic effects. Since plant-derived drugs may be less expensive and possess less toxicity than conventional therapeutic agents it search from the flora of Brunei would help reduce risks associated with toxicity.
There are abundance of Garcina species in the tropical forest of Brunei which has not be explored for their medicinal values. The species have being reported to contained useful bioactive compounds with antimicrobial activities (Kaikabo et al 2009, 2011; Cowan 2007), and possibly anticancer agents.
Therefore the aims of this project is to screen some Garcinia species from the forest of Brunei and characterized for their anticancer and antimicrobial activities.
2.0 RATIONALE AND AIMS
Due to the growing cases of cancer as the leading cause of death worldwide. Although some of the conventional drugs used in treatment are toxic. There is increasing emergence of multidrug resistance organisms that resist treatment with commonly used antimicrobial and makes therapeutic interventions unsuccessful. There is the need to explore and search for useful bioactive compounds from plants that could serve as a lead for the development of anticancer and antimicrobial drugs in future. Also, the study would provide baseline useful information on the medicinal relevance of Garcinia species from the tropical flora of Brunei.
3.1 Plant materials and identification
Garcinia species would be identified and collected from the tropical Forest of Brunei. The plant would be authenticated by a plant taxonomist, voucher specimen would be deposited at the herbarium of the Department of Biology University of Brunei, Darussalam.
Powdered plant material would be extracted with appropriated extraction solvent as described (Eloff, 2004). An aliquot of 3g of powdered plant material will be extracted in 30ml of acetone. Tubes will be vigorously shaken, the supernatant will be filtered through Whatmann No.1 filter paper into a pre-weighed glass vial and place under a stream of cool air to dry. After drying the weight of each extract would be determined. The extract would be kept at 4°C until used.
3.3 Bioassay guided isolation of bioactive compounds
3.3.1 Thin layer chromatography fingerprinting
In this step a 10mg/ml concentration of the plant extract would be prepared from the stock. An aliquot of 10μl would be loaded onto a 10 by 10 centimeter cut thin layer chromatography plate (TLC) (Silica gel 60 F254, Merck) from the edge above a distance of 1cm and a gap 0.4cm between extract spot the plate will be developed in a solvent with different mobile phase polarity as described (Kotze and Eloff, 2002).
3.3.2 Test microorganisms
A type culture collection (ATCC) of bacteria would be obtained and maintained for the subsequent studies.
The TLC plates from 3.3.1 would be evenly spread with an active culture of growing bacteria from 3.3.2 and incubated at 37°C for 24 hours under 100% relative humidity. After overnight incubation the plate is sprayed with an aqueous solution of 2mg/ml of p-idonitetrozolium violet (INT, Sigma). The clear zones against red background indicates inhibition of microbial growth by the bioactive compounds in the extract (Hamburguer and Cordell, 1987). Compounds from the clear zones can now be isolated purified and structurally elucidated.
3.3.3 Microdilution assay
This technique will be used to evaluate the inhibitory activity of the extract. A two-fold serial dilution micro plate method (Eloff, 1988) would be used to determine the minimum inhibitory concentration of the extracts.
3.4 Solvent-solvent Fractionation
After bulk extraction, about 20 grams of the extract will be subjected to solvent-solvent fractionation, according Suffness and Douros (1979). This involves 5 fractionation steps to afford fractions for subsequent testing and product isolation. The steps are:
Step 1: Chlroform-Water extraction to obtain chloroform fractions
Step 2: Water-Butanol extraction this would afford butanol fractions
Step 3: Hexane extraction would afford hexane and water-MeOH fractions
Step 4: Carbon tetrachloride afford carbon tetrachloride and water-MeOH fractions
Step 5: Chloroform extraction afford chloroform and water-MeOH fractions
3.5 TLC finger printing and bioautography of the eluted fractions
This will be performed as described (Kotze and Eloff, 2002);which has be highlighted in 3.3.1 above. And bioautography as described (Hamburguer and Cordell, 1987) and highlighted in subsection 3.3.3
3.6 Column chromatography of the fractions
A particular fraction that is active based on the TLC fingerprinting above 3.5 will be subjected to column chromatography using the mobile phase as in solvent-solvent fraction as described (Suffness and Douros, 1979). Any fraction that has an impurity would be cleaned by hexane or sephadex treatment.
3.7 Characterization NMR
Isolated fraction which is the target compound would be chemically characterized using Nuclear Magnetic Resonance (NMR) spectroscopy. This tool has been extensively used in drug discovery (Neeraj Umpumanyu et all 2007). The spectral data obtained will be used to identify and named the isolated compound. This would take into account various parameters (Prestgard, 2000).
3.8 In vitro Anticancer activity
Some cancer cell lines such as: MCF-7, A549, SGC-7901, HCT-8, HO-4980, Hela, HepG2, PC-3, LNCap, Vero, MDCK will be purchased from a reputable source. All the cancer cell lines will be maintained in Roswell Park Memorial Institute 1640 (RPMI 1640) medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin. Vero and MDCK will be maintained in DMEM medium supplemented with 10% fetal bovine serum and 100 U/mL penicillin and 100 μg/mL streptomycin. The cells will be kept at 37°C in a humidified atmosphere containing 5% CO2 until required.
3.9 Cytotoxicity assay
Inhibition of cell proliferation by the most active compound isolated will be measured by the MTT assay (Jing et al., 2010). Briefly, cells will be plated in 96-well culture plates (1-105cells/well) separately. After 24 h incubation, cells will be treated with isolated active fraction (0, 3.13, 6.25, 12.5, 25, 50 and 100μM, eight wells per concentration) for 72 h, MTT solution (5 mg/mL) will then be added to each well. After 4 h incubation, the formazan precipitate will be dissolved in 100μM Dimethyl sulfoxide, and then the absorbance will be measured in an ELISA reader at 570 nm. The cell viability ratio was calculated by the following formula: Inhibitory ratio (%) = (Optical density control (OD control) - Optical density control (OD treated)/ Optical density control (OD control) -100%). Therefore IC50 value is the concentration of active fraction inhibiting cancer cells growth by 50%.
4.0 EXPECTED BENEFITS OF THE OUTCOME OF THE STUDY
It is expected that at the end of the study possible antimicrobial and anticancer bioactive compounds will be isolated and characterized from Garcinia species in Brunei. The study would contribute to knowledge by filling the gap of information on the medicinal properties of these species. A possible novel compound may be isolated which would be a lead for the development of antimicrobial and anticancer drugs in future, these may be patented. The scholar would be awarded with a PhD degree and the scholarship will further enhanced the bilateral relationship of the two countries; the awardees and the recipients.