Plants have being used worldwide for the intervention of diseases. These are a beginning of bioactive compounds which may hold mending belongingss. Medicative workss may supply new curative solutions in the signifier of infusions or compounds which may be active against pathogens ( Kaikabo, 2009 ) and malignant neoplastic diseases.
Many anti-cancer agents have been isolated from assorted works. Scientists are still trying to research the bioavailability of anti-cancerous compounds in undiscovered works species. Medicative workss have proved to successfully help in assorted complaints taking to mass testing for their curative constituents. As at presently, the hunt for workss derived compounds rich in anticancer and antimicrobic belongingss has been high because of their medicative importance and application in interventions of malignant neoplastic disease and infections. Cancer has been adjudged as the current prima cause of decease. It has been showed that about two-thirds of anticancer drugs approved worldwide up to 1994 were of works beginning [ 3 ] .
Due to rapid happenings of multiple drug immune strains of pathogens to bing antimicrobic. There is the demand to take bold attack and pressing hunt for new disinfectants from medicative workss. Many medicative workss have been screened extensively for their antimicrobic possible worldwide [ 4-6 ] . Any substance that can either suppress the growing or kill infective beings with comparatively low toxic effects to host cells are considered good campaigners as possible antimicrobic agents [ 1 ] .
However, bulk of chemically derived compounds used as antimicrobic and anticancer are non without toxic effects. Since plant-derived drugs may be less expensive and possess less toxicity than conventional curative agents it search from the vegetation of Brunei would assist cut down hazards associated with toxicity.
There are copiousness of Garcina species in the tropical wood of Brunei which has non be explored for their medicative values. The species have being reported to contained utile bioactive compounds with antimicrobic activities ( Kaikabo et al 2009, 2011 ; Cowan 2007 ) , and perchance antineoplastic agents.
Therefore the purposes of this undertaking is to test some Garcinia species from the wood of Brunei and characterized for their anticancer and antimicrobic activities.
2.0 RATIONALE AND AIMS
Due to the turning instances of malignant neoplastic disease as the taking cause of decease worldwide. Although some of the conventional drugs used in intervention are toxic. There is increasing outgrowth of multidrug opposition beings that resist intervention with normally used antimicrobic and makes curative intercessions unsuccessful. There is the demand to research and seek for utile bioactive compounds from workss that could function as a lead for the development of anticancer and antimicrobic drugs in hereafter. Besides, the survey would supply baseline utile information on the medicative relevancy of Garcinia species from the tropical vegetation of Brunei.
3.1 Plant stuffs and designation
Garcinia species would be identified and collected from the tropical Forest of Brunei. The works would be authenticated by a works taxonomer, voucher specimen would be deposited at the herbarium of the Department of Biology University of Brunei, Darussalam.
Powdered works stuff would be extracted with appropriated extraction dissolver as described ( Eloff, 2004 ) . An aliquot of 3g of powdery works stuff will be extracted in 30ml of propanone. Tubes will be smartly shaken, the supernatant will be filtered through Whatmann No.1 filter paper into a pre-weighed glass phial and topographic point under a watercourse of cool air to dry. After drying the weight of each infusion would be determined. The infusion would be kept at 4 & A ; deg ; C until used.
3.3 Bioassay guided isolation of bioactive compounds
3.3.1 Thin bed chromatography fingerprinting
In this measure a 10mg/ml concentration of the works infusion would be prepared from the stock. An aliquot of 10?l would be loaded onto a 10 by 10 centimetre cut thin bed chromatography home base ( TLC ) ( Silica gel 60 F254, Merck ) from the border above a distance of 1cm and a spread 0.4cm between extract topographic point the home base will be developed in a dissolver with different nomadic stage mutual opposition as described ( Kotze and Eloff, 2002 ) .
3.3.2 Test micro-organism
A type civilization aggregation ( ATCC ) of bacteriums would be obtained and maintained for the subsequent surveies.
The TLC plates from 3.3.1 would be equally spread with an active civilization of turning bacteriums from 3.3.2 and incubated at 37 & A ; deg ; C for 24 hours under 100 % comparative humidness. After nightlong incubation the home base is sprayed with an aqueous solution of 2mg/ml of p-idonitetrozolium violet ( INT, Sigma ) . The clear zones against ruddy background indicates suppression of microbic growing by the bioactive compounds in the infusion ( Hamburguer and Cordell, 1987 ) . Compounds from the clear zones can now be isolated purified and structurally elucidated.
3.3.3 Microdilution check
This technique will be used to measure the repressive activity of the infusion. A double consecutive dilution micro home base method ( Eloff, 1988 ) would be used to find the minimal repressive concentration of the infusions.
3.4 Solvent-solvent Fractionation
After bulk extraction, approximately 20 gms of the infusion will be subjected to solvent-solvent fractional process, harmonizing Suffness and Douros ( 1979 ) . This involves 5 fractional process stairss to afford fractions for subsequent testing and merchandise isolation. The stairss are:
Measure 1: Chlroform-Water extraction to obtain chloroform fractions
Measure 2: Water-Butanol extraction this would afford butyl alcohol fractions
Measure 3: Hexane extraction would afford hexane and water-MeOH fractions
Measure 4: Carbon tetrachloride afford C tetrachloride and water-MeOH fractions
Measure 5: Chloroform extraction afford trichloromethane 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 subdivision 3.3.3
3.6 Column chromatography of the fractions
A peculiar fraction that is active based on the TLC fingerprinting above 3.5 will be subjected to column chromatography utilizing the nomadic stage as in solvent-solvent fraction as described ( Suffness and Douros, 1979 ) . Any fraction that has an dross would be cleaned by hexane or sephadex intervention.
3.7 Characterization NMR
Isolated fraction which is the mark compound would be chemically characterized utilizing Nuclear Magnetic Resonance ( NMR ) spectrometry. This tool has been extensively used in drug find ( Neeraj Umpumanyu et all 2007 ) . The spectral informations obtained will be used to place and named the stray compound. This would take into history assorted parametric quantities ( Prestgard, 2000 ) .
3.8 In vitro Anticancer activity
Some malignant neoplastic disease 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 beginning. All the malignant neoplastic disease cell lines will be maintained in Roswell Park Memorial Institute 1640 ( RPMI 1640 ) medium supplemented with 10 % foetal bovine serum and 100 U/mL penicillin and 100 ?g/mL streptomycin. Vero and MDCK will be maintained in DMEM medium supplemented with 10 % foetal bovine serum and 100 U/mL penicillin and 100 ?g/mL streptomycin. The cells will be kept at 37 & A ; deg ; C in a humidified ambiance incorporating 5 % CO2 until required.
3.9 Cytotoxicity assay
Inhibition of cell proliferation by the most active compound isolated will be measured by the MTT check ( Jing et al. , 2010 ) . Briefly, cells will be plated in 96-well civilization home bases ( 1-105cells/well ) individually. After 24 h incubation, cells will be treated with stray active fraction ( 0, 3.13, 6.25, 12.5, 25, 50 and 100?M, eight Wellss per concentration ) for 72 H, MTT solution ( 5 mg/mL ) will so be added to each well. After 4 h incubation, the formazan precipitate will be dissolved in 100?M Dimethyl sulfoxide, and so the optical density will be measured in an ELISA reader at 570 nanometer. The cell viability ratio was calculated by the undermentioned expression: Inhibitory ratio ( % ) = ( Optical denseness control ( OD control ) – Optical denseness control ( OD treated ) / Optical denseness control ( OD control ) -100 % ) . Therefore IC50 value is the concentration of active fraction suppressing malignant neoplastic disease cells growing by 50 % .
4.0 EXPECTED BENEFITS OF THE OUTCOME OF THE STUDY
It is expected that at the terminal of the survey possible antimicrobic and antineoplastic bioactive compounds will be isolated and characterized from Garcinia species in Brunei. The survey would lend to knowledge by make fulling the spread of information on the medicative belongingss of these species. A possible novel compound may be isolated which would be a lead for the development of antimicrobic and antineoplastic drugs in future, these may be patented. The bookman would be awarded with a PhD grade and the scholarship will further heighten the bilateral relationship of the two states ; the awardees and the receivers.