The Anticancer and Other Bioactvity Investigations on the Extract and Some Compounds of Croton Membranaceus [Euphorbiaceae]

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2007-08-15
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Cancer is still responsible for many deaths worldwide. However, cancer prevention is almost impossible due to its numerous aetiological factors. Therefore, the best solution for cancers remains early detection and cure or treatment. Meanwhile, the current treatment options available for cancer including; surgery, radiation therapy, bone marrow transplant and chemotherapy are not only extremely expensive and unaffordable especially for third world countries such as Ghana, but are plagued with treatment failures due to toxic, side effects, drug resistance and intolerance. As such the need to search for new, potent, effective, relatively safer, reliable and affordable remedies for the treatment and management of cancer is still paramount. Research into plants has increased in recent years as they have been a source of numerous bioactive compounds including very potent anticancer agents. In Ghana, several plants are used by herbalists and traditional healers for the treatment and/or management of various cancers generally called "kokram" (Twi). However, the efficacies of these plant products as anticancer agents are often ill defined. This research project therefore, sought to establish a scientific basis for the justification and validation or otherwise of the traditional uses of a selection of Ghanaian medicinal plants in the treatment and management of cancers, and to identify and select suitable candidate(s) for further anticancer investigations and other possible bioactivities including, antimalarial and antimicrobial effects. In this study, the methanolic extracts of ten plant species were evaluated for cytotoxicity and growth inhibitory activities against three human cancer cell lines, DLD-1, MCF-7 and Ml4, using the MTT assay. The extracts of Adenia lobata root, Clerodendrum capitatum leaves, Garcinia kola stem bark, Plumbago zeylanica leaves and Vernonia conferta root, showed relatively low cytotoxic activities (IC50 values above 68 ng/ml), while extracts of Ficus asperifolia leaves, Paullinia pinnata root and Thonningia sanguinea root exhibited moderate activity (IC50 values 40 - 52 jig/ml) against at least one of the three cell lines. Croton membranaceus root extract exhibited markedly higher cytotoxic activities, particularly against the DLD-1 and MCF-7 cells (IC50 = 16.0 and 17.4 Hg/ml, respectively), while Zanthoxylum xanthoxyloides bark extract was 2-3 fold more active against DLD-1 cells (IC50 = 16 fig/ml) than against the other cell lines. These results lent some support for the use of these species, especially C. membranaceus and Z xanthoxyloides in traditional medicines for the treatment of cancer and further work on these plants were deemed worthwhile. The bioassay guided fractionation of the methanolic extract of Croton membranaceus root (CMR) revealed that the cytotoxic activity resided mostly in the ethyl acetate fraction. Separation of this fraction using column chromatography resulted in the isolation of six compounds including; a novel furano-clerodane diterpenoid [ 12-oxo-15,16-epoxy- 3,13( 16), 14-clerodatrien-17,18-dioic acid dimethyl ester] for which was suggested the trivial name "crotomembranafuran" (1), the glutarimide alkaloid, [2,-7V-(2- methylbutanoyl)-A^-phenylethyl-glutarimide] commonly called julocrotine (2), p- sitosterol (3), p-sitosterol-3-D-glucoside (4), the labdane diterpenoid [laB&a- 8(17),13E-dien-6a, 15-di-O-glucopyranoside] commonly called gomojoside H (5) and DL-butane-1,2,3,4-tetraol (DL-threitol) (6). Apart from julocrotine (2), which has previously been obtained from this plant species, this is the first report of the isolation of compounds 1, 3, 4, 5 and 6 from Croton membranaceus. Three of the compounds, crotomembranafuran (1), p-sitosterol-3-D-glucoside (4) and DL-threitol (6) were active against human prostate cancer (PC-3) cells, with IC50 values of 4.1, 9.7 and 6.6 jig/ml, respectively, which indicated that the novel compound (1) was significantly the most active. In the antimalarial testing, (3-sitosterol (3), P-sitosterol-3-D-glucoside (4) and DL-threitol (6) exhibited little or no in vitro antiplasmodial activity on Plasmodium falciparum strain 3D7, with IC50 values above 100 fig/ml. However, crotomembranafuran (1), julocrotine (2) and gomojoside H (5) had weak activity giving IC50 values of 43.61, 44.62 and 46.11 jug/ml, respectively. In the antimicrobial investigations, gomojoside H (5) showed significant antimicrobial activity against Staphylococci aureus, Bacillus subtilis and Pseudomonas aeruginosa, giving minimum inhibitory concentrations (MICs) of less than 10 ng/ml. The other compounds had little or no antimicrobial activity with MICs above 200 fig/ml. The results of the study established that Croton membranaceus root has a number of bioactive compounds including labdane and clerodane type diterpenoids, glutarimide alkaloids and phytosterols. Whilst gomojoside H (5) is a potent antibacterial agent, crotomembranafuran (1) is a novel clerodane diterpenoid with significant cytotoxic (anticancer) properties especially on human prostate cancer (PC-3) cells, and its presence together with the previously known P-sitosterol-3-D-glucoside (4) and DL- threitol (6) could explain, at least in part, the success claimed for the use of the Croton membranaceus root extract in the treatment and management of prostate and other related cancers.
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A thesis submitted to the College of Health Sciences in fulfilment for the Degree of Doctor of Philosophy, 2007
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