ISOLATION AND CHARACTERISATION OF CYTOTOXIC COMPOUNDS FROM Caesalpiniabenthamiana(BAILL.) HEREND. & ZARUCCHI (LEGUMINOSAE) AND CombretumracemosumP. BEAUV. (COMBRETACEAE)

Abstract

The incidence of multi-drug resistant cancer cells and the adverse effects associated with available chemotherapy have necessitated the search for new drug candidates. Several traditional healers in Africa utilise plants to treat cancer, but few anticancer drug leads have been reported from African medicinal plants. This study was designed to isolate and characterise the cytotoxic constituents from selected plants used in Nigerian ethnomedicine. Ethnobotanical survey on plants used for the management of different cancers was conducted in three local government areas of Ekiti State, Nigeria. Nine plants selected from the survey were macerated in methanol. Preliminary cytotoxic activity of the plant extracts was assessed using in vitro 3-(4,5-Dimethylthiazolyl-2)-2,5-diphenyltetrazolium bromide (MTT) viability assay in human breast adenocarcinoma (MDA-MB-453) and normal kidney (HEK-293) cell lines with methotrexate as standard. Extracts of the most active plants, Caesalpiniabenthamiana (CB) leaf (FHI-110847) and Combretum racemosum(CR) root bark (FHI-109781) were successively partitioned into n-hexane and dichloromethane. The two fractions were subjected to MTT viability,cyquant direct cell proliferation and caspase-3/7 green detection assays using human breast (MCF-7), lung (A549) and prostate (PC3) carcinoma cells. Active fractions were subjected to column and preparative thin layer chromatographic separations to isolate bioactive compounds. Cytotoxic activity of isolated compounds was further investigated with MTT viability assay using human MCF-7, A549 and PC3 carcinoma cells. Structures of the isolated compounds were determined by spectroscopic techniques (infrared, mass spectrometry and nuclear magnetic resonance) and compared with literature data. The IC50 were determined by fitting the data to a sigmoid dose-response curve. One way ANOVA and Dunnett’s Multiple Comparison test was conducted at α0.05 Ethnobotanical survey revealed 57 plants utilised by respondents in the study area. Methanol extracts with the highest cytotoxic activity were CB (IC50 = 3.21±0.09 and 0.09±0.08 μg/mL) and CR (IC50 = 0.71±0.11 and 0.26±0.20 μg/mL) on MDA-MB-453 and HEK-293 cells, respectively, compared to methotrexate (0.94±0.30 and 0.02±0.13 μg/mL). The dichloromethane fractions of CB (IC50 = 8.45±0.11 – 27.43±0.17 μg/mL) andCR (IC50= 9.76±0.09 – 30.47±0.03 μg/mL) displayed the highest cytotoxic activity, respectively, compared to methotrexate (IC50 = 9.97±0.08 – 12.98±0.07 μg/mL) on tested cell lines. Cyquant direct cell proliferation assay showed significant reduction in live carcinoma cells, while caspase-3/7 green detection assay showed significant increase in number of dead carcinoma cells. Methyl gallate (simple phenol),benthamianoate and 2-methoxyacrylic acid (phenolic acids), benthamianin (alkaloid),benthamiacone and benthamianol (terpenoids) were isolated from CB, while racemonoate (phenolic acid) was isolated from CR. The CB terpenoid, benthamiacone exhibited the highest cytotoxic activity [(IC50 = 13.23±0.10 (R2=0.70) – 21.97±0.06 μg/mL (R2=0.83)]. Cytotoxic activity of benthamiacone was not significantly different from methotrexate (p>0.05). The cytotoxic activity displayed by benthamiacone isolated from Caesalpiniabenthamiana and racemonoate isolated from Combretumracemosum validated the folkloric use of the plants in cancer treatment. The two plants could serve as potential templates and leads for the development of new chemotherapeutic agents.