BIOASSAY-LED ISOLATION OF CYTOTOXIC COMPOUNDS FROM EXTRACTS OF AFRAMOMUM MELEGUETA (ROSCOE) K. SCHUM. SEEDS AND STROPHANTHUS HISPIDUS DC. WHOLE PLANT

Abstract

Cancer is the second leading cause of death globally. Several chemotherapeutic agents used for the treatment of cancer have many side effects. This necessitated a search for new potent chemotherapeutic agents with high selectivity in order to minimise the occurrence of debilitating side effects. This led to the ethnobotanical survey of plants used for treatment of cancer in Akinyele Local Government area of Oyo State. This study was aimed at evaluating the cytotoxicity of the selected plants, isolate and characterise their active constituents. Different morphological parts of six plants selected from the survey were macerated separately in redistilled methanol at room temperature (26-33 oC) and evaluated for preliminary in vitro cytotoxicity using Brine Shrimp Lethality (BSL) assay and MTT (3(4,5-dimethylthiazol-2-yl)-2diphenyltetrazoliumbromide) colorimetric assay against human carcinoma Rhabdomyosarcoma (RD), breast (AU565), cervix (Hep-2C) and African green monkey kidney epithelial (normal) cell lines. Cyclophosphamide and doxorubicin were used as standards. The two most active plants, Aframomum melegueta seeds (Ams) FHI 112374 and Strophanthus hispidus whole plant (Shw) FHI 112443 were selected based on their significant cytotoxic activities. The Methanolic extracts of Ams and Shw were successively partitioned into n-hexane, dichloromethane and ethyl acetate fractions and screened against cancer cell lines. The active fractions of Ams and Shw were then subjected to various chromatographic techniques (Vacuum Liquid Chromatography, column chromatography, HPLC) for isolation of bioactive compounds. Isolated compounds were characterised using spectroscopic techniques (UV, IR, and 1D and 2D NMR). Data were analysed using one-way ANOVA and Dunnet Multiple Comparison test at α0.05. The Methanolic extracts of Ams and Shw showed significant BSL activity at LC50 of 7.22±0.01 and 9.23±0.12 µg/mL, respectively. The Ams hexane fraction had the best cytotoxic activity with LC50 of 2.27±0.18 µg/mL against brine shrimp. Strophanthus hispidus whole plant (CC50 of 2.94±0.01; 1.66±0.03 µg/mL) and Ams (CC50 = 5.18±2.10 and 9.74±0.16 µg/mL) had good cytotoxic activity on RD and Hep -2C, respectively. The Ams hexane (CC50 of 0.98±0.1 µg/mL) showed significant cytotoxicity on RD cell, with a good selectivity of 98.97 compared to iii cyclophosphamide (32.32). The n-hexane and dichloromethane fractions of Shw showed cytotoxicity of 10.72±0.18 and 1.26±0.06 µg/mL on AU565 with selectivity of 1.31 and 1.92, respectively. The Ams hexane afforded compounds 6-shogaol (1), 6- paradol (2), 1-dehydrogingerdione (3), while Ams dichloromethane afforded zingerone (4), 6-gingerol (5), [6]-isodehydrogingerdione (6), (Z)-3-hydroxy-1-(4-hydroxy-3- methoxyphenyl) dec-3-en-5-one (7). The Shw hexane afforded Daucosterol (8), stigmasterol (9) and a novel compound; 2-(4-ethoxy-3-methoxyphenyl)-5-hydroxy 3,7,8-trimethoxy-4H-chromen-4-one (10), while Shw dichloromethane afforded pityriacitrin (11), ursolic acid (12), strophanthidin digitoxoside (13) and urs-12-en-28- oic acid, 2,3,23,-trihydroxy-(2β,3β,4α) (14). Compound 1 showed significant cytotoxicity (CC50 = 0.11±0.01 µg/mL) on RD with selectivity of 215.45, while ursolic acid (CC50 = 8.07 ± 0.02 µM) and strophanthidin-digitoxoside (CC50 = 11.42 ± 0.60 µM) had highest cytotoxicity on AU565 both with selectivity ˃100. Aframomum melegueta seeds and Strophanthus hispidus whole plant exhibited significant cytotoxic properties. Ursolic acid, strophanthidin-digitoxoside isolated from Strophanthus hispidus and 6-shogaol isolated from Aframomum melegueta could serve as leads in anticancer drug discovery.