Isolation of Metabolites and Screening for Antimicrobial Activity of Calodendrum Capense Thunb. (Rutaceae).
Okwemba, Ronald Ing’ahu
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In tropical countries many indigenous communities maintain traditional medicine practices. Efforts have been made to identify and elucidate specific bioactive plant metabolites that exhibit significant physiological and pharmacological effects. Advances have been made to identify virulence factors in pathogenic microbes and their novel inhibitors from natural products, due to emerging resistance of pathogens to frequently used synthetic medicines. Phytochemical studies of plant species of the Rutaceae family have led to isolation of a variety of compounds; alkaloids, flavonoids, coumarins and terpenoids exhibiting significant bioactivities. In this study, extracts from the plant species of Calodendrum capense Thunb. (Rutaceae) were subjected to isolation and phytochemical screening of metabolites. The organic crude extracts of leaves, stem bark, fruit pericarp and seeds of this plant species were sequentially extracted starting with hexane, DCM, EtOAc and MeOH. Antimicrobial assay was carried out against bacterial strains; Staphylococcus aureus, Escherichia coli and Bacillus subtilis and antibiotic chloramphenicol was used as standard with zone of inhibition 24 mm. The stem bark hexane and EtOAc extracts showed MIC and zones of inhibitions of 2500 μg/ml (14 mm) and 1250 μg/ml (8 mm), respectively, leaves hexane extract showed MIC of 1250 μg/ml (7 mm) against Staphylococcus aureus. The hexane, DCM, EtOAc and MeOH leaves extracts showed MIC and zones of inhibitions of 1250 μg/ml (11 mm), 2500 μg/ml (10 mm), 2500 μg/ml (12 mm) and 2500 μg/ml (9 mm) respectively, while the fruit pericarp hexane, DCM, EtOAc and MeOH extracts showed 2500 μg/ml (11mm), 2500 μg/ml (9 mm), 1250 μg/ml (13 mm) and 2500 μg/ml 13 mm) respectively against Bacillus subtilis. Fungal strains used were; Candida albicans, Aspergillus niger, Trichophyton mentagrophytes and Penicillium citrinium and bioactivities were compared with those of antibiotic fluconazole as standard with zone of inhibition 28 mm. The leaves EtOAc and MeOH extracts exhibited MIC of 1250 μg/ml and 2500 μg/ml respectively, fruit pericarp extract showed MIC of 2500 μg/ml against Penicillium citrinium and were inactive against E. coli. Cytotocity assay of crude extracts of Calodendrum capense were done on vero cells 199 and E6. The hexane extract from the fruit pericarp was slightly toxic to vero cells 199 (IC50 81.48 μg/ml) and vero cell E6 (IC50 85.84 μg/ml) in comparision to standard podophylotoxin (IC50 65.13 μg/ml) which were toxic to vero cells. Isolation and purification of compounds was carried out using TLC, CC, VLC and preparative TLC. Structure elucidation was done by analysis of 1H NMR, 13C NMR, DEPT, HSQC, HMBC, COSY, NOESY, IR spectroscopic and mass spectrometric data. The isolated compounds were; 7-O-dimethylallyl demethylenedictamnine (148), confusameline (149), limonin (150), limonin diosphenol (151) and 5-methoxypsolaren (152). Compound (148), (149) and (152) were isolated for the first time from this plant. The compound (148) showed MIC and zone of inhibition of 2500 μg/ml (12 mm) against Penicillium citrinium. The compounds isolated can be used in vivo based on the antimicrobial and cytotoxicity assay results.