Evaluation of antimicrobial potential of the leaf and stem bark extracts of euclea crispa (thunb.) and its possible synergism with standard antibiotics

Abstract

This study assays for preliminary phytochemical screening of leaf and stem bark extract of Euclea crispa, determines functional groups present in each potent fraction partitioned from the extracts, evaluates intensive antimicrobial properties of the extracts, assesses membrane attack capability of the fractions and evaluates drug-drug interaction between the most active fractions and selected antibiotics. The phytochemical screening was determined following conventional approach while functional groups were determined using FT-IR analysis. Agar-well diffusion was used for sensitivity test, agar dilution and broth-micro dilution were employed to determine minimum bacteriostatic and bactericidal concentrations, while time-kill kinetics was evaluated at different concentrations over a period of 2 h. Impact of the extracts against cell membrane was assessed via scanning electron microscopy and determination of the amount of proteins and nucleotides leakages. Evaluation of drug-drug interaction was carried out using time-kill assay at different concentrations of drugs combination against multi-drug resistant isolates. Presence of tannins, saponins, flavonoids, cardiac glycosides, reducing sugars, steroids and absence of alkaloids were common to both extracts. Moreover, some functional groups viz; alkanes, alkenes, alkynes, alcohol, phenol, aldehyde, aromatics, sulfoxides, nitrile, amides and amines were also detected in the active fractions. The largest zone of inhibition (26±0.50 mm) was shown by ethyl acetate fraction of the leaf extract against Aeromonas hydrophila at 10 mg/ml. The lowest minimum inhibitory concentration (MIC) of 0.08 mg/ml is exhibited by the fractions partitioned into n-butanol, ethyl acetate and water against test bacterial isolates while the range of MIC against yeast is 0.31–1.25 mg/ml. Absolute mortality was achieved by n-butanol fraction against Bacillus pumilus and Klebsiella pneumoniae after 90 and 120 min respectively at 1×MIC and by n-hexane fraction at 2 × MIC. Ethyl acetate fraction achieved absolute mortality against both representative bacteria after 120 min at 2 × MIC in addition to Escherichia coli (1323) under similar condition. n-Hexane fraction achieve total mortality against Candida albicans after 120 min at 1 × MIC. Maximum zone of inhibition (22±0.58 mm) was observed for the fractions partitioned into n-butanol and ethyl acetate from the stem bark extract at 10 mg/ml. The lowest MIC for that of n-butanol (0.31 mg/ml) is against Enterococcus faecalis while the lowest for that of ethyl acetate, n-hexane and water is 0.63 mg/ml against a number of test isolates. After 120 min of contact time only ethyl acetate fraction is able to eliminate both Listeria sp. and Salmonella Typhimurium at 1×MIC. Maximum amount of proteins released by fractions of the leaf extracts from Bacillus pumilus (0.53±0.005 μg/ml) is by the fraction partitioned into water after 120 min of treatment at 2 × MIC while that from Klebsiella pneumoniae (0.57±0.001 μg/ml) is by n-butanol fraction and C. albicans (0.54±0.002 μg/ml) was by n-hexane fraction. Furthermore, maximum nucleotides leakage of 45.8±0.03 and 44.3±0.03 μg were obtained from Bacillus pumilus and C. albicans by n-hexane fraction at 2×MIC respectively while the maximum nucleotides leakage from Klebsiella pneumoniae is 40.7±0.06 μg by n-butanol fraction. On the other hand from the stem bark extracts, ethyl acetate fraction released maximum amount of proteins from Listeria sp. (0.625±0.004 μg/ml) and it was n-hexane fraction from S. Typhimurium (0.789±0.001 μg/ml) at 3 × MIC after 120 min. The maximum amount of nucleotides leakage (47.9±0.12 μg) was from Listeria sp. by the fraction partitioned into n-butanol at 3 × MIC after 120 min. Images of SEM reveal a level of structural damage in the membrane of test isolates which ultimately results in leakage of intracellular components. While determining possible synergism, out of 130 different combination tests between the leaf extract and antibiotics, 91.5% express synergy while 8.5% are indifferent. On the other hand, 88.5% of the same number of combination tests is synergistic between the stem bark extract and standard antibiotics with no record of antagonism in both cases. The extracts of E. crispa exhibit significant antimicrobial properties which in a way confirm the plant a good source of bioactive compounds with membrane-active components and as well may serve to enhance potency of the available standard antibiotics and equally provide alternative therapy in combating infectious diseases locally.