Criblage antibactérien de six plantes médicinales du Cameroun vis-àvis des bactéries Gram Négatif multirésistantes : Identification des principes actifs de Acacia polyacantha (Euphorbiaceae) et leur mode d’action

Abstract

Bacterial infections, especially those caused by multidrug-resistant Gram-negative bacteria are a real public health problem around the world. Faced with this scourge, natural substances derived from medicinal plants could constitute an alternative to antibiotics, given the rich biodiversity of the plant world. The purpose of the present study is to evaluate the antibacterial activity of six medicinal plants from Cameroon (Acacia polyacantha, Alchornea cordifolia, Eremomastax speciosa, Laportea aestuans, Pennisetum purpureum and Spathodea campanulata), against multi-resistant Gram-negative bacteria as well as the identification of the active ingredients of Acacia polyacantha (Euphorbiaceae) and their mode of action. The crude extracts of the plants were obtained by maceration in methanol. Standard phytochemical methods enabled us to determine the main groups of secondary metabolites present in plant extracts. The microdilution method in liquid medium allowed us to determine the inhibition parameters including minimum inhibitory concentrations (MIC) and minimum bactericidal concentrations (CMB) on 15 Gram negative bacterial strains namely: Escherichia coli (ATCC8739; AG102; AG100ATet ; ATCC10536), Providencia stuartii (ATCC29916; NEA16), Pseudomonas aeruginosa (PA01; PA124), Klebsiella pneumoniae (ATCC11296; KP55; KP63) and Enterobacter aerogenes (ATCC13048; EA-CM64; EA27; EA289). At the end of antibacterial screening, the crude extract of Acacia polyacantha leaves which showed the best antibacterial activities on the strains was subjected to column chromatography. In addition, the effect of the most active constituent (3-O-[􀀁-galactopyranosyl-(1→4)-􀀁-Dgalactopyranosyl]-oleanolic acid: compound 8) was evaluated on the plasma membrane, ATPase pumps and bacterial growth kinetics on the P. stuatii ATCC29916 strain (because the crude extract of the leaves of A. polyacantha and its most active constituent simultaneously showed good antbacterial activity). The in vitro activity in the presence and absence of an efflux pump inhibitor, Phenylalanine Arginine-β-Naphthylamide (PAβN) was determined. Acute oral toxicity was assessed according to the limit test method described by OECD code 425. Phytochemical analysis showed the presence of polyphenols, tannins, triterpenes and sterols in all the extracts studied. The bioguided purification of the leaves of A. polyacanhta made it possible to isolate stigmasterol (1), 􀀁-amyrin (2), 3-O-􀀁-Dglucopyranosylstigmasterol (3), 3-O methyl-D-chiro-inositol (4), epicatechin (5), quercetin-3-O-glucoside (6), 3-O- [􀀁-D-xylopyranosyl-(1→4)-􀀁-D-galactopyranosyl] oleanolic acid (7) and 3-O-[􀀁-galactopyranosyl- (1→4)-􀀁-D-galactopyranosyl] oleanolic acid (8). The crude plant extracts tested individually had a broad spectrum of antibacterial activity on all the strains studied. The best antibacterial activities (MIC varying between 64 and 1024 􀀃g/ml) were obtained with: the extract of the leaves (73.3%) of A. polyacantha; extracts of the leaves (93.3%), bark (86.7%) and roots (80%) of Alchornea cordifolia as well as extracts of Laportea aestuans (86.7%) and of Pennisetum purpureum (66.7 %). The lowest MIC value (8μg/ml) was obtained with extracts of the leaves and bark of A. Polyacantha against E.aerogene ATCC13048 and P. Stuartii ATCC29916. The fractions of A. polyacantha (APFb and APFd) were active on 88.9% of the bacterial species tested. Compound 8 inhibited the growth of 88.9% of the bacteria tested. PAβN markedly enhanced the activity of A. polyacantha leaf extract (APF), fractions (APFb, APFd) and compound 8 on all bacteria tested. Synergistic effects were obtained when the leaf extract of A. polyacantha and compounds 7 and 8 were combined with erythromycin (ERY), gentamycin (GEN), ciprofloxacin (CIP) and norfloxacin (NOR). The extract of the leaves of A. polyacantha and compound 8 act on the bacterial kinetics of P. stuartii in the latency phase. These block the bacteria's proton pumps and destroy the plasma membrane. The LD50 of A. polyacantha leaf extract is greater than 5000 mg/kg. The present study provides in-depth information on the possible use of the plants studied, in particular A. polyacantha and its compounds to fight infections caused by multidrug-resistant Gram-negative bacteria.