Activités des extraits de Crinum jagus [J. Thomps.] Dandy (Amaryllidacées) sur les diarrhées motrices, sécrétoires et à Shigella flexineri induites chez les rats

Abstract

Crinum jagus is a plant traditionally used in Africa and particularly in Cameroon to treat diarrhea. The present study was undertaken to evaluate the effects of the whole plant extracts on secretory diarrhea, motor diarrhea, and Shigella-induced diarrhea in rats. Crude extracts were prepared by maceration of Crinum jagus powder in water, ethanol or ethanol/water mixture. The plant‟s flavonoids, alkaloids and saponins fractions were also prepared using appropriate techniques. In primary screening assays, the extracts and fractions were tested for microbial susceptibility in vitro using disc diffusion method in Muller-Hinton agar medium, serial tube dilution technique (for determination of the minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) values, and in vivo in a castor oil–induced secretory diarrhea rat model. Cytotoxicity of the extracts and fractions was also assessed on monkey kidney epithelial cell cultures (LLC-MK2). Among the extracts and fractions, ethanol/water extract (EWCj) and the flavonoids fraction were found to be more effective on castor oil-induced diarrhea (P <0.01), with EWCj being the most effective on motor and secretory diarrhea, and on in vitro bacterial growth inhibition, and was therefore selected for further investigations of the antidiarrheal activity and safety. The EWCj antidiarrheal effect was evaluated on rats with normal intestine motility, carbachol-accelerated intestinal motility, serotonin-accelerated intestinal motility, or pretreated with different antagonists of receptors which modulate intestinal transit (naloxone, glibenclamide, and yohimbine) and with isosorbide dinitrite, a NO donor. Antisecretory effect of EWCj was assessed in castor oil and prostaglandin E2 –induced enteropooling in rats. Shigellosis was induced in rats by oral administration of 9 x 108 CFU of Shigella flexineri and treated twice daily with C. jagus hydro ethanolic extract for seven consecutive days, with ciprofloxacin serving as positive control. The latter animals were sacrificed and blood collected for determination of interleukins (IL-2, INF-γ), immunoglobulins (IgA, IgM), motilin and intestinal vasoactive peptide levels using ELISA method; and for blood cell count using hematological automate. The antioxidant potential of WECj was studied using its 1.1-Diphenyl-2-picrylhydrazyl (DPPH) free radical scavenging activity, ferrous ions chelating ability and nitric oxide radical inhibiting activity. Assessment of EWCj safety was done through acute toxicity in rats. Microbial sensitivity tests on WECj revealed CMB/MIC ratios of 6.0, 1.67, 6.0 and 3.0, on S. dysenteriae A1, E. coli, S. typhi, and S. aureus, respectively. The CMB/MIC ratios were 3.00 and 3.33 for flavonoids on S. aureus and on C. albicans, respectively. All extracts and fractions including WECj, did not show any significant toxicity or reduction in LLC-MK2 cell viability. Moreover, WECj LD50 in rats was >5000 mg/kg body weight, suggesting inocuity of the extract. Phytochemical studies of WECj revealed the presence of tannins, alkaloids, saponins, flavonoids, polyphenols, triterpenes, coumarins, anthocyanidins, sterols and polysaccharides. The EWCj significantly (P <0.01) inhibited the action of carbachol and serotonin on intestinal transit stimulation. Animal pretreatment with naloxone, glibenclamide or isosorbide dinitrite, showed significant reduction (P <0.01) of the spasmolytic activity of WECj whereas pretreatment with yohimbine did not affect the activity of the extract. Crinum jagus also inhibited (P <0.01) castor oil/prostaglandin E2-induced intestinal secretions. The extract also reduced (P <0.01) diarrheal stool emission, Shigella flexineri stool density, and lowered IL-2, INF-γ, IgA, IgM and motilin blood levels whereas it increased (P <0.01) the vasoactive intestinal peptide level. In diarrheal rats, C. jagus extract restored the decreasing white blood cells and hemoglobin and reduced the density of mucus-filled goblet cells. The extract exhibited DPPH free radical scavenging activity, ferrous ions chelating ability and nitric oxide radical inhibiting activity, which in part, could explain its antidiarrheal activity. From this work, it is clear that C. jagus inhibits bacterial growth, intestinalmotility and secretion, and could then be used for effective treatment of shigellosis, motor and secretory diarrhea.