Phytochemical composition, safety and in vitro anthelmintic activity of Ziziphus mucronata barks extract against Haemonchus contortus

Abstract

Helminthic infections are some of the most prevalent diseases affecting livestock and human beings in developing countries. In Chad, nematode infections are considered as a major health problem for the animal and human population. The current drugs of choice against these nematodes are known to have some limitations including toxicity, high cost and emergence of anthelmintic resistance. In Chad, the barks of Ziziphus mucronata are commonly used for treatment of helminthosis, although its activity has not been authenticated. Therefore, the current study aimed at determining the phytochemicals, safety and anthelmintic activity of Z. mucronata barks against Haemonchus contortus. Z. mucronata barks were obtained from Deli in Chad and extraction undertaken at JKUAT, Kenya. Qualitative phytochemical screening for the aqueous and methanolic extracts of Z. mucronata barks was done using standard methods. The in vitro anthelmintic activity of the plant extracts against H. contortus was evaluated using the egg hatch assay, larval mortality assay and adult worm mortality assay according to World Association for the Advancement of Veterinary Parasitology (WAAVP) guidelines. In accordance with the Organization of Economic Co-operation and Development (OECD) guidelines, the safety of the plant extracts was assessed in mice by acute oral toxicity test in dosages ranging from 500 to 2000mg/kg body weight. Probit analysis, paired-samples t-test and ANOVA using Turkey post hoc tests were used to determine whether there were significant differences. Phytochemical screening of Z. mucronata barks extracts showed the presence of saponins, tannins, glycosides, flavonoids and steroids. Anthelmintic activity of methanolic extract in egg hatch assay showed a higher activity with IC50 value of 3.9 mg/ml as compared with aqueous extract which had an IC50 value of 14.7 mg/ml. Albendazole exhibited IC50 value of 0.05 mg/ml in egg hatch assay. There were significant (P<0.05) differences in egg hatch inhibition of extracts compared to Albendazole. Results of larval mortality assay showed that a xvii concentration of 2.6 mg/ml of methanolic extract and 7.5 mg/ml of aqueous extract inhibited 50% (EC50) of infective larvae of H. contortus. The EC50 value against larval mortality by Albendazole was 0.04 mg/ml. For larval mortality assay, the EC50 of the extracts were significantly (P<0.05) lower compared to that of Albendazole. However, for adult worms mortality assay, a maximum concentration of 2.4 mg/ml of methanolic extract, 5.0 mg/ml of aqueous extract and 0.04 mg/ml of Albendazole was required to induce mortality of 50% (EC50) of adult H. contortus worms after 24 hours post exposure. For adult worms assay, the EC50 of the extracts were significantly (P<0.05) lower compared to that of Albendazole. Acute oral toxicity test revealed that the mean body weight of mice treated with Z. mucronata extracts did not differ significantly (p>0.05) from those of control mice. The mean values of Alanine transaminase (ALT) (in mice treated with methanolic extracts) and blood urea nitrogen (BUN) (of treated mice with both extracts) did not differ significantly (P>0.05) with those of the normal mice. In conclusion, this study demonstrated that methanolic extracts of Z. mucronata barks at dosages used were not toxic when used in the mice model and thus could be safe in mammalian hosts. Further, both extracts had an anthelmintic activity against all stages of H. contortus and can thus be considered for further development as an anthelmintic. The study recommends that quantification of the phytochemicals, identification and isolation of the bioactive compounds responsible for anthelmintic activity and chronic toxicity of the extracts of Z. mucronata should be undertaken.