Ethnopharmacology, phytochemical composition and toxicity of medicinal plants used against livestock helminths in drylands
Medicinal plants are important natural resources in drylands for communities on which their livestock and other forms of livelihoods have been sustained based on traditional knowledge. However, lack of scientific basis of their biological activity has led to inadequate recognition of the value and acceptance of herbal medicine in livestock healthcare. The main goal of this study was to identify and document ethnopharmacological practices, determine efficacy, phytochemical composition and toxicity of medicinal plants used in treatment of livestock helminths by the agropastoral communities in Nakasongola district of Uganda. A two stage sampling ethno-pharmacological study was carried out with agro-pastoralists in Nakasongola district of Uganda. Participatory methods were used to establish a livestock disease inventory, ethno-diagnosis and medicinal plants used. Qualitative phytochemical screening was conducted on Zathoxylum chalybeum, Euphorbia heterophylla, Rhoicissus tridentata, Secamone africana and Vernonia amygdalina, using standard chemical analysis procedures and thin layer chromatography. In vitro anthelmintic activities on adult worm motility of 70% ethanol and water plants extracts were determined using five serial dilutions with positive and negative controls. Ascaris suum model was used. Acute oral toxicity of E. heterophylla ethanolic extract was conducted using single graded doses ranging from 1500 to 4000mg/kg body weight in mice. Histopathology was carried out using Meyer's hematoxylin and eosin stain. Generalised Linear Models were performed to determine treatment effects on worms and the extracts' median effective dose, in addition to determining the median lethal dose of E. heterophylla in mice. The analysis was conducted using graph pad prism version 5.01 computer programme. Trypanosomosis (20.9%), East coast fever (15.5%) and helminthosis (12.8%) were the most prevalent of the 25 diseases prevalent in the study area. Thirty seven plants species belonging to 28 genera and 24 families were reportedly used in treatments against helminthosis. The frequently used plant parts were leaves (54.1 %) and roots (29.7%). Water extraction (81.1 %) was the main method of preparation and oral drenching was the main route of administration. About 65% of the population used combination of traditional and conventional Veterinary medicine. Ethno-diagnosis compared well with veterinary medicine diagnosis. Women were more knowledgeable in disease diagnosis and herbal treatment than the equivalent proportion of men. The five plants studied contained; tannins, saponins, flavonoids, alkaloids, reducing sugars, coumarins derivatives, anthracenones, anthocyanins with Siafricana lacking saponins and reducing sugars. There was significant difference in the mean yields obtained with ethanol and water plants extracts (p < 0.05) in Z.chalybeum and R.tridentata. Thin layer chromatography characterization with antimony chloride and vanillin sprays indicated presence of polyphenols and sapogenins in all the plants. Chloroform: Methanol (9: 1, v/v) was found the best solvent for separation of compound in all the plants. The study also revealed plants' similarities in a number of compounds under different observation conditions. The highest concentration of the aqueous extracts of the z. chalybeum, E.heterophylla, R. tridentata, Siafricana and Viamygdalina at 48 hours, caused percent mean worm motility inhibition of 100%, 100%, 90%, 93.3% and 90% respectively while the ethanol extracts of the same plants inhibited the motility by 93%, 100%, 80.0%, 93.3% and 90% respectively in a dose-dependent response compared with negative control. The median effective doses of ethanolic extract of the plants in the same order were found to be 30.85mg/ml, 26.85mg/ml, 12.32mg/ml, 40.08mg/ml and 5.94mg/ml respectively. The median effective dose of aqueous extracts of Z. chalybeum, E. heterophylla, R. tridentata, S africana and Vamygdalina were 6.28 mg/ml, 4.60 mg/ml, 23.52mg/ml, 25.41 mg/ml and 13.70mg/ml respectively. The study also revealed the dosage adopted by the agro-pastoral community on all plants was lower than what would produce beneficial results. Irrespective of solvent used for extraction of bioactive ingredients, there was highly significant differences in the motility inhibition count that was dose-dependent in all dose levels' effect of all the plants species crude extracts when compared with negative control: Z.chalybeum (F (5,53) = 10.62, P = 0.001; R2 = 0.92); E.heterophylla (F (5,53) = 4.41, P= 0.003; R2 = 0.92); R.tridentata (F (5,53) = 4.14, P =0.005; R2 = 0.90); Siafricana (F (5.53) = 8.00, P = 0.001; R2 = 0.92) and Vamygdalina (F (5,53) = 14.01, P = 0.001; R2 = 0.93). There was a significant difference in motility inhibition by the different methods of extraction: Z. chalybeum (F(2, 53) = 323.80, p= 0.001); Eheterophylla (F (2,53) = 140.43, p= 0.001); R.tridentata (F (2,53) = 213.66, p= 0.001); S.africana (F (2.53) =183.26, p= 0.001); Vamygdalina (F (2, 53) =257.43, p=O.OOl). The potency of the plants extracts and albendazole also significantly differed (p< 0.001). Therefore, the efficacy of the plants could be attributed to the chemical compounds working individually or in combinations. The potency of the extract could be species specific that is enhanced by solvent used to extract the active ingredients. This study also revealed that E.heterophylla ethanolic extract induced varying toxicity signs in dose dependent manner in the mice with major histopathological lesions including hemorrhages, congestion, peri-vascular degeneration and necrosis in liver, brain, kidney and lung in the groups that received 2000 to 4000mg/kg body weight. There was also erosive hemorrhagic enteritis and ulceration in the stomach and intestines. The clinical manifestation of mice prior to death included excessive urination, circling, paralysis, excessive thirst, loss of appetite, loss of gait, tachypnea, dehydration and stupor. The 24 hour median lethal dose was 2831 mg/kg body weight and the 95% confidence interval of median lethal dose was 2490 to 3218 mg/kg body weight and R2 is 0.96 indicating E heterophylla is of low toxicity. The results therefore revealed the validity of agro-pastoral community claims of anthelmitic potential of the most used plants and support their use although under-dosing was unearthed. The phytochemical constituents of the five plants obtained indicate that there is a rationale in the use of these plants as anthelmitics by the Nakasongola agro-pastoralists, Acute oral toxicity study of Euphorbia heterophylla demonstrated the toxicity potential that may be associated with uncontrolled use of anthelmitic plants by the communities. There is therefore a need for structural elucidation to isolate and determine the structure of active compounds in these plants as well as the efficacy in vivo and long term effect of administration of the potent plants' extracts. More studies involving other species of animals need to be undertaken while the communities also need sensitization on appropriate dosage.