Fractionation Coupled to Molecular Networking: Towards Identification of Anthelmintic Molecules in Terminalia leiocarpa (DC.) Baill.

Terminalia leiocarpa is a medicinal plant widely used in ethnoveterinary medicine to treat digestive parasitosis whose extracts were shown to be active against gastrointestinal nematodes of domestic ruminants. The objective of our study was to identify compounds responsible for this activity. Column fractionation was performed, and the activity of the fractions was assessed in vitro on Haemonchus contortus and Caenorhabditis elegans as well as their cytotoxicity on WI38 fibroblasts. Two fractions were the most active on both nematode models and less cytotoxic. LC-MS/MS analysis and manual dereplication coupled to molecular networking allowed identification of the main compounds: ellagic acid and derivatives, gallic acid, astragalin, rutin, quinic acid, and fructose. Other potentially identified compounds such as shikimic acid, 2,3-(S)-hexahydroxydiphenoyl-D-glucose or an isomer, quercetin-3-O-(6-O-galloyl)-ß-D-galactopyranoside or an isomer, and a trihydroxylated triterpenoid bearing a sugar as rosamultin are reported in this plant for the first time. Evaluation of the anthelmintic activity of the available major compounds showed that ellagic and gallic acids were the most effective in inhibiting the viability of C. elegans. Their quantification in fractions 8 and 9 indicated the presence of about 8.6 and 7.1 µg/mg ellagic acid and about 9.6 and 2.0 µg/mg gallic acid respectively. These concentrations are not sufficient to justify the activity observed. Ellagic acid derivatives and other compounds that were found to be positively correlated with the anthelmintic activity of the fractions may have additive or synergistic effects when combined, but other unidentified compounds could also be implicated in the observed activity.

In vivo anticoccidial activity and immune response of Moringa oleifera and Vernonia amygdalina leaves against Eimeria magna and Eimeria media in rabbits.

Introduction: The aims of this study were to determine the immune response and the anticoccidial activity induced by Moringa oleifera and Vernonia amygdalina leaves in rabbits infected with Eimeria magna and Eimeria media. Methods: Thirty-five-day-old rabbits, free from coccidia, were infested with 2.103 oocysts of Eimeria magna and Eimeria media, then received the acetone extract of the leaves of Moringa oleifera and Vernonia amygdalina at different doses by oral gavage. Results and discussion: The inhibition of the excretion of oocysts was evaluated by the McMaster technique and the levels of cytokines (IL-4 and IL-12) and immunoglobulin IgG were assayed by the ELISA method. The in vivo efficacy on E. magna and E. media oocysts was 95.43% and 96.53% for Moringa oleifera and Vernonia amygdalina at 1000 mg/kg bw against 98% for the positive control. Interestingly the plant extracts increased the production of interleukin (IL) and immunoglobulins (Ig) compared to controls. Plasma IL-4 levels (pg/ml) in rabbits were 128.94 and 131.38; those of IL-12 (pg/ml) were 395.55 and 426.56, and then for those of IgG (µg/ml) were 14.70 and 13.94 respectively with the acetone extracts of Moringa oleifera and Vernonia amygdalina on D14 PT at 1000 mg/kg bw. This study indicates that Moringa oleifera and Vernonia amygdalina can be used as an alternative to synthetic anticoccidials. These plants could be used to increase the resistance of the immune system of rabbits to infestations of Eimeria species in rabbit farms.

Anthelmintic Activity, Cytotoxicity, and Phytochemical Screening of Plants Used to Treat Digestive Parasitosis of Small Ruminants in Benin (West Africa).

Medicinal plants continue to be used alone or in combination with veterinary drugs to treat animal ailments, especially in developing countries where livestock farmers often lack access to modern veterinary services and drugs. In addition, digestive parasitosis remain a major constraint for small ruminant livestock. The objective of this study was to screen the anthelmintic activity of the main plants used in the treatment of the digestive parasitosis of small ruminants in Benin. A total of 40 extracts were prepared using the successive maceration of 10 plants in four solvents of increasing polarity. The phytochemical screening of the plants was performed, and the anthelmintic activity of the extracts was evaluated on L3 larvae of Haemonchus contortus. The cytotoxicity of the 40 extracts was determined on WI38 noncancerous fibroblast cells using the MTT assay, and the total phenol content (TPC), total flavonoid content (TFC), and condensed tannin content (CTC) were quantified in the most effective extracts using colorimetric methods. The results show that the plants contained tannins, flavonoids, and triterpenoids which may, in part, justify their anthelmintic activities. All plants gave active extracts at the highest concentration tested (1200 µg/mL). Methanol (MeOH) extracts were, in general, more effective than the hexane (HEX), dichloromethane (DCM), and aqueous (H2O) ones in inhibiting larval migration, with the MeOH extracts of Terminalia leiocarpa, Adansonia digitata, and Momordica charantia being the most effective. Nevertheless, the MeOH extract of M. charantia was highly cytotoxic at the concentration of 100 µg/mL. The anthelmintic activity of M. charantia, Vitex doniana, and Caesalpinia bonduc was studied on H. contortus for the first time. These results provide scientific information that can be used for better valorization of the anthelmintic potential of the studied plants and to initiate the process of the identification of new anthelmintic molecules.

Ethnoveterinary knowledge of sheep and goat farmers in Benin (West Africa): effect of socioeconomic and environmental factors.

Sheep and goats are two of the main animal species raised in Benin and one of the main sources of income for people living in rural areas. Faced with the inaccessibility of synthetic veterinary products and their low purchasing power, the majority of breeders use ethnoveterinary practices to treat small ruminants diseases. The specific objectives of the current study were (1) to document the traditional knowledge regarding the disorder groups treated and the medicinal plants used in the health and zootechnical management of small ruminants in Benin and (2) to assess the effect of gender, ethnicity, agro-ecological zone and herd size associated with them. To achieve these objectives, an ethnoveterinary survey was conducted in different agro-ecological zones from September 2018 to February 2019. A questionnaire was administered to 506 breeders. The data were analyzed through calculation of the Fidelity Level (FL), Cultural Importance Index (CI) and Informant Consensus Factor (ICF). Ten disorder groups were treated by the people surveyed. These were mainly digestive disorders (D) and reproductive disorders (W), both presenting a ICF value of 0.8. A total of 101 medicinal plants belonging to 42 families and 90 genera were recorded. Spondias mombin, Zanthoxylum zanthoxyloides and Khaya senegalensis were the most important plants with CI values of 0.208, 0.125 and 0.121, respectively. Gender, ethnicity, agro-ecological zone and flocks size were the socioeconomic and environmental factors that significantly influenced the level of ethnoveterinary knowledge. Chemical and biological analysis are needed on less studied plants such as, Striga hermonthica, Crossopteryx febrifuga, Elaeis guineensis and Momordica charantia.

In vitro effects of tannin and extracts of Bridelia ferruginea and Mitragyna inermis on the exsheathment of infective larvae of Haemonchuscontortus.

Bridelia ferruginea (Euphorbiaceae) and Mitragyna inermis (Rubiaceae) are two plants of the beninese pharmacopeia used in vivo for the control of gastrointestinal nematodes (GINs) in small ruminants. The objective of the present study is to explore the mechanism of bioactive compounds involved in the action of these two plants on the third-stage infective larvae (L3s) of Haemonchus contortus. Thus, sheathed L3s of H. contortus were incubated with acetone extracts of B. ferruginea and M. inermis at concentrations of 0, 150, 300, 600 and 1200 µg/mL for 3 h at 25°C. The L3s were then washed and artificially submitted to exsheathment in the presence of sodium hypochlorite solution. The role of tannins was verified by adding a tannin inhibitor, polyvinyl polypyrrolidone (PVPP), to the acetone extracts of these two plants for 2 h at 25°C. Acetone extracts from B. ferruginea and M. inermis inhibited the exsheathment of H. contortus larvae (p < 0.001) and this inhibitory effect was dose-dependent for M. inermis at the concentrations tested. Treatment of B. ferruginea and M. inermis extracts with PVPP was associated with a partial restoration of the exsheathment kinetics of H. contortus larvae (p < 0.001), confirming the predominant role of tannins but also the residual role of other secondary metabolites. These in vitro results suggest that these plants are endowed with anthelmintic (AHs) properties and therefore likely to be used as alternatives to synthetic molecules.