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.

Antitrypanosomal activity of Crossopteryx febrifuga and phytochemical profiling using LC-MS/MS analysis coupled to molecular network and SIRIUS.

Crossopteryx febrifuga (Rubiaceae) is a plant widely used in traditional African medicine to treat tryapnosomiasis. The aim of our study was to evaluate the antitrypanosomal activity of C. febrifuga extracts and to identify the compounds responsible for this activity. We prepared 4 extracts by successive maceration of plant leaf powder in n-hexane, dichloromethane, methanol and water. The antitrypanosomal activity of the extracts was assessed on Trypanosoma brucei brucei and their selectivity on Leishmania mexicana mexicana and human non cancer WI38 fibroblast cells. The dichloromethane extract, the most antitrypanosomal (IC50 of 9.3 ± 0.8 µg/mL) was fractionated on an Open Column Chromatography to give 14 fractions. Fractions 6-9 were the most active with an IC50 ranging from 1.3 to 2.1 µg/mL. All fractions were analyzed by UPLC-ZenoTOFHRMS, followed by manual dereplication of metabolites detected in the most active fractions. Manual dereplication was aided by the Molecular Network (MN) and SIRIUS. Metabolic profiling of fractions 6-9 has enabled us to identify 33 compounds, most of which were reported for the first time in C. febrifuga. These include buddlenol C (6), naringenin (7), maslinic acid (22), corosolic acid or isomer (24), asperphenamate (25), hydroxyoctadecenoic acid (29), sumaresinolic acid or isomer I and II (30 and 31), glycyrrhetinic acid (32) and oleanolic acid (36). The bioactivity-based approach identified maslinic (22), corosolic (24), and oleanolic (36) acids as linked to the antitrypanosomal activity. The data obtained support the traditional use of C. febrifuga in the traditional treatment of trypanosomiasis. Further studies are required to verify the activity observed in vivo.

Ethnoveterinary survey of trypanocidal medicinal plants of the beninese pharmacopoeia in the management of bovine trypanosomosis in North Benin (West Africa).

Cattle breeding is of great socio-economic importance for Benin's cattle farmers in general and those of North Benin in particular. The objective of this study is to inventory the natural products of medicinal plants of the Beninese pharmacopoeia for the management of trypanosomes in cattle in North Benin. The methodology consisted of individual and semi-structured interviews with cattle farmers on the use of medicinal plants. A total of 360 cattle farmers were selected and interviewed in twelve villages in four municipalities (Tchaourou, N'dali, Bembèrèkè and Gogounou) in northern Benin. Different quantitative ethnobotanical indices were calculated to determine the level of use of plant species. The Relative Frequency of Citation (RFC), the Informant Consensus Factor (ICF = 0.918) and the Generic Coefficient (Rg = 1.04) were evaluated. The knowledge of medicinal plants was influenced by the level of education and the main activity of those who practiced animal husbandry. The results yielded 48 medicinal plants for veterinary use belonging to 46 genera and 28 families. The Leguminosae family (12.50%) was the most represented. The most cited plants with a RFC above 10% were K. senegalensis, P. africana, K. africana, M. inermis, S. latifolius, M. polyandra. The parts used were leaves (46.15%); barks (24.62%) and roots (15.38%). Decoction (53.23%), plundering (32.26%) and maceration (11.26%) were the main methods of preparation. The administration was mainly by oral route. The calculated indices show a high diversity of medicinal plants with trypanocidal properties in the control of cattle trypanosomosis in the Sudanese and Sudano-Guinean zones of northern Benin. Plant species with high citation and RFC values should be selected for comprehensive pharmacological and phytochemical research to validate this ethnomedical knowledge in the management of cattle trypanosomosis.

Typology of rabbit rearing systems in tropical areas: A case of Benin (West Africa).

The breeding of rabbits in tropical areas is confronted with enormous difficulties related to feeding and health, which limit the expansion and viability of these farms. The objective of this study is to carry out a typology of rabbit farms in tropical conditions to characterize the structure and function of this rearing in order to better understand their production results. A sample of 600 rabbit farms located throughout Benin was selected. The typology was carried out using multiple correspondence analysis (MCA) followed by Hierarchical Cluster Analysis (HCA) according to the Ward's aggregation algorithm and Euclidean distance, which identified five typological groups. Group 1 covered 45.7% of the farms and included small-scale production (fewer than 20 does) by professional breeders using traditional parasite control methods. Group 2 covered 3.3% of the rearing and includes a larger number of semi-extensive farms using self-produced feed. Group 3 (14.7%) was characterized by farms with fewer than 20 does that were semi-extensive and used more phytotherapy. In Group 4 (9.7% of the farms), the extensive mode was the most used, with veterinary medicine being the most used. Group 5 concentrated 26.7% of the farms and was marked by semi-extensive breeding. These farms did not record any cases of parasitosis. The typology carried out allowed a better understanding of the operating modes of these farms, their problems and the main limiting factors.

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.