Acacia farnesiana pods (plant: Fabaceae) possesses anti-parasitic compounds against Haemonchus contortus in female lambs.

Acacia farnesiana pods are rich in secondary metabolites and their biological activities have been recorded as antibacterial, antioxidant and anthelmintic. Previously, an in vitro bioguided study showed the important ovicidal and larvicidal effects of an organic fraction (EtOAc-F) from a hydroalcoholic extract of A. farnesiana pods against Haemonchus contortus. The present study aimed to assess the in vivo anthelmintic effect of EtOAc-F from A. farnesiana pods on the H. contortus faecal egg elimination in female lambs and on the infective larvae (L3) population reduction in coprocultures. The EtOAc-F was obtained from a hydroalcoholic extract from A. farnesiana pods through chromatographic procedures; additionally, some secondary compounds were identified using high-performance liquid chromatography (HPLC). Twenty-one 'Katahdin' crossbred female lambs ranging from three to four months of age, with body weights 21.9 ± 0.39 kg were used. Animals were orally infected with H. contortus (L3) by a single dose of 350 L3/kg BW. Three experimental groups (n = 7) were assigned as follows: 1) Control (untreated), 2) Albendazole, as a positive control (at 7.5 mg/kg BW, unique dose) and 3) EtOAc-F (at 100 mg/kg BW, once every third day, with three applications in total). Individual faecal samples were collected once a week for 5 weeks (at days 38, 45, 52, 59 and 66) post-treatment, to measure the faecal egg counts (FEC) and to obtain the H. contortus (L3) population from faecal cultures. The highest FEC reduction caused by EtOAc-F was 67.7%; meanwhile, albendazole showed a total FEC reduction after the second week post-treatment (day 45). On the other hand, the fraction caused an important reduction in the larval population in coprocultures (54.3-68.5%). The phytochemical analysis revealed the presence of galloyl derivatives and flavonoids as major compounds. The A. farnesiana pods could serve as a natural anthelmintic for the control of H. contortus, and perhaps for controlling other parasites of veterinary importance.

Galloyl flavonoids from Acacia farnesiana pods possess potent anthelmintic activity against Haemonchus contortus eggs and infective larvae.

ETHNOPHARMACOLOGY RELEVANCE: Acacia farnesiana (L.) Willd is a shrub legume used as condiment, medicinal plant and bioactive herbage. This species is used in traditional medicine of several countries to relieve the symptoms of gastrointestinal diseases, diarrhoea, stomach pain and typhoid as well as astringent, antidysenteric and anthelmintic. Some studies have shown that this plant displayed anthelmintic activity against several gastrointestinal nematode parasites of livestock, and also against parasites of human beings, such as malaria. AIM OF THE STUDY: This work describes the isolation and chemical identification of the anthelmintic compounds of Acacia farnesiana pods against eggs and infective larvae of the sheep parasitic nematode Haemonchus contortus. The bio-guided chemical fractioning of A. farnesiana pods using ethyl acetate against H. contortus eggs and infective larvae allowed for the identification of naringenin 7-O-(6″-galloylglucoside) (flavonol group) as the compound responsible for the anthelmintic activity against this important parasitic nematode. MATERIALS AND METHODS: Anthelmintic activity was assessed using the egg hatching inhibition assay (EHI) and mortality tests. A complete hydroalcoholic extract (HA-E) at 12.5-50 mg/mL, an aqueous fraction (Aq-F) at 3.12-25 mg/mL and an ethyl acetate fraction (EtOAc-F) at 3.12-25 mg/mL were analysed in the first selection phase. The purification of compounds through the chromatographic separation of the organic fraction resulted in nine less complex mixtures (C1F1, C1F2, C1F3, C1F4, C2F1, C2F2, C2F3, C2F4 and C2F5) that were assessed at 0.62-5 mg/mL concentrations. In addition, thiabendazole (0.6 mg/mL) and ivermectin (5 mg/mL) were used as positive controls. Likewise, distilled water and 4% methanol were used as negative controls. The bioactive compounds of EtOAc-F were obtained and characterised through chromatographic processes like open column chromatography, thin layer chromatography (TLC), high performance liquid chromatography (HPLC), ultra-performance liquid chromatography (UPLC) and gass chromatography-mass detection (GC-MS). Bioactive compounds were identified by spectroscopy (1H and 13C NMR) and mass spectrometric analysis. Additionally, the H. contortus eggs and infective larvae exposed to the bioactive compounds were observed through environmental scanning electron microscopy (ESEM) and confocal laser scanning microscopy (CLSM). Data were analysed based on a completely randomised design using ANOVA through a general linear model. RESULTS: The EtOAc-F fraction showed the highest ovicidal and larvicidal activities, at close to 100% at 3.12 and 6.25 mg/mL, respectively. The treatments C1F2, C1F3 and C2F3 displayed the main ovicidal activity (80-100%) at 2.5 mg/mL. The major compounds found in these sub-fractions were identified as galloyl derivatives and flavanones, including gallic acid (1), methyl gallate (2), ethyl gallate (3), naringin (4), naringenin 7-O-(4″, 6″-digalloylglucoside) (5), naringenin 7-O-(6″-galloylglucoside) (6) and naringenin (7). Likewise, the ESEM and CLSM images showed that the assessed compounds adhered to the eggshell and the external cuticle of the larvae. CONCLUSION: These results indicate that A. farnesiana pods contain nematocidal compounds and might be promising natural anthelmintic agents against H. contortus. This leguminous plant could be used as a nutraceutical food source for the control of gastrointestinal nematodes in small ruminants.