Isotonic, aqueous-based media as simple and suitable test media for short-term Haemonchus placei adult worm motility assay.

The solvating power of test media used in anthelmintic assays is critical to the validity of assay results, especially when evaluating plant extracts. High solutes in media lowers its solvating power, altering the range of concentrations available for investigation and assay performance. To identify simplified, well-tolerated media for adult Haemonchus placei with improved solvating power, we investigated the impact of varying solutions of pH (2.5-8.5), salinity (19-154 mM), and normal saline (NS) incorporating dissolution enhancers (acetone, propylene glycol, DMSO and Tween-80; 10-40% v/v) on the nematode over 3 h at room temperature. The performance of identified media, NS and 20% Tween-80 in NS, were evaluated by preparing sample extracts (acetone extract Sarcocephalus latifolius, AESL20&10; and chloroform extract Vernonia amygdalina, CEVA20&10) stock solutions (20 and 10 mg/mL) in them, assessed their apparent dissolution, and each highest stock solution that dissolves the extracts evaluated for anthelmintic activity against H. placei. We found isotonicity to be the critical-to-worm survival factor as H. placei survived 100% in pH solutions 3.5-8.5, and saline solutions 39-154 mM. The dissolution enhancers, at 40%, gave no survival. At 30% and 20%, only Tween-80 gave 92.5% and 100% survival, respectively. At 10%, Tween-80, acetone, DMSO and propylene glycol gave 100%, 100%, 87.5% and 0% survival, respectively. In 20% Tween-80 in NS, AESL20&10 and CEVA20&10 dissolved, furnishing wider concentration range (20-0 mg/mL); whereas only AESL10 dissolved in NS (narrower concentration range, 10-0 mg/mL). The LC50s (mg/mL) of 7.67 (AESL10, NS) and 7.48 (AESL20, Tween-80 in NS) were not significantly different (p > 0.05), while CEVA20 (Tween-80 in NS) gave 2.67. Our findings show that NS and 20% Tween-80 in NS, as isotonic, aqueous-based media, are suitable, and well-tolerated as test media for adult H. placei in a short-term motility assay. Up to 30% Tween-80 could be used to enhance dissolution where necessary.

Anthelmintic activity and non-cytotoxicity of phaeophorbide-a isolated from the leaf of Spondias mombin L.

ETHNOPHARMACOLOGICAL RELEVANCE: Helminthosis (worm infection) is a disease of grazing livestock, with significant economic implications. Increasing resistance to existing synthetic anthelmintics used to control helminthosis and the unwanted presence of residues of the anthelmintics reported in meat and dairy products present a serious global health challenge. These challenges have necessitated the development of novel anthelmintics that could combat drug resistance and exhibit better safety profiles. Spondias mombin L. (Anacardiaceae) is a plant that has been used traditionally as a worm expeller. AIM OF STUDY: The aim of the work reported herein was to isolate and characterise anthelmintic compound(s) from S. mombin leaf, establishing their bioactivity and safety profile. MATERIALS AND METHODS: Adult Haemonchus placei motility assay was used to assess anthelmintic bioactivity. Bioassay-guided chromatographic fractionation of acetone extract of S. mombin leaf was carried out on a silica gel stationary phase. The structure of the compound was elucidated using spectroscopy (1H and 13C NMR) and Liquid Chromatography-Mass Spectrometry (LC-ESI-MS). Screening to exclude potential cytotoxicity against mammalian cells (H460, Caco-2, MC3T3-E1) was done using alamar blue (AB) and CellTitreGlo (CTG) viability reagents. RESULTS: The acetone extract yielded an active fraction 8 (Ethyl acetate: methanol 90:10; anthelmintic LC50: 3.97 mg/mL), which yielded an active sub-fraction (Ethyl acetate: Methanol 95:5; anthelmintic LC50: 53.8 µg/mL), from which active compound 1 was isolated and identified as phaeophorbide-a (LC50: 23.0 µg/mL or 38.8 µM). The compound was not toxic below 200 µM but weakly cytotoxic at 200 µM. CONCLUSIONS: Phaeophorbide-a (1) isolated from S. mombin leaf extract and reported in the plant for the first time in this species demonstrated anthelmintic activity. No significant toxicity to mammalian cells was observed. It therefore represents a novel anthelmintic pharmacophore as a potential lead for the development of novel anthelmintics.

Bioactivity and cytotoxicity profiling of vincosamide and strictosamide, anthelmintic epimers from Sarcocephalus latifolius (Smith) Bruce leaf.

ETHNOPHARMACOLOGICAL RELEVANCE: The leaf of Sarcocephalus latifolius is known to be used traditionally by the Fulanis in Nigeria to deworm animals. As helminthosis remains a major constraint to profitable livestock production worldwide, a precarious situation aggravated by the advent of resistant parasites, the discovery of new anthelmintics is a priority, necessitating exploration of medicinal plants for their anthelmintic principles. AIM OF THE STUDY: To identify and characterise compounds with anthelmintic activity from the leaf of Sarcocephalus latifolius. MATERIALS AND METHODS: Powdered S. latifolius leaves were extracted by successive maceration with n-hexane, chloroform and acetone. The dried extracts were evaluated for anthelmintic activity against Haemonchus placei adult worms, and the most active extract was subjected to bioassay-guided chromatographic separations. The isolated compounds were evaluated for cytotoxicity against the mammalian HeLa and MC3T3-E1 cell lines, using alamar blue and CellTitreGloTM to quantify cell viability. LC50 values were computed from the in vitro anthelmintic activity data by fitting to a non-linear regression equation (variable slope). Isolated compounds were characterized using spectroscopic and mass spectrometric analyses. RESULTS: Anthelmintic activity LC50 values for n-hexane, chloroform and acetone extracts were 47.85, 35.76 and 5.72 (mg/mL), respectively. Chromatographic separation of acetone extract afforded two bioactive epimers, identified as vincosamide (LC50 14.7 mg/mL) and strictosamide (LC50 12.8 mg/mL). Cytotoxicity evaluation showed that, below 200 µg/mL (400 µM), neither compound was toxic to the HeLa or MC3T3-E1 cells. CONCLUSION: Vincosamide and strictosamide could serve as novel scaffolds for the development of anthelmintic derivatives with improved potency and helminth selectivity.