The use of H-NMR Metabolomics to Optimise the Extraction and Preliminary Identification of Anthelmintic Products from the Leaves of Lysiloma latisiliquum.

Introduction: Tannin-rich forages are recognised as an important alternative for the control of gastrointestinal nematodes in small ruminants. Lysiloma latisiliquum, a forage commonly consumed by goats and sheep, has shown anthelmintic activity when tested against Haemonchus contortus. However, to date, the metabolites responsible for the activity are not known.

Objective: To use H-NMR metabolomics in the extraction and identification of anthelmintic metabolites from L. latisiliquum.

Methodology: Eight different solvent systems were compared for the optimum extraction of anthelmintic metabolites from L. latisiliquum. H-NMR spectra of the tannin-free extracts were measured in methanol-d using trimethylsilylpropanoic acid (TSP) as internal standard. Extracts were also evaluated for their anthelmintic activity using the larval exsheathment inhibition assay against H. contortus. These data were correlated by multivariate analysis [principal component analysis (PCA) and orthogonal projections to latent structures discriminant analysis (OPLS-DA)] and analysed. To validate the results obtained after the OPLS-DA, a bioassay-guided isolation of bioactive metabolites was conducted.

Results: The PCA of the H-NMR data allowed the identification of hydrophilic solvents as those best suited for the extraction of anthelmintics from L. latisiliquum and indicated that the bioactive metabolites are high-polarity, glycosylated products. Similarly, OPLS-DA of the data enabled the detection of activity-related signals, assigned to the glycosylated metabolites quercitrin and arbutin obtained from the bioassay-guided purification of the extract.

Conclusion: The results of this investigation confirm metabolomics as a useful tool in the detection of bioactive metabolites in plants without previous phytochemical studies. Copyright © 2017 John Wiley & Sons, Ltd.