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

Abstract

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)-β-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.

Keywords: Caenorhabditis elegans; Haemonchus contortus; Terminalia leiocarpa; anthelminthic activity; ellagic acid; gallic acid; molecular networking.

Figure 1

Molecular network of fractions 1 to 9 of Terminalia leiocarpa leaves MeOH extract showing the major compounds in fractions 8 (green), 9 (red), and other fractions (blue). The numbers indicate the identification code of the compounds and asterisks show the compounds whose identification was confirmed by injection with the standard. (A) triterpenic derivatives, (B) O-glycosylated flavonoids, (C) ellagic acid derivatives, (D) fatty acids, (E) ellagic acid, (F) sugar, (G) tannin, (H) glycosylated galloylated flavonoids. Edge widths are proportional to the level of similarity (cosine score). The size of the nodes is proportional to the sum of quasi-molecular ion intensity of fractions 1 to 9. * compounds identified by standard comparison.

Figure 2

Base peak intensity (BPI) chromatogram of fraction 8 of the MeOH extract of Terminalia leiocarpa showing the main compounds identified. (5) 2,3-(S)-hexahydroxydiphenoyl-D-glucose or isomer; (8) gallic acid; (9) ellagic acid derivative; (18) quercetin-3-O-(6-O-galloyl)-β-D-galactopyranoside or isomer; (19) ellagic acid; (23) astragalin, and (29) rosamultin or isomer.

Figure 3

Base peak intensity (BPI) chromatogram of fraction 9 of the MeOH extract of Terminalia leiocarpa showing the main compounds identified. (3) Quinic acid; (8) gallic acid; (9) ellagic acid derivative; (18) quercetin-3-O-(6-O-galloyl)-β-D-galactopyranoside or isomer; (19) ellagic acid; (20) quercetin-3-O-glucuronide; (23) astragalin, and (29) rosamultin or isomer.

Figure 4

Molecular network of fractions of MeOH extract of Terminalia leiocarpa leaves showing compounds significantly positively (green nodes) or not significantly (yellow nodes) correlated with the anthelmintic activity on C. elegans. The numbers indicate the identification code of the compounds and asterisks show the compounds whose identification was confirmed by the injection of a standard. (A) triterpenic derivatives, (B) O-glycosylated flavonoids, (C) ellagic acid derivatives, (D) fatty acids, (E) ellagic acid, (F) sugar, (G) tannin, (H) glycosylated galloylated flavonoids. Edge widths are proportional to the level of similarity (cosine score). The size of the nodes is proportional to the sum of quasi-molecular ion intensity of fractions 8 and 9. * compounds identified by standard comparison.

Figure 5

Heatmap of viability inhibition rate (%) (minus that of the negative control) of young Caenorhabditis elegans adults treated with the compounds at different concentrations.