Neglected tropical diseases (NTDs) are a group of 20 diseases considered as the most devastating infections in developing countries. They comprise both chronic parasitic and bacterial infections, but the most common causative agents of NTDs are parasitic helminths. Holigarna caustica (Dennst.) Oken is used by the tribes of Northeast India for the treatment of intestinal problems. Therefore, the present study was undertaken to investigate the active principles of this plant responsible for its anthelmintic activity, using bioassay-guided fractionation. An ethanol extract of H.caustica fruit was fractionated on a silica gel column, followed by HPLC, while nematicidal activity was followed throughout on C.elegans as a model organism.
The nematicidal assay was carried out in a 96-well microplate (flat-bottom). Fresh L4 stage larvae were collected in M9 buffer and adjusted to approximately 5000 larvae/mL. The wells were filled with 19 ul of overnight cultured E. coli OP50 (O.D. 600=0.5) and subsequently 10ul of worm suspension was added to get ~45 L4 larvae per well. Subsequently, 1ul of test sample (stock solution or dilution thereof in DMSO at different concentrations) was added to each well; DMSO (1ul) was used in a separate well as a solvent control. The movement of worms in the 96-well plate was recorded for 20 h at 20ºC using a WMicrotracker device. The percentage of the average movement over 20 h in the presence of test samples with the extract, compared with the DMSO control (0.5%), was used to estimate the relative nematicidal activity. Levamisole (final concentration 50uM) was systematically used as a positive control. After 20 h of incubation, all wells were observed under a microscope to distinguish nematicidal from non-nematicidal activity.
Only fraction 3 (F-3), which elutes with ethyl acetate/hexane, 50:50, had a clear nematicidal effect. Thus, F-3 was further separated on HPLC to isolate the active compounds. The tentative identification by MS was confirmed by NMR as linoleic acid by comparison of its spectroscopic data. The study demonstrates that linoleic acid is the major compound responsible for the nematicidal properties from the ethanolic extracts of H.caustica fruit. This data suggests that fruits of this tree may be a good choice for developing an anthelmintic botanical preparation.
Biomolecules . 2020 Jul 14;10(7):E1043. doi: 10.3390/biom10071043.
Panda SK, Das R, Mai AH, De Borggraeve WM, Luyten W.