Mode of action of 3-butylidene phthalide as a competent natural pesticide

In this study, the biological activities and mode of action of 3-butylidene phthalide (3-BPH) were studied. 3-BPH had a superior efficiency against microsclerotia of Macrophomina phaseolina compared to the commercial fungicide tricyclazole. The microsclerotia formation and pigmentation were inhibited at 100 mu g/mL. Moreover, the fungicide exhibited in silico affinity toward trihydroxy naphthalene reductase (3HNR). Both 3-BPH and tricyclazole showed congruence in the orientation and interaction within the 3HNR active site. 3-BPH displayed a strong interaction with SER-164, TYR-178, and TYR-223, with estimated binding energy and inhibition constant of -6.78 kcal mol(-1), and Ki = 12.6 mu M, respectively. Furthermore, it showed in vitro and in silico inhibitory activity against Drosophila melanogaster acetylcholinesterase in a concentration-dependent manner with IC50 = 730 mu g/mL. It also impaired Galleria mellonella phenol oxidase enzyme, which corresponds with the insect's immune system. Phytotoxicity of 3-BPH was evident against Lemna minor at 1000 mu g/mL; nevertheless, it was nontoxic at the concentrations inhibiting M. phaseolina microsclerotia and dark pigments suggest that it may be safe for use on other plants at low doses. Further assays are wanted to develop 3-BPH as a novel crop protection compound.