Antiparasitic Activity and Potential Active Compounds from Azadirachta Indica Revealed by Affinity Ultrafiltration Chromatography-Mass Spectrometry with Acetylcholinesterase and Lactate Dehydrogenases

Azadirachta indica (A. indica) has traditionally been used to treat parasitic diseases, while its antiparasitic compounds and their mechanism remain ambiguous. Acetylcholinesterase (AChE) and lactate dehydrogenases (LDH) are the key target enzymes in the survival of parasites. At present, an integrated method combining affinity ultrafiltration-liquid chromatography-mass spectrometry (UF-LC-MS) with molecular docking was established to screen antiparasitic compounds targeting AChE and LDH from A. indica. The alamarBlue (R) and Ellman method, which revealed that ethyl acetate (EA) fraction exhibited powerful antitrypanosomic and AChE inhibitory activity, was submitted to UF-LC-MS and molecular docking. Afterward, d-(+)-catechin and (-)-epicatechin were considered potential AChE inhibitors with binding degree (BD) values of 38.04-38.43% and binding energy (BE) values from - 8.29 to - 7.76 kcal/mol. Carnosol was identified as a potential AChE and LDH dual inhibitor with BD values of 42.01% and 26.02%, and BE values of - 9.62 and - 9.06 kcal/mol, respectively. Finally, carnosol, d-(+)-catechin, and (-)-epicatechin displayed significant AChE inhibitory activity with the IC50 values of 40.66 +/- 3.10, 54.98 +/- 9.65, and 72.93 +/- 1.32 mu mol/L, respectively. Collectively, the integrated strategy is effective to screen and identify AChE and LDH inhibitors from A. indica, and d-(+)-catechin, (-)-epicatechin, and carnosol are expected to be natural lead compounds for the treatment of parasitic diseases. Besides using UF-LC-MS to screen for antiparasitic compounds from A. indica, this study offers a strategy for screening active components of medicinal plants.