In vitro and in silico studies of the larvicidal and anticholinesterase activities of berberine and piperine alkaloids on Rhipicephalus microplus

Rhipicephalus microplus is responsible for high economic losses in livestock and its control has become difficult due to the establishment of tick populations resistant to commercial acaricides. This study aimed to evaluate the in vitro larvicidal effect of the alkaloids berberine and piperine, and also to investigate their inhibitory mechanisms against the acetylcholinesterase enzyme. The effects of the alkaloids on larvae were observed through the immersion test at the following concentrations: 1.5; 3; 6; 12; 16 and 24 mM. Berberine and piperine presented larvicidal activity greater than 95 %, not differing from 100 % for the positive fipronil control (p > 0.05). Of the two alkaloids, piperine had a lower effective concentration (EC), with an EC50 of 6.04 mM. The acetylcholinesterase enzyme used in the study was obtained from R. microplus larvae (RmAChE) and the anticholinesterase activity was determined spectrophotometrically. The highest anticholinesterase activity, measured as inhibition concentration (IC), was observed for berberine (IC50 = 88.13 mu M), while piperine showed lower activity (IC50 > 200 mu M). Docking studies in RmAChE, followed by 10 ns molecular dynamics simulation, suggest that berberine stabilizes the RmAChE at lower Root-Mean-Square Deviation (RMSD) than Apo protein. Few hydrogen-bond interactions between berberine and RmAChE residues were balanced by hydrophobic and pi-type interactions. Berberine fills preferentially the peripheral anionic site (PAS), which correlates with its non-competitive mechanism. These results suggest that berberine and piperine alkaloids have an in vitro acaricidal action on R. microplus larvae, and the likely mechanism of action of berberine is related to RmAChE inhibition when accessing the PAS residues. These findings could help the study of new natural products that could inhibit RmAChE and aid in the development of new acaricides.

Automated summary

This study evaluated the larvicidal effect of berberine and piperine against R. microplus larvae and their inhibitory mechanisms against the acetylcholinesterase enzyme. The results showed strong larvicidal activity of both alkaloids, with berberine exhibiting better inhibition of the acetylcholinesterase enzyme. Docking studies suggested that berberine may stabilize RmAChE and fill the peripheral anionic site to exert its inhibitory effect.