Acetylcholinesterase inhibitors from Conocarpus lancifolius Engl. (Combretaceae)

Abstract Conocarpus lancifolius Engl. (Combretaceae) has several potential health-promoting effects, such as antidiabetic, antimicrobial, antioxidant, and cytotoxic effects. Phytochemical study of the ethyl acetate fraction of the leaf extract of this plant led to the isolation and identification of eight compounds viz., gallic acid (1), dihydromyricetin (2), myricetin (3), daucosterol (4), syringetin 3-O-β-D-glucopyranoside (5), quercetin 3-O-β-D-glucoside (6), gallocatechin (7), and (-)-epigallocatechin-3-O-gallate (8). Their acetylcholinesterase (AChE) in vitro and in silico inhibitory activities were evaluated. Daucosterol (4) showed the highest activity (IC50 0.316 μM) which was further validated by the superimposed docking orientation with the co-crystallized inhibitor, donepezil. Graphical Abstract


Acetylcholinesterase inhibition
Tacrine was the first AChE inhibitor approved by the FDA for the treatment of AD (Peterson and Graff-Radford 2022). However, its routine prescription was limited due to its hepatotoxic effect at the normal therapeutic dose. Safer AChE inhibitors, including donepezil and galantamine, were discovered later that are currently used to alleviate the symptoms of non-progressive AD (Grossberg 2003). Therefore, there is a necessity to discover new effective AChE inhibitors with no side effects. The isolated compounds from C. lancifolius were evaluated for their potential in vitro AChE inhibitory activity using donepezil as a reference standard. The results showed that donepezil showed greater inhibition with an IC 50 value of 0.057 lM. Regarding the isolated compounds, gallocatechin (7), daucosterol (4), and myricetin (3) showed better AChE inhibitory activity with IC 50 values of 0.309, 0.316, and 0.376 lM, respectively, compared to other isolated compounds. Further, myricetin showed a higher inhibition than dihydromyricetin indicating the importance of C-ring unsaturation for the activity. Moreover, the glycosides (5 and 6) showed lower AChE inhibitory activities (5.331 and 4.681 lM, respectively) compared to myricetin (3) and gallocatechin (7), indicating that flavonoid aglycones are more active as AChE inhibitors than glycosides.

Molecular docking interactions
AChE is a potential protein target for Alzheimer's disease (AD). The structure of AChE protein is characterized by the presence of a deep, long, and narrow gorge leading to the active site, which is coated by fourteen conserved aromatic amino acid residues (Dvir et al. 2010). The active site is hydrophobic comprising several distinguishable subsites: the esteratic subsite (catalytic triad: Ser203, His447, and Glu334), the oxyanion hole, the anionic subsite, the acyl binding pocket, and the peripheral anionic subsite (Dvir et al. 2010).
The top docking score (-8.9578 kcal/mol) was obtained for daucosterol (4). The orientation of (4) was very similar to that of the docked structure of the co-crystallized ligand, donepezil ( Figure S28). AChE-daucosterol complex was formed through H-bonding interaction with Gly122 in the oxyanion hole and pi-H stacking with Trp286 of the peripheral anionic subsite (Table S4 and Figure S27(d)). The presence of more sp 3 carbon atoms in the structure of (4) forms a flexible cyclic structure that fills the narrow-elongated gorge. The glucose moiety occupies the catalytic pocket and the sterol skeleton extended outwards to fill the peripheral pocket of 4EY7 causing its blockade resulting in inhibition of binding to the ACh substrate (Dvir et al. 2010). This result may explain the high in vitro AChE inhibitory activity of compound 4 (IC 50 À 0.316 lM).

Conclusions
This study described the isolation and characterization of eight known compounds from Conocarpus lancifolius. Out of them, daucosterol, myricetin, and gallocatechin showed remarkable in vitro and in silico AChE inhibitory activities.

Disclosure statement
No potential conflict of interest was reported by the author.

Funding
The author(s) reported there is no funding associated with the work featured in this article.