Analysis of Bioactive Compounds Piper crocatum as Inhibitors of Acetylcholinesterase In Silico and In Vitro

Abstract

Alzheimer’s disease, the leading cause of dementia in older adults, involves memory loss and cognitive decline, with β-amyloid plaques and neurofibrillary tangles (NFTs) as key features. Acetylcholinesterase (AChE), an enzyme that breaks down acetylcholine, plays a role in the formation of these plaques and tangles. AChE is a promising target for the development of small molecule inhibitors in Alzheimer’s disease (AD) treatment. Indonesia’s native red betel (Piper crocatum) contains bioactive compounds that inhibit AChE activity, as shown in previous research. This study aims to evaluate the AChE inhibitory potential of bioactive compounds from P. crocatum extracts (water, n-hexane and ethyl acetate) using an in silico approach (molecular docking) with 3 different docking software programs. Donepezil served as the reference compound, and the results were compared with in vitro AChE inhibition assays. The potential AChE inhibitors, based on molecular docking using PyRx, Autodock Vina and YASARA Structure, from each extract are SM05 (n-hexane extract), SM15 (water extract) and SM18 (ethyl acetate extract), with the most negative ΔGbind values, measuring −8.8, −9.2 and −11.2 kcal/mol (more negative than Donepezil’s ΔGbind values). SM15 and SM18 Compounds show promise, based on its ΔGbind values, interactions with AChE, favorable pharmacokinetic properties, bioavailability, bioactivity and toxicity positioning both compounds as strong candidates for AD therapy. Both compounds were docked to the AChE substrate binding pockets (6O4W), forming hydrogen bonds with His447 and Phe297 at the “gorge” active site, and hydrophobic interactions with key amino acids in the peripheral anionic site (PAS) and substrate-binding sites (Tyr124, Phe297 and Phe338).  This is consistent with in vitro assay results, which show that the ethyl acetate extract has strong inhibition, with an IC50 of 16.7908 ppm, while the water infusion extract yields a 26.621 % inhibition of AChE enzyme activity. In addition, the DIY extract exhibited the strongest AChE inhibitory activity with an IC50 of 40.799 ppm.

HIGHLIGHTS

• Piper crocatum (red betel leaf) shows potential as a natural acetylcholinesterase (AChE) inhibitor, with compounds SM05 (2-(3,4-Dimethoxyphenyl)-6-ethoxy-7-methoxy-1-naphthol), SM15 (Columbin) and SM18 (Flemiphilippinin A) identified as promising candidates through in silico molecular modeling, as indicated by the most negative binding free energy values generated through 3 molecular docking methods (Virtual Screening with PyRx, Autodock Vina and YASARA Structure), especially compound SM18 (Flemiphilippinin A) from ethyl acetate extract (−11.2 kcal/mol). This aligns with the in vitro assay results, which show that the ethyl acetate extract has the strong inhibition, with an IC₅₀ of 16.7908 ppm, as provided by Nurinsani et al. [10].
• The red betel leaves water extract from DIY exhibited the most potent acetylcholinesterase (AChE) inhibitory activity with the lowest IC₅₀ value (40.799 ppm), highlighting the significant inhibitory activity of the extract.
• Flemiphilippinin A (SM18) showed the strongest binding affinity to AChE (ΔGbind −2 kcal/mol), supported by favorable pharmacokinetic properties and bioavailability, as indicated by the fulfillment of Lipinski’s Rule of 5, ADME and bioactivity scores. It acts as a Nuclear receptor ligand (0.53), while Columbin (SM15) exhibits bioactivity as a Nuclear receptor ligand [0.66] and as an Enzyme inhibitor (0.57), suggesting that both compounds have potential as therapeutic candidates for Alzheimer’s disease (AD).
• Virtual screening and molecular docking revealed robust interactions of SM05, SM15 and SM18 with critical AChE active site residues, indicating their capability to inhibit AChE activity and potentially prevent AD progression.
• The 3 compounds from red betel leaf fractions are safe and have potential as oral drugs for Alzheimer’s therapy, with oral toxicity similar to donepezil (toxicity class IV, LD50 505 mg/kg) based on ProTox3 Prediction (with toxicity class IV for SM15, toxicity class V for SM05 and SM18).
• Pharmacokinetic evaluation confirmed the 3 compounds met Lipinski’s rule of 5 and showed high gastrointestinal absorption, with SM15 excelling in drug-likeness and bioactivity parameters critical for AD therapy.

GRAPHICAL ABSTRACT