In-depth analysis of the acetylcholinesterase inhibitors of Ganoderma amboinense based receptor-ligand affinity coupled with complex chromatography

Abstract

Given the abundance and intricate nature of the chemical components found in natural food ingredients, functional food research has long grappled with two significant technical challenges: rapid and precise screening of active substances and targeted preparation. This study outlines a novel methodology to enhance the efficiency of preparing natural products, presenting new extraction processes, biological activity screening, and the preparation of acetylcholinesterase inhibitors from Ganoderma amboinense. acetylcholinesterase inhibitors were rapidly screened using ultrafiltration coupled with liquid chromatography and mass spectrometry. The inhibitory effect of active compounds on acetylcholinesterase was investigated via enzymatic reaction kinetics, with the mechanism also explored. Additionally, molecular docking and molecular dynamics simulations were used to verify the anti-alzheimer's disease activity of active compounds. Following this, the biological activity of ligands identified were separated and purified using complex chromatography (consecutive-CCC and semi-preparative HPLC). We used n-hexane: ethyl acetate: methanol: water (4.0:6.0:4.0:6.0, v/v/v/v), which optimized by a universal quasichemical functional group activity coefficient-based mathematical model and semi-preparative HPLC, 25–60% A (0–50 min), 60–100% A (50–60 min). Twelve active ingredients viz. ganoderic acids I, C2, G, B, K, A, D, F, J, ganoderenic acid A, deacetyl ganoderic acid F, and lucidenic acid F with the binding affinity for AChE were identified and isolated from natural foods. We enhance the efficiency and purity of active compounds preparation by incorporating enzyme inhibition assays and computerized virtual screening with compound chromatography. With this approach, we seamlessly incorporate fast discovery, screening, and focused preparation of active ingredients, as well as delving into the mechanisms of action of active monomers and proteases, and the molecular-level interactions between active ingredients and proteases. This approach effectively mitigates the occurrence of false positives attributable to a single technique, thereby providing valuable data to underpin future advances in enzyme inhibitor screening and isolation.