NEUROPROTECTIVE POTENTIAL OF GC–MS CHARACTERIZED PHYTOCONSTITUENTS FROM ALSTONIA VENENATA LEAVES: INTEGRATED IN-SILICO AND IN-VITRO EVALUATION TARGETING THE PI3K/AKT SIGNALLING PATHWAY

Abstract

Alzheimer’s disease (AD) is a neurodegenerative disorder that advances gradually over a long period, where amyloid-beta aggregates cause oxidative stress-related disease conditions, neuroinflammation, dysfunction of mitochondria, and neuronal apoptosis. Current pharmacotherapies provide merely symptomatic relief and have been implemented with blood-brain barrier inhibitory effects as well as adverse side effects. Therefore, a novel need for multi-target neuroprotective natural agents is increasing. This paper sought to assess the neuroprotective capacity of Alstonia venenata leaf extract following an overall in-silico, in-vitro, and mechanistic methodology. The analysis of the ethanol extract was done using GC-MS to profile the phytochemicals. Swiss ADME was used to assess drug-likeness and blood-brain barrier permeability. The analyses included target prediction, network pharmacology, protein-protein interaction, Gene ontology and KEGG pathway. Molecular docking was done towards Akt1, GSK-3β, and β-amyloid. The neurotoxicity induced by SH-SY5Y cells by AlCl3 was evaluated by using cell viability through MTT and caspase-3 ELISA measures. Nine out of 27 identified phytoconstituents met the BBB permeability and Lipinski criteria. The network analysis found 355 overlapping AD targets with hub genes being AKT1, CASP3, GSK3B, and MAPK1. KEGG analysis also revealed the PI3K-Akt pathway to be the most prominent neuroprotective mechanism. Molecular docking demonstrated that β-carotene, sitosterol, squalene, and quinic acid have high affinities with Akt1 and GSK-3β. In-vitro response demonstrated that the cell viability increased with a dose up to 400 µg/mL, which provided the protection of nearly 95% and had a significant effect on caspase-3 activity. Alstonia venetata presents strong neuroprotective activity, which activates PI3K-Akt-GSK-3b-caspase signalling pathway, which justifies its application as a valuable anti-Alzheimer therapeutic target. The major bioactive compounds identified included β-sitosterol (C₂₉H₅₀O), squalene (C₃₀H₅₀), quinic acid (C₇H₁₂O₆), α-linolenic acid (C₁₈H₃₀O₂), and β-carotene (C₄₀H₅₆), which were further evaluated for drug-likeness, molecular docking, and biological activity.