Synergistic neuroprotective effects of Vitexin and Thymol against Okadaic acid-induced neurotoxicity: Computational...

Alzheimer's disease (AD) is a neurodegenerative disorder characterized by dementia and inexorable loss of neurons. Despite extensive research, the currently approved drugs offer only limited efficacy which highlights the need for exploring novel synergistic natural compounds capable of mitigating multiple targets of AD.

This study evaluated the neuroprotective potential of the synergistic combination of Vitexin and Thymol using computational and in vitro model systems in N2a cells. Computational pharmacokinetic screening revealed the blood-brain barrier (BBB) permeability of thymol and 0.55 bioavailability score for Vitexin. Molecular docking studies showed higher binding affinity of Vitexin with JNK and Thymol with COX-1 and CAMK-II and 100 ns molecular dynamics simulation exhibited stable binding with sustained hydrogen bond and hydrophobic interactions. Based on the IC₅₀ values [Vitexin (135.24 µg/ml, 312.9 µM) and Thymol (36.91 µg/ml, 245.7 µM)] and combination index (CI < 1) determined by the acetylcholinesterase (AChE) inhibition assay and checkerboard assay (performed with 15 different combinations) respectively, the effective combination (Vitexin 45.08 µg/ml; Thymol 7.382 µg/ml; CI = 0.53) was fixed for further studies. In okadaic acid (OA) induced neurotoxicity model, pre-treatment with the combination significantly increased the cell viability (88.36 ± 3.73%) (n = 3). Real-time PCR results revealed the upregulation of PP1 gene expression and modulation of MAPK family (MEK1/2, ERK1/2 and JNK), and other tau related kinases (GSK3β, CAMKII and P70 s6).

The above findings demonstrate that the combination of Vitexin and Thymol effectively protect the neuronal cells from OA induced cytotoxicity and modulate the hyperactivation of kinases, suggesting its potential in preventing tau hyperphosphorylation in AD conditions.