Identification of anticholinesterase active compounds from ethylacetate fraction of hydroalcoholic extract of Itrifal Sana using TLC-bioautography-MS and its validation using in silico molecular approach

Traditional formulations are used extensively throughout the world due to their holistic approach to health and wellness with the fewest possible adverse effects. Itrifal Sana is a traditional unani polyherbal formulation, a unique combination that makes it synergistically potent, capable of providing dual benefits for health and well-being. Even though the formulation is frequently utilised, there is no scientific evidence to support its therapeutic efficiency. The present study was designed to detect and identify bioactives responsible for acetylcholinesterase inhibitory activity by TLC-bioautography-MS and its validation using an in silico molecular approach. Authentication of the formulation was done using macroscopy and powder microscopy. Quality control was done using UPLC-MS fingerprint analysis. TLC-bioautography-MS was done to detect the bioactives responsible for acetylcholinesterase inhibitory activity and the findings were validated using in-silico approach. The TLC-MS bioautography revealed the presence of rosmarinic acid, kaempferol, and apigenin as potential bioactive anticholinesterase metabolites. UPLC-MS analysis demonstrated the separation of 48 phytocompounds in the best active fraction of formulation. In silico analysis of identified metabolites showed acetylcholinesterase inhibitory activity of ten identified metabolites, moreover, rosmarinic acid and lobeline showed the best potential activity. Our findings indicated that Itrifal Sana, which was investigated for the first time, has an enormous potential for managing AD caused by acetylcholinesterase enzyme inhibition. It was derived through successfully attempted TLC-bioautography-MS and in silico approach; however, further research on their full efficacy using in vitro cell line studies, in vivo studies, pharmacokinetics studies, and toxicity studies is still needed. TLC-bioautography-MS and in silico molecular approach offer much more effective, accurate, and reliable results than the conventional methods in the identification and validation of bioactive components from IS, a polyherbal formulation helps to advance the development of natural product-based therapeutics for cholinesterase dysfunctional diseases.