Identification of acetylcholinesterase inhibitory metabolites from hydroalcoholic extract of Itrifal Muqawwi Dimagh using thin-layer chromatography‒bioautography‒mass spectroscopy and its validation using in silico molecular approach

Traditional formulations owing to their holistic approach to health and wellness with minimum side effects are widely used all over the globe. Itrifal Muqawwi Dimagh (IMD) is a traditional polyherbal formulation used in the Unani system of medicine conventionally claimed to support and enhance cognitive function. The formulation is widely used by a large population due to its alleged therapeutic efficacy. Despite being widely used, the formulation lacks scientific validation and justification in establishing its therapeutic potential. The present study was designed to detect and identify bioactives responsible for acetylcholinesterase inhibitory activity by thin-layer chromatography (TLC)‒bioautography‒mass spectroscopy (MS) and its validation using in silico molecular approach. Quality control was done using ultraperformance liquid chromatography (UPLC)‒MS fingerprint analysis. The authentication of individual constituents of the formulation was done by using powder microscopy. Ethyl acetate and hydrolyzed ethyl acetate fraction of hydroalcoholic extract of IMD were prepared, and in vitro acetylcholinesterase (AChE) inhibitory activity by Ellman’s method was performed. The TLC‒MS bioautography revealed the presence of scopoletin, tannic acid, ellagic acid, and catechin as potential bioactive anticholinesterase metabolites. In silico analysis of the identified metabolites from IMD showed AChE activity of ten identified metabolites, moreover, catechin and naringenin showed the best of potential activity. UPLC‒MS analysis demonstrated separation of 33 phytocompounds in the best active fraction of formulation. Hence, based on our findings, it can be concluded that IMD has a great potential to overcome cholinergic deficiency and can be used for neuroprotection and other neurological disorders after successful in vivo pharmacokinetic and toxicity validation.