Sustainable instrumental thin-layer chromatography-based methodology for standardization of neuroprotective components in propolis collected from India

Bee propolis is a heterogeneous mixture of phytoactive metabolites with reported antioxidant, anti-inflammatory, and neuroprotective properties. The chemical composition is highly variable, complex and presents the greatest challenge for standardization. So, the study was aimed at standardization of selected phytochemicals in propolis collected from a region in India (IP). A thin-layer chromatography (TLC) method was developed for five markers with prior evidence of neuroprotective activity using orthogonal detection and subsequently validated as per the International Council for Harmonisation ICH Q2 (R1) guidelines. The methodology was assessed for greenness using different metrices and then applied to marketed samples. Furthermore, antioxidant and anticholinesterase activity were evaluated directly using TLC‒bioautography technique. The method was developed using toluene‒ethyl acetate‒formic acid (7.4:2.6:0.5, V/V) as the mobile phase, with R_F values of 0.235, 0.353, 0.552, 0.606, and 0.655 for luteolin, p-coumaric acid, chrysin, galangin, and pinocembrin, respectively. The optimized method was found to be precise (%RSD ≤ 2.0%), accurate (90‒110%), linear over the concentration ranges (R² ≥ 0.995), sensitive and robust. Pinocembrin (2.30 ± 0.12% w/w) and galangin (5.78 ± 0.30% w/w) were found in the highest concentration. TLC‒bioautography analysis confirmed the contribution of the selected phenolics in anticholinesterase and antioxidant activity. The analytical eco-score value of 76, analytical greenness calculator (AGREE) score of 0.62 and other assessed analytical pictograms indicated the methodology developed had low environmental impact. Overall, a simple, rapid, precise, and green TLC analytical methodology has been developed for IP for quality control and preliminary screening of neuroprotective activity.