Unveiling the Bioactive Potential of Shilajit: Integrated Chemical Profiling and Antimicrobial Evaluation via Chromatographic, Spectroscopic, and In Silico Approaches.

Shilajit is a herbomineral exudate widely used in traditional medicine and composed mainly of humic substances and low-molecular-weight organic constituents. However, the chemical composition and biological relevance of its low-molecular-weight fraction remain poorly understood. In this study, sequential fractionation was employed to obtain humins (HM), humic acid (HA), fulvic acid (FA), and a low-molecular-weight residual fraction (RF). FTIR analysis supported successful fractionation, with HA and FA exhibiting characteristic humic-like functional groups, while the RF showed reduced polymeric features consistent with enrichment in smaller organic molecules. Comprehensive chemical profiling using multi-solvent GC-MS following derivatization enabled the detection of structurally diverse metabolites. The RF exhibited a broader metabolite profile than whole Shilajit, with 36 low-molecular-weight compounds tentatively identified, including phenolics, aromatic acids, esters, flavanol derivatives, and sterol-related compounds. SEM-EDS analysis revealed heterogeneous morphology and elemental composition. In silico ADMET predictions suggested favorable gastrointestinal absorption and low predicted toxicity for most compounds. Antimicrobial assays demonstrated stronger antibacterial activity of the RF compared to whole Shilajit, while antifungal activity remained limited. Overall, the findings highlight the biological significance of low-molecular-weight constituents in Shilajit.