NOVEL PARAMETERS TO DEFINE BIO-EFFICACY OF SHILAJIT RESIN AND ITS VALIDATION

Shilajit, a herbo-mineral blackish brown resin, extracted from humic rocks, is attributed to several therapeutic claims in Ayurveda like antiaging, health-promoter, immune- booster, urogenital problems and bio-enhancer. The lack of strong parameters for its quality control and standardization; and the knowledge gap to explain its mechanism of action are the important factors behind its restricted use. Here, for the first time, we have used a High-Resolution Advance Mass Spectrophotometer (HRAMS) to visualize the metabolites of shilajit and further identified novel metabolites for its characterization. Seven brands of shilajit, available in the market (5 resins and 2 capsules), were purchased and used to validate our findings in terms of molecular weight, abundance, retention time and structural characteristics. We found 14 novel metabolites and six of them were acidic, eight were non-acidic. Among them, only 4 metabolites were common in all seven samples, which were (a) NP-020214, (b) 3-tert- Butyladipic acid, (c) Dodecanedioic acid and (d) Azelaic acid. However when only resins were compared, then additional nine common metabolites were identified as (1) 4-Acetamidobenzoic acid, (2) Phenylacetylglycine, (3) Benzamide,3-(Sulfooxy)benzenepropanoic acid,   (4)   4-(2-Hydroxyethyl)phenyl hydrogen sulfate, (5) N-Benzylformamide, (6) p-Cresol, (7) 2- Hydroxyhippuric acid, (8) thymol sulfate and (9) Prostaglandin-B1. Thus, the first 4 metabolites may be considered as universal novel marker compounds in addition to existing compounds, for standardization purposes. However, the remaining 9 can only be used for pure resin preparations of shilajit. The bioinformatics-based analysis indicated that all 14 metabolites passed the drug-likeness, but only 7 of them could cross the Blood blood-brain barrier (BBB). Further, the presence of heavy metals and trace elements were analyzed by Induction Coupled Plasma Atomic Emission Spectroscopy (ICP-AES) and the presence of no heavy metals (Pb, As, Cd and Hg) were detected in any sample, but all of them showed a high amount of essential trace elements but in variable amount. Aqueous solutions of shilajit showed high conductance and solubility (92-95%) at normal pH. Thus, here we could identify the additional 14 novel molecules, which could be used for characterization and quality control of shilajit.