Chromatographic Fingerprinting of Bioactive Constituents of Herbal Drugs In Herbo-Mineral Regimen

Abstract

Shilajit along with Triphala has been one of the most widely used herbal drug combinations used for the symptomatic treatment of hyperthyroidism, as a hepato-protective and adaptogenic agent. Here, a reliable HPTLC and HPLC method was developed for the analysis of the mentioned drug combination for the detectable and quantifiable detection of Quercetin, Myricetin, Gallic acid, Ellagic acid, Tannic acid, Ascorbic acid and Humic acid. After several trials, the HPTLC analysis was performed using precoated silica gel plates as the stationary phase with Tol., E.A, F.A. and Meoh at the ratio of 3:3:0.8:0.2%v/v/v/v as the mobile phase. As result separation of Quercetin, Myricetin, Tannic acid found good separation with that similar Rf of Gallic and Ellagic acid required involvement of 2D-TLC.The constituents were detected at 254 nm. Although, humic acid and ascorbic acid were not detected and hence a different mobile phase consisting of Tol.: E.A: F.A. in the ratio of 6:3:1%v/v/v. The detection wavelength selected was 254nm and 366 nm respectively. A robust HPLC analytical was intended to be developed for the same purpose.After successful method development Qbd approach applied for method optimization. Here, a reversed phase chromatographic set up was employed with a C18 column and Meoh:KH2PO4 buffer, pH 2.5 (65:35) and ACN: KH2PO4 buffer pH 3. The analyte under consideration were analyzed using a PDA detector. According to ICH criteria, the developed methodology was validated. Parameters including linearity, accuracy, prescision, range, specificity, robustness, LOD, LOQ, and system appropriateness were tested and found to be in agreement with the given values.Upon successful method development of the two methods, it was established that the bioactive constituents of Shilajit in combination with Triphala can be successfully analyzed by a HPTLC and HPLC methods.