Simultaneous estimation of bioactive markers using validated HPTLC method and assessment of hepatoprotective effect...

A new, precise, and reproducible HPTLC method was developed and validated for the estimation of andrographolide (AG), gallic acid (GA), and kutkin (KT2) in an ayurvedic formulation, HEPASAVE Syrup. The quantification of the three biomarkers was performed on TLC aluminum plates pre-coated with silica gel 60F₂₅₄ as stationary phase. Linear ascending development was carried out in a Camag twin trough glass chamber saturated with toluene: ethyl acetate: formic acid: methanol (3:3:0.8:0.4, v/v/v/v) at room temperature (25 ± 2 °C). Camag TLC scanner III was used for simultaneous spectro-densitometric scanning and analysis in absorbance mode at 254 nm. The system was found to give compact spots for AG, GA, and KT2 with the respective R_f values of 0.74, 0.66, and 0.21. The data for calibration plots showed a good linear relationship for AG with r² = 0.995, GA with r² = 0.993, and KT2 with r² = 0.992 in the concentration range of 200 ng to 800 ng, 80 ng to 320 ng and 2000 ng to 6000 ng, respectively to the peak area. The amount of AG, GA, and KT2 in the formulation was quantified and found to be 1.27 %w/v, 1.15 %w/v, and 0.014 %w/v, respectively. Statistical analysis of the data showed that the method is reproducible for the quantification of AG, GA, and KT2, simultaneously in HEPASAVE Syrup. It also significantly reduced the levels of SGPT, SGOT, and ALP in animals.