High-Performance Thin Layer Chromatography Methods for Estimation of Chemical Markers in Stachytarpheta indica Vahl.

Background: Stachytarpheta indica Vahl. (Verbenaceae family), commonly known as Indian Snakeweed, is a valuable medicinal plant. A systematic phytochemical analysis of bioactives of this plant is lacking till date. Objectives: The present study is aimed to develop validated High-Performance Thin-Layer Chromatography (HPTLC) methods for quantification of 6 marker compounds, β-sitosterol, lupeol, ursolic acid, apigenin, caffeic acid and verbascoside in S. indica plant. Materials and Methods: Normal phase chromatography was used to develop mobile phase systems that could aid in the identification and quantification of the phytochemicals in the entire plant. Several enhancements were made to the basic method of thin-layer chromatography to automate the different steps, to increase the resolution achieved and to allow more accurate quantitative measurement. The methods were validated as per the International Council for Harmonization (ICH) guidelines. Results: The linear regression analysis data for the calibration plots showed a good linear relationship (r2=0.9873-0.9979) in the concentration range of 1-5 μg for β-sitosterol, lupeol and ursolic acid; 0.5-5 μg for apigenin, 1-3 μg for caffeic acid; and 3-8 μg per spot for verbascoside with respect to area. The average recoveries for phytoconstituents were 98.32 to 98.97%, indicating the good reproducibility. Verbascoside 1.3598±0.014% w/w was present at high concentration and ursolic acid 0.0027±0.0007% w/w was present at low concentration in the whole plant powder. Conclusion: The validated TLC-densitometric methods were simple, precise, accurate, specific and can be used for the routine quality control of raw materials of S. indica.