A validated phytochemical marker based HPTLC method for the segregation of Bauhinia vahlii Wight and Arn. from geologically different samples of Indian zones

Bauhinia vahlii Wight and Arn. is a commonly used climber in traditional medicinal practices. The present work introduces a validated high-performance thin-layer chromatography (HPTLC) method for identifying the superior B. vahlii species from various regions in India, such as Pune, Jhansi, and Ranikhet, by analyzing phytochemical markers. The current study identified and estimated lupeol and β-sitosterol in leaf extracts of B. vahlii, detecting them as magenta and violet grey bands using anisaldehyde‒sulfuric acid reagent at retention factor (R_F) ~0.68 and R_F ~0.53, respectively, at 514 nm. This was achieved using a mobile phase consisting of toluene‒ethyl acetate‒formic acid (8:2:0.2, V/V). The method was validated for parameters such as linearity, limits of detection, and quantification and precision, as per the International Council for Harmonisation (ICH) guidelines Q2R1. The calibration plots of the analyzed data exhibited a good linear relationship with R² = 0.99 ± 0.02 in the concentration range 200‒1400 ng per spot for lupeol and 600‒1300 ng per spot for β-sitosterol with respect to the peak area. Lupeol was quantified as 61.61, 37.10, and 63.14 µg in 100 mg leaf extracts of B. vahlii collected from Pune, Jhansi, and Ranikhet regions of India, respectively. Meanwhile, β-sitosterol was determined to be 116.80, 105.13, and 90.25 µg from Pune, Jhansi, and Ranikhet, respectively, in 100.0 mg of the sample. Among the three locations, Ranikhet exhibited the highest lupeol content, while Pune showed the highest β-sitosterol estimation. The validated HPTLC method employed was simple, accurate, and reproducible for the rapid screening of B. vahlii collected from diverse Indian topographies. This study may be utilized as a valuable quality assessment tool for stakeholders, aiding adherence to Good Manufacturing Practice standards.

Graphical abstract