SEPERATION AND QUANTIFICATION OF PHARMACOLOGICALLY ACTIVE MARKERS OLEANOLIC ACID, 20-HYDROXYECDYSONE AND Β- SITOSTEROL FROM ACHYRANTHES ASPERA AND FROM MARKETED FORMULATION BY HIGH PERFORMANCE THIN LAYER CHROMATOGRAPHY

Objective – To develop simple, accurate, precise and reproducible High Performance Thin Layer Chromatographic (HPTLC) method for simultaneous quantification of Oleanolic acid, 20-hydroxyecdysone and β-sitosterol in the whole plant extract of Achyranthes aspera. Methodology – In the present work a precise, accurate and reproducible HPTLC method is developed and validated for simultaneous quantification of three pharmacologically active markers Oleanolic acid, 20-hydroxyecdysone and β-sitosterol from the whole plant of Achyranthes aspera and from marketed Cystone® tablets of Himalaya herbal healthcare. These markers were extracted from the whole plant and from Cystone® tablets using Soxhlet extraction followed by chromatographic separation on HPTLC silica gel 60 F254 pre-coated plates using double development method. The analysis was carried out using a double development method. The first development up to 50 mm was carried out using a mixture of dichloromethane- ethanol (8:2) (v/v) as mobile phase and the second development up to 80 mm using a mixture of petroleum ether- ethyl acetate- methanol (8.5:1:1) (v/v/v), as mobile phase with chamber saturation of 20 minutes. Quantification was carried out at 540 nm for all the three markers. Results – Linear responses for Oleanolic acid, 20-hydroxyecdysone and β-sitosterol were obtained over the concentration ranges of 20-400 μg/mL , 40-400 μg/mL and 40-400 μg/mL respectively. Conclusion – This HPTLC method can be used as a quality control tool for quantification of these markers simultaneously from raw material as well as marketed formulation.