REVERSE PHASE-HIGH PERFORMANCE LIQUID CHROMATOGRAPHY METHOD FOR ESTIMATION OF BIOACTIVE COMPOUNDS FROM DIFFERENT FRACTIONS OF NEW POLYHERBAL FORMULATION OF CLINACANTHUS NUTANS AND ELEPHANTOPUS SCABER

Background: Polyherbal formulation is the trend of modern pharmacognosy and holistic approach in the treatment of uncurable diseases such as cancer. Medicinal plants contain many bioactive compounds that give different pharmacological activities. Therefore, it is important to investigate the quality of new polyherbal formulation by identification of bioactive markers. Objective:  To identify apigenin (1) , luteolin (2), b-sitosterol (9), 3’-hydroxy-5, 6, 7, 4’- tetra methoxy flavone (8), quercetin (3), vanillin(7), gallic acid (5), benzoic acid (6) and rutin (4) as biomarker from different fractions of the new polyherbal formulation of Clinacanthus nutans and Elephantopus scaber by RP-HPLC gradient method.   Methods:  A Shimadzu HPLC was utilized to perform the analysis which was equipped with an autosampler, column oven, and UV/VIS detector. An HPLC column used was Merck Licrochart Purospher Start RP 18 column (250mm, 4.6 mm i.d, 5μm pore size). The temperature was maintained at 40.0 ºC throughout the study. The mobile phase isocratic method with acetonitrile 100%. The flow rate was 0.5 mL min⁻¹. Absorbance was observed at λ = 360 nm. Results: After identification of bioactive compounds it was established that 3’-hydroxy-5, 6, 7, 4’- tetra methoxy flavone, gallic acid, benzoic acid and vanillin was found in a polyherbal formulation that was not present before in Clinacanthus nutans and Elephantopus scaber. Other bioactive compounds such as Apigenin. Luteolin, b-sitosterol, quercetin, and rutin were present in both medicinal herbs as well as inside polyherbal fractions. Vanillic acid was the oxidative product of vanillin that was found in Elephantopus scaber. In the polyherbal formulation, vanillin was found that shows the transformation of vanillic acid into vanillin. Conclusion:  Formation of new bioactive compounds from a polyherbal formulation that was not present before in both medicinal plant. Identification of biomarkers helps to increase the quality of the new product for future.