Determination of total triterpenoid content

In each well of the microtiter plate, 10 μL of 1 mg/mL concentration extracts dissolved in 100% methanol, 15 μL of vanillin-glacial acetic acid solution (5% w/v), and 50 μL of perchloric acid solution were introduced. The microtiter plate was then subjected to heating at 60°C for 45 minutes, followed by cooling on ice to room temperature. Before recording absorbance, 225 μL of glacial acetic acid was added to each well. A control experiment was prepared, including all the aforementioned components, with 100% methanol replacing the sample. For the construction of the calibration curve, ursolic acid dissolved in 100% methanol at concentrations ranging from 0.0078 to 1 mg/mL was used instead of the extract. Each measurement was conducted in triplicate. The absorbance was determined at 548 nm using a microtiter plate reader, specifically the Multiscan Sky, Thermo Scientific, Finland. The total triterpenoid content in the samples was computed based on the calibration curve equation and expressed as milligrams of ursolic acid equivalents (UAE) per gram of dry extract – mg UAE/g dry extract.