Quantitative Determination of Triterpenes

The total triterpenoid content of the Water-Vbr and EtOH70%-Vbr extracts was determined using a colorimetric method [58 (link)] with modifications. Briefly, 100 μL of the extract was mixed with vanillin/glacial acetic acid (150 μL, 5% (p/v)) and perchloric acid solution (500 μL). The sample solutions were heated at 60 °C for 45 min and then cooled to room temperature. Subsequently, 2.25 mL of glacial acetic acid was added, and the absorbance was measured at 765 nm using a spectrophotometer Hitachi U-2000 (Tokyo, Japan). Oleanolic acid was used as the standard control. Measurements were obtained in triplicate, and the mean and standard deviation values were adjusted using a straight-line equation (Y = 0.0013x + 0.0858 and R² = 0.9991). The results are presented as the mean (±SD) of oleanolic equivalents per gram of the extract.