2.9. Triterpenoid HPLC Analysis

Triterpenoids (oleanolic acid and ursolic acid) were extracted using a previously reported method with slight modifications [28]. Fine powders (100 mg) of different parts (roots, stems, and leaves) of L. pubescens plants were suspended in 2 mL of methanol (95% v/v) containing 1% HCl and then sonicated for 60 min. The sonicated samples were then centrifuged and the resulting supernatants collected. The remaining sludge was re-extracted twice in the same manner. The combined supernatants were dried using nitrogen gas and then dissolved in methanol (0.5 mL). HPLC analysis was performed using an Agilent 1200 series HPLC system incorporating a Welch Ultisil PAH column (4.6 × 250 mm, 5 μm, Welch Materials, West Haven, CT, USA) to separate the oleanolic and ursolic acids. The mobile phase consisted of acetonitrile and water (85:15) at a flow rate of 1 mL/min. The oven temperature was set to 30 °C, and the detection wavelength was 210 nm. Calibration curves were constructed by plotting six different concentrations of oleanolic and ursolic acids. The linear equations for oleanolic acid and ursolic acid were y = 1.1342 x + 0.5792 (R² = 0.9999) and y = 1.7683 x + 0.0763 (R² = 0.9998), respectively.