One gram of each sample was extracted with 20 mL of methanol/water (9:1) and chloridric acid 1%, solution at room temperature (25 ± 2 °C) and in darkness for 24 h. The obtained solutions were filtered in a Büchner funnel and residual methanol from the solution was removed by nitrogen automated rapid evaporation (Rapid Mini of the Crescent Scientific, Mumbai, Maharashtra India). The obtained solution was purified using a Bond Elut C18 (500 mg 6 mL) column (Agilent, Santa Clara, CA, USA) according to the manufacturer’s instructions. The solid samples obtained were dissolved in 1 mL of methanol/water solution (1:1) with a final concentration of 30 mg/mL. HPLC-DAD analyses were carried out in duplicate and performed using an Agilent 1100 Infinity (Agilent, Santa Clara, CA, USA), equipped with a diode array detector (DAD) and with a 250 × 4.6 mm i.d., 5 µm Symmetry Shield RP 18 column; the mobile phases: 100% organic solvent (MeOH) in isocratic conditions; total time 35 min. The column temperature was maintained at 25 °C. The flow was 1 mL/min and the injection volume was 10 μL. The chromatogram profiles were recorded from 190 to 500 nm and monitored at 210, 280 nm ± 2 nm. HPLC-grade solvents, methanol, water, anhydrous sodium sulfate and chloridric acid were obtained from Carlo Erba Reagenti (Milano, Italy). The reference compound (betulinic acid) was obtained from PhytoLab GmbH & Co. (Vestenbergsgreuth, Germany).
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