Approximately 100 mg of powdered materials were soaked in 5 mL of methanol. They were ultrasonically processed for 1 h at ambient temperature using an ultrasonicator (Branson 1510E-MT 42 kHz, Danbury, CT, USA) for sample extraction. Before submitting the sample solutions for UHPLC analysis, they were filtered through a membrane with a pore size of 0.22 microns and diluted to a volume of 10 mm using methanol. Madecassoside (87.5%), madecassic acid (88.1%), asiatic acid (92.6%) and asiaticoside (88.8%) were obtained from ChromaDex Inc (Los Angeles, CA, USA). The solvents used in the UHPLC analysis were of analytical HPLC grade (Merck, Darmstadt, Germany). Whatman membrane filters (0.22 μm pore size; PTFE; P/N E252, Buckinghamshire, England) were used for the filtration of sample solutions.
It was found that the primary bioactive components in pennywort leaves were asiatic acid (AA), madecassoside (MS), madecassic acid (MA) and asiaticoside (AS). In methanol, 1000 g/mL solutions of MA, MS, AA and AS, were made to be used as standards. To obtain calibration curves for MS, AS, MA and AA, an adequate volume of each standard solution was mixed and diluted with methanol at six (6) concentrations ranging from 100 to 500 μg/mL.
It was found that the primary bioactive components in pennywort leaves were asiatic acid (AA), madecassoside (MS), madecassic acid (MA) and asiaticoside (AS). In methanol, 1000 g/mL solutions of MA, MS, AA and AS, were made to be used as standards. To obtain calibration curves for MS, AS, MA and AA, an adequate volume of each standard solution was mixed and diluted with methanol at six (6) concentrations ranging from 100 to 500 μg/mL.