HPLC analysis of total triterpenoids

Qualitative analysis of total triterpenoids was performed according to the procedures of a previously published method [12]. Briefly, 0.1 mg of the A. camphorata fruiting body was dissolved in 10 mL of 100% methanol and then filtered through a 0.22 μm membrane to prepare the sample solution. The 10 μL sample solution was analyzed using HPLC (Model L-2130, Hitachi, Japan). The detection of the absorbance using a photodiode array detector at 275 nm was carried out. The model of the analytical column was Intertsil ODS-3 C-18 C18 column, 25 cm × 4.6 mm i.d., 5 μm. The mobile solution eluded, containing of 0.2% formic acid and 100% acetonitrile at a flow rate of 1.0 mL per min was performed. A colorimetric method was used to determine the total triterpenoid content of A. camphorata fruiting bodies. Briefly, under reflux in 5 mL of 95% ethanol for 30 min, the extraction of 0.5 g sample was gained. The extraction was then filtered and centrifuged at 3000×g for 10 min. One mL of the diluted solution (1/10) was dried, and 0.2 mL of 5% (w/v) vanillin–glacial acetic acid and 0.08 mL of perchloric acid were added sequentially after the extraction was done. The mixture was shaken in a 60°C water bath for 20 min. After being added to 3.72 mL of glacial acetic acid against a standard oleanolic acid curve, a 550 nm absorbance (UV visible spectrophotometer, Model U-2800, Hitachi, Japan) was used to read the concentration of total triterpenoids.