Test Methods and Standards
Preparation of methanolic extracts
Test samples were dried in an oven at 50℃ for 2 d. Five grams of each dry sample was refluxed 4 times with methanol for 6 h; the extract was filtered and evaporated; and 10 mg of each dry extract was diluted with 1 ml methanol immediately before use.
HPLC system
HPLC was performed on an Agilent 1100 series with DAD detection. The detection wavelength was set to 210 nm. Separation was obtained with a reversed-phase column (Cosmosil 5C18- AR-II, 250 × 4.6 mm, Kyoto, Japan) eluted at a flow rate of 1 ml min-1 with a linear solvent gradient elution of A (0.0085% H3PO4 in H2O) and B (acetonitrite), and the column was eluted according to the following profile: 0~65 min, 30~47% B, 65~100 min, 47~47% B, 100~140 min, 47~100% B, 140~170 min, 100~100% B, 170~175 min, 100~30% B, 175~200 min, 30~30% B. The column temperature was set to 30℃. The injection volume was 20 µL. Ten known compounds (with purities of > 99% for each compound) including 5 ergostane triterpenoids (antcins C and K, and zhankuic acids A, B, and C), 4 lanostane triterpenoids (sulphurenic acid, dehydrosulphurenic acid, eburicoic acid, and dehydroeburicoic acid), and 1 monophenyl (4,7-dimethoxy-5-methyl-1,3-benzodioxole) were used as the detected components. Their retention times and patterns of detecting wavelength (210 nm) were based on our previous studies（Shen CC, Kuo YC, Huang RL, Lin LC, Don MJ, Chang TT, Chou CJ. 2003. New ergostane and lanostane from Antrodia camphora. J Chin Med 14:247-58.；Chang TT,Wang WR,Chou CJ.2011. Differentiation of mycelia and basidiomes of Antrodia cinnamomea using certain chemical components.Taiwan J For Sci26(2):125-33. ）. Each component and its retention times (in min) in parenthesis were as follows: antcin K (23.1 and 24.3), 4,7-dimethethoxy-5-methyl-1,3-benzodioxole (35.8), antcin C (59.7 and 62.5), zhankuic acid C (62.6 and 64.0), zhankuic acid B (83.4 and 84.5), dehydrosulphurenic acid (83.5), sulphurenic acid (87.5), zhankuic acid A (93.9 and 95.0), dehydroeburicoic acid (140.1), and eburicoic acid (141.4).