A cytochrome P 450 monooxygenase responsible for the C- 22 hydroxylation step in the Paris polyphylla steroidal saponin biosynthesis pathway Author Yin, Yan School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China & School of Chinese Materia Medica, Beijing University of Chinese Medicine, Beijing 100102, PR China Author Gao, Linhui School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China & State Key Laboratory of Breeding Base Dao-di Herbs, National Resource Center for Chinese Materia Medica, China Academy of Chinese Medical Sciences, Beijing 100700, Author Zhang, Xianan School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China xnzhang@ccmu.edu.cn Author Gao, Wei School of Traditional Chinese Medicine, Capital Medical University, Beijing 100069, PR China text Phytochemistry 2018 2018-12-31 156 116 123 http://dx.doi.org/10.1016/j.phytochem.2018.09.005 journal article 10.1016/j.phytochem.2018.09.005 1873-3700 10484444 5.9. HPLC method for detecting steroid saponins in P. polyphylla roots For HPLC, an aliquot of the same fresh plant root material that was used for expression analysis was freeze-dried at −50 ̊C for 8 h, and then pulverized using a ball mill. 0.5 g of the resulting powder were extracted in 10 mL of 75% ethanol, sonicated at 100 Hz for 50 min and filtered. The filtrate's solvent was evaporated to dryness, and the residue was reconstituted with 10 mL of methanol. The extracts were filtered through a 0.22 μm PTFE filter and analyzed on a 1200 HPLC system (Agilent, USA ) equipped with an Eclipse Plus C-18 column ( 4.6 mm × 250 mm , 5 μm, Agilent, Santa Clara, CA, USA ) at a detection wavelength of 203 nm . The injection volume was 10 μL. Water and acetonitrile were used as solvent A and B, respectively. The gradient program comprised a linear gradient from 30% to 60% B (0–40 min), followed by a linear gradient from 60% to 30% B (40–50 min). The flow rate of the mobile phase was 1 mL/min, and the column temperature was kept at 30 ̊C. Polyphyllin I, polyphyllin II, polyphyllin VI, and polyphyllin VII were used as standard compounds to create standard curves for quantification, and their retention times were 24.8, 22.0, 15.8, and 13.2 min, respectively. Three biological replicates were prepared for each sample.