Paris polyphylla, SMITH

Yin, Yan, Gao, Linhui, Zhang, Xianan & Gao, Wei, 2018, A cytochrome P 450 monooxygenase responsible for the C- 22 hydroxylation step in the Paris polyphylla steroidal saponin biosynthesis pathway, Phytochemistry 156, pp. 116-123 : 122

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.