Lc 10at vp pumps

The decoction was prepared by extracting CR with boiling water. Briefly, dried crude drugs of CR (25 g) were immersed in 500 mL of water for half an hour and then heated on a gas stove. After boiling, gentle heating was continued until the volume reduced to half volume and filtered while hot. The filtrate was concentrated to make 50 mL to afford a concentration of 0.5 g/mL and divided into aliquots, then frozen at −30 °C for later use.
For characterization, CR decoction (300 μL) was mixed with 700 μL of MeOH. After vortexed and centrifuged, the supernatant (140 μL) was mixed with 60 μL of daidzein (100 μg/mL as internal standard) and 20 μL was subject to HPLC analysis. The system was equipped with a Shimadzu SIL-10AD VP automatic sample injector, a Shimadzu SPD-10AVP Detector and a Shimadzu LC-10AT VP pumps. Reversed-phase separation was carried out using an Apollo C18 column (4.6 × 250 mm, 5 μm) and equipped with a guard column (4.6 × 50 mm, 5 μm) (Alltech Associates Inc., USA). The mobile phase consisted of acetonitrile and 0.1% phosphoric acid (25:75) and the flow rate was 1.0 mL/min. The detection wavelength was set at 270 nm.

The hairy roots were picked out of the culture flasks with a pair of forceps, rinsed thoroughly with distilled water, blotted dry by a paper towel, and then dried at 40-45°C in an oven to attain the constant dry weight (Dw). The dried root samples were ground into powder and then extracted with methanol (20 mg roots/mL) under sonication for 30 min. After removal of the solid, the extract was evaporated to dryness and redissolved in methanol, and applied to high-performance liquid chromatography (HPLC) for the analysis of the rutin and quercetin content. The HPLC system was equipped with two LC-10ATvp pumps and a SPD-M10Avp diode-array detector (Shimadzu, Kyoto, Japan), and using a C₁₈ column (4.6 × 250 mm, 5 μm, Phenomenex, Torrance, CA, USA). The separation was performed using a mixture of acetonitrile and distilled water (0.2% H₃PO₄) with a gradient elution: (0-8 min, 20% acetonitrile; 8-13 min, 20-40% acetonitrile; 13-29 min, 40% acetonitrile; 29-30 min, 40-20% acetonitrile; 30-35 min, 20% acetonitrile). The flow rate was set at 1.0 mL/min, and UV detection at 365nm. The temperature of the column was set at 30°C, and the sample injection volume was 20 μL as reported by Zhao et al.[24 (link)] The rutin and quercetin were detected and quantified with the standards obtained from the Institute for Identification of Pharmaceutical and Biological Products (Beijing, China).