Millex 0.2 μm nylon membrane syringe filters

Before extraction, fresh samples were lyophilized for 72 h to remove water. Dried roots were ground and passed through a 40-mesh screen. Root powder (~ 500 mg) was extracted with methanol in a Soxhlet extractor for 12 hours. The extract solution was condensed under vacuum, transferred into a 50 mL volumetric flask and diluted to volume with methanol. The concentration of the solution was equivalent to 10 mg/mL of crude herb. All the extract solutions were filtered through Millex 0.2 μm nylon membrane syringe filters (Millipore Co., Bedford, MA) and stored at −20°C until HPLC analysis.

HPLC analysis was conducted on a Waters 2960 instrument with a Waters 996 photodiode array detector (Milford, MA, USA). The separation was carried out on an Alltech Ultrasphere C18 column (5 μm, 250 × 3.2 mm I.D.) (Deerfield, IL, USA) with a guard column (Alltech Ultrasphere C18, 5 μm, 7.5 × 3.2 mm I.D.). Acetonitrile (solvent A) and water (solvent B) were used. Gradient elution started with 18% solvent A and 82% solvent B, changed to 21% A for 20 min; to 26% A for 3 min and was held for 19 min; to 36% A for 13 min; to 50% A for 9 min; to 95% A for 2 min and was held for 3 min and finally was changed to 18% A for 3 min and held for 8 min. The flow rate was 1.0 ml/min and the detection wavelength was set to 202 nm. All the tested solutions were filtered through Millex 0.2 μm nylon membrane syringe filters (Millipore Co., Bedford, MA, USA) before use. The levels of ginsenosides in each sample were calculated using standard curves of ginsenosides.