3.3.3. High-Performance Liquid Chromatography (HPLC) Analysis of Panasenoside and Kaempferol

Panasenoside and kaempferol contents of ginseng leaves were determined using HPLC. The equipment was an HPLC system (CBM-20A; Shimadzu Co, Ltd., Kyoto, Japan) with 2 gradient pump systems (LC-20AT; Shimadzu, Japan), an auto sample injector (SIL-20A; Shimadzu), a UV detector (SPD-20A; Shimadzu), and a column oven (CTO-20A; Shimadzu). Each sample extract (0.1 mg) was dissolved in 1 mL of 80% methanol (*v*/*v*) and filtered through a 0.22 μm membrane filter before sample injection into the HPLC system. HPLC separation was performed on an Inertsil ODS-SP C18 column (250 mm × 4.6 mm, 5 μm; GL Sciences, Tokyo, Japan). The injection volume of samples was 10 μL. The gradient running phase was programmed with the combination of solvent A (water with 0.1% trifluoroacetic acid) and solvent B (acetonitrile), in which solvent B was sequentially increased from 14% to 18% (0 to 10 min), 18% to 30% (10 to 20 min), 30% to 60% (20 to 30 min), 60% to 65% (30 to 33 min), 65% to 100% (33 to 40 min), and 100% to 100% (40 to 50 min) and then finally adjusted from 100% to 14% (50 to 65 min). The operating temperature was set at 35 ◦C. The flow rate of the mobile phase was kept at 1.0 mL per min. The detector was set at 355 nm for monitoring panasenoside and kaempferol.

#### 3.3.4. HPLC analysis of Ginsenosides

The ginsenoside contents were determined by HPLC, the same equipment as mentioned before in Section 3.3.3. The prepared sample solution was filtered through a 0.22 μm membrane filter. The injection volume was 10 μL. A Kinetex C18 column (100 mm × 4.6 mm, 2.6 μm; Torrance, CA, USA) was used. The gradient running phase was programmed with the combination of solvent A (water) and solvent B (acetonitrile), in which solvent B was sequentially increased from 17% to 23% (0 to 30 min), 23% to 24% (30 to 35 min), 24% to 32% (35 to 45 min), 32% to 44% (45 to 48 min), 44% to 44% (48 to 52 min), 44% to 55% (52 to 65 min), 55% to 100% (65 to 85 min), and 100% to 100% (85 to 95 min) and finally adjusted from 100% to 17% (95 to 105 min). The operating temperature and flow rate were the same as those in the procedure mentioned in Section 3.3.3. The detector was set at 203 nm for monitoring ginsenosides.

#### *3.4. Determination of Antioxidant Activities*

The ginseng leaf powder (90 mg) was weighed, added to 30 mL of 80% methanol solution in a 50 mL tube, and extracted at 30 ◦C for 30 min with sonication. Then, the sample was centrifuged at 3500 r/min for 15 min. The supernatant was collected and filtered through a 0.22 μm membrane filter. The DPPH radical-scavenging activity was measured, as described by Eom et al. [17] with some modifications. Roughly, 1 mL of the sample solutions at different concentrations were mixed with 3 mL of DPPH solution. After the reaction for 30 min in the dark, the absorbance was measured at 517 nm. Next, the ABTS radical-scavenging ability was measured, as described by Lim et al. [45] with some modifications. Briefly, 0.5 mL of sample solutions at different concentrations were taken in a test tube and 5 mL of the prepared ABTS solution was added. After the reaction at room temperature for 10 min in the dark, the absorbance was measured at 734 nm. IC50 values denote the concentration of the sample, which is required to scavenge 50% of DPPH and ABTS free radicals.
