2.4. HPLC Analysis of Antioxidant Compounds

HPLC analysis (Agilent 1200 series; Agilent Technologies, Santa Clara, CA, USA) of the phenol-rich fraction was conducted according to Kim et al. [17]. CLF1 and CLF2 (5 g) were redissolved in water and then fractionated with a mixture of ethyl acetate and ether (1:1 = v/v). Each fraction was dissolved in methanol (10 mg/mL each) following concentration under reduced pressure.

We applied the modified gradient conditions for the HPLC analysis according to Kim et al. [17]. For qualitative and quantitative analysis, a reverse phase system with synergistic fusion RP column (250 × 4.6 mm, 4 μm; Phenomenex, Torrance, CA, USA) was used at 35 °C. The mobile phase consisted of 0.5% acetic acid in water and acetonitrile. The standard solutions were prepared at a constant volume with methanol and water. The elution program was as follows (B%): 2%, 5 min; 2–5%, 12 min; 5–8%, 17 min; 8–30%, 65 min; 30%, 68 min; 50%, 78 min; 100%, 100 min. The injection volume, flow rate, and wavelength were 10 μL, 1.0 mL/min, and UV 280 nm, respectively. Gallic acid, chlorogenic acid, homogentisic acid, caffeic acid, catechin, ferulic acid, ρ-coumaric acid, naringin, quercetin, and cinnamic acid were used as standards (Sigma-Aldrich). All solutions were filtered through a polyvinylidene difluoride (PVDF, 0.22-μm) membrane (Pall Co., Port Washington, NY, USA).