Rhizobioside

All reagents, HPLC-grade phenolic standards (proanthocyanidins, catechins, epicatechin, chlorogenic acid, rutin, rhizobioside, quercetin, quercitrin, P-hydroxybenzoic acid, quercetin triglycoside kaempferol glycoside, ferulic acid, quercetin rhamnoside and P-coumaric acid) were also purchased from Sigma–Aldrich (St. Louis, MO, USA). Methanol, acetonitrile and formic acid were purchased from Tedia Company, Inc. (Fairfield, CT, USA). Folin-Ciocalteu reagent and all other chemicals were obtained from Sinopharm Chemical Reagent Co. (Shanghai, China).

Protocatechin, catechin, epicatechin, rhizobioside, quercetin, centaureidin-3-O-galactoside, and centaureidin-3-O-glucoside standards were purchased from Sigma-Aldrich (St. Louis, MO, USA). Chromatography-grade methanol was purchased from Tedia (USA). All flavonoid standard stock solutions (1 mg ml^–1) were stored at a low temperature and protected from light, and the standard curve was constructed using the external standard method. High-performance liquid chromatography (HPLC, USA, Agilent, 1260) was performed under the following conditions: the chromatographic column was an XDB-C18 column (150 × 4.6 mm, 5 μm); the mobile phase A was 1% aqueous acetic acid solution and the mobile phase B was methanol solution; the elution program was 0–10 min, 5–30%, 10–25 min, 30–50%, 25–35 min, 50–70%, and 35–40 min, 70–5% (all mobile phase B); the flow rate was 0.8 ml/min; the sample volume was 20 μl; the column temperature was 30°C; and the wavelength was 280 nm. Qualitative and quantitative analyses of flavonoids in ‘XJ4’ RAFE were performed according to the above methods and conditions.