Glycitin

Extraction and HPLC analyses of isoflavones and kaempferol glycosides were performed using previously published methods (Lim et al., 2021 (link)). The standards of eight isoflavone—two aglycones (daidzein and genistein; LC Laboratories, Woburn, MA, USA), three β-glycosides (daidzin, glycitin, genistin; LC Laboratories), and three malonyl glycosides (malonyl daidzin, malonyl glycitin, malonyl genistin; GenDEPOT, Katy, Texas, USA)—were used with a serial concentration (2.5, 5, 10, and 20 mg/L) to calculate the standard curve for quantifying isoflavones in HPLC analysis based on an external standard method. Kaempferol glycosides were quantified with robinin (kaempferol-3-O-robinoside-7-O-rhanmoside), a kaempferol triglycoside with λmax 265.0 and 347.1 nm. The robinin standard isolated from kudzu and purified as described in Eom et al. (2018) (link) was used with a serial concentration (1.25, 2.5, 5, and 10 mg/L) to calculate the standard curve for quantifying kaempferol glycosides in this experiment.

The standards for anthocyanins comprised delphinidin 3-O-glucoside chloride, petunidin 3-O-glucoside chloride, cyanidin chloride (Extrasynthese, Genay, France), and cyanidin 3-O-glucoside chloride (SinoStandards Bio-Tech Co., Ltd., Chengdu, China). The standards for flavan-3-ols consisted of catechin hydrate (Sigma Aldrich Co., St Louis, MO, USA) and epicatechin (Chemfaces, Wuhan, China). The standards for isoflavones comprised daidzein, glycitein, genistein, daidzin, glycitin, genistin (LC Laboratories, Woburn, MA, USA), acetyl daidzin, acetyl glycitin, acetyl genistin (Nacalai Tesque, Kyoto, Japan), malonyl daidzin, malonyl glycitin, and malonyl genistin (GenDEPOT, Katy, TX, USA).

Among 56 phenolic compounds, isoflavonoids, phenylpropanoids, and stilbenoid standards were purchased from Sigma-Aldrich, Inc. (St. Louis, MO, USA), along with dimethyl sulfoxide (99.9%). The anthocyanin standards were purchased from Extrasynthese Impasse Jacquard, (Rue Jacquard, Genay, France). Biochanin A, daidzein, daidzin, formononetin, genistein, genistin, and glycitin were obtained from LC Laboratories (New Boston St. Woburn, MA, USA), while glycitein was purchased from Wako Pure Chemical Industries, Ltd. (Chuo-ku, Osaka, Japan), and polydatin (Piceid) and trans-resveratrol was obtained from Cayman Chemicals (Ann Arbor, MI, USA). HPLC grade methanol, absolute ethanol, and acetonitrile were purchased from Fisher Scientific (Seongnam-Si, Gyeonggi-Do, Korea), while formic acid (>98%) was obtained from Junsei Chemical Co., Ltd. (Chuo-ku, Tokyo, Japan). A 0.1 N standard solution of hydrochloric acid was purchased from Dae-Jung Chemicals & Metals Co., Ltd. (Shiheung-city, Gyeonggi-do, Korea), and deionized water with a resistivity of 18.2 MΩ was obtained from a PURELAB Option-Q System (Lane End, High Wycombe HP14 3BY, UK).