Rutin

Chromogenic substrate S-2238 was purchased from Chromogenix (Milan, Italy); bovine thrombin and argatroban were purchased from Sigma-Aldrich (St. Louis, MO, USA). The phosphate buffer saline (PBS) (pH 7.2-7.4, 0.01 M) and dimethyl sulfoxide (DMSO) were purchased from Solarbio (Beijing, China). Standard substance of galangin, kaempferol, astragaline, fisetin, quercetin, quercitrin, isoquercitrin, rutin, isorhamnetin, isorhamnetin-3-O-neohespeidoside, typhaneoside, myricetin, myricitrin, baicalein, baicalin, wogonin, wogonoside, hispidulin, scutellarein, genkwanin, hydroxygenkwanin, luteolin, luteoloside, 6-methoxyluteolin, dihydroquercetin, dihydromyricetin, naringenin, naringin, narirutin, hesperetin, hesperidin, neohesperidin, L-epicatechin, (-)-epigallocatechin, (-)-epicatechin gallate, proanthocyanidin B1 with 98% purity on the basis of HPLC analysis were obtained from Chengdu Herbpurify Co., Ltd. (Chengdu, China). Herbacetin and morin were obtained from Chengdu Must Bio-Technology Co., Ltd. (Chengdu, China). chrysin, apigenin, diosmetin and kaempferol-7-O-β-d-glucopyranoside were purchased from Chengdu Desite Bio-Technology Co., Ltd. (Chengdu, China).

Fresh leaf samples (0.2–0.5 g) were ground into powder in liquid nitrogen, then immersed in acidified methanol (0.3% HCl (v/v), 5 mL) at 4 °C for 24 h in the dark, and then centrifuged (5000× g) for 20 min at low temperature (4 °C). Supernatant was assayed for measurements of the total anthocyanidin by using an ultraviolet-visible spectrophotometer (UV-6000PC, Shanghai Metash, Shanghai, China) at 530 nm. A colorimetric method was used to analyze the total flavonoid content as described by Ren et al. [25 (link)]. Rutin (Chengdu Herbpurify Co., Ltd., Chengdu, China) solution was used as standard. The JA content in leaves was measured using a quantitative enzyme immunoassay kit (Meimian Biotech Co., Ltd., Yancheng, China). For measurement of anthocyanidin, total flavonoid, and JA content, each sample was replicated three times. ANOVA with a Student–Newman–Keuls test was performed in IBM SPSS Statistics (version 20.0 software for Windows; SPSS, Chicago, IL, USA) to analyze the data.