Myricitrin

The samples collected in May, August, and October were dried and ground into powder. Powder samples (0.5 g) were treated for 30 min by ultrasonic extraction after soaking in petroleum ether (150 mL) for 30 min. The homogenates were filtered, and residues were immersed in 70% alcohol (20 mL) for 30 min and extracted with 70% alcohol by ultrasonic treatment for 30 min. For each extract, 2 mL was dissolved in 3.0 mL of methanol and filtered through a 0.45 µm microporous film. Then, 20 µL of filtrate was run on a Dionex-P680 HPLC (Dionex, Pliening, Germany) system with an Agilent TC-C18 column (250 mm × 4.6 mm, 5 µm, Agilent Technologies Inc., Palo Alto, AR, USA). The mobile phase consisted of acetonitrile (A solvent) and 0.1% phosphate solution (B solvent), used according to the following gradient elution program: 0 min, 0%A/90%B; 15 min, 15%A/85%B; 45 min, 15%A/85%B, 10 min, 25%A/75%B; 15 min, 25%A/75%B and 15 min, 10%A/90%B. The detection wavelength was set at 280 nm, and flow rate was 1 mL/min. The chemical standards included dihydromyricetin (15092141), myricitrin (11060222), and myricetin (14101531), which were purchased from Shanghai Yuanye Biotechnology Co., LTD, Shanghai, China, and quercetrin (20140318), which was purchased from Shanghai Jinsui Biotechnology Co., LTD.

LC-MS-grade methanol was purchased from Merck (Darmstadt, Germany). Formic acid was purchased from Fisher Scientific (LC-MS grade, Thermo Scientific, Carlsbad, CA, USA), and ammonium formate was obtained from Macklin (HPLC grade, purity: ≥99%). Sakuranetin was purchased from Sigma-Aldrich (Sigma-Aldrich, Inc., St. Louis, MO, USA, CAS number: 2957-21-3); quercitrin (CAS number: 522-12-3), myricitrin (CAS number: 17912-87-7), kaempferol (CAS number: 520-18-3), luteolin (CAS number: 491-70-3), rutin (CAS number: 153-18-4), hyperoside (CAS number: 482-36-0), kaempferol-3-O-rutinoside (CAS number: 17650-84-9), catechin (CAS number: 225937-10-0), and catechin gallate (CAS number: 130405-40-2) were purchased from Shanghai Yuanye Bio-Technology Co., Ltd. (Shanghai, China). Ultrapure water was purified using a Milli-Q system (18 MΩ*cm, Millipore, Burlington, MA, USA).

Chemical reagents like ethanol, methanol, acetonitrile, and formic acid were all of chromatographic grade from Thermo Fisher Scientific Inc. (Waltham, MA, USA). Using a Milli-Q Plus instrument from Merk KGaA, 18.2 MΩ.cm water was made (Darmstadt, Germany1). Folin–Ciocalteu and aluminum nitrate were supplied by Solarbio Science & Technology Co., Ltd. (Beijing, China). Chemical standards including chlorogenic acid, ferulic acid, vitexin, rutin, gallic acid, myricitrin, morin, glycitein, wogonin, kaempferol-3-O-sophoroside, liquiritigenin, and butin were obtained from Yuanye Biological Technology Co., Ltd. (Shanghai, China), with >98% purity. The extract was concentrated on Bond Elut PPL 6 cc, 500 mg C18 solid phase extraction (SPE) cartridge that was purchased from Agilent Technology Co., Ltd. (Beijing, China). High-glucose (4.5 g/L) Dulbecco’s modified eagle’s medium (DMEM), which contains 10% (v/v) heat-inactivated fetal bovine serum, 100 µg/mL of streptomycin, and 100 U/mL of penicillin, was obtained from Gibco Laboratories (Carlsbad, CA, USA). Escherichia coli 0111: B4 LPS was a Sigma product (St. Louis, MO, USA).