Flavonoids

Barley seeds were purchased from Jiangsu Yanjiang Institute of Agricultural Sciences, China, in July 2019. Ethyl acetate, linol, methanol, glutamic acid, and sodium hypochlorite were purchased from Sinopharm Chemical Reagent Company (Shanghai, China). methanol (chromatographic grade), glacial acetic acid, acetonitrile, phenolic acids, and flavonoids were purchased from Sigma-Aldrich Chemical Co. (Shanghai, China). Trichloroacetic acid (TCA), 2-thiobarbituric acid (TBA), 3-mercaptopropionic acid (3-MP), 2,2′-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), ATP, nicotinamide adenine dinucleotide phosphate (NADPH), ethylene diamine tetraacetic acid (EDTA), β-mercaptoethanol, coenzyme A (CoA), L-phenylalanine, trolox, GABA, dimethylaminobenzenesulfonyl chloride (derivatizing agent), and polyvinyl pyrrolidone (PVP) were purchased from Maclean Biochemical Technology Co., Ltd. (Shanghai, China). Hydrochloric acid and acetone were purchased from Nanjing Chemical Reagent Co., Ltd. (Nanjing, China).

Flavonoids (rutin and quercetin) were purchased from Sigma-Aldrich, USA. Different concentrations of standard were prepared in 100% methanol. A series of solutions (0, 10, 20, 50, 100, 200 and 300 µg/mL) was prepared to determine the limit of detection (LOD) and linearity of detector (response).

About 100 g of air-dried powder aerial parts of the two samples of H. curassavicum (Coastal and Inland) were extracted in 70% hydro-methanol at room temperature (27 ± 2°C), filtered, and dried under vacuum to give dark black gum (1.6 and 1.85 g, respectively).
The standard phenolic, gallic, cinnamic, chlorogenic, ferulic, coffeic, syringic, ellagic, coumaric acids, vanillin, caffeine, propyl gallate, and flavonoids, quercetin, rutin, catechin, pyrocatechol, naringenin, 4‘.7-dihydroxy isoflavone, were purchased from Sigma-Aldrich (Germany). Trifloroacetic acid and acetonitrile (HPLC gradient grades) were purchased from Sigma-Aldrich (Germany). The used di-distilled water in HPLC was obtained by Hamilton water distillation apparatus (Hamilton Laboratory Glass Ltd., Kent, England).
HPLC analysis was performed using an Agilent 1260 series. The separation was carried out using a C18 column (4.6 × 250 mm i.d., 5 μm). The mobile phase contained water (A) and 0.02% trifloroacetic acid in acetonitrile (B) with a flow rate of 1 mL min⁻¹. The mobile phase was automated successively in a linear gradient as follows: 0 min (80% A), 0–5 min (80% A), 5–8 min (40% A), 8–12 min (50% A), 12–14 min (80% A), and 14–16 min (80% A). The multi-wavelength detector was monitored at 280 nm. The injection volume was 10 μL for each of the sample solutions. The column temperature was maintained at 35 °C.