Catechin

Methanol, acetone, acetic acid (glacial) 100%, 2-thiobarbituric acid (TBA), hydrochloric acid ACS (37%), 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ), butylated hydroxyanisole (BHA), and 3,5-Di-tert-4butylhydroxytoluene (BHT) were purchased from Merck (Darmstad, Germany). Iron (III) chloride (97%), sodium acetate anhydrous (≥99%), sodium phosphate monobasic monohydrate ACS (≥98%), sodium phosphate dibasic ACS (≥99%), sodium carbonate ACS (≥99.5%), Folin and Ciocalteu’s phenol reagent 2 N, trichloroacetic acid (TCA) ACS (≥99%), 1,1,3,3-Tetraethoxypropane (≥96%), gallic acid, flourescein sodium salt, (±)-6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid (Trolox), 2,2′-azobis(2-methyl-propionamidine) dihydrochloride (AAPH), phenolic acids (gallic, syringic, ferulic, chlorogenic, caffeic, and p-coumaric), and flavonoids (catechin, rutin, quercetin, luteolin, kaempferol, epicatechin, myricetin, and isorhamnetin) were purchased from Sigma-Aldrich (St. Louis, MO, USA)

BGH was kindly supplied by Talingchan Bean Sprouts (Bangkok, Thailand). Analytical-grade reagents of gallic acid, (+)-catechin, 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), caffeic acid, ferulic acid, gentisic acid, protocatechuic acid, syringic acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), Folin–Ciocalteu phenol reagent, 2,2-azinobis-3-ethylbenzothiazoline-6-sulfonic acid (ABTS), 2,4,6-tri(2-pyridyl)-s-triazine (TPTZ), 3,5-dinitrosalicylic acid (DNS), vitexin, diosgenin, p-nitrophenyl α-D-glucopyranoside (pNPG), and porcine pancreatic α-amylase (α-Amy) (EC 3.2.1.1, type VI) were purchased from Sigma Chemical Co. (St. Louis, MO, USA). Analytical-grade solvents of Acarbose and α-glucosidase (α-Glu) were products from Thermo Scientific Chemicals Co. (Shanghai, China) and Megazyme Ltd. (Bray, Ireland), respectively. HPLC-grade solvents, including glacial acetic acid, acetonitrile, and sulfuric acid, were purchased from QRëc chemical (Auckland, New Zealand). Other chemicals involving starch-soluble NaNO₂, Na₂CO₃, NaOH, vanillin, AlCl₃, FeCl₃, NaCl, MgCl₂, CaCl₂, K₂S₂O₈, D-glucose, and CH₃COONa were analytical-grade reagents from KemAus Co. (Cherrybrook, Australia).

In this study, most of the chemicals, reagents, and standards were analytical grade and purchased from Sigma-Aldrich (Castle Hill, NSW, Australia). Gallic acid, L-ascorbic acid, vanillin, hexahydrate aluminium chloride, Folin-Ciocalteu’s phenol reagent, sodium phosphate, iron(III) chloride hexahydrate (Fe[III]Cl₃.6H₂O), hydrated sodium acetate, hydrochloric acid, sodium carbonate anhydrous, ammonium molybdate, quercetin, catechin, 2,2′-diphenyl-1-picrylhy-drazyl (DPPH), 2,4,6tripyridyl-s-triazine (TPTZ), and 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS) were purchased from the Sigma-Aldrich (Castle Hill, NSW, Australia) for the estimation of polyphenols and antioxidant potential. Sulfuric acid (H₂SO₄) with 98% purity was purchased from RCI Labscan (Rongmuang, Thailand). HPLC standards including Gallic acid, p-hydroxybenzoic acid, caftaric acid, caffeic acid, protocatechuic acid, sinapinic acid, chlorogenic acid, syringic acid, ferulic acid, coumaric acid, catechin, quercetin, quercetin-3-galactoside, diosmin, quercetin-3-glucuronide, epicatechin gallate, quercetin-3-glucoside, kaempferol and kaempferol-3-glucoside were produced by Sigma-Aldrich (Castle Hill, NSW, Australia) for quantification proposes. HPLC and LC-MS grade reagents including methanol, ethanol, acetonitrile, formic acid, and glacial acetic acid were purchased from Thermo Fisher Scientific Inc. (Scoresby, VIC, Australia). To perform various in vitro bioactivities and antioxidant assays, 96 well-plates were bought from the Thermo Fisher Scientific (VIC, Australia). Additionally, HPLC vials (1 mL) were procured from the Agilent technologies (VIC, Australia).

The anthocyanins were determined using high-performance liquid chromatography (HPLC) as previously described (Qi et al., 2013 (link)). For extraction of other flavonoids, freeze-dried flowers were finely ground and 50mg was extracted in 500 μl of MeOH for 48h at 4 °C in darkness. After samples were centrifuged, the supernatants were transferred to fresh tubes and the pellet was resuspended and incubated in 500 μl of 1% MeOH at 4 °C for 24h, and then the supernatant was combined for further HPLC analysis. HPLC was performed as previously described (Qi et al., 2013 (link)). Cyanidin, cyanidin-galactoside, dihydroquercetin, dihydrokaempferol, (+)-catechin, (–)-epicatechin, luteolin, naringenin, and quercetin were obtained from Sigma-Aldrich China (Shanghai). Standards of afzelechin, (–)-epiafzelechin, (+)-gallocatechin, and (–)-epigallocatechin were purchased from BioBioPha (Yunnan, China). The delphinidin chloride (ChromaDex, Santa Ana, CA, USA), petunidin chloride (ChromaDex), and other flavonoids such as dihydromyricetin (YiFang S&T, Tianjin, China) equivalents were used as standards for quantification. Mean values and SDs were obtained from three biological replicates.

For the phytochemical analysis by LC-MS/MS, a number of 25 polyphenols were used as standards. The following standards were purchased from Sigma (Sigma-Aldrich Chemie GmbH, Schnelldorf, Germany): caftaric acid (≥97%), chlorogenic acid (≥95%), caffeic acid (≥98%), p-coumaric acid (≥98%), rutin (≥94%), apigenin (≥95%), kaempferol (≥97%), luteolin (≥98%), gentisic acid (≥98%), myricetol (≥96%), fisetin (≥98%), (+)-catechin (≥96%), (−)-epicatechin (≥90%), quercetin (≥95%), quercitrin (quercetin 3-rhamnoside) (≥78%), isoquercitrin (quercetin 3-d-glucoside) (≥98%), hyperoside (quercetin 3-d-galactoside) (≥97%), patuletin (≥98%), protocatechuic acid (3,4-dihydroxybenzoic acid) (≥97%), syringic acid (≥95%), vanillic acid (≥97%), rosmarinic acid (≥96%). Gallic acid (≥98%) was acquired from Merck (Darmstadt, Germany). Ferulic acid (≥99%) and sinapic acid (≥98%) were obtained from Roth (Karlsruhe, Germany). Solvents used for extraction and separation were of HPLC analytical-grade (methanol, ammonium acetate, acetonitrile) or analytical-grade (petroleum ether, chloroform, hydrochloric acid, acetic acid, potassium hydroxide). These solvents and Folin-Ciocâlteu reagent were purchased from Merck (Darmstadt, Germany). 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Alfa-Aesar (Karlsruhe, Germany).