Pepsin from porcine gastric mucosa

Individual polyphenol standard, green tea Catechin mix (G-016): ((+)—Catechin, (−)—Catechin-3-gallate, (−)—EpiCatechin, (−)—EpiCatechin-3-gallate, (−)—EpigalloCatechin-3-gallate, (−)—GalloCatechin, (−)—GalloCatechin-3-gallate), pancreatin from porcine pancreas (P1750), pepsin from porcine gastric mucosa (P6887), and bile from bovine and ovine (B8381) were purchased from Merck (Darmstadt, Germany). Methanol, ethyl acetate, acetone, hexane, and acetonitrile with HPLC grade were purchased from PanReac ApliChem (Barcelona, Spain). Kreon 25,000 U was purchased in a pharmacy with the corresponding permission. Six monoglycosides mixture (pelargonidin 3-glucoside, cyanidin 3-glucoside, peonidin 3-glucoside, delphinidin 3-glucoside, petunidin 3-glucoside, and malvidin 3-glucoside), malvidin, and malvidin 3,5-diglucoside were purchased from Biolink Group-Polyphenols AS (Sandnes, Norway). All other reagents were purchased from PanReac ApliChem (Barcelona, Spain).

Pepsin from porcine gastric mucosa, porcine bile extract, and pancreatin and lipase from porcine pancreas were obtained from Sigma (St. Louis, MO, USA). Solvents (HPLC-grade) were provided by Romyl (Teknokroma, Barcelona, Spain), and the purified water was obtained from a Milli-Q water purification system (Millipore, Milford, MA, USA).

Ethanol 96% (v/v) extra pure, sodium bicarbonate (NaHCO₃), magnesium chloride hexahydrate (MgCl₂·6H₂O), ammonium carbonate ((NH₄)₂CO₃), and hydrochloric acid (HCl) 37% (v/v) were purchased from Scharlau (Barcelona, Spain). Potassium chloride (KCl), potassium phosphate monobasic (KH₂PO₄), sodium chloride (NaCl), calcium chloride dihydrate (CaCl₂·2H₂O), and sodium hydroxide (NaOH) 1M were purchased from Panreac (Barcelona, Spain). Alpha-amylase used for in vitro simulated gastrointestinal digestion was purchased from MP Biomedicals (Aurora, Ohio, USA), and pepsin from porcine gastric mucosa, pancreatin from porcine pancreas (8USP), and bile bovine were purchased from Sigma Aldrich (Steinheim, Germany).

The dried HBJ flowers (300 g, moisture content was ca. 10%) were harvested from Tongxiang, Zhejiang province, China. The obtained samples were ground into powders, which were selected and divided into five sieve fractions (850, 425, 250, 180, and 150 μm corresponding to 20, 40, 60, 80, and 100 meshes, respectively). The representative external standards with the purity of 98% were all provided by Chengdu Must Bio‐Technology Co. Ltd., namely, 3‐CQA, 4‐CQA, 5‐CQA, 3,4‐CQA, 3,5‐CQA, 4,5‐CQA, linarin, apigenin, acacetin, rutin, luteolin, apigenin‐7‐O‐glucoside, and luteolin‐7‐O‐glucoside. Acetonitrile (ACN) and methanol were purchased from Merck of chromatographic grade. Hydrochloric acid (HCl), ethyl acetate, acetone, potassium peroxodisulfate (K₂S₂O₈), ethyl alcohol, acetic acid, monopotassium phosphate (KH₂PO₄), disodium hydrogen phosphate (Na₂HPO₄), and ferric chloride with analytical grade were bought from Sinopharm Chemical Reagent Co., Ltd. The ultrapure water (18.2 MΩ•cm) was produced using a Milli‐Q system (Millipore). α‐Amylase, pepsin from porcine gastric mucosa (enzymatic activity of 250 U/mg protein), and pancreatin from porcine pancreas (enzymatic activity of 24 U/mg of lipase) were purchased from Sigma Chemical Co.

Quinoa was planted in the open field at Wanggeertang town (East longitude 102.847758, North latitude 35.216012, Altitude 2500 m), Xia he County, Gan nan Tibetan Autonomous Prefecture, China in May. Commercial mature quinoa was harvested in September of the same year. Within 3 h of mechanical shelling, quinoa was packed in woven bags and transported to the laboratory. Samples were stored at 4 °C for later use. Pepsin from porcine gastric mucosa (power, ≥250 units/mg solid) was purchased from Sigma-Aldrich (St. Louis, MO, USA). Pepsin (Potency: 1:3000) was purchased from Shanghai Yuanye Biological Technology Co., Ltd., (Shanghai, China). The rest of the chemicals used are of analytical grade.

Chitosan was obtained from Tokyo Chemical Industry, while WPI and MCTs were provided by Shanghai Yuanye Bio-Technology Co., Ltd. EO was purchased from J&K Scientific Ltd. CO was purchased from Innochem Co., Ltd. Apigenin, glycerol, and mucin from the porcine stomach, pepsin from porcine gastric mucosa (250 units/mg solid), bile salts and lipase from porcine pancreas (100–650 units/mg protein) were purchased from Sigma-Aldrich (Sigma Chemical Co. St. USA). All the solvents and reagents were of analytical grade. The double-distilled water used in this study was obtained from a water purification system (Direct-Pure-EDI 15UV).