Bovine hemoglobin

Insulin receptors are highly expressed in the brain, mainly distributed in the cortex, hippocampus, and hypothalamus [31 (link)]. Therefore, we chose the mouse hippocampal neuron HT22 cells. The cell lines were purchased from Shanghai Institute of Cell Biology, Chinese Academy of Sciences. The culture condition was DMEM medium containing 10% fetal bovine serum. Cells were incubated in incubators at 37°C and 5% CO₂. Bovine hemoglobin (Sigma, USA) was dissolved in complete medium (25 μmol/L) and incubated to establish SAH model in vitro.

Standards of phenolic acids and flavonoids (apigenin, 2,3-dihydroxybenzoic acid, caffeic acid, catechin, chlorogenic acid, chrysin, ferulic acid, galangin, gallic acid, hesperidin, m-hydroxybenzoic acid, p-hydroxybenzoic acid, 5-hydroxyferulic acid, kaempferol, methyl p-coumarate, morin, myricetin, naringenin, naringin, p-coumaric acid, pinocembrin, quercetin, quercitrin, rosmarinic acid, rutin, salicylic acid, salicylic acid glucoside, sinapic acid, syringic acid, trans-cinnamic acid, vanillic acid, p-coumaric acid-d₆, and salicylic acid-d₄) were purchased from Sigma-Aldrich (Steinheim, Germany).
The sodium molybdate dihydrate, sodium nitrite, sodium hydroxide, sodium carbonate, hydrogen peroxide, sodium ascorbate, dimethyl sulfoxide (DMSO), and bovine hemoglobin were also purchased from Sigma-Aldrich (Steinheim, Germany). The LC (liquid chromatography) grade methanol, analytical grade orthophosphoric acid, hydrochloric acid, aluminum chloride, sodium acetate, ethanol, and Folin–Ciocalteu reagent were purchased from Merck (Darmstadt, Germany). The DPPH (2,2-diphenyl-1-picrylhydrazyl) was obtained from Alfa-Aesar (Karlsruhe, Germany). All spectrophotometric data were acquired using a Jasco V-530 UV-Vis spectrophotometer (Jasco International Co., Ltd., Tokyo, Japan).

P. lutzii (American Type Culture Collection—ATCC MYA-826—Pb01) and P. brasiliensis (ATCC 32069—Pb18) were maintained in Brain Heart Infusion (BHI) solid broth supplemented with glucose 4% (w/v) at 36 °C. To obtain cell wall proteins extracts, P. lutzii yeast cells were cultured in BHI liquid medium, supplemented with glucose 4% (w/v) for 72 h at 36 °C and under shaking at 120 rpm. Next, 5 × 10⁶ cells/mL were transferred to McVeigh and Morton modified medium (MMcM) chemically defined liquid medium [35 (link)] without iron, supplemented with 50 µM of the iron chelator bathophenanthrolinedisulfonic acid (BPS—Sigma-Aldrich, St. Louis, MO, USA) and were maintained in this condition for 36 h in order to establish intracellular iron depletion. Finally, yeasts cells were transferred to MMcM containing bovine hemoglobin 10 µM (Sigma-Aldrich, St. Louis, MO, USA) or BPS 50 µM and maintained for 48 h.

All the reagents used were of analytical grade. Cocaine, 7-nitroindazole, and other chemicals, sucrose, Tris, DL-dithiothreitol, phenylmethylsulfonyl fluoride, potassium phosphate, calcium chloratum (CaCl₂), magnesium chloratum (MgCl₂), L-arginine, L-valine, bovine hemoglobin, beta-nicotinamide adenine dinucleotide 2′-phosphate reduced tetrasodium salt (NADPH), ethylenediaminetetraacetic acid (EDTA), bovine serum albumin (fraction V), reduced glutathione (GSH), oxidized glutathione (GSSG), glutathione reductase (GR), cumene hydroperoxide, Percoll, and MTT (3-[4,5-dimethylthiazol-2-yl]-2,5-diphenyltetrazolium bromide), were purchased from Sigma Chemical Co. (Taufkirchen, Germany). 2,2′-Dinitro-5,5′-dithiodibenzoic acid (DTNB) and D-glucose were obtained from Merck (Darmstadt, Germany). Dimethyl sulfoxide (DMSO) was purchased from Valerus (Sofia, Bulgaria).

Bovine hemoglobin was obtained from Sigma Chemical (Sigma, China). Hemoglobin concentration was calculated on the basis of the hemin monomer. Hemoglobin solutions were filter sterilized with 0.45-μm Millipore filters. E. coli strains were grown on LB medium (Sigma-Aldrich, Product Number: L3522) aerobically at 37 °C. The solid media contained 1.5% Difco agar. R. anatipestifer strains were cultured on LB agar supplemented with 5% sheep blood or in TSB liquid medium (Solarbio, China) at 37 °C. When necessary, the medium was supplemented with appropriate antibiotics at the following concentrations: ampicillin (Amp), 100 μg/ml; kanamycin (Kan), 50 μg/ml; cefoxitin (Cfx), 1 μg/ml; erythromycin (Erm) 1 μg/ml; and spectinomycin (Spec), 60 μg/ml.

Maltodextrin, bovine hemoglobin, L-leucine, L-tyrosine, pepsin from porcine gastric mucosa, bovine serum albumin (BSA), Folin–Ciocalteu phenol reagent, and 2,4,6-trinitrobenzenesulfonic (TNBS) acid were purchased from Sigma-Aldrich (St Louis, MO, USA). Acetone, HCl, sodium sulfite, and sodium hydroxide were purchased from LabScan (RCI Labscan Ltd., Bangkok, Thailand). Trichloroacetic acid (TCA), potassium sulfate (K₂SO₄), sodium chloride (NaCl), magnesium chloride (MgCl₂), and lithium chloride (LiCl) were procured from Loba Chemi (Mumbai, Maharashtra, India). Electrophoresis chemicals were acquired from BioRad (Richmond, VA, USA). Whey protein isolate was purchased from ProflexTM (Power Corp. Co., Ltd., Bangkok, Thailand). Threadfin bream (24–48 h after capture) were bought from a local market. Kidney bean protein isolate (KBPI) was prepared as described by Gulzar et al. [15 (link)] with the aid of the pH shift process.