Sodium chloride (nacl)

Thermus thermophilus strains HB8 (ATCC 27634) and HB27 (ATCC BAA-163) were grown in Thermus medium (TM medium) composed of 4.0% (w/v) BBL™ Polypeptone™ Peptone (Becton–Dickinson, United States), 2.0% (w/v) Bacto™ Yeast Extract (Becton–Dickinson), 1.0% (w/v) sodium chloride (Nacalai tesque, Japan) and 0.1% Castenholz’ basal salt solution under strong aeration in baffled flask (i.e., rotation at 180 rpm) at 70 °C [27 (link), 28 (link)]. TM medium containing silicic acid was prepared using a 1000 ppm sodium orthosilicate stock solution in 10 mM NaOH. Prior to use, the medium was pH-adjusted to 7.2 with HCl and autoclaved [11 (link)]. Hygromycin (100 µg/mL) was added to liquid or agar medium as needed.

Arabidopsis thaliana plants, ecotype “Columbia” (Col-0), were used as the plant material. Plants were grown on mineral nutrient medium (Supplemental Tables S1 and S2) [29 (link)] containing 1% sucrose and 0.8% agarose at 22 °C under constant illumination (40 µmol m⁻² s⁻¹). For NaCl treatment, plants were vertically grown on medium supplemented with 0, 5, 10, 20, 40, or 60 mM NaCl (Sodium Chloride; Nacalai Tesque, Kyoto, Japan). Treatments with KCl (Potassium Chloride; Nacalai Tesque) and NaNO₃ (Sodium Nitrate; Nacalai Tesque) were performed similarly.

Dibasic sodium phosphate, sodium dihydrogen phosphate, D,L-glyceraldehyde, human recombinant aldose reductase (HR-AR), AG, quercetin, critric acid monohydrate, natrium carbonicum, sodium azide, gelatin and sulphuric acid were purchased from Wako Pure Chemical Industries, Ltd. (Osaka, Japan). Sodium bicarbonate, sodium chloride, potassium dihydrogen phosphate and ethanol were supplied by Nacalai Inc. (Kyoto, Japan). Tween 20, bovine serum albumin, glucose, O-phenylenediamine dihydrochloride, phosphate buffered saline, fetal bovine serum (FBS), steroyl myristoyl phosphatidylcholine glycine (SMPC Gly), 3-(4, 5-dimethylthazol-2-yl)-2, 5-diphenyl tetrazolium bromidetetrazolium salt (MTT) were purchased from Sigma-Aldrich company, Ltd. (St. Louis, MO, U.S.A.). NADPH was provided by Oriental Yeast Co., Ltd. (Tokyo, Japan). Anti-AGE antibody and goat anti-mouse IgG, HRP conjugate-secondary antibody Millipore (Merck U.S.A) were purchased from Transgenic Inc. (Hyogo, Japan). Silica gel 60 F₂₅₄ TLC plates (Merck, U.S.A), silica gel 60 N (100–200 μm) and ODS were purchased from Kanto Chemical (Tokyo, Japan). DMSO was purchased from Wako Pure Chemical Industries, Ltd (Japan). Kaempferol-3-O-β-D-glucopyranoside was isolated from liquorice, and was identified by NMR and MS data [11 (link)].

SH end group PVA (PVA-SH: 100% hydrolyzed, average Mw = 198,000) was obtained from Kuraray Co., Ltd., Tokyo, Japan. Sodium p-styrenesulfonate (SSS) was purchased from Tosoh Co., Tokyo, Japan. 2,2′-Azobis(2-methylpropionamidine) dihydrochloride (V-50) and analytical grade glutaraldehyde (GA) (25 wt.% solution in water) were purchased from Wako Pure Chemical Industries, Osaka, Japan. Sodium chloride, hydrochloric acid, and potassium chloride (all analytical grade) were purchased from Nacalai Tesque, Kyoto, Japan.

PEG solution was prepared by dissolving 30 g of polyethylene glycol 6000 (Nacalai Tesque, Inc., Kyoto, Japan) and 14.61 g of sodium chloride (Nacalai Tesque) in 100 mL of distilled water. The mixture was stirred and then autoclaved at 121 °C for 15min, and stored at room temperature until use.

Total lipids were extracted from brown rice powder by using the Folch method^{46 (link)}. Briefly, samples (50 mg) were extracted with 1 mL of a chloroform–methanol mixture (2:1, v/v) at room temperature by shaking in a mixer mill (Retsch MM400) with 300 mg of quartz sand (Wako Pure Chemical Industries, Ltd.) for 2 min at a frequency of 30 Hz and then stirred on a rotator (25 rpm; RT-50, Taitec Co., Saitama, Japan) at room temperature for 20 min. The mixture was centrifuged at 12,000 × g for 3 min and the supernatant was transferred to a new 2-mL microtube (Eppendorf AG). Extraction was repeated with 0.5 mL of the extraction solvent, and the supernatants were combined. Sodium chloride (0.3 mL; 0.9% in ultrapure water; Nacalai Tesque Inc., Kyoto, Japan) was then added, mixed by vortexing, and the sample was centrifuged at 12,000 × g for 3 min. The upper phase was removed and the remaining lower phase was lyophilized in a centrifugal concentrator (CC-105) for 1 h. Methanol (300 μL) was added to the lyophilized lipid extract, and the sample was vortexed, sonicated, and centrifuged at 12,000 × g for 3 min. The supernatant was filtered through a hydrophilic polytetrafluoroethylene membrane filter unit (Millex-LG). An aliquot of the filtrate (5 μL) was used for HPLC analysis to determine the γ-oryzanol content.