Phytic acid

Chitosan (deacetylation degree ≥ 95%; viscosity, 100 to 200 mPa·s; Aladdin), sodium dihydrogen phosphate (Aladdin), sodium tripolyphosphate (Aladdin), gelatin (porcine skin, type A, 300 Bloom, Sigma-Aldrich), phytic acid (Sigma-Aldrich), and other chemicals (Sinopharm Chemical Reagent Co. Ltd.) were purchased and used without further purification.

Chitosan (medium molecular weight, 85% deacetylation), RB, aniline, phytic acid [50 weight % (wt %) in water], APS, PCL, CSA, hexafluoroisopropanol, acetic acid, and phosphate buffer tablets were all purchased from Sigma-Aldrich and used without further purification. Chemicals for the ex vivo experiments were purchased from either VWR International or Sigma-Aldrich.

Enzymatic hydrolysis of phytic acid (Sigma, Poznań, Poland) was performed as described elsewhere [14 (link)] with modifications. The reaction was conducted in a total volume of 5 mL containing 100 mg of phytate and 50 mg of wheat phytase (Sigma, Poznań, Poland) at a pH of 5.15 and a temperature of 55 °C. The reaction was stopped after two hours by heating (100 °C for 5 min). Next, the obtained hydrolysate of phytic acid (hPA120) was cooled down on ice and precipitated enzymatic proteins were centrifuged (12,000× g, 10 °C, 10 min). The supernatant was collected, filter sterilized (filter pore 0.22 µm), aliquoted, and stored at −20 °C. Directly before use, the hydrolysate was defrosted and diluted twice with McCoy 5A medium without antibiotics. Before applying onto colonocytes, the hPA120 was 10-fold diluted in a medium appropriate for investigated cell lines.
The composition of inositol phosphates in phytate hydrolysate was determined by applying HPLC-MS (LC-20, Shimadzu, Kyoto, Japan; QTRAP 5500 mass spectrometer, AB SCIEX, Vaughan, Canada) and identified using real standards comprised the retention time and the presence of the respective parent and daughter ion (negative) pairs (Table S1) [15 (link)].

The antibodies to GPX4 and β-actin were obtained from R&D systems (Abingdon, UK) and Thermo Scientific (Dartford, UK) respectively. Curcumin, quercetin, rutin, EGCG, tannic acid and phytic acid were obtained from Sigma-Aldrich Company Ltd. (Dorset, UK) (Curcumin cat. no. C7727; EGCG cat. no. E3768). Erastin was purchased from Bertin Bioreagent (Montigny-le-Bretonneux, France).

Hexane, acetone, ethanol, methanol, HPLC-grade methanol, sodium carbonate, 2,20-azino-bis (3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 2,2-diphenyl1-picrylhydrazyl (DPPH), Folin–Ciocalteu reagent, gallic acid solution, hydrochloric acid, sodium nitrite, aluminum chloride, sodium hydroxide, iron (III) chloride, sulfosalicylic acid, phytic acid and potassium persulfate were purchased from Sigma Aldrich (Schnelldorf, Germany). The phenolic acid (gallic acid, vanillic acid, chlorogenic acid, caffeic acid, ferulic acid) and flavonoid (catechin, epicatechin, quercetin, and kaempferol) standards were purchased from Sigma Aldrich (Schnelldorf, Germany).

DHA-95E, an ethyl all cis-4,7,10,13,16,19-docosahexaenoic acid, was obtained from Harima Chemicals (Tokyo, Japan). Tricosanoic acid, linoleic acid, α-linolenic acid, arachidonic acid, eicosapentaenoic acid and other fatty acids, phytic acid, bipyridine were purchased from Sigma Chemical Co. (St Louis, MO, USA). All other materials of the highest grade were obtained commercially.

Yeast strain, the vector, and antibiotics were supplied from Invitrogen (USA). Phytic acid and sodium salt were purchased from Sigma-Aldrich (USA). Sodium acetate, sodium carbonate, sodium thiosulphate, ammonium heptamolybdate tetrahydrate, sulfuric acid 95–98%, glacial acetic acid, acetone, and glycine were supplied from Merck-Millipore (Germany). The plasmid DNA extraction kit, yeast genomic DNA extraction kit, and RNA extraction kit were obtained from SinaClon (Iran). Restriction enzymes were purchased from Thermo Scientific (USA). Commercial phytase was from Novozyme (Denmark).