Folin cioc lteu reagent

Baobab flour (BF) was acquired in the North of Benin, and wheat flour (WF) type 650 was acquired at the Profil supermarket, Timisoara (Romania).
The reagents used were DPPH solution and HCl (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany), Ethyl alcohol (SC Chimreactiv SRL, Bucharest, Romania), Folin-Ciocâlteu reagent (Sigma-Aldrich Chemie GmbH, Munich, Germany), and Na₂CO₃ (Geyer GmbH, Renningen, Germany). The equipment used in the study was Specord 205 (Analytik Jena AG, Jena, Germany), Chopin Mixolab (Chopin Technologies, Paris, France), the Varian 220 FAA spectrophotometer (Palo Alto, CA, USA), and Thermostat INB500 (Memmert GmbH, Schwabach, Germany).

First, 1 g FW of sample was homogenized in a solution containing 50% acetone and 50% water (1:10). The samples were vortexed and incubated for 15 h at 20 °C. Then, 100 μL of supernatant was mixed with 0.5 mL of Folin–Ciocâlteu reagent (Sigma-Aldrich, Italy) and 6 mL of distilled water. Next, 1.5 mL of Na₂CO₃ (20%) was added, before incubating at 20 °C for 2 h. The absorbance was read at 765 nm. The concentration of total phenolic compounds was calculated as a function of the values obtained from the gallic acid standard curve (0, 50, 100, 250, and 500 mg·L⁻¹) (R² = 0.9954). The total phenolic content was expressed as mg·100 g⁻¹ gallic acid equivalent.
The antioxidant activity was determined using DPPH. About 1 g of fresh weight was mixed with 1.5 mL of methanol solution (80%), sonicated for 30 min, and centrifuged (10 min, 5 °C, 5000× g). Then, 0.01 mL of supernatant was mixed with 1.4 mL of 150 μM DPPH solution in methanol and water (95:5), before incubating for 30 min in the dark. The sample was read at 517 nm. The antioxidant activity was calculated as a function of the values obtained from the Trolox standard curve (0 to 0.5 mg·mL⁻¹) (R² = 0.9995). DPPH scavenging activity values were expressed as Trolox equivalent antioxidant activity (mg TE·100 g⁻¹).

Folin-Ciocâlteu reagent, sodium carbonate, gallic acid, aluminium chloride, sodium acetate, and rutin were purchased from Sigma-Aldrich (Darmstadt, Germany). Flavonoid references used for HPLC-DAD analysis (saponarin—purity ≥ 98%, isoorientin—purity ≥ 99%, and isovitexin—purity ≥ 99%) were obtained from Extrasynthèse (Lyon, France). Human cancerous cell lines (HeLa, A549) and human healthy cell line (WI-38) were obtained from the Laboratory of Medical Chemistry (GIGA Center, University of Liège, Liège, Belgium). All reagents used for cell culture assays were purchased from Bio-Whittaker-Lonza (Verviers, Belgium). Cell proliferation reagent (WST-1) was purchased from Roche (Bâle, Switzerland). Reagents for antioxidant assays were purchased from Sigma-Aldrich (Darmstadt, Germany).

Aluminum chloride (AlCl3), Folin-Ciocâlteu reagent, indomethacin, carboxymethylcellulose, o-phthalaldehyde, Lambda carrageenan type IV, sodium carbonate (Na₂CO₃), ABTS (diammonium 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonate), DPPH (2,2-Diphenyl-1-(2,4,6-trinitrophenyl) hydrazyl), and TPTZ (2,4,6-Tris(2-pyridyl)-s-triazine), were purchased from Sigma–Aldrich (Taufkirchen, Germany). 2-thiobarbituric acid and Bradford reagent were obtained from Merck KGaA (Darmstadt, Germany) and ELISA tests for cytokines (TNF-α, IL-6) were purchased from Elabscience (Houston, TX, USA). The Bradford total protein assay was obtained from Biorad (Hercules, CA, USA). All HPLC reagents and standards were of analytical grade and were acquired from Sigma–Aldrich (Germany) and Decorias (Rediu, Romania), respectively.

The total phenol content was determined by the Folin–Ciocâlteu method with minor modifications [71 (link)]. Thus, a quantity of 0.5 mL was taken from each extract prepared as described above, over which 1.25 mL of Folin–Ciocâlteu reagent (Sigma-Aldrich Chemie GmbH, Munich, Germany) was added, diluted 1:10 with distilled water. The samples were incubated for 5 min at room temperature. After incubation, 1 mL of Na₂CO₃ (Geyer GmbH, Renningen, Germany; 60 g/L aqueous solution) was added. The samples were incubated for 30 min at 50 °C, using the thermostat (INB500, Memmert GmbH, Schwabach, Germany). The absorbance was read at 750 nm using a UV–VIS spectrophotometer (Specord 205; Analytik Jena AG, Jena, Germany). As a control, we used 70% ethanol (Sigma-Aldrich; Merck KGaA, Darmstadt, Germany). The calibration curve was obtained with gallic acid (2.5–250 μg/mL). Results were expressed in mg GAE per kg dry matter (d.m.). All determinations were made in triplicate.

Analytical grade acetone, methanol, ethanol and water (VWR International s.r.l., Milan, Italy) were used for the extraction of phenolic and flavonoid compounds. Trolox (6-hydroxy-2,5,7,8-tetramethilchroman-2-carboxylic acid, 97%), DPPH (2,2-Diphenyl-1-picrylhydrazyl, 95%), ABTS (2,2′-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt, potassium persulphate, potassium acetate, aluminium chloride and Folin-Ciocâlteu reagent were obtained from Sigma-Aldrich (Milan, Italy).
Analytical standard grade vanillic acid (97%), ellagic acid (97%), p-coumaric acid (98%), ferulic acid (99%), quercetin (98%), gallic acid (98%) were also purchased from Sigma-Aldrich (Milan, Italy). HPLC gradient grade methanol and water (VWR International s.r.l., Milan, Italy) was used as chromatographic mobile phase. Formic acid (99%; Sigma Aldrich, Milan, Italy) was used to acidify the aqueous mobile phase, prepared with high-purity water. All samples were filtered through a syringe filters in PVDF, pore size 0.45 µm (Millipore, SER.DIA s.r.l., Reggio Calabria, Italy).

The fermentation involved two probiotic strains, Lactobacillus fermentum (LMG 6902) and Lactobacillus rhamnosus (LMG 25626), which were obtained from BCCM/LMG Bacteria Collection. Dried de Man, Rogosa, and Sharpe (MRS) growth media were acquired from HIMEDIA (Einhausen, Germany).
The commercial soy beverage originated by Dennree (Allemagne, Germany), was purchased from a food store (Cluj-Napoca, Romania). The raw materials of the soy beverage were water and 8% soy from ecologic farming, having 1.5% fat, 0.9% carbohydrates, and 3% proteins. The product was UHT pasteurized. Xylitol, and inactivated dried organic Chlorella vulgaris powder were acquired from specialized stores in Cluj-Napoca, Romania. The country of origin this powder was China, having 60% proteins, 12% fibers, 9.5% carbohydrates, and 7.9% fat.
The enzymes used for the simulation of gastrointestinal digestion, pepsin from porcine gastric mucosa (P6887), pancreatin from porcine pancreas (P7545), and bovine bile extract (B8631), were acquired from Sigma-Aldrich (Taufkirchen, Germany). The chemicals for the biological characterization, DPPH (1,1-diphenyl-2-picrylhydrazyl), Trolox, and Folin–Ciocâlteu reagent were also purchased from Sigma-Aldrich. All the materials and chemicals used in the experiment were of analytical grade.