Folin denis reagent

P-cresol (Acros Organics, NJ, 99+%) was dissolved in methanol. For the detection of tri-p-cresol phosphate (TCP, Pfaltrz & Bauer, CT) in solution, it was dissolved in methanol. Folin–Denis Reagent was purchased from Sigma-Aldrich (St. Louis, MO, USA). All D.I. water used was from a Millipore Direct-Q water purification system (EMD Millipore, Bedford, MA, USA, with a resistivity of 18 MΩcm⁻²).

Methyl alcohol 99.9%, n-hexane 95%, chloroform 99%, n-butanol 99%, dimethyl sulfoxide (DMSO) 99.7%, aluminum chloride 99.9%, sodium acetate >99%, quercetin, acetone >99.5%, picric acid ≥98.0%, dextrose 99.5%, sodium carbonate 99.5%, phosphate buffered saline (pH 7.2 at 25 °C), potassium ferricyanide (III), trichloroacetic acid, α-Cyano-4-hydroxycinnamic acid (for MALDI-TOF MS), acetonitrile ≥99.0%, iron (III) chloride 99%, ascorbic acid ≥99%, sulfuric acid 99.9%, sodium phosphate 96%, ammonium molybdate, potassium persulfate ≥99.0%, 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) diammonium salt (ABTS), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox) 97%, Folin–Denis’ reagent, gallic acid, tannin and chemical standards were purchased from Sigma-Aldrich.
EMEM and DMEM incubation media, L-glutamine, essential amino acids mixture, penicillin-streptomycin 100x lyophilized mixture, 0.25% trypsin solution and 0.02% versene solution were purchased from PanEco, Ltd. (Moscow, Russia), fetal bovine serum—from BioWest (Nuaillé, France), and MTT (3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyl-tetrazolium bromide)—from Diaem, Ltd. (Moscow, Russia).

The total phenolic content of the sample was determined by the Folin–Denis assay, as described previously [8 (link)]. Briefly, 20 μL of sample or gallic acid (Sigma-Aldrich, Singapore) were mixed with 100 μL of Folin–Denis reagent (Sigma-Aldrich, Singapore) and 1880 μL of 7.5% aqueous sodium bicarbonate solution for 30 min at room temperature. The reaction was measured at 765 nm. The total phenolic content was calculated and the activity compared with the gallic acid standard curve. The result was expressed as mg gallic acid equivalent/g sample.

The total polyphenol content was measured following the method described by Fedeli et al. [68 (link)]. Briefly, 100 mg of ground samples were homogenized in 4 mL of 70% (v/v) acetone (Carlo Erba, Cornaredo, Milan, Italy) and then centrifuged at 4000 rpm (PK110 centrifuge, Alc International S.r.l., Cologno Monzese, Milan, Italy) for 5 min. The supernatant (0.5 mL) was added to 3 mL of deionized water, 0.125 mL of Folin–Denis reagent (Sigma-Aldrich), 0.750 mL of saturated Na₂CO₃ (Carlo Erba, Cornaredo, Milan, Italy), and finally 0.950 mL of deionized water. Samples were placed in an oven at 37 °C for 30 min; afterward, they were centrifuged at 4000 rpm (PK110 centrifuge, Alc International S.r.l., Cologno Monzese, Milan, Italy) for 5 min, and their absorbance was read at 765 nm using a UV-Vis spectrophotometer (8453, Agilent, Santa Clara, CA, USA). For the quantification, the absorbance of the samples was referred to a calibration curve (5–20 µg mL⁻¹) of gallic acid (Sigma-Aldrich, USA) used as standard. Results were expressed as mg of gallic acid equivalent on a dry weight basis (mg GAE g⁻¹ dw).

Folin-Denis reagent, sodium carbonate, aluminum chloride, potassium acetate, potassium persulfate, 1,1-diphenyl-1-picrylhydrazyl (DPPH), 2,2′-azinibis 3-ethyl benzothiazoline-6-sulfonic acid (ABTS), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), iron(III) chloride hexahydrate, gallic acid, acetic acid, lipopolysaccharide (LPS), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), ascorbic acid (Vit C), and quercetin were purchased from Sigma-Aldrich (St. Louis, MO, USA). Iron (II) sulfate heptahydrate (FeSO₄) was purchased from Junsei (Tokyo, Japan). Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), phosphate-buffered saline (PBS), penicillin-streptomycin (P/S), and trypsin-EDTA were obtained from Gibco (Waltham, MA, USA). The xanthine oxidase (XO) assay kit was purchased from Abcam (Cambridge, MA, USA). The other reagents used were of analytical grade.

Total phenolics content (TPC) of each extract was determined using the Folin–Denis method [37 (link)]. Briefly, 160 μL of sample was dissolved in EtOH (5, 10 mg/mL) and mixed thoroughly with 160 μL of Folin–Denis reagent (Sigma-Aldrich Co., St. Louis, MO, USA) for 3 min at RT. After adding 160 μL of 10% (w/v) sodium carbonate, the mixture was allowed to stand at RT for a further 60 min in the dark. After centrifugation at 10,000 rpm for 10 min, the supernatant (100 μL) was collected and its absorbance was measured at 700 nm. A standard curve was prepared using gallic acid (Sigma-Aldrich, St. Louis, MO, USA) with a concentration range from 0 to 500 μg/mL. Total phenolic content was expressed as mg gallic acid equivalents (GAE)/g of sample.

Sodium carbonate, potassium chloride, potassium persulfate, ferric chloride, sodium acetate, potassium acetate, aluminum chloride, and hydrochloric acid were purchased from Qualigens (Mumbai, India). 2,2-azinobis (3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), 2,4,6-tripyridyl-s-triazine (TPTZ), orthophosphoric acid, Folin-Ciocalteu reagent, formic acid, acetonitrile and methanol were purchased from Merck KGaA (Darmstadt, Germany). Ascorbic acid, tannic acid, Folin-Denis reagent and all phenolic standard compounds (rutin hydrate, phloridzin dihydrate, p-coumaric acid, (+)-catechin hydrate, gallic acid, 3-hydroxybenzoic acid, 4-hydroxybezoic acid, ellagic acid, vanillic acid, caffeic acid, m-coumaric acid, ferulic acid, trans-cinnamic acid and chlorogenic acid) and anthocyanins (malvidin, cyanin, cyanidin, delphinidin and pelargonidin) were procured from Sigma-Aldrich (St. Louis, Missouri, United States). All chemicals were of analytical and HPLC grade and the solutions were prepared with methanol and lab ultra-pure water (Rions India Lab Water Systems, India).