Pyrogallol

All cell lines were seeded in 96 well plates (Costar) at 5 × 10³ cells/well and incubated for 24 h prior to treatment. Cells were treated with afuresertib (Selleckchem), isorhamnetin (Merck), formononetin (Merck), pyrogallol (Merck) and taxifolin (Merck) in a 16-point dose response (n = 6) over 72 h, with concentrations ranging from 1 to 500 μM. Appropriate vehicle (0.1% DMSO) and positive cell death controls (10% DMSO) were used. Viability was assessed by adding 1:10 PrestoBlue (ThermoFisher) over 45 min and measuring the resulting fluorescence at 544/590 nm (excitation/emission) on a FLUOstar Omega microplate reader (BMG LABTECH). Fluorescence intensity was normalized against that of control wells to determine percentage viability of drug-treated cells.

Thiobarbituric acid (TBA), reduced glutathione (GSH), oxidized glutathione (GSSG), and dithiobis-2-nitrobenzoic acid (DTNB) were purchased from Sigma Chemical Co., St. Louis, MO, USA. Pyrogallol (PG), diethylene triamine pentaacetic acid (DTPA), and trichloroacetic acid (TCA) were purchased from Merck, Germany. RNase, ethanol, bromophenol blue, ethidium bromide, 100 bp DNA ladder, and Taq polymerase were purchased from Fermentas, USA; mouse oligonucleotide primers for interleukin 1 (IL 1), interleukin 10 (IL 10), tumour necrosis factor beta (TNF β), and beta actin (β actin) were procured from Eurogentec, Belgium; RPMI-1640 from Himedia, Mumbai, India; RT² SYBR green ROX q PCR master mix, RNeasy lipid tissue mini kit, Qiazol lysis reagent, and RT² first strands kit were from Qiagen, Hilden, Germany, GenElute mammalian genomic DNA Miniprep kit was from Sigma/Aldrich, USA. Mouse T and B cell selection kit was from Stem Cell Technologies Inc., Canada. ³H-thymidine was from BRIT, India. All the other chemicals used were of analytical grade and were purchased from qualified local vendors.

Analytical standards (Zeaxanthin, Lutein, β-carotene, Gallic acid, Protocatechuic acid, 4-Hydroxy-benzoic acid, Vanillic acid, Chlorogenic acid, Caffeic acid, p-coumaric acid, Ferulic acid, Sinapic acid, Rutin, Kaempferol, IsoQuercetin, Astragalin, Myricetin, Quercetin, α-tocopherol, γ-tocopherol, δ-tocopherol, 5-methyltetrahydrofolate, tetrahydrofolate, 5-formyltetrahydrofolic acid, l-ascorbic acid) were obtained from Sigma Aldrich (Poznan, Poland). 10-formylfolic acid and pteroyltri-γ-L-glutamic acid were obtained from Schirck’s Laboratories (Jona, Switzerland). α-amylase obtained from Sigma Aldrich (Poznan, Poland) and γ-glutamyl hydrolase obtained from rat blood plasma (Europa Bioproducts Ltd., Cambridge, UK). Other reagents (acetonitrile, triethylamine, ethyl acetate, pyrogallol, ethanol, potassium hydroxide, orthophosphoric acid, methanol, nitric acid, lanthanum chloride, potassium dihydrogen phosphate) were purchased from Merck (Warsaw, Poland).

All chemicals, reagents, and standards used in our study were of analytical grade. Usnic acid standard 98.1% purity, DPPH, and Poloxamer 407 were purchased from Sigma-(Sigma-Aldrich Chemie GmbH., Taufkirchen, Germany); 65% HNO₃, 30% H₂O₂, Folin-Ciocâlteu reagent, Pyrogallol, acetone, and ethanol were supplied by Merck (Merck KGaA, Darmstadt, Germany).
Artemia Brine Shrimp Eggs and Artemia salt (Dohse Aquaristik GmbH & Co. Gelsdorf, Germany) were bought online from https://www.aquaristikshop.com/ (accessed on 5 February 2022). Canola oil (TAF PRESOIL SRL, Cluj, Romania) was purchased from the manufacturer’s distribution units.

Phenolic standards, namely, catechin, catechol, syringic acid, chlorogenic acid, cinnamic acid, ellagic acid, kaempferol, ferulic acid, gallic acid, rutin, caffeic acid, naringenin, benzoic acid, o-coumaric acid, p-hydroxybenzoic acid, pyrogallol, p-coumaric acid, quercetin, quinol, rosmarinic acid, and vanillic acid were bought from Merck KGaA (Darmstadt, Germany). The purities of standards were up to 99%. The solvents used were of analytical grade, dimethylsulfoxide (DMSO; Alfa Aesar GmbH & Co KG, Massachusetts, United States), orthophosphoric acid (H₃PO₄), ethanol, methanol, and acetonitrile HPLC-grade (Fisher Scientific International, Inc., Hampton, New Hampshire, United States).