Ascorbic acid

Inside the anaerobic chamber (Bactron VI, Shellab, Sheldon Manufacturing, Incorporation using a mixed gas of 5% hydrogen, 5% carbon dioxide, and 90% nitrogen) at 38 °C a volume of 100 μL of mix culture of SRB was inoculated and enriched in culture standard medium. The Postgate standard medium was used for the growth of the SRB; for the maintenance a sulfate poor Postgate medium, agar was used and it is referred as the modified Postgate medium. Sodium lactate was used as a carbon source. Blackening of the medium due to the formation of iron sulfides was indicative of positive growth.
The Postgate standard medium contained per L: agar, 2.0 g (Difico); KH₂PO₄, 0.5 g (Merk); NH₄Cl, 1.0 g (J.T. Backer); Na₂SO₄, 1.0 g (Merk); CaCl₂, 1.0 g (Vetec); MgCl₂·6H₂O, 1.83 g (Merk); yeast extract, 1.0 g (Merk); ascorbic acid, 0.1 g (Merk); sodium thioglycolate, 0.013 g (Vetec); sodium citrate, 6.38 g (Synth); sodium lactate 1.75 mL; NaCl (Quemis) 3.5%, resazurin, 2.0 mL (Vetec) 0.025% P/V, FeSO₄·7H₂O, 0.5 g (Merk). All the components were dissolved in distilled water and the pH was adjusted to 7.5–8.0 using 5 M HCl. After this, the solution was homogenized by agitation and later sterilized at 121 °C for 30 min.

HPLC-grade methanol (MeOH) was purchased from Merck (Darmstadt, Germany). All the standards (UPLC-ESI-MS/MS) were purchased from Shanghai ZZBIO Co., Ltd. (Shanghai, China) and Extrasynthese (Lyon, France). Hydrochloric acid was bought from Xinyang Chemical Reagent (China). HPLC standards were purchased from Chengdu Biopurify Phytochemicals Ltd. (Chengdu, China; catalogue numbers can be found at: https://www.phytopurify.com/). Gallic acid, chlorogenic acid, quercetin, Folin-Ciocalteu reagent, 2,4,6-Tri(2-pyridyl)-s-triazine (TPTZ), formic acid, 2-thiobarbituric acid, and 2,2-diphenyl-1-picrylhydrazyl (DPPH) were purchased from Sigma-Aldrich (São Paulo, Brazil). Ascorbic acid, ethylenediaminetetraacetic (EDTA), and absolute ethanol were purchased from Vetec (Duque de Caxias, Brazil). The cell lines (A549, HepG2, HCT8, and EA.hy926) were obtained from the Rio de Janeiro Cell Bank (Rio de Janeiro, Brazil).

The following substances were used: 2,2-diphenyl-1-picrylhydrazyl, 5,5′-dithiobis (2-nitrobenzoic acid), bovine serum albumin, glutathione, butylated hydroxytoluene, Griess reagent, MTT, pyrogallol, xylenol orange (all from Sigma, St. Louis, USA), absolute ethanol, acetic acid, ascorbic acid, ferrous ammonium sulfate, hydrochloric acid, formaldehyde, hydrogen peroxide, methanol, sodium acetate, trichloroacetic acid (Vetec, Rio de Janeiro, RJ, Brazil), sulfuric acid, dimethyl sulfoxide and N,N-dimethylformamide (DMSO, Synth, Diadema, SP, Brazil), Dulbecco's Modified Eagle Medium (DMEM, Vitrocell, Campinas, SP, Brazil), fetal bovine serum (FBS, Gibco), and dextran sulfate sodium (Alfa Aesar, Heysham, Lancashire, UK).