Pyridoxine hydrochloride

For sample extraction, 0.5 g of sample was extracted with 1 mL of ultra-pure H₂O for 20 min in an ultrasonic bath (SONOREX, Bandelin, RK 103H, Darmstadt, Germany) at room temperature. The samples were centrifuged (Microcentrifuge Hettich D-78532, Germany) at 11,000× g rpm for 2 min, then the supernatant was filtered through a 0.45 µm cellulose filter.
A Vanquisher H UHPLC (ultra-high performance liquid chromatography) system from Dionex (Thermo Fisher Scientific, Germany) with a DAD detector was used for the analysis of the following vitamins: thiamine (B1), riboflavin (B2), nicotinamide (B3), pyridoxine (B6), cyanocobalamin (B12). The mobile phase was composed of ultra-pure H₂O with 1% acetic acid and MeOH in gradient with a flow of 0.3 mL/min. The chromatographic column used was a Accucore aQ (100 × 2.1 mm, 2.6 mm) from Thermo Fisher kept at 25 °C. The injection volume was 8 µL and the detector was set at 270 nm. Thiamine hydrochloride, riboflavin, niacinamide, pyridoxine hydrochloride, and cyanocobalamin standards were purchased form Sigma-Aldrich, Inc. (Sydney, NSW, Australia). The dilutions were made in ultra-pure water with a 0.1–100 µg/mL linearity range. The quantification limit was 0.1 and the detection limit was 0.03 µg/mL for all analyzed vitamins.

Seven B vitamin standards (thiamine for vitamin B_1, riboflavin for vitamin B₂, nicotinamide for vitamin B₃, pantothenic acid for vitamin B₅, pyridoxine hydrochloride for vitamin B₆, biotin for vitamin B₇, and folic acid for vitamin B₉) were purchased from Sigma Aldrich Corporation (St. Louis, MO, USA). To make a 100,000 ng/mL stock B vitamin cocktail, 10 mg of each of the seven different B vitamins were mixed into 100 mL of 1% formic acid/H₂O. The standard was divided into small tubes for single use, covered with aluminum foil and stored in a freezer at −80 °C. Portions of the stock B vitamin cocktail were later diluted into various concentrations for constructing the standard curves and also for assessing limit of detection.

Fluoxetine hydrochloride, Imipramine hydrochloride, and Duloxetine hydrochloride were obtained from Sigma‐Aldrich (US). Tris‐hydroxymethyl aminomethane, 2‐thiobarbituric acid, 5, 5‐dithio nitrobenzoic acid (DTNB), and trichloroacetic acid (TCA) were purchased from Sigma‐Aldrich (US). Pyridoxine hydrochloride, 2‐pyrrolidone‐5‐carboxylic acid, N‐acetyl cysteine, 1, 1‐3, 3‐tetramethoxypropane, and glutathione (GSH) powder were purchased from Sigma‐Aldrich (USA). The kits for liver biochemistry assay (ALT, AST, alkaline phosphatase (ALP), and total bilirubin) were obtained from Sigma‐Aldrich (US).

3-MP (sodium salt) was purchased from Santa Cruz Biotechnology; lead (II) acetate, pyridoxine hydrochloride, l-cysteine, thiosulfate, GSH, and β-ME were from Sigma–Aldrich.

Human hepatocarcinoma cells (HepG2) (ATCC: HB8065) were obtained from the Banco de Células do Rio de Janeiro (BCRJ). The cells were grown in sterile culture flasks of 75 cm² containing Dulbecco’s Modified Eagle Medium (DMEM) enriched with L-glutamine (Gibco^®, Billings, MT, USA), pyridoxine hydrochloride (Sigma-Aldrich^®, Darmstadt, Germany), sodium pyruvate (Sigma-Aldrich^®, Darmstadt, Germany), antibiotic solution with 1% streptomycin (Sigma-Aldrich^®, Darmstadt, Germany), 0.6% penicillin G (10 mL/L) (Sigma-Aldrich^®, Darmstadt, Germany), and 10% serum fetal bovine (SFB) (Gibco^®, Billings, MT, USA). The cells were incubated (carbon dioxide incubator, Panasonic^®, Kadoma, Japan) at 37 °C and 5% carbon dioxide (CO₂) until 80–90% confluence. Cell subculturing was carried out with a minimum of 3 passages and a maximum of 10 passages.

Yeast bioassays for vitamin B₆ content were performed according to a method previously established with the Saccharomyces carlsbergensis American Type Culture Collection 9080 strain (Tambasco‐Studart et al., 2005). The amount of plant material required for vitamin B₆ extraction varied between organs: leaves (50 mg), roots (100 mg), dry seeds (50 mg), fresh seeds (20 mg), embryo (20 mg). Frozen ground tissues were re‐suspended in 20 mm sulfuric acid (ratio: 100 mg tissue/1 mL extraction buffer), incubated for 30 min in the dark at room temperature and the extract was sterilized for 1 h at 100°C. After extraction, the solution was adjusted to pH 5.7 using 3 m sodium acetate and centrifuged. The supernatant was then treated with acid phosphatase (0.2 U/10 μL in 50 μL plant extract) (Sigma‐Aldrich, St. Louis, MI, USA) and β‐glucosidase (0.2 U/10 μL in 50 μL plant extract) (Sigma‐Aldrich, St. Louis, MI, USA) for 12–15 h at 37°C to convert phosphorylated and glucosylated forms of vitamin B₆ to free forms. Total vitamin B₆ content was calculated using the linear range of a dose−response curve constructed with known amounts of commercial pyridoxine hydrochloride (Sigma‐Aldrich, St. Louis, MI, USA).