4 hydroxybenzaldehyde

To estimate the chemical profile of the subfractions under study, normal- and reverse-phase thin-layer chromatography (TLC) assays were performed using commercially available standards (rutin, quercetin, glucose, glycine, alanine, serine, valine, leucine, asparagine, aspartic acid, lysine, glutamic acid, and arginine). The systems dichloromethane:methanol (7:3 v/v), ethyl acetate:methanol:water:glacial acetic acid (7:2:2:0.5 v/v/v/v), and n-butanol:acetone:glacial acetic acid:water (35:35:10:20 and 70:70:20:10 v/v/v/v) were used as solvents for normal-phase TLC, while water:acetonitrile (7:3 v/v) was used for reverse-phase TLC. Once the plates were developed, spots were visualized with 4-hydroxybenzaldehyde (Merck, Darmstadt, Germany) and aminoethanol dimethylborate (Sigma-Aldrich, St. Louis, MO, USA) for flavonoids, and ninhydrin (Merck) for amino acids, following the supplier’s directions.

Analytical grade ferric chloride, ferrous chloride, ammonia solution (25 wt %), thiophene, 4-hydroxybenzaldehyde, acetonitrile, potassium permanganate, 4-aminophenol, 3-bromothiophene, 1,6-dibromohexane, N-bromosuccinimide, acetic acid, sodium hydrogen bicarbonate, potassium iodide, potassium carbonate, tetrahydrofuran, methanol, hydrochloric acid, acetone and ethyl acetate were purchased from Merck (Darmstadt, Germany). acetone was procured from Fisher Scientific (Loughborough, UK). thiophene carboxaldehyde, polyvinyl alcohol and n-butyllithium (2.0 M in cyclohexane) were obtained from Sigma Aldrich (Milwaukee, WI, USA). Magnesium sulfate anhydrous, ethanol denatured and hexane were received from J. Kollins (Parkwood, Australia), while dimethyl sulfoxide-d₆ (DMSO-d₆) and phthalate esters were purchased from Acros Organics (Geel, Belgium). Ultrapure water was prepared by a model Aqua Max-Ultra ultra-pure water purification system (Zef Scientific Inc., San Diego, CA, USA). Stock solutions of 1000 mg·L⁻¹ of standards were prepared by dissolving appropriate amounts of compounds in methanol, which remain stable for three months if stored in a refrigerator at 4 °C. Working standard solutions were prepared daily by diluting the stock standard solution to the required concentrations.

Standards rutin, quercetin, glucose, valine, proline, glycine, arginine, leucine, asparagine, lysine, isoleucine, aspartate, and glutamate; reagents Naphthol, and aminoethanol dimethylborate, 3-isobutyl- 1-methylxanthine (IBMX), dimethyl sulfoxide (DMSO), dexamethasone, insulin, 3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyl- tetrazoliumbromide (MTT), dihydroethidium (DHE), silimaryn, where obtained from Sigma-Aldrich (St. Louis, MO, USA). High-glucose Dulbecco’s Modified Eagle’s Medium (DMEM) and calf bovine serum were purchased from ATCC (Rockville, MD, USA). Reagents 4-hydroxybenzaldehyde and ninhydrin was from Merck Millipore (Burlington, MA, USA). The pierce Coomassie Bradford Protein Assay Kit, Fetal bovine serum (FBS), Gentamicin was obtained Thermo Fisher Scientific (Waltham, MA, USA). Reagents dichloromethane, methanol, ethyl acetate, glacial acetic acid, n-butanol, acetone, acetonitrile, sulfuric acid (H₂SO₄) were purchased from J.T. Baker (Madrid).