Apigenin

HPLC-grade water was obtained from a LC-Pak™ Millex system (Millipore Corporation, Billerica, MA, USA). Formic acid, MS grade methanol, quercetin, apigenin, pinobaskin, crysin, pinocembrin, and galangin were obtained from PhytoLab, Vestenbergsgreuth, Germany.

Stock solutions of analytical standards luteolin and apigenin (Lot. 2,578 and 2,968, Phytolab, Vestenbergsgreuth, Germany) were prepared at 1,000 mg/mL in MeOH (Tedia, Rio de Janeiro, Brazil) in volumetric flasks. Six concentration of work solutions (1; 4; 8; 12; 16, and 20 μg/mL) were done on the day for calibration curves of each compound. The solutions were filtered in a 0.45 μm PTFE filter (Merck Millipore, Darmstadt, Germany) before analyses by HPLC-DAD-UV. Injections of 20 μL were performed in triplicate to obtain the calibration curves from the areas corresponding to the peaks of luteolin and apigenin. The analytical curve (1–20 μg/mL) of the standards was plotted based on the UV-Vis signal at 254 nm: luteolin content (μg/mL) = (Abs (mAu) + 21,030)/83,557; R2 = 0.9995 and apigenin content (μg/mL) = (Abs (mAu) + 27,059)/77,296; R2 = 0.996. Flavones amounts were calculated in mg/g of dry extract. The following dilution factors were used for luteolin and apigenin quantitative analysis: 147.06 and 11.47, respectively.

The total flavonoid content (TFC) was evaluated by the spectrophotometric method as described in the Romanian Pharmacopoeia (1993) at 430 nm with AlCl₃ as a coloring agent. The flavonoids were determined quantitatively using a Shimadzu Nexera-i HPLC equipped with Fortis C18 columns (150 × 2.1 mm × 3 µm) and a UV–Vis DAD detector at 360 nm. The elution was performed with a solvent gradient (see Supplementary Table S1). The standards for the identification of apigenin, kaempferol, luteolin, and quercetin were obtained from Phytolab (Germany), while calibration curves were required for their quantitation (see Table 3). The antioxidant capacity of the BC-GTE was evaluated using methods such as FRAP (Ferric reducing antioxidant power), CUPRAC (cupric reducing antioxidant capacity), superoxide radical, and xanthinoxidase inhibition [49 (link),50 (link),51 (link)]. FRAP and CUPRAC are spectral methods to evaluate the antioxidant capacity. FRAP is the ferric-reducing ability of plasma, while CUPRAC is the cupric ion-reducing antioxidant capacity. All assessments were performed in technical triplicates.

Orientin, isoOrientin, vitexin, isovitexin, schaftoside, isoschaftoside, luteolin, apigenin, and quercetin (>95%, reference substances) were purchased from Phytolab (Vestenbergsgreuth, Germany). Caco-2 cells were obtained from the ATCC (American Type Culture Collection, Manassas, VA, USA). Acetonitrile (LiChrosolv^®), trifluoroacetic acid (TFA), dimethyl sulfoxide (DMSO), formic acid, sodium acetate, sulfatase (from Helix pomatia, type H-1, lyophilized, >10,000 U/g), β-glucuronidase (from bovine liver, type B-10, 10,100 U/g) methanol (LiChrosolv^®), 3-(4,5-dimethylthiazolyl)-2,5-diphenyl tetrazolium bromide (MTT), and sodium dodecyl sulfate (SDS) were purchased from Merck (Darmstadt, Germany). Hanks’ balanced salt solution (HBSS) was obtained from Biowest (Nuaillé, France). Dulbecco’s modified Eagle medium (DMEM) was purchased from Life Technologies (Carlsbad, CA, USA). Phosphate-buffered saline (PBS), fetal bovine serum (FBS), trypsin-ethylenediaminetetraacetic acid (trypsin-EDTA), nonessential amino acids (NEAAs), penicillin, and streptomycin were purchased from Biochrom AG (Berlin, Germany). Transwell^® plates (insert diameter 12 mm, pore size 3.0 µm, membrane growth area 1.12 cm²) were obtained from Corning Costar (Cambridge, MA, USA), and 96-well plates were purchased from TPP (Trasadingen, Switzerland).

Formic acid (>98% purity), acetonitrile (LC-MS grade), methanol, rutin, quercetin, apigenin and kaempferol were purchased from Merck (Darmstadt, Germany). apigenin 7-O-glucoside, quercetin 3-O-glucoside, kaempferol 3-O-rutinoside and luteolin were purchased from Extrasynthese (Genay Cedex, France). Cynarin, chlorogenic acid, 1,5-dicaffeoylquinic acid, 3,5-dicaffeoylquinic acid, 4,5-dicaffeoylquinic acid, luteolin 7-O-glucuronide, apigenin, 7-O-glucoside, luteolin 7-O-glucoside and diosmetin were purchased from Phytolab (Vestenbergsgreuth, Germany). Bond Elut Jr 500 mg SPE-C18 cartridge was from Agilent (Santa Clara, California, USA). A Milli-Q purification system (Millipore, Bedford, MA, USA) was used to obtain the deionised water (18.2 MΩ cm). All chemicals used for biological assays were obtained as pure commercial products from Sigma Chemical Co (St. Louis, USA) and used without further purification. Spectrophotometric determinations were obtained with an Ultrospec 2100 spectrophotometer (Biochrom Ltd, Cambridge, England) using a 1 cm length path cell.

S. bachtiarica aerial parts were used for polyphenolic compound determination based on standards including gallic acid, vanillic acid, caffeic acid, ferulic acid, p-coumaric acid, quercetin, luteolin, and apigenin (Phytolab, Germany, 98% purity). Methanolic extraction was applied, in which 2g of leaf samples was used and after grinding were extracted using 20 mL of methanol (80%), and shaken at 25 °C for 20 h. The extraction was repeated twice and the air-dried extract was dissolved in HPLC solvent A (1 mL), filtered (0.22 μm disk), and 20 μL was injected to an Agilent 1090 system with detection range of 260 and 350 nm. A 250 × 4.6 mm, 5 μm, symmetry C18 column (Waters Crop., Milford, MA, USA) was applied in this experiment. The mobile phase included formic acid (99.9:0.1) as a solution (A) and acetonitrile/formic acid (99.9:0.1) as a solution (B) with gradient elution at 25 °C and flow rate of 0.8 mL min⁻¹. The gradient program started from A: B (90:10) for 1 min, followed by 10–26% B for 40 min, 26–65% B for 30 min, and finally 65–100% B for 5 min followed by equilibration with 0–90% A for 4 min. Polyphenolic components were determined by comparing UV spectra and the retention times with pure standards, and the amount was reported in mg per 100 g of dry sample weight.

Low-molecular-weight chitosan (LCH, viscosity 108 cP, 1% in 1% acetic acid, deacetylation degree 83%), medium-molecular-weight chitosan (MCH, viscosity 407 cP, 1% in 1% acetic acid, deacetylation degree 75%), and pentasodium tripolyphosphate (TPP) were purchased from Sigma-Aldrich (Madrid, Spain).
Ethanol (99.5% purity) was purchased from Panreac (Barcelona, Spain). Formic acid (99%) was obtained from Acros Organics (Madrid, Spain) and acetonitrile HPLC grade was obtained from Macron Fine Chemicals (Madrid, Spain). Phenolic compounds standards (HPLC purity ≥95%), such as rosmarinic acid and eriodyctiol, were obtained from Sigma-Aldrich (Madrid, Spain). Lithospermic acid, salvianolic acid, apigenin, apigenin 7-O-β-glucuronide, and vicenin II were obtained from Phytolab (Madrid, Spain). Ethyl gallate, apigenin 7-O-glucoside, caffeic acid, and luteolin 7-O-β-glucuronide were obtained from Extrasynthese S.A. (Genay, France); arbutin was obtained from TCI (Belgium); and taxifolin was obtained from Fluka analitycal (Madrid, Spain).