Apigenin

The standard compounds and reagents (all of analytical grade): MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide), DMSO (dimethyl sulfoxide), gallic acid, Na₂CO₃, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), Folin-Ciocalteu’s reagent, resazurin, tetracycline, chloramphenicol, kanamycin, acetonitrile (LC-MS grade, gradient grade), formic acid, 4-methoxybenzoic acid, 4-hydroxy-3-methoxycinnamaldehyde, caffeic acid, quercetin, luteolin and pinostrobin PBS were purchased from Merck (Darmstadt, Germany). The standards of 4-hydroxybenzoic acid, p-coumaric acid, ferulic, isoferulic acid, pinobanksin, chrysin, sakuranetin, naringenin, apigenin, kaempferol, isorhamnetin, acacetin and pinocembrin were obtained from Extrasynthese (Genay, France) and galangin from Alfa Aesar, (Haverhill, MA, USA). The antibiotics: ampicillin, oxacillin, teicoplanin, gentamicin, amikacin, fusidic acid, erythromycin, mupirocin, rifampicin, levofloxacin, norfloxacin, linezolid, bacitracin were purchased from Argenda (Poland). Methanol, ethanol, vanillin, benzoic acid, cinnamic acid were purchased from (POCH, Gliwice, Poland). Ultrapure H₂O (18.0 MΩ) was obtained with a Milli-Q Advantage A10 system (Millipore, Billerica, MA, USA). The absorbance of the reaction mixture in Folin-Ciocalteu, DPPH assays were measured using a Genesys 20 spectrophotometer (Thermo Scientific, Waltham, MA, USA).

Caffeic, ferulic, p‐coumaric, gallic, and chlorogenic acids, catechin, quercetin, 3‐hydroxytyrosol, spiraeoside, oleuropein, daidzein, luteolin, naringenin, formononetin, 2,2‐diphenyl‐1‐picrylhydrazyl (DPPH), HPLC‐grade ethanol, 4‐methyl‐2‐pentanol, and acetonitrile were purchased from Sigma‐Aldrich. Apigenin and hyperoside were purchased from Extrasynthese. Ethanol was obtained from Romil. Ultrapure water from a Milli‐Q system (Millipore) with a resistivity at 25°C of 18 MΩ ∗ cm was used throughout the analyses. Helium (Rivoira) at a purity of 99.999% was the GC carrier gas. The SPME glass vials and the fibers were from Supelco; the capillary GC‐MS column HP‐Innowax (30 m × 0.25 mm × 0.5 μm) was purchased from Agilent J&W (Agilent Technologies Inc.).

Reversed-phase thin-layer chromatography aluminum-backed silica plates (RP-18F 254, 20 × 20 cm, Merck, Darmstadt, Germany) were used to fractionate the ethyl acetate extract of T. brownii leaves. Ellagic acid (E-2250, Sigma-Aldrich, Gillingham, UK), gallic acid (G-7384, Sigma-Aldrich, China), apigenin (Extrasynthese 286 Genay, France), corilagin (Sigma-Aldrich, Darmstadt, Germany), luteolin (Sigma-Aldrich, Darmstadt, Germany) and quercetin (Merck Art. 7546, 285 Darmstadt, Germany) were used as reference pure compounds, since many of them were previously known from Terminalia. A total of 20 µL of extract and compounds at 50 mg/mL and 5 mg/mL, respectively, was applied equidistantly 1.5 cm from the bottom of the TLC plate. The plate was developed in a mobile phase of methanol: water: orthophosphoric acid (50:50:1, v:v:v). The spots were visualized using a Camag Video documentation System (Camag Reprostar 3 TLC Visualizer) and two UV wavelengths at 366 and 254 nm.
The developed TLC plates (RP-18 F254_s Merck, Darmstadt, Germany) containing the ethyl acetate extract of the T. brownii leaves were in situ subjected to qualitative antioxidant analysis using 0.2% w/v of the 2,2-Diphenyl-1-picrylhydrazyl reagent (DPPH, Sigma-Aldrich D9132-1G, Schnelldorf, Germany). All compounds with antioxidant properties were revealed with yellow color in visible light.

All the standards, reagents, and solvents used through experiments were of analytical grade. Apigenin, diosmetin, hyperoside, rutin, quercitrin, luteolin-7-O-glucoside, Apigenin-7-O-glucoside, p-coumaric acid, caffeic acid, rosmarinic acid, and chlorogenic acid standards were purchased from Extrasynthese (Genay, France), and avicularin from Chromadex (Santa Ana, CA, USA). Acetic acid and acetonitrile were purchased from Sigma-Aldrich GmbH (Buchs, Switzerland), and ethanol from Vilniaus degtine, AB (Vilnius, Lithuania). Potassium persulfate, sodium acetate trihydrate, iron (III) chloride hexahydrate, and 2,4,6-tri(2-pyridyl)-S-triazine (TPTZ), 2,2-diphenyl-1-picrylhydrazyl (DPPH·) radical, and 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS) were obtained from Sigma-Aldrich (Steinheim, Germany) and Trolox from Fluka Chemika (Buchs, Switzerland). Folin-Ciocalteu reagent, gallic acid monohydrate, aluminum chloride hexahydrate, sodium carbonate, hexamethylenetetramine, copper chloride, and neocuproine were purchased from Sigma-Aldrich GmbH (Buchs, Switzerland). Deionized water used in HPLC and for the samples preparation produced by the Crystal E high-performance liquid chromatography (HPLC, Adrona SIA, Riga, Latvia) water purification system.

Acetonitrile (AcN), methanol (MeOH), and Folin–Ciocalteu reagent were purchased from Merck (Darmstadt, Germany), phosphoric acid from Sigma-Aldrich (St. Louis, MO, USA), and acetic acid, hydrogen peroxide, and nitric acid from VWR International S.A.S. (Fontenay-sous-Bois, France). Apigenin, Apigenin-7-O-glucoside, catechin, hydroxytyrosol, luteolin, luteolin-7-O-glucoside, oleuropein, rutin, tyrosol, and verbascoside were purchased from Extrasynthese (Genay, France). All chemicals were of analytical grade purity and were used without further purification.
Deionised water was obtained using a Hydrolab 10 SP purification system (Hydrolab, Straszyn, Poland).

Phospholipon90G (P90G; phosphatidylcholine content ≥ 94%) was purchased from Lipoid GmbH (Ludwigshafen, Germany). Propylene glycol (PG) were purchased from Galeno Srl (Comeana, Prato, Italy). LC-MS grade acetonitrile, formic acid, methanol, 85% w/w phosphoric acid, 2,2-diphenyl-1-picrylhydrazyl (DPPH), 6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tris(pyridin-2-yl)-1,3,5-triazine (TPTZ), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), 2′,7′-dichlorofluorescein diacetate (DCFH-DA), dimethyl sulphoxide (DMSO), and all the other reagents, if not otherwise specified, were purchased from Sigma-Aldrich/Merck (Milan, Italy). Standards of 5-O-caffeoylquinic (chlorogenic acid), 1,3-dicaffeoylquinic acid (cynarin), 3,5-dicaffeoylquinic acid (isochlorogenic acid A), 3,4-dicaffeoylquinic acid (isochlorogenic acid B), 4,5-dicaffeoylquinic acid (isochlorogenic acid C), ferulic acid, apigenin, hispidulin, and luteolin were purchased from Extrasynthese (Genay, France) and TransMIT (Giessen, Germany). Ultrapure water (18 MΩ·cm) was obtained with a Milli-Q Advantage A10 System apparatus (Millipore, Milan, Italy).