Aluminum chloride hexahydrate

Folin–Ciocalteu reagent, gallic acid, and quercetin standards were obtained from Sigma-Aldrich (Poznan, Poland). Aluminum chloride hexahydrate, methanol, and sodium carbonate were obtained from Sigma-Aldrich (Poznan, Poland). The total phenolic was measured using spectrophotometry with a modified Folin–Ciocalteu method [35 (link)]. Total phenol content, expressed as milligrams of gallic acid equivalent (GAE) per gram of extract (GAE mg/g), was calculated on the basis of a standard calibration curve of gallic acid (Y = 0.1157x + 0.087, R² = 0.9749). Total flavonoid content of the plants fractions crud extracts was determined by colorimetric method [36 ,37 ]. The concentration of total flavonoid content in the test samples was calculated from the calibration plot (Y = 1.2308x + 0.0151, R² = 0.9775) and expressed as mg quercetin equivalent (QE)/g of dried extract. The extracts were dissolved in dimethyl sulfoxide (DMSO) to make a stock solution of 20 mg/mL.

ABTS (2,2′-azinobis-[3-ethylbenzthiazoline-6-sulfonic acid] diammonium salt), Trolox (6-Hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid), catechin, Folin and Ciocalteu’s phenol reagent, sodium carbonate (Na₂CO₃), gallic acid, vanillin, catechin, sodium nitrite (NaNO₂), aluminum chloride hexahydrate (AlCl₃ × 6 H₂O), DTT (dithiothreitol), NEM (N-ethylmaleimide), 2,2′-dipyridyl, iron chloride hexahydrate (FeCl₃ x 6 H₂O), ascorbic acid, as well as all HPLC-grade solvents were purchased from Sigma–Aldrich (Milan, Italy). High purity standards for the qualitative-quantitative determination of fatty acids (palmitic, stearic, oleic, linoleic, punicic), organic acid (citric, malic, succinic), sugars (glucose and fructose) were purchased from Sigma–Aldrich (Milan, Italy). Anthocyanin (cyaniding 3-glucoside, cyaniding 3,5-diglucoside, delphinidin 3-glucoside, delphinidin 3,5-diglucoside) standards were purchased from Extrasynthase (GenayCedex, France).

Sigma-Aldrich Aldrich (Castle Hill, NSW, Australia) provided most of the chemicals utilized for extraction and characterization. Gallic acid, Folin–Ciocalteu’s phenol reagent, L-ascorbic acid, sodium phosphate, vanillin, aluminum chloride hexahydrate, iron(III) chloride hexahydrate (FeCl₃·6H₂O), sodium phosphate monobasic monohydrate, sodium phosphate dibasic heptahydrate, trichloroacetic acid, hydrochloric acid, ethylenediaminetetraacetic acid (EDTA), ferrozine, ammonium molybdate, 3-hydroxybenzoic acid, catechin, iron (II) sulfate heptahydrate, potassium ferrocyanide(III), DPPH, 2,4,6 tripyridyl-s-triazine (TPTZ), and ABTS were purchased from Sigma Aldrich (Castle Hill, NSW, Australia). From Chem-Supply Pty Ltd. (Adelaide, SA, Australia), we purchased sodium carbonate anhydrous and hydrogen peroxide (30%), and we also purchased 98% H₂SO₄ from RCI Labscan (Rongmuang, Thailand). Thermo Fisher Scientific Inc. supplied methanol, ethanol, acetonitrile, formic acid, glacial acetic acid, iron (III) chloride anhydrous, and HPLC and LC-MS-grade chemicals (Scoresby, VIC, Australia). Thermo Fisher Scientific provided 96-well plates for various in vitro bioactivities and antioxidant tests (Scoresby, VIC, Australia). HPLC vials (1 mL) were also purchased from Agilent equipment (Melbourne, VIC, Australia).

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.

Butylmethoxydibenzoylmethane and vinylpyrrolidone copolymer were purchased from Pharma Special (São Paulo, Brazil). Ethylhexyl methoxycinnamate and ethylhexyl dimethyl 4-aminobenzoic acid (ethylhexyl dimethyl PABA) were obtained from Fragon (São Paulo, Brazil). Phenoxyethanol/parabens and anhydrous citric acid were obtained from Vital Especialidades (São Paulo, Brazil). 2,2-Diphenyl-1-picrylhydrazyl (DPPH•), Folin-Ciocalteu reagent, aluminum chloride hexahydrate (AlCl₃, 99%), gallic acid, (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-carboxylic acid-Trolox^®, 2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-4H-1-benzopyran-4-one and 3,3′,4′,5,6-pentahydroxyflavone (quercetin ≥ 95%) were from Sigma-Aldrich (São Paulo, Brazil). Sodium hydroxide, sodium carbonate, acetic acid and methanol were purchased from Synth (São Paulo, Brazil). Ethanol 96% (v/v) was purchased from LS Chemicals (São Paulo, Brazil), and distilled.

MilliQ water purged with nitrogen to remove carbon dioxide and L- $\alpha$ -amino acids were used throughout this work. Di-sodium succinate, sodium aspartate monohydrate, sodium glutamate monohydrate, aluminum chloride hexahydrate (AlCl₃ · 6H₂O; ≥99.0%), magnesium chloride (MgCl₂ · 6 H₂O; ≥99.0%), and sodium carbonate (Na₂CO₃; ≥99.5%) were purchased from Sigma-Aldrich. Perchloric acid (HClO₄; 60%) was purchased from Riedel-de haën. Sodium hydroxide (NaOH; 1M) was purchased from Corto Erba reagents groups. All products were used as received without any further treatment.

For phytochemical analysis, the AP leaves and stalks were crushed and homogenized. The total polyphenol content defined as gallic acid equivalent and the total flavonoid content defined as quercetin equivalent were analyzed according to [15 ] by using Folin-Ciocalteu phenol reagent and aluminum chloride hexahydrate (both Sigma-Aldrich, Sternheim, Germany), respectively.
The individual compounds of AP were determined in cooperation with the Department of Pharmacognosy and Molecular Basis of Phytotherapy, Medical University of Warsaw. The plant compounds were extracted with hot deionized water and analyzed with UHPLC-DAD-MS as described previously [12 (link)]. The HPLC analyses were performed using an Ultimate 3000 series system (Dionex, Idtsein, Germany), and a UV–vis in the range of 200–450 nm and MS spectra.