Neocuproine

A sample of A. adstringens (Schltdl) bark used for this study was obtained from a local market in the state of Querétaro, México, and identified as IBUNAM:MEXU:24829. Methanol (anhydrous 99.8%), ethanol (ACS reagent 99.5%), choline chloride (99%), betaine hydrochloride (98%), glycerol (ACS reagent, 99.5%), sucrose (99.5%), gold III chloride trihydrate (99.9%), Folin and Ciocalteu phenol reagent (2 M), gallic acid monohydrate (acs reagent, >98%), sodium carbonate (anhydrous, >99.5%), sodium hydroxide (>97%, pellets), quercetin (>95%), 2,2.diphenyl-1-picrylhydracyl (DPPH), neocuproine (>98%), ammonium acetate (>98%), (±)-6-hydroxy-2,5,7,8-tetramethylcromane-2-caboxylic acid (97%), and 2,2-azino-bis(3-ethylbenzothiazoline-6-sulfonic acid) were supplied by Merck KGaA, Darmstadt, Germany.

Methanol (pro-analysis), Folin–Ciocalteu phenol reagent, aquadest, aquabidest, GA, ammonium acetate buffer, CuCl₂, K₂S₂O₄, ammonium acetate buffer, neocuproine, AlCl₃, FeCl₃, HCl, and quercetin were obtained from Merck-Millipore (Darmstadt, Germany). Trolox, ABTS, sodium carbonate, glacial acetate acid, and DPPH were obtained from Sigma-Aldrich (St. Louis, MO, USA). 2,4,6-tripydyl-s-triazine (TPTZ) and acetic acid were obtained from Sisco Research Laboratories Pvt. Ltd. (Maharashtra, India).

The Cupric reducing antioxidant capacity (CUPRAC) method [24 (link)] was used in order to establish the antioxidant activity (AC) of the final products. Briefly, 1 mL CuCl₂ 0.01 M solution (S.C. Chemical Company S.A., Bucuresti, Romania), was mixed with 1 mL neocuproine (7.5 × 10⁻³ M) (2,9-Dimethyl-1,10-phenanthroline) and 1 mL acetate buffer (Merck, Darmstadt, Germany). The resulting solution was mixed with 1.1 mL alcoholic extract (preparation see Section 3.2) and incubated for 30 min at 20 °C. The solution absorption was measured at 450 nm using an ultraviolet–visible (UV-VIS) spectrophotometer (Analytic Jena Specord 205, Jena, Germany); Trolox (6-hydroxy-2,5,7,8-tetramethilcroman-2-carboxylic acid) (Merck, Darmstadt, Germany) was used as the reference. All experiments were carried out in triplicate.

In the cupric ion reducing antioxidant capacity (CUPRAC) experiments, the reduction power of tea samples was determined using the procedure described by Apak et al. [68 (link)]. In short, 1.00 mL of 10 mmol L^–1 cupric chloride (Merck, Germany), 1.00 mL of 7.5 mmol L^–1 neocuproine in ethanol (Merck, Germany), and 1.00 mL of ammonium acetate buffer (38.54 g of ammonium acetate dissolved in 500 mL of DIW, pH 7.0) were added to a glass reaction tube. Next, 0.25 mL of the diluted tea extract (DIW for blank sample) and 0.875 mL of DIW were added. The mixture was shaken gently and left standing for 1 h at room temperature prior to absorbance measurement at 450 nm. The CUPRAC activity was presented as equivalent amount of Trolox in mg per gram dried mass sample (mg TE g^–1 DM) based on the Trolox standard curve.

The substances used in the preparation of the systems, including fisetin (FIS) (>95%) and Poly(1-vinylpyrrolidone-co-vinyl acetate (CPV)), were obtained from Sigma Aldrich Chemie (Berlin, Germany).
Trifluoric acid and methanol HPLC grades were provided by Merck (Darmstadt, Germany). High-quality pure water was prepared using a Direct-Q 3 UV purification system (Millipore, Molsheim, France).
Activity testing reagents: 2,2-Diphenyl-1-picrylhydrazyl, iron (III) chloride hexahydrate, 2,2′-casino-bis (3-ethylbenzothiazoline-6-sulfonic acid), neocuproine, 2,4,6-Tri(2-pyridyl)-s-triazine, and Trolox were supplied by Sigma-Aldrich (Schnelldorf, Germany). Sodium chloride and sodium hydrogen phosphate were purchased from Avantor Performance Materials (Gliwice, Poland). Ammonium acetate (NH₄Ac) and methanol were supplied by Chempur (Piekary Śląskie, Poland). Cupric chloride dihydrate, ethanol (96%), isopropanol (99%), acetic acid (99.5%), and sodium acetate trihydrate were obtained from POCH (Gliwice, Poland).

Soy lecithin and neem oil were purchased from Galeno srl (Carmignano, Prato, Italy). Sodium hyaluronate was purchased by DSM Nutritional Products AG Branch Pentapharm (Dornacherstrasse 112 CH-4147 Aesch BL/Switzerland). Argan oil, 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), gallic acid, ferrous sulphate, 1,1-diphenyl-2-picrylhydrazyl radical (DPPH), (±)-6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid (Trolox), 2,4,6-tris(2-pyridyl)-1,3,5-triazine (TPTZ), 2,2′-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid (ABTS), neocuproine (2,9-dimethyl-1,10-phenanthroline) hydrochloride, Folin-Ciocalteu’s reagent, sodium carbonate, ferric chloride, ammonium acetate, copper chloride dihydrate, potassium persulphate, copper sulphate pentahydrate and all the other reagents were of analytical grade and were purchased from Sigma-Aldrich (Milan, Italy). Ultrapure water (18 MΩ·cm) was obtained with a Milli-Q Advantage A10 System apparatus (Millipore, Milan, Italy). Reagents and plastics for cell culture were purchased from Life Technologies Europe (Monza, Italy).