Neocuproine

Antioxidant (DPPH and ABTS radical scavenging, reducing power (CUPRAC and FRAP), phosphomolybdenum, and metal chelating (ferrozine method)) and enzyme inhibitory activities [cholinesterase (ChE) Elmann’s method], tyrosinase (dopachrome method), α-amylase (iodine/potassium iodide method), and α -glucosidase (chromogenic PNPG method)) were determined using the methods previously described by Zengin et al. (2014) (link) and Dezsi et al. (2015) (link).
For the DPPH (1,1-diphenyl-2-picrylhydrazyl) radical scavenging assay: Sample solution (1 mg/mL; 1 mL) was added to 4 mL of a 0.004% methanol solution of DPPH. The sample absorbance was read at 517 nm after a 30 min incubation at room temperature in the dark. DPPH radical scavenging activity was expressed as millimoles of trolox equivalents (mg TE/g extract).
For ABTS (2,2′-azino-bis(3-ethylbenzothiazoline) 6-sulfonic acid) radical scavenging assay: Briefly, ABTS+ was produced directly by reacting 7 mM ABTS solution with 2.45 mM potassium persulfate and allowing the mixture to stand for 12–16 in the dark at room temperature. Prior to beginning the assay, ABTS solution was diluted with methanol to an absorbance of 0.700 ± 0.02 at 734 nm. Sample solution (1 mg/mL; 1 mL) was added to ABTS solution (2 mL) and mixed. The sample absorbance was read at 734 nm after a 30 min incubation at room temperature. The ABTS radical scavenging activity was expressed as millimoles of trolox equivalents (mmol TE/g extract) (Mocan et al., 2016a (link)).
For CUPRAC (cupric ion reducing activity) activity assay: Sample solution (1 mg/mL; 0.5 mL) was added to premixed reaction mixture containing CuCl₂ (1 mL, 10 mM), neocuproine (1 mL, 7.5 mM) and NH₄Ac buffer (1 mL, 1 M, pH 7.0). Similarly, a blank was prepared by adding sample solution (0.5 mL) to premixed reaction mixture (3 mL) without CuCl₂. Then, the sample and blank absorbances were read at 450 nm after a 30 min incubation at room temperature. The absorbance of the blank was subtracted from that of the sample. CUPRAC activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For FRAP (ferric reducing antioxidant power) activity assay: Sample solution (1 mg/mL; 0.1 mL) was added to premixed FRAP reagent (2 mL) containing acetate buffer (0.3 M, pH 3.6), 2,4,6-tris(2-pyridyl)-S-triazine (TPTZ) (10 mM) in 40 mM HCl and ferric chloride (20 mM) in a ratio of 10:1:1 (v/v/v). Then, the sample absorbance was read at 593 nm after a 30 min incubation at room temperature. FRAP activity was expressed as milligrams of trolox equivalents (mg TE/g extract).
For phosphomolybdenum method: Sample solution (1 mg/mL; 0.3 mL) was combined with 3 mL of reagent solution (0.6 M sulfuric acid, 28 mM sodium phosphate and 4 mM ammonium molybdate). The sample absorbance was read at 695 nm after a 90 min incubation at 95°C. The total antioxidant capacity was expressed as millimoles of trolox equivalents (mmol TE/g extract) (Mocan et al., 2016c (link)).
For metal chelating activity assay: Briefly, sample solution (1 mg/mL; 2 mL) was added to FeCl₂ solution (0.05 mL, 2 mM). The reaction was initiated by the addition of 5 mM ferrozine (0.2 mL). Similarly, a blank was prepared by adding sample solution (2 mL) to FeCl₂ solution (0.05 mL, 2 mM) and water (0.2 mL) without ferrozine. Then, the sample and blank absorbances were read at 562 nm after 10 min incubation at room temperature. The absorbance of the blank was sub-tracted from that of the sample. The metal chelating activity was expressed as milligrams of EDTA (disodium edetate) equivalents (mg EDTAE/g extract).
For ChE inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with DTNB (5,5-dithio-bis(2-nitrobenzoic) acid, Sigma, St. Louis, MO, United States) (125 μL) and AChE [acetylcholines-terase (Electric ell AChE, Type-VI-S, EC 3.1.1.7, Sigma)], or BChE [BChE (horse serum BChE, EC 3.1.1.8, Sigma)] solution (25 μL) in Tris–HCl buffer (pH 8.0) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of acetylthiocholine iodide (ATCI, Sigma) or butyrylthiocholine chloride (BTCl, Sigma) (25 μL). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (AChE or BChE) solution. The sample and blank absorbances were read at 405 nm after 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the cholinesterase inhibitory activity was expressed as galanthamine equivalents (mgGALAE/g extract) (Mocan et al., 2016b (link)).
For Tyrosinase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with tyrosinase solution (40 μL, Sigma) and phosphate buffer (100 μL, pH 6.8) in a 96-well microplate and incubated for 15 min at 25°C. The reaction was then initiated with the addition of L-DOPA (40 μL, Sigma). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (tyrosinase) solution. The sample and blank absorbances were read at 492 nm after a 10 min incubation at 25°C. The absorbance of the blank was subtracted from that of the sample and the tyrosinase inhibitory activity was expressed as kojic acid equivalents (mgKAE/g extract) (Mocan et al., 2017 (link)).
For α-amylase inhibitory activity assay: Sample solution (1 mg/mL; 25 μL) was mixed with α-amylase solution (ex-porcine pancreas, EC 3.2.1.1, Sigma) (50 μL) in phosphate buffer (pH 6.9 with 6 mM sodium chloride) in a 96-well microplate and incubated for 10 min at 37°C. After pre-incubation, the reaction was initiated with the addition of starch solution (50 μL, 0.05%). Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-amylase) solution. The reaction mixture was incubated 10 min at 37°C. The reaction was then stopped with the addition of HCl (25 μL, 1 M). This was followed by addition of the iodine-potassium iodide solution (100 μL). The sample and blank absorbances were read at 630 nm. The absorbance of the blank was subtracted from that of the sample and the α-amylase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) (Savran et al., 2016 (link)).
For α-glucosidase inhibitory activity assay: Sample solution (1 mg/mL; 50 μL) was mixed with glutathione (50 μL), α-glucosidase solution (from Saccharomyces cerevisiae, EC 3.2.1.20, Sigma) (50 μL) in phosphate buffer (pH 6.8) and PNPG (4-N-trophenyl-α-D-glucopyranoside, Sigma) (50 μL) in a 96-well microplate and incubated for 15 min at 37°C. Similarly, a blank was prepared by adding sample solution to all reaction reagents without enzyme (α-glucosidase) solution. The reaction was then stopped with the addition of sodium carbonate (50 μL, 0.2 M). The sample and blank absorbances were read at 400 nm. The absorbance of the blank was subtracted from that of the sample and the α-glucosidase inhibitory activity was expressed as acarbose equivalents (mmol ACE/g extract) (Llorent-Martínez et al., 2016 (link)).
All the assays were carried out in triplicate. The results are expressed as mean values and standard deviation (SD). The differences between the different extracts were analyzed using one-way analysis of variance (ANOVA) followed by Tukey’s honestly significant difference post hoc test with α = 0.05. This treatment was carried out using SPSS v. 14.0 program.

The DPPH assay was conducted according to Studzińska-Sroka et al. [35 (link)] with modifications. Briefly, 25 μL of the dry extracts dissolved in DMSO (Sigma-Aldrich, Saint-Louis, MO, USA) at different concentrations (0.3125–5 mg/mL) were mixed with a 175 μL of DPPH (Sigma-Aldrich, St. Louis, MO, USA) solution (39 mg 50 mL⁻¹ of MeOH; the final assay concentrations were 78.13–625 μg mL⁻¹). The reaction mixture was shaken and incubated in the dark, at room temperature for 30 min. Absorbance was measured at 517 nm against the blank (25 μL of DMSO and 175 μL of MeOH). The control contains 25 μL of DMSO and 175 μL of DPPH solution. The inhibition of the DPPH radical by the sample was calculated according to the following formula: DPPH scavenging activity (%) = (A0−A1)/A0 × 100%, where A0 is the absorbance of the control and A1 is the absorbance of the sample. Analyses were performed in six replicates. Vitamin C was used as a standard (7.5–80 μg mL⁻¹; the final assay concentrations were 0.9375–15 μg mL⁻¹). The results were expressed as the IC₅₀ value corresponds to the concentration of the extract required to inhibit DPPH radical formation by 50% and was determined from the linear regression analysis.
The FRAP assay was performed according to Tiveron et al. [36 (link)] with some modifications. The stock solutions of FRAP reagent included 300 mM acetate buffer (pH 3.6), 10 mM 2,4,6-Tris(2-pyridyl)-s-triazine (TPTZ, Sigma-Aldrich, Saint-Louis, MO, USA) solution in 40 mM HCl, and 20 mM FeCl₃·6H₂O (Sigma-Aldrich, Saint-Louis, MO, USA) solution. The working FRAP solution was freshly prepared by mixing 25 mL of acetate buffer, 2.5 mL of TPTZ solution, and 2.5 mL of FeCl₃·6H₂O solution, and then warmed at 37 °C before usage. Briefly, 25 μL of the tested extracts were dissolved in DMSO at different concentrations (0.0125–3.2 mg of extract/mL) was mixed with 175 μL of the FRAP solution (the final assay concentrations were 3.125–400 μg mL⁻¹), shaken and incubated at 37 °C for 30 min in the dark condition. Then the absorbance was read at 593 nm. The analysis was performed in six replicates. Vitamin C was used as a standard (0.0125-0.2 mg mL⁻¹; the final assay concentrations were 0.195–3.125 μg mL⁻¹). The results were expressed as the IC_0.5, which corresponds to the extract concentration required to produce 0.5 O.D. value.
The CUPRAC assay was conducted according to Apak et al. [37 (link)] with modifications. The stock solutions of the CUPRAC reagent included equal parts of acetate buffer (pH = 7.0), 7.5 mM neocuproine (Sigma-Aldrich, St. Louis, MO, USA) solution in 96% ethanol, and 10 mM CuCl₂·H₂0 (Avantor Performance Materials, Gliwice, Poland) solution. Briefly, 50 μL of the dry tested extracts dissolved in DMSO at different concentrations (0.0125–1.6 mg mL⁻¹), were mixed with 150 μL of CUPRAC solution (the final assay concentrations were 3.125–400 μg mL⁻¹), shaken and incubated at room temperature for 30 min in the dark condition. Then the absorbance was read at 450 nm. The analysis was performed in six replicates. Vitamin C was used as a standard (0.0125–0.1 mg mL⁻¹; the final assay concentrations were 3.125–25 μg mL⁻¹). The results were expressed as the IC_0.5 which corresponds to the extract concentration required to produce 0.5 O.D. value.

Neocuproine (Aladdin, China) was selected as an indicator for detecting in-situ Cu⁺ production. Briefly, 1.04 mg of neocuproine was dissolved in 5 mL of ethyl alcohol and then diluted five times with ultrapure water. Subsequently, the buffer solution at pH 6.5 was prepared by dissolving KH₂PO₄ and NaOH in ultrapure water. CpBT dispersion at a concentration of 400 μg mL^-1 was prepared by dissolving itself in ultrapure water, and then the final working solution, consisting of 0.75 mL of buffer solution, 1.0 mL of CpBT solution, and 0.8 mL of neocuproine solution, was irradiated under sonication for 0-12 min (1 MHz, 1.0 W cm^-2, 50% duty cycle). Finally, the absorption of the reaction solution was measured by a UV-vis absorbance spectrometer at 452 nm.