2 2 diphenyl 1 picrylhydrazyl (dpph)

Measurements were performed according to Brand–Williams et al., with some modifications [44 (link)]. The assay is based on the measurement of the scavenging capacity of the antioxidants towards it. The odd electron of a nitrogen atom in DPPH is reduced by receiving a hydrogen atom from the antioxidants to the corresponding hydrazine [45 (link)]. The antioxidant activity of tested CDs was determined by spectrometric determination of the reduced form of DPPH as percent of maximal antioxidant activity of ascorbic acid (1 mM).
A solution of a stable free radical, DPPH (1,1–diphenyl–2–picrylhydrazyl; Sigma–Aldrich, Germany) in concentration 200 µM/L in ethanol was stored in the dark for 2 h. Solutions of selected CDs in ethanol at the concentration of 4 mg/mL were prepared and diluted with ethanol to obtain concentrations: 0.125, 0.25, 0.5, 1, and 2 mg/mL. To 20 µL of the tested CDs or solvent, 180 µL of the DPPH solution were added. The mixtures were vortexed and allowed to stand for 20 min in the dark at room temperature (25 °C). Absorbance was measured at 517 nm against ethanol as a blank. Figure 14 represent % of ascorbic acid antioxidant effect.

All reagents were obtained from commercial sources and were of guaranteed reagent. Methanol, chloroform, pyridine, and ribitol for GC-MS analysis were purchased from Wako Pure Chemicals Industries, Ltd. (Osaka, Japan). Methoxyamine hydrochloride and N-methyl-N-(trimethylsilyl)trifluoroacetamide (MSTFA) for GC derivatization were purchased from Sigma-Aldrich (St. Louis, MO, USA) and GL Sciences (Tokyo, Japan), respectively. DPPH and 2-morpholinoethanesulfonic acid (MES) for measuring DPPH activity were purchased from Sigma-Aldrich and TCI (Tokyo, Japan), respectively. Fluorescein and 2,2′-azobis(2-amidinopropane) dihydrochloride for use in the ORAC assay were purchased from Sigma-Aldrich and Wako, respectively. Trolox (Sigma-Aldrich) was used as a standard for DPPH and ORAC assays.

To evaluate the antioxidant activity, the method of reduction of the 1,1-diphenyl-2-picrylhydrazyl (DPPH) radical was used [28 ]. The methanolic extracts were evaluated at concentrations of 3.125 to 1000 ppm. The DPPH (Sigma-Aldrich) was prepared to 125 μM in methanol, 100 μL of each sample was taken, and 100 μL of DPPH was added; the samples were allowed to stand for 30 min protected from light. The absorbance at 517 nm was measured using a spectrophotometer (Jenway 320d). As a positive control, a solution of tocopherol (Sigma-Aldrich) was used and as negative control EtOH; the reduction percentage was calculated using
$\begin{array}{c}\%\text{Reduction}=\frac{\text{Absorbancy\hspace{0.17em}negative\hspace{0.17em}control}–\text{Absorbancy\hspace{0.17em}sample}}{\text{Absorbancy\hspace{0.17em}negative\hspace{0.17em}control}}\times 100.\end{array}$

Antiradical capacity was analyzed with an assay using DPPH, as proposed by Brand-Williams et al. [58 (link)]. A solution of DPPH (600 μM) (Sigma-Aldrich, St. Louis, MO, USA) in absolute methanol (CTR, Monterrey, Nuevo Leon, Mexico) was prepared, with absorbance adjusted to 0.700 nm with the UV–Vis spectrophotometer at 515 nm. The standard curve was prepared with Trolox^® (Sigma-Aldrich, St. Louis, MO, USA) with concentrations of 0.1, 0.2, 0.4, 0.6, 0.8, 1, and 1.2 mM. Subsequently, 975 μL of the DPPH solution was mixed with 25 μL of standard or extract solution, and incubated in the dark for 30 min. The free radical-scavenging capacity was determined based on the change in absorbance at 515 nm, according to the standard curve. The free radical scavenging capacity of each sample was determined according to the volume used in each reaction and then extrapolated to the total volume extracted per gram of dry weight material. Finally, it was expressed in mmol TE/g DW. Each reaction was done in triplicate.

The 1,1-diphenyl-2-picrylhydrazyl radical (DPPH •) has been used to evaluate the free radical scavenging activity of antioxidants (Panico et al., 2009 (link)). The DPPH solution was prepared by adding 2.37 mg DPPH (Sigma-Aldrich, Munich, Germany) in 2 ml 99% ethanol. One hundred milligrams of fresh leaf samples were grinded in 1 ml of extraction solution (1:1 Ethanol:Water). After centrifugation at 12,000 g for 10 min at 4°C, 50 μl supernatant was used in a reaction cell which contained 50 μl freshly prepared 3 mM DPPH solution and 900 μl ethanol (99%). After incubation for 10 min in a dark room at 25°C, the absorbance was determined at 515 nm. A reference solution contained 50 μl DPPH solution and 950 μl of ethanol (99%). The decline in absorbance at 515 nm was recorded for each sample and the quenching percentage of the DPPH radical was calculated based on the observed decrease in absorbance using the formula:
% Inhibition = [(A₀-A₁)/A₀] × 100, where A₀ is the absorbance value of the DPPH • blank solution and A₁ is the absorbance value of the sample solution.

After removing the medium, the cells were used to determine intracellular ROS generation while using the fluorescent dye 2’,7’-Dichlorofluorescin diacetate (DCFDA) cellular ROS detection assay kit (Abcam), according to manufacturer’s instruction. DCFDA assay was performed in triplicate for each sample (n = 3). Briefly, the cells were washed with buffer and incubated with 25 μM DCFDA at 37 °C for 1 h. 50 μM of tert-butyl hydrogen peroxide (TBHP) was used as ROS positive control. After incubation, fluorescence intensity was determined by the excitation and emission = 485/535 nm.
The DPPH (Sigma-Aldrich, St. Louis, MO, USA) radical scavenging activity of SP was measured according to Lim et al. [19 (link)]. 200 μM DPPH solution (dissolved in methanol, 100 μL) and the same volume of diluted extract (0, 1.56, 3.12, 6.25, 12.5, 25, 50, and 100 μg/mL) were mixed in 96-well microplate and reactive at room temperature for 30 min. The DPPH radical scavenging activity for each sample (n = 3) was determined in triplicate. The absorbance was measured at 520 nm with a blank containing DPPH and methanol. The DPPH radical scavenging activity was calculated according to the equation.

The antioxidant capacity of flavonoids was measured in three separate tests: ABTS, DPPH, and ferric ion reducing antioxidant power (FRAP) assays (ABTS, DPPH, and FRAP reagents from Sigma-Aldrich). All tested compounds were dissolved in DMSO. All determinations were performed in triplicate. Standard curves were prepared for all assays using different concentrations of Trolox (6-hydroxy-2,5,7,8-tetramethylchroman-2-carboxylic acid) (Sigma-Aldrich). Trolox is a water-soluble analogue of vitamin E with high antioxidant activity, commonly used as a control and a reference in studies of antioxidant activity [19 (link)]. The results were expressed as the equivalent of μmol Trolox per μmol of the test compound. The absorbance was measured using a UV-2401 PC spectrophotometer (Shimadzu, Kyoto, Japan). Concentrations of the tested samples were in the range from 50 µg/mL to 10 mg/mL.