# Evaluation of _Sambucus nigra_ L. Biopotential as an Unused Natural Resource
## 2 Materials and Methods
### Determination of Biological Activity  
#### 2.7.1 Antioxidant Properties  
Radical Scavenging Activity  
The radical scavenging properties of the extracts were estimated by described method [24] against 1,1-diphenyl-2-picrylhydrazyl radical (DPPH) and 2,2\({}^{\prime}\)-azinobis(3-ethylbenzothazoline)-6-sulfonic acid radical cation (ABTS). These UV-vis-based methods are based on the decolorization of free radicals by plant sample. In brief, for DPPH assay, 20 uL of extract was dissolved in methanol in several concentrations, was mixed with 180 uL of 0.1 mM DPPH solution. Discoloration of mixtures was measured at 517 nm after 30 min. For the ABTS assay, ABTS radicals are produced by the reaction of 7 mM ABTS solution and 2.45 mM potassium persulfate at room temperature. Before the reaction started, the ABTS solution was diluted with methanol to an absorbance of 0.700 \(\pm\) 0.02 at 734 nm. Afterward, extract (500 uL) was added to the ABTS (1 mL) and mixed. The sample absorbances were read at 734 nm after 30 min incubation at room temperature. Resultswere expressed as milligrams Trolox equivalents per gram of extract (mg TE/g E). On the other hand, a test of nitric oxide radical scavenging capacity was determined according to previously described procedures [25].  
Reducing Power Activity  
The cupric ion reducing capacity (CUPRAC) and the ferric reducing antioxidant power (FRAP) assays were performed to evaluate the reducing power of the extracts. These assays are based on reducing the cupric and ferrous ions. For CUPRAC experiments, prepared extracts (250 \(\mu\)L) was added to the reaction mixture containing CuCl\({}_{2}\) (500 \(\mu\)L, 10 mM), neocuproine (500 \(\mu\)L, 7.5 mM), and NH\({}_{4}\)Ac buffer (500 \(\mu\)L, 1 M, pH 7.0). Then, preparation of the blank was carried out by adding extracts (250 \(\mu\)L) to the reaction mixture (1.5 mL) without CuCl\({}_{2}\). Finally, the sample and blank absorbances read at 450 nm after 30 min incubation at room temperature. The absorbance of the blank was subtracted from the absorbance of samples. In the FRAP test, the extract (50 \(\mu\)L) was added to FRAP reagent (1 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 absorbances were read at 593 nm after a 30 min incubation at room temperature. Results were expressed as milligrams Trolox equivalents per gram of extract (mg TE/g E) [24].  
The total antioxidant activity of the extracts was examined by phosphomolybdenum method. Using this method, an aliquot of 20 \(\mu\)L of the extracts was combined with 200 \(\mu\)L of reagent solution in an Eppendorf tube including 0.6 M sulfuric acid, 28 mM sodium phosphate, and 4 mM ammonium molybdate. The samples absorbances were read at 695 nm after 90 min incubation at 95 \({}^{\circ}\)C. Total antioxidant capacity was expressed as equivalents of Trolox as determined by the equation of the standard Trolox curve [24].  
The metal chelating activity of the extracts was estimated using ferrous ions method as antioxidant activity. In this method, the chelation ability of the natural compounds is a sign of their antioxidant potential and can therefore be determined by this test. In brief, 1 mL of extract was added to FeCl\({}_{2}\) solution (25 \(\mu\)L, 2 mM). The reaction was initiated by the addition of 5 mM ferroazine (0.2 mL). Similarly, a blank was prepared by adding sample solution (1 mL) to FeCl\({}_{2}\) solution (25 \(\mu\)L, 2 mM) and water (100 \(\mu\)L) without ferroazine. Then, the sample and blank absorbances were read at 562 nm after 10 min incubation at room temperature. The absorbance of the blank was subtracted from the absorbance of samples. Results were expressed as milligrams EDTA equivalents per gram of extract (mg EDTA/g E) [24].