# Olive Paste-Enriched Cookies Exert Increased Antioxidant Activities
## 2 Materials and Methods
### In Vitro Antioxidant Activity of Cookies  
The antioxidant activity of cookies before and after baking was evaluated by the DPPH, ABTS, FRAP, and CUPRAC assays. The capacity of methanolic extract of cookies to scaveng the free radical of DPPH was evaluated by the method of [PERSON] et al. [18] with minor modifications. An aliquot of the extract (10 to 100 \(\mu\)L) or appropriate standard solution of Trolox was diluted with methanol up to 0.9 mL. Then, 0.1 mL of 0.6 mM DPPH reagent in methanol was added, followed by vigorous stirring. After 15.0 min in the dark, the absorbance was measured at 515 nm against a reference sample containing methanol. The results were expressed as Trolox equivalents in \(\mu\)mol per g of cookie.  
Determination of ABTS radical scavenging activity of samples was performed by the method of [PERSON] et al. [19] with minor modifications. ABTS radical cation (ABTS\({}^{\bullet+}\)) was produced by the oxidation of ABTS with potassium persulfate (K\({}_{2}\)S\({}_{2}\)O\({}_{8}\)). The ABTS\({}^{\bullet+}\) was generated by reacting 7 mmol/L stock solution of ABTS with potassium persulphate in a final concentration equal to 2.45 mmol/L. The ABTS\({}^{\bullet+}\) working solution was prepared by dilution of the stock solution using distilled water to give an absorbance of 0.700 at 734 nm. Aliquots of cookie extracts (10 to 100 \(\mu\)L) or appropriate amounts of Trolox standards were dried under a stream of nitrogen followed by the addition of 1.0 mL working ABTS\({}^{\bullet+}\) solution and were vigorously stirred. Samples remained for 15.0 min in the dark at ambient temperature and the absorbance was measured at 734 nm. The ability of the extracts to scavenge the ABTS\({}^{\bullet+}\) was evaluated relative to a reference sample that did not contain any quantity of extract. The results were expressed as Trolox equivalents in \(\mu\)mol per g of cookie.  
The reducing potential of the samples was determined using the FRAP assay as described by [PERSON] and [PERSON] [20]. The method is based on the reduction of the Fe\({}^{3+}\)-tripyridtriazine complex to its ferrous-colored form at low pH in the presence of antioxidants. The FRAP reagent was freshly prepared and contained 0.2 mL of a 10 mM TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl plus 0.2 mL of 20 mM FeCl\({}_{3}\)\(\bullet\)6H\({}_{2}\)O plus 0.2 mL of 3.0 M acetate buffer, pH 3.6. Aliquots of cookie extracts (0.01 to 0.10 mL) were transferred in glass test tubes, dried under a stream of nitrogen, and dissolved in distilled water to a final volume of 900 \(\mu\)L, followed by addition of 300 \(\mu\)L of FRAP solution and vigorous stirring. The samples were incubated for 10 min in a 37 \({}^{\circ}\)C water bath and the absorbance was measured at 593 nm. A standard curve was prepared using Trolox. The antioxidant activity of the cookie extracts was expressed as Trolox equivalents in \(\mu\)mol per g of cookie.  
The reducing capacity of the samples was also determined using the CUPRAC assay according to [PERSON] et al. [21]. Amounts of the samples in the range of 10 to 100 \(\mu\)L were transferred in test tubes and were dried under a stream of nitrogen. After that, 10 mM CuCl\({}_{2}\)\(\bullet\)2H\({}_{2}\)O, 7.5 mM neocuproine, and 1 mM CH\({}_{3}\)COONH\({}_{4}\) buffer solution with pH = 7.0 were added in 300 \(\mu\)L each, followed by the addition of distilled water up to the volume of 1200 \(\mu\)L. The samples were well stirred and remained at room temperature for 30 min. The absorbance of the samples was then measured at 450 nm. A standard curve was prepared by the same procedure using Trolox. The antioxidant activity of samples was expressed as Trolox equivalents in \(\mu\)mol per g of cookie.