Photochem apparatus

About 200 mg of powdered cookies were extracted with 80% methanol according to methodology described by Przygodzka et al. [26 (link)]. Then extracted samples were stored in −80 °C before prior analysis of antioxidant and reducing capacity (DPPH, PCL, and FRAP assays).
Therefore, DPPH assay was established to measure antioxidants (extracted from cookies) scavenging ability against DPPH radicals [39 (link)]. The measurement were performed at a microplate reader (Tecan M1000 Infinite PRO) with the wavelength established at 517 nm. Results were presented as mmol Trolox per gram of sample. PCL method was used to measure ability of antioxidants from cookies to scavenge superoxide anion radicals (O₂^−•). The measurement was performed using PHOTOCHEM apparatus (Analytik Jena, Jena, Germany) according to protocols elaborated by Zieliński et al. [40 (link)]. The extracts of cookies were determined in two methodologies: for lipophilic antioxidants (PCL ACL) and hydrophilic (PCL ACW) methodology extracts of were expressed as µmol Trolox per gram of sample. The reducing power was determined using FRAP assay according to Horszwald and Andlauer [39 (link)]. The mixture’s absorbance was measured at 593 nm after 5 min reaction with microplate reader (M1000 Infinite PRO, Tecan, Männedorf, Switzerland). The FRAP method is based on the reduction of ferric ion by antioxidant compounds.

Indicators of antioxidant activity were evaluated in breast muscle, liver and serum. Photochem® apparatus (Analytik Jena, Leipzig, Germany) was used to evaluate the antioxidant capacity of water-soluble substances (ACW) and the antioxidant capacity of lipid-soluble substances (ACL) using a photoluminescent (PCL) method based on the scavenging activity against the superoxide anion radical [19 (link)]. PCL ACW and PCL ACL kits were obtained from Analytik Jena Ag (Jena, Germany).
Ferric reducing antioxidant potential (FRAP) assay was carried out according to microplate method described by Horszwald and Andlauer, [12 (link)] and the results were expressed as milligram of Trolox per gram of dry matter of breast/liver or milligram of Trolox per millilitre of serum.
Additionally, thiobarbituric acid reactive substances (TBARS) levels were determined in liver for measuring lipid peroxidation according to the method described by Ognik and Wertelecki, [20 (link)]. Results were expressed in micromole of malondialdehyde (MDA) per kilogram of tissue. The reduced glutathione (GSH) and oxidized glutathione (GSSG) concentrations were also determined in liver by an enzymatic recycling method described by Rahman et al. [21 (link)].

The DPPH scavenging activity was determined with a method described by Brand-Williams et al. [42 (link)]. A decrease in the absorbance of the solution obtained was monitored at 517 nm using an Infinite M1000 PRO plate reader (Tecan Group AG, Männedorf, Switzerland). Results were presented as µmol Trolox per gram of sample. The ABTS test described by Horszwald and Andlauer [41 (link)] was used to determine bee product extract’s antioxidant activity. Measurements were carried out using the plate reader. The antioxidant activity was expressed as mmol Trolox/g sample. The PCL method with the Photochem apparatus (Analytik Jena, Leipzig, Germany) was used to measure antioxidants’ effectiveness against superoxide anion radicals. Antioxidant activity was analyzed using the ACW (antioxidative capacities of water-soluble compounds) and ACL (antioxidative capacities of lipid-soluble compounds) kits. The assay was conducted as previously described by Sawicki et al. [10 (link)]. The data obtained was presented as µmol Trolox per gram of sample.
The reducing power was determined using the FRAP assay according to Horszwald and Andlauer [41 (link)]. The mixture’s absorbance was measured at 593 nm after 5 min reaction using a microplate reader. The FRAP method is based on the reduction of ferric ion by antioxidant compounds.