2.12.1. Reagents

All the solvents used were of analytical grade. Methanol was purchased from Sigma-Aldrich Co (St. Louis, MO, USA). Sodium carbonate, L-ascorbic acid, 2,2-diphenylpicrylhydrazyl (DPPH.), 2,4,6-tris(2-pyridyl)-s-triazine (TPTZ), gallic acid (97.5%), fructose (>99.5%), glucose (>99.5%), maltose monohydrate (>99%), sucrose (>99.5%), Sigma-Aldrich Co (St. Louis, MO, USA). Folin–Ciocalteu (2N) phenol reagent, Iron (III) chloride hexahydrate and Iron (II) Sulfate heptahydrate (FeSO4·7H2O) were from Honeywell-Fluka (Harvey St., Muskegon, MI, USA).

## 2.12.2. Sample Preparation

Five grams of honey was dissolved in 50 mL of distilled water. To eliminate the interferences of reducing sugars, the blank measurements were performed using an artificial honey containing: 40% fructose, 30% glucose, 8% maltose, 2% sucrose and 20% water [42]. All measurements were performed in triplicate (*n* = 3).

### 2.12.3. Total Phenolic Content (TPC)

The total phenol content was determined by a modified colorimetric assay using the Folin–Ciocalteu reagen<sup>t</sup> [43,44]. Briefly, honey samples were dissolved in distilled water (0.1 g/mL), until a clear solution was obtained. Then, 100 μL of the honey solution was added to 1000 μL of Folin–Ciocalteu reagen<sup>t</sup> previously diluted 1:10 with distilled water. After one minute, 300 μL of saturated sodium carbonate (Na2CO3) solution was added. The mixture was vortexed for 2 min, and the content was transferred into a 1.5 mL cuvette; absorbance was determined after one hour of incubation in the dark at 750 nm against a blank solution. A calibration curve was constructed with gallic acid (0.02–0.2 mg/mL) and the results were expressed as mg of gallic acid equivalents (GAE) per kg of honey.

### 2.12.4. Ferric Reducing Antioxidant Power (FRAP)

The reducing capacity of honey was estimated by the FRAP assay. This method involves the reduction of the ferric 2,4,6-tripyridyl-s-triazine complex (Fe+3-TPTZ) to its ferrous, colored form (Fe+2-TPTZ) in the presence of antioxidants [45]. The FRAP reagen<sup>t</sup> consists of 2.5 mL of 10 mM of TPTZ (2,4,6-tripyridyl-s-triazine) solution in 40 mM HCl, 2.5 mL of 20 mM of FeCl3 and 25 mL of 0.3 mM of acetate buffer, pH 3.6. Aliquots of 100 μL of the honey solution (0.1 g/mL) were mixed with 900 μL of FRAP reagen<sup>t</sup> and the absorbance of the reaction mixture was measured at 593 nm after incubation at 37 ◦C for 30 min. A standard aqueous solution of FeSO4-7H2O (0.05–0.5 mM) was used for the construction of the calibration curve and the results were expressed as mmol Fe+2/Kg of honey.

### 2.12.5. Antiradical Activity (DPPH)

The radical scavenging activity of honey was estimated spectrophotometrically using the stable free radical 2,2-diphenyl-1-picryl-hydrazile (DPPH). The principle of the method is based on the discoloration (purple) of DPPH solution in the presence of an antioxidant [46]. In short, 0.75 mL of DPPH solution (136 μM) was mixed with 0.375 mL of the honey solution (0.1 g/mL). The mixture was shaken vigorously and then incubated for 60 min at 25 ◦C in the dark; the absorbance of the remaining DPPH was determined at 517 nm against a blank solution. The scavenging activity was expressed as a percentage of absorbance reduction (RSA%) according to the following equation: RSA % = [(At=0 − At = 60)/At=0] × 100, where At=0 is the absorbance of the solution at t = 0 min and At = 60 is the absorbance of the DPPH solution after 60 min of incubation [47]. All measurements of standards and samples were performed in triplicate.
