*2.5. Phytochemical Characterization*

For the phytochemical characterization, all the samples were diluted with methanol (Scharlab, Spain).

#### 2.5.1. Total Phenolic Compounds Determination

The phenolic compounds were determined by Folin–Ciocalteu's colorimetric method [14,15], using gallic acid as the standard. Initially, 450 μL of distilled water were mixed with 50 μL of each sample or gallic acid (Sigma-Aldrich, USA) solution. Then, 2.5 mL of Folin–Ciocalteu's reagen<sup>t</sup> (Sigma-Aldrich, USA) (0.2 N) were added, being the mixtures left for 5 min before the addition of 2 mL of aqueous Na2CO3 (Sigma-Aldrich, USA) (75 g/L). The reaction mixtures were incubated for 90 min at 30 ◦C. After incubation, the content in total phenolic compounds was determined by colorimetry at 765 nm [14,15].

A standard curve was prepared using methanolic solutions of gallic acid at 500, 300, 250, 200, 150, 100, and 50 mg/<sup>L</sup> (y = 0.0010x; R<sup>2</sup> = 0.9612). The total phenolic compounds content was expressed as g of gallic acid equivalents (GAE)/100 g of sample (honey, propolis, and mixtures of honey with propolis) [14,15].

#### 2.5.2. Flavonoid Determination

The aluminum chloride colorimetric method was used to determine the flavonoids content according to a previously implemented method [14,15]. To 500 μL of each solution, either the samples or the quercetin (Sigma-Aldrich, USA) (used as standard), 1.5 mL of methanol, 0.1 mL of aluminum chloride (Sigma-Aldrich, USA) 10% (w/v), 0.1 mL of 1 M potassium acetate (Sigma-Aldrich, USA) and 2.8 mL of distilled water were added. These solutions remained for 30 min at room temperature and then the absorbances were measured using a spectrophotometer (Helios–Omega, Thermo Scientific, USA) at 415 nm [14,15].

To construct the calibration curve, eight quercetin solutions were prepared in methanol with a concentration of 200, 175, 150, 100, 75, 50, 25, and 12.5 μg/mL (y = 0.0146 x; R<sup>2</sup> = 0.9887). The flavonoids content was expressed as g of quercetin equivalents (QE)/100 g of sample (honey, propolis, and mixtures of honey with propolis) [14,15].

#### *2.6. Antioxidant Activity Evaluation*

For the antioxidant activity evaluation all the samples were diluted with methanol.

#### 2.6.1. DPPH Free Radical Scavenging Assay

The DPPH (2,2-diphenyl-1-picrylhydrazyl) free radical scavenging assay was used to evaluate the antioxidant activity of the samples [16]. Briefly, to 100 μL of each sample, 3.9 mL of a 0.1 mM DPPH (Sigma-Aldrich, USA) methanolic solution were added, being this mixture stirred until complete homogenization. The control solution consisted in 100 μL of methanol with 3.9 mL of the DPPH solution. The reaction mixtures were kept at room temperature in the absence of light for 30 min, time after which the absorbances were read at 517 nm using a spectrophotometer (Helios–Omega, Thermo Scientific, USA) [16].

The percentage of inhibition (%Inhibition) of DPPH free radical by the samples was determined using the equation %Inhibition = [(Abscontrol − Absample)/Abscontrol] × 100, where Abscontrol corresponds to the absorbance of the control and Abssample is the absorbance of each sample [14,15]. The results were expressed as %Inhibition/100 g sample (honey, propolis, and mixtures of honey with propolis).

#### 2.6.2. β-Carotene Bleaching Test

The β-carotene bleaching test was also employed to evaluate the antioxidant properties of the samples [16]. Firstly, a β-carotene (Sigma-Aldrich, USA) solution in chloroform with a concentration of 20 mg/mL was prepared. To 500 μL of this solution 40 μL of linoleic acid (TCI Europe N.V., Belgium), 400 μL of Tween 40 (Riedel-de H¨aen, Germany) and 1 mL of chloroform (Scharlab, Spain) were added. This mixture was transferred to a round bottom flask and subjected to a rotary evaporation system at 45 ◦C to ensure complete evaporation of the chloroform. After this, 100 mL of water saturated with oxygen were added to the mixture, forming an emulsion. Secondly, 300 μL of each sample were transferred to test tubes and 5 mL of the previously prepared β-carotene emulsion were added. The tubes were stirred until complete homogenization and were placed in a water bath at 50 ◦C for 1 h. Using a spectrophotometer (Helios–Omega, Thermo Scientific, USA), the absorbances of the samples were measured at 470 nm at the initial (t = 0 h) and final time (t = 1 h). The antioxidant activity was determined as percentage of inhibition of β-carotene's oxidation (%Inhibition) using the following equation, %Inhibition = [(Abssample t = 1 h − Abscontrol t = 1 h)/(Abscontrol t = 0 h − Abscontrol t = 1 h)] × 100, where Abscontrol corresponds to the absorbance of the control and Abssample is the absorbance of each sample [14,15]. The results were expressed as %Inhibition/100 g sample (honey, propolis, and mixtures of honey with propolis).

#### *2.7. Assessment of In Vitro Anti-Inflammatory Activity*

The anti-inflammatory activity was determined by evaluating the capacity of the samples to inhibit protein denaturation [17]. Initially, a solution of bovine serum albumin (BSA) (Sigma-Aldrich, USA) at 1% (w/v) in phosphate bu ffer saline (PBS) solution was prepared. The pH of this solution was adjusted to 6.8 using glacial acetic acid (Scharlab, Spain). Then, 100 μL of the samples diluted in methanol were mixed, in test tubes pre-heated at 37 ◦C, with 900 μL of the BSA solution previously prepared. The control was composed of distilled water. The tubes were then incubated for 10 min at 72 ◦C and after this period cooled in ice for another 10 min. Finally, measurements of the absorbances were performed using a spectrophotometer (Helios–Omega, Thermo Scientific, USA) at 620 nm. The percentage of inhibition of protein denaturation (%Inhibition) was determined applying the following equation, %Inhibition = 100 − [(Abssample × 100)/Abscontrol], where Abscontrol corresponds to the absorbance of the control and Abssample is the absorbance of each sample [17]. The results were expressed as %Inhibition/100 g sample (honey, propolis, and mixtures of honey with propolis).

#### *2.8. Evaluation of the In Vitro Wound-Healing Activity*

#### 2.8.1. Cell Culture

Normal human dermal fibroblasts (NHDF) cell line was maintained in RPMI-1640 culture medium (Sigma-Aldrich, USA) supplemented with 10% fetal bovine serum (FBS) (Sigma-Aldrich, USA), 1% mixture of antibiotic/antimycotic (Sigma-Aldrich, USA), 0.01 M of HEPES (Sigma-Aldrich, USA), 0.02 M of *<sup>L</sup>*-glutamine (Sigma-Aldrich, USA) and 0.001 M of sodium pyruvate (Sigma-Aldrich, USA). Subsequently, the cells were incubated at 37 ◦C in an air incubator with a humidified atmosphere with 5% CO2 [18].

#### 2.8.2. Wound Scratch Assay

The samples were tested for wound-healing activity by using the wound scratch assay [13,19]. NHDF cells were seeded in 12-well plates (4 × 10<sup>4</sup> cells/well) and cultured until a monolayer confluence was reached. After the adhesion of the cells, the medium was removed from the wells and the cell monolayer was scraped in a straight central line using a p200 micropipette tip, creating a scratch, with reference points being marked in the plates. The wells were washed with PBS to remove floating cells and cell debris. Then, the PBS was removed, the samples were prepared in RPMI-1640 and sonicated, then they were added to the wells. Supplemented RPMI-1640 culture medium was added to the control wells. After this, the plates were placed under a phase-contrast microscope and images were acquired at the initial moment (t = 0 h). Then, the plates were incubated at 37 ◦C (5% CO2) and examined once again under the microscope after 2, 24, and 36 h [13,19].

The size of the scratch zones was assessed manually using a digital image analysis tool (IC Measure software version 2.0.0.161) (The Imaging Source, Germany) that allowed the estimation of the distance between the injury margins. Using the IC Measure, the distance between the margins of the lesion in the control at 0 h was estimated, which was considered the initial one and was used to scale all other measurements to more easily compare the estimated distances of the injuries between the samples and the control.

#### *2.9. Statistical Analysis*

The results were presented as mean values ± standard deviation. To determine the reproducibility of the measurements, each assay was performed at least in triplicate. The calculated distance between the margins of the injury were analyzed using the statistical program IBM SPSS Statistics 25 (https://www.ibm.com/analytics/spss-statistics-software) (IBM, Armonk, NY, USA). The significant di fference among means was analyzed by Student's *t*-test (assuming the normal distribution of the continuous variables). A level of *p*-value < 0.05 was considered significant.

#### **3. Results and Discussion**

#### *3.1. FTIR Analysis of the Samples of Honey, Propolis, and Propolis Extracts*

Regarding the honey samples and given the flora that exists at the place of harvest, it is expected that Honey 1 was produced mainly during the autumn from species such as *Arbutus unedo*, *Castanea sativa*, *Quercus faginea*, and *Pinus pinaster*. Honey 2 was produced during the summer from flowers such as *Rosa* spp., *Dahlia* spp., *Hydrangea* spp., and flowers from fruit trees like *Prunus avium*, *Malus* spp., *Pyrus communis*, and *Prunus spinosa*. Finally, Honey 3 was produced during the spring, when most wildflowers and aromatic plants bloom (*Papaver rhoeas*, *Chrysanthemum coronarium*, *Lavandula stoechas*, *Rosmarinus <sup>o</sup>*ffi*cinalis*, *Baccharis trimera*, and *Thymus mastichina*).

The propolis samples subsequently collected did not show a direct correlation with the honey samples, because bees repair their hive continuously throughout the year, whenever this need arises, so the conditions under which propolis is produced will not necessarily be the same conditions under which honey is produced.

The FTIR spectra of the samples of honey, propolis, and propolis extracts were recorded (Figure 1).

**Figure 1.** FTIR spectra of the samples.

The spectra of honeys show a water band at approximately 3300 cm<sup>−</sup><sup>1</sup> and 1650 cm<sup>−</sup>1. Near 2900 cm<sup>−</sup><sup>1</sup> it is possible to observe a band that may be associated with groups present in amino acids. The bands between 1450 and 750 cm<sup>−</sup><sup>1</sup> may correspond to organic acids and to sugars commonly present in honey, such as sucrose, glucose, and fructose. Even though the honey samples have been produced in different seasons, they present quite similar spectra.

The spectra of propolis samples show approximately the same bands but with grea<sup>t</sup> dissimilarity in terms of the intensity of the signal. In these spectra, it can be observed a water band at around 3400 cm<sup>−</sup>1, and very pronounced bands at nearly 2900 and 2850 cm<sup>−</sup><sup>1</sup> that may correspond to some aliphatic compounds. The bands observed between 1650 and 1600 cm<sup>−</sup>1, as well as the bands between 1550 and 1400 cm<sup>−</sup><sup>1</sup> may be caused by the presence of flavonoids and other aromatic compounds. The band at 1150 cm<sup>−</sup><sup>1</sup> may be due to the presence of hydroxyflavonoids. Some of the bands were not considered and may be related to wax and other debris present in the samples. The spectra of propolis extracts show approximately the same bands, but with lower intensity than the propolis samples.

The water band observed in both propolis samples and extracts is less pronounced than the band registered in the honeys. In contrast, the bands identified as possible aromatic compounds and flavonoids in propolis samples and extracts spectra are more pronounced. Finally, the sugar bands in honey spectra are more evident and better outlined.

The results now obtained are in agreemen<sup>t</sup> with other previously published results for other honey and propolis samples [20,21].
