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 officinalis, 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).
The spectra of honeys show a water band at approximately 3300 cm−1 and 1650 cm−1. Near 2900 cm−1 it is possible to observe a band that may be associated with groups present in amino acids. The bands between 1450 and 750 cm−1 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 great dissimilarity in terms of the intensity of the signal. In these spectra, it can be observed a water band at around 3400 cm−1, and very pronounced bands at nearly 2900 and 2850 cm−1 that may correspond to some aliphatic compounds. The bands observed between 1650 and 1600 cm−1, as well as the bands between 1550 and 1400 cm−1 may be caused by the presence of flavonoids and other aromatic compounds. The band at 1150 cm−1 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 agreement with other previously published results for other honey and propolis samples [
20,
21].
3.2. Phytochemical Characterization
Plant polyphenols present at least 8000 distinct known structures, being the most important class of natural bioactive compounds, which exhibit various biological activities [
22]. Honey presents three classes of flavonoids with analogous structure: flavonols, flavones, and flavanones. Flavonoids are responsible for the color, taste, and flavor of the honey and they also improve its beneficial health effects [
22]. Furthermore, the floral sources used by bees to produce honey, whose predominance depends on seasonal and environmental issues, influences the phenolic composition and antioxidant activity of honey.
The results of the phytochemical characterization of the samples regarding total phenolics and flavonoids contents are presented in
Table 1.
Total phenolic compounds content of honey samples ranged from 0.029 to 0.107 g GAE/100 g sample, the values observed for propolis extracts ranged from 21.747 to 28.947 g GAE/100 g sample, and finally for the mixtures of honey with propolis ranged from 1.219 to 3.506 g GAE/100 g sample. The honey that presented the highest content in phenolic compounds is Honey 1 and the lowest content can be found in Honey 3. These differences may be related with the different seasons in which the honeys were produced, as mentioned above.
Propolis extracts showed a much higher concentration of phenolic compounds than honey samples, with the highest content found in Propolis Extract 1 followed by Propolis Extract 3 and Propolis Extract 2. An increase in phenolic content was observed with the addition of higher concentrations of propolis extracts to honey, and the highest value was obtained with H1PE3 at 0.5%.
Flavonoids were almost absent from honey samples. The only one that presents flavonoids in its composition is Honey 1, but even this sample has a very low content. In contrast, the flavonoids determined in the propolis extracts ranged from 1.786 to 5.494 g QE/100 g sample, and in the mixtures ranged from 0.054 to 0.452 g QE/100 g sample. Propolis extracts showed a higher concentration of flavonoids than honey samples, with the highest content in Propolis Extract 1 followed by Propolis Extract 3 and Propolis Extract 2. An increase in flavonoid content was observed in all samples with the addition of higher concentrations of propolis extract to honey, as expected.
The values of total phenolic compounds and flavonoids determined in the present work are very similar to the ones obtained for selected Czech honeys [
23].
3.3. Antioxidant and Anti-Inflammatory Activities
Honey is an important natural source of antioxidants and has potential therapeutic value in several inflammatory diseases and in the treatment of heart disease, cancer, and cataracts, in addition to its sweetening capacity and lower glycemic load [
24]. The biological properties of honey comprise antioxidant, antimicrobial, anti-inflammatory, and wound-healing activities [
24].
In this work, the antioxidant activity of the samples was evaluated by two different methods that measure distinct antioxidant properties (
Table 2). The DPPH free radical scavenging assay is based on the capacity of the samples to scavenge free radicals, while the β-carotene bleaching test allows the indirect evaluation of the capacity of the samples to inhibit the lipid peroxidation [
25].
The honey that presented the highest antioxidant activity measured by the DPPH assay was Honey 1, opposed to Honey 3 that showed no relevant activity measured by this method. Propolis extracts revealed extremely high levels of antioxidant activity across all samples, with Propolis Extract 3 presenting the highest value. An increase in the antioxidant activity was observed in all samples with the addition of propolis extract to honey, however adding a higher concentration of propolis did not result in a considerable rase in activity in most cases.
Concerning the results of β-carotene bleaching test, the honey that revealed the highest antioxidant activity was once again Honey 1. The antioxidant activity of this honey measured by both methods is related with the presence of great amounts of phenolic compounds, as previously mentioned. Propolis extracts revealed high levels of antioxidant activity measured by β-carotene bleaching test as it was also verified by DPPH assay.
Analyzing the data obtained throughout the different assays it was possible to verify that dark-brown honey (H1) presented a higher content in phenolic compounds and flavonoids, followed by red honey (H2) and finally by light-yellow honey (H3). These results were consistent with the bioactive activity of the different samples. Since Honey 1 presented better phytochemical results, it was used in all the mixtures of honey with propolis.
The anti-inflammatory activity was evaluated using an in vitro assay that studied the ability of the samples to inhibit protein denaturation using a BSA solution (
Table 2). It was noted that propolis extracts reveal a higher anti-inflammatory activity than honeys. The honey that presented the highest activity was Honey 2, and among the Propolis Extracts, PE3 revealed the highest activity. Generally, an increase in the anti-inflammatory activity was observed in all samples when adding propolis extract to honey. In a previous work involving Malaysian honeys, the authors concluded that the anti-inflammatory activity may be attributed, at least in part, to the phenolic compounds [
24].
3.4. Wound-Healing Activity
In the present study, NHDF cells were used in a scratch assay. Although all the preliminary characterization data showed that the propolis extracts always presented better results than the other samples under study, it was decided to also evaluate the wound-healing activity for all the samples including the mixtures of honey with propolis.
It is already known that honey is not toxic against normal cells but is extremely cytotoxic to the tumor or cancer cells, as it was previously described [
26]. Similar results were found in propolis extracts, which demonstrated cytotoxicity in human fibrosarcoma and colon adenocarcinoma cells while presenting no cytotoxic action in normal human skin fibroblasts [
27].
By using the microscopic images, it was possible to evaluate the evolution of the gap created in the confluent cell monolayer in the presence of the samples (
Table 3,
Table 4, and
Table 5).
Analyzing the different images and comparing them to the control samples it is possible to say that the honey that shows better results after 36 h is Honey 2 (
Table 3), while the propolis extract that presented better results was Propolis Extract 2 at 0.5% (
Table 4); the best mixture is the honey with propolis—H1PE3 0.3% (
Table 5).
Examining all the images, it is possible to observe that the cells continue alive when incubated with the samples. Moreover, it is clear that the samples promote cell migration, demonstrating the wound-healing potential of honey and propolis.
Furthermore, by estimating the distance between the margins of the scratch (
Table 6) the conclusions were the same. For all the samples, except for Honey 3 at 2 h, a significant (
p-value < 0.05) reduction of the scratch was observed when compared to the control at the same time of incubation. In general, the samples that showed the best results were the mixtures of honey with propolis. However, the sample that presented the maximum activity was the Propolis Extract 2 at 0.5%.
In opposition to what was previously observed [
1], in the present work the obtained results suggest that the effect of combining propolis with honey is not synergistic but just the combined effect of honey and propolis. This may be due to the chemical composition of each particular honey that will directly influence its bioactivities. In the honey samples now studied, the concentration in total phenolic compounds is relatively lower than in other samples of honey [
1]. Moreover, flavonoids were not detected in the honey samples. These observations may explain the additive results observed for the mixtures of honey with propolis, suggesting the contribution of the propolis compounds to the biological activities.
Considering all the obtained results, it is possible to verify that the samples that presented higher cell migration levels also presented higher bioactivity.
During the inflammation process, honey promotes the release of inflammatory cytokines (TNF-α IL-6, IL-1β, and NO) by monocytes, which might stimulate collagen synthesis by fibroblasts, playing important roles in the initiation and amplification of this process [
10]. The modulation of the severity of inflammation can be associated with the anti-inflammatory properties of the polyphenols present in honey. Honey initiates an active but controlled inflammation but does not let the inflammation to develop in a chronic or exaggerated state, modulating the inflammatory phase of wound-healing [
10]. The anti-ulcerous activity of honey and propolis can be attributed to flavonoids that can act alone or in combination with other compounds such as sterols, terpinens, saponins, gums, and mucilage [
4].
A recently published paper, in which the potential wound-healing properties of propolis was evaluated, demonstrated that propolis promoted a marked increase in the wound repair capacity of keratinocytes [
28]. It was also proved that the regenerative properties of propolis are mainly due to H
2O
2 (which is extracellularly released and passes across the plasma membrane) is able to modulate intracellular mechanisms [
28].