Evaluation of the In Vitro Wound-Healing Activity and Phytochemical Characterization of Propolis and Honey

Honey and propolis are natural substances produced by Apis mellifera that contain flavonoids, phenolic acids, and several other phytochemicals. The aim of this study was to phytochemically characterize three different types of honey and propolis, both separately and mixed, and to evaluate their wound-healing activity. Total phenolic compounds and flavonoids were determined using the Folin–Ciocalteu’s and aluminum chloride colorimetric methods, respectively. The antioxidant activity was evaluated by both the DPPH free radical scavenging assay and β-carotene bleaching test, and the anti-inflammatory activity was determined by a protein denaturation method. To evaluate the wound-healing activity of the samples, NHDF cells were subjected to a wound scratch assay. The obtained results showed that dark-brown honey presents a higher concentration of phenolic compounds and flavonoids, as well as higher antioxidant and anti-inflammatory activities. Propolis samples had the highest concentrations in bioactive compounds. Examining the microscopic images, it was possible to verify that the samples promote cell migration, demonstrating the wound-healing potential of honey and propolis.


Introduction
Honey is a natural substance produced by the western honeybee (Apis mellifera) from nectar and exudates of flowers and trees. Honey contains flavonoids, phenolic compounds, and numerous sugars in its composition, mainly glucose and fructose. It also contains small amounts of minerals, vitamins, proteins, and enzymes [1][2][3][4][5].
The color and flavor of both honey and propolis vary according to the plant species used in their production, health state of the bees, season, and the environmental conditions to which the beehive is exposed [2][3][4][5]. The color of the honey can vary from deep brown to yellow, and propolis

Fourier-Transform Infrared Spectroscopy (FTIR)
FTIR was used to obtain spectra of the samples of honey, propolis and propolis extracts. These spectra were obtained with 64 scans and a 4 cm −1 resolution, between 4000 and 600 cm −1 using a Nicolet iS10 smart iTRBasic (Thermo Fisher Scientific, Waltham, MA, USA) model.

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

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 reagent (Sigma-Aldrich, USA) (0.2 N) were added, being the mixtures left for 5 min before the addition of 2 mL of aqueous Na 2 CO 3 (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].

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].

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

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 = [(Abs control − Abs ample )/Abs control ] × 100, where Abs control corresponds to the absorbance of the control and Abs sample 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).

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 = [(Abs sample t = 1 h − Abs control where Abs control corresponds to the absorbance of the control and Abs sample 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).

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 buffer 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 − [(Abs sample × 100)/Abs control ], where Abs control corresponds to the absorbance of the control and Abs sample is the absorbance of each sample [17]. The results were expressed as %Inhibition/100 g sample (honey, propolis, and mixtures of honey with propolis).

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 4 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% CO 2 ) 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.

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 difference 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.

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 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].

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 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].

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].

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].

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%. Table 3. Microscopic images obtained from the scratch wound-healing assay with the honeys (magnification: 100×).

Representative Image of the Cells at the Initial Moment (0 h)
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. Control 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. 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. 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. 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. 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. 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. 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. 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. 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 (TNFα 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 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 (TNFα 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 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 (TNFα IL-6, IL-1β, and NO) by monocytes, which might stimulate collagen synthesis by fibroblasts, Table 4. Microscopic images obtained from the scratch wound-healing assay with the propolis extracts (magnification: 100×).

Samples 2 h 24 h 36 h
PE1 0.3% 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]. PE2 0.5% 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]. PE2 0.5% 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]. 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]. PE2 0.5% 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]. PE2 0.5% 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]. 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]. 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]. 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]. 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]. 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]. 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 H2O2 (which is extracellularly released and passes across the plasma membrane) is able to modulate intracellular mechanisms [28].

H1PE3 0.5%
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 H2O2 (which is extracellularly released and passes across the plasma membrane) is able to modulate intracellular mechanisms [28].

H1PE3 0.5%
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 H2O2 (which is extracellularly released and passes across the plasma membrane) is able to modulate intracellular mechanisms [28].
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 2 O 2 (which is extracellularly released and passes across the plasma membrane) is able to modulate intracellular mechanisms [28].

Conclusions
This work demonstrated the biological potential of honey and propolis, particularly the wound-healing activity, which is related with their antioxidant and anti-inflammatory properties. Further studies should be performed to clarify the mechanism of action of honey and propolis by which cell migration is stimulated.