*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%.

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


**Table 4.** Microscopic images obtained from the scratch wound-healing assay with the propolis extracts (magnification: 100×).


**Table 5.** Microscopic images obtained from the scratch wound-healing assay with the mixtures of honey with propolis (magnification: 100×).

In opposition to what was previously observed [1], in the present work the obtained results sugges<sup>t</sup> 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 H2O2 (which is extracellularly released and passes across the plasma membrane) is able to modulate intracellular mechanisms [28].

**Table 6.** Calculated distance between the margins of the injury.


Results expressed as mean ± standard deviation; \* Indicates a significant result (*p*-value < 0.05).
