**1. 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 [1–5].

Propolis is a resinous substance produced by the same species of bees, being composed of wax, resinous secretions from plants, tree buds, pollen, and salivary enzymes. Propolis contains essential oils and organic compounds such as phenolic acids and flavonoids [4,6–8]. Similarly to honey, propolis also contains several minerals, vitamins, and enzymes [9]. This substance is used to protect the beehive, acting as a natural sealant, antiseptic, and embalming agen<sup>t</sup> [1,4,6–8,10].

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 [2–5]. The color of the honey can vary from deep brown to yellow, and propolis

can be found in variations of green, red, or brown [6,7,11]. Di fferences in their composition result in di fferent biological activities [1,4,7,12]. Honey and propolis have been used in traditional medicine for thousands of years mainly in burns and infected lesions [4,10]. Propolis was used primarily as an oral antiseptic and in dermal lesions. In ancient Egypt, propolis was also employed in mummification [6].

The main mechanism of action of honey derives from its antioxidant and anti-inflammatory activities [1,2,10]. The antioxidant potential of honey is due to the presence of phenolic compounds, namely gallic acid and flavonoids, that promote the free radical scavenging [2,10]. Its anti-inflammatory activity is associated with the ability to increase cellular proliferation, autolytic debridement, and to stimulate the immune system, reducing edema and pain [10]. Propolis has a higher content in flavonoids and phenolic acids than honey, which suggests that propolis should have a higher antioxidant and anti-inflammatory activities [1,7].

Currently, an increasing interest in mixing propolis with honeys was verified, as products including propolis are already available on the market. The commercial achievement of these products is dependent of the level of acceptance and expectations of the consumers [1]. However, the potential benefits of adding propolis to honeys is still to be scientifically validated.

Wounds are responsible for the consumption of large amounts of healthcare resources to ameliorate the quality of life of patients, and so they represent a considerable health challenge. Several scientific experiments have been performed to find novel compounds that possess wound-healing properties, particularly from natural sources [13]. Honey was employed for the repair of battle wounds in both World War I and in modern history. The healing activity of honey in infected wounds was initially described in Europe and USA in mid-20th century [10]. Moreover, evidences sugges<sup>t</sup> that propolis has therapeutic activity, through quantitative and qualitative analyses of collagen types I and III expression and degradation in wounds matrix, which suggests the favorable biochemical environment supporting re-epithelization of propolis [11].

The first aim of this study was to phytochemically characterize di fferent samples of honey and propolis together with mixtures of propolis extracts with honey in di fferent concentrations, determining the contents in total phenolic compounds and flavonoids, as well as evaluating the antioxidant and anti-inflammatory activities. The second purpose of this work was to evaluate the *in vitro* wound-healing activity of the samples using normal human dermal fibroblasts (NHDF) by means of the scratch assay.

#### **2. Materials and Methods**

#### *2.1. Honey and Propolis Samples*

Honey and propolis samples were collected from a *Langstroth* beehive in a mountainous forest in the north central Portugal, specifically in the region of Sabugal (Guarda) during September 2018 (GPS coordinates: 40◦2333.5" N 7◦0345.4" W).

Three di fferent samples of honey and propolis were collected from the same beehive to maintain consistent the conditions of production. Each honey sample was collected from a di fferent box inside the hive and was then filtered individually until there was no visible debris or particles. Propolis samples were scraped from the boxes and frames of the hive and then separated according to the box from which they were collected. All samples were stored individually in an appropriate sterilized container.

Dark-brown honey was considered as Honey 1 (H1), red honey was labeled as Honey 2 (H2), and light-yellow honey was identified as Honey 3 (H3). Propolis samples were named according to the box from which the honey samples were collected as Propolis 1 (P1), Propolis 2 (P2), and Propolis 3 (P3).
