*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.


**Table 1.** Total phenolic compounds and flavonoids content of the samples.

Results expressed as mean ± standard deviation.

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].



1Results expressed as mean ± standard deviation.

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 grea<sup>t</sup> 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].
