Antioxidant activity of honey supplemented with bee products

The aim of this work was to evaluate the influence of supplementation of multiflower honey with bee products on the phenolic compound content and on antioxidant activity. Average total phenolic and flavonoids contents in the multiflower honeys were 36.06 ± 10.18 mg GAE/100 g and 4.48 ± 1.69 mg QE/100 g, respectively. The addition of royal jelly did not affect significantly the phenolic compound content and antioxidant activity. Supplementation of honey with other bee products, i.e. beebread, propolis, pollen, resulted in significant increase in the total phenolic and flavonoids contents, and in antiradical activity and reducing power, with the largest effect found for addition of beebread. Significant linear correlations between the total phenolic and flavonoids contents and antiradical activity and reducing power were found.


Introduction
One of the valuable components of the human diet is bee products, which are rich source of biologically active compounds (Oroian 2012). Their preventive and therapeutic properties result from their antioxidant activity. Bee products can also be an attractive health promoting food ingredients and components of functional food (Viuda-Martos et al. 2008;Salimi et al. 2013). They exhibit a very broad spectrum of antibacterial, antiviral, antiradical, antioxidant ABSTRACT The aim of this work was to evaluate the influence of supplementation of multiflower honey with bee products on the phenolic compound content and on antioxidant activity. Average total phenolic and flavonoids contents in the multiflower honeys were 36.06 ± 10.18 mg GAE/100 g and 4.48 ± 1.69 mg QE/100 g, respectively. The addition of royal jelly did not affect significantly the phenolic compound content and antioxidant activity. Supplementation of honey with other bee products, i.e. beebread, propolis, pollen, resulted in significant increase in the total phenolic and flavonoids contents, and in antiradical activity and reducing power, with the largest effect found for addition of beebread. Significant linear correlations between the total phenolic and flavonoids contents and antiradical activity and reducing power were found. and antitumor properties and are used for the prevention and treatment of many diseases (Mercan et al. 2007;Isidorov et al. 2009). Honey and bee products such as beebread, royal jelly and propolis can be used as components of functional foods due to their significant antioxidant properties (Sahinler & Kaftanoglu 2005;Viuda-Martos et al. 2008;Kruszewski et al. 2014;Socha et al. 2015). They contain a significant number of components which exhibit antioxidant activity (Jasicka-Misiak et al. 2012;Ramanauskiene et al. 2012;Maurya et al. 2014). Although the main components responsible for antioxidant activity of honey are phenolic acids and flavonoids, the synergistic effects of all bioactive compounds present in honey and bee products are not without a significance (Viuda-Martos et al. 2008). Bee products are gaining more and more appreciation of consumers due to the presence of bioactive compounds. Among these compounds, the polyphenolic ones which are natural antioxidants constitute a significant group. The most natural way of introducing bee products such as royal jelly, pollen, propolis and beebread to the human diet appear to be an addition to honey. Therefore, the aim of this work was to assess the effect of addition of bee products to natural honeys on their antioxidant properties.

Results and discussion
Results of the determination of phenolic acids and flavonoids contents and antiradical activity and reducing power of the honey are presented in Table 1. The natural multiflower honeys contained from 21.73 to 50.12 mg GAE/100 g of phenolic compounds, with the mean value being 36.06 mg GAE/100 g. The values obtained are similar to those reported in the literature for multiflower honeys (Maurya et al. 2014). Addition of a royal jelly to the honey not significantly affected the concentration of phenolic compounds, which was 39.88 mg GAE/100 g. A significant increase in the amount of phenolic compounds was found for honeys supplemented with the other bee products. In the case of supplementation with pollen and propolis, the average concentration of phenolic compounds was 84.22 and 114.82 mg GAE/100 g, respectively, while the highest concentration of phenolic compounds (179.96 mg GAE/100 g) was determined in the honeys supplemented with beebread (Table 1). Antioxidant activity of beebread related to the presence of a wide variety of substances with antioxidant properties has been previously reported Table 1. Total phenolic and flavonoid content, antiradical activity and reducing power of natural and supplemented with bee products honeys. by Isidorov et al. (2009). Noteworthy is, however, a large variation of the samples of that group, where RSD value was as much as 71%. This variability is due to a variation in the amount of added beebread, which was from 10 to 60%, as declared by the manufacturers. The other groups of honeys were characterised by a much smaller variation in supplementation with bee products, and it was 0.5-0.8%, 5-10% and 0.5-1% for honeys supplemented with royal jelly, pollen and propolis, respectively. The lack of appropriate standards results in a considerable discretion over the supplementation of honey with bee products. Noteworthy, however, is that in this study, the honey samples supplemented with propolis in much smaller amount than with beebread were characterised by only slightly lower phenolic content than the last ones. This demonstrated the great antioxidant activity and phenolic compounds content in propolis (Socha et al. 2015). A similar relationship was observed for flavonoid content. The natural multiflower honeys contained from 3.61 to 7.13 mg QE/100 g of flavonoids, with the mean value being 4.48 mg QE/100 g (Table 1). These values are confirmed by the literature data (Khalil et al. 2012;Šarić et al. 2012;Perna et al. 2013). In the presence of royal jelly, the mean flavonoid content of honey was significantly reduced, while addition of the other bee products caused increase in concentration of flavonoids. That increase was relatively the greatest for samples containing beebread, but with high variability in this group of samples (RSD = 57%), resulting from very different levels of supplementation. Slightly less flavonoid content was determined in the honeys with propolis (Table 1), because they contained much lower bee product added as compared to the samples with beebread. Supplementation of honeys with royal jelly did not affect their ability to deactivate free radicals, both DPPH • and ABTS •+ , since the ranges and mean values of the antiradical activity were similar to those determined for the natural honey (Table 1). A significant increase in the ability to deactivate free radicals was found for honeys supplemented with the other bee products. Similarly to previous results, the highest antiradical activity was exhibited by the honeys supplemented with beebread. Analogous results were found for reducing power. An addition of royal jelly did not affect a reducing power of the honeys. That characteristic was significantly increased in the honeys supplemented with the other bee products (Table 1). The honeys supplemented with beebread showed the highest reducing power, however, due to the different levels of supplementation with bee products, the variability of that parameter was significant in this group of samples (RSD = 75%). A lower reducing power was determined in honeys supplemented with propolis or pollen.

Type of sample
Significant linear correlations were found between the total phenolic content and the antiradical activity against DPPH • (r = 0.9453) and ABTS •+ (r = 0.9031), and reducing power (r = 0.9719), as well as between total flavonoids content and antiradical activity against DPPH • (r = 0.8787) and ABTS •+ (r = 0.8855), and reducing power (r = 0.9437). Literature data confirm the existence of such relationships (Khalil et al. 2012;Gambacorta et al. 2014).
The results of principal component analysis ( Figure. S1) partly confirmed the variability of the determined characteristics of the honeys in the groups of samples (Table 1). The first two reduced factors explain more than 96% of the variance, and two groups of samples with similar properties can be clearly distinguished among all the honey samples. The first group is of natural multiflower honeys and these with royal jelly, while the second one is of the honeys with pollen. Some similarity between each other of the samples was also found for the honeys with propolis; however, dissimilarity of two samples does not allow them to group. The most diverse group of samples was the group of honeys with beebread that is confirmed by the values of variability of the determined characteristics for these samples (Table 1).

Conclusions
Supplementation of honeys with bee products is the most natural way of using their health promoting potential in the prevention of many diseases and of delivering a wide range of bioactive compounds into the human body. Particularly remarkable product is propolis, an addition of which to a honey in small quantities, even at levels below 1%, results in a significant increase in the phenolic compound content and improves the antioxidant activity of a honey. Using the bee products in a supplementation of honey requires further studies on the effect of these additives on the merchantable quality and sensory attractiveness of natural honeys.

Disclosure statement
No potential conflict of interest was reported by the authors.

Supplemental data and research materials
Experimental details relating to this article are available online http://dx.doi.org/10.1080/14786419. 2015.1057582, alongside Figure S1.