Interactions of resveratrol with other phenolics and activity against food‐borne pathogens

Abstract The aim of this study was to investigate the antibacterial activity of individual phenolics and their binary mixtures with resveratrol against selected food‐borne pathogens. The antibacterial activity was quantified using the broth microdilution method by the determination of minimal inhibitory concentrations (MICs). Interactions between compounds in the binary phenolic mixtures were determined by calculating the fractional inhibitory concentration index (FICI). The influence of the number of OH groups in the phenols’ structure on their antibacterial activity was assessed by principal component analysis (PCA). The most effective compounds were flavone luteolin and flavonol rutin, while the weakest antimicrobial activity was observed for phenolic acid and flavan‐3‐ols (catechin and epicatechin). The synergistic effect (FICI ≤0.5) of equimolar mixture of resveratrol with kaempferol was confirmed against Staphylococcus aureus, Bacillus cereus, and Escherichia coli, while the mixture of rutin with resveratrol proved synergistic only against S. aureus. The increasing concentrations of resveratrol in the mixtures with kaempferol and rutin resulted in a loss of synergism which indicates that only selected phenolic mixtures, with optimal concentrations of their individual components, result in synergistic antibacterial activity. We did not find an association between total number of OH groups and antibacterial activity of either individual phenolics or their mixtures.

The antibacterial activity of phenolic compounds is related to their structures and the type of microorganism (Kumar & Pandey, 2013;Shan, Cai, Brooks, & Corke, 2007). Due to large variability of the reactive groups in phenolics' structure, their antimicrobial effects may be mediated by different mechanisms. In addition, different targets on the microorganism cells may be affected (Skandamis, Koutsoumanis, Fasseas, & Nychas, 2006;Xie, Yang, Tang, Chen, & Ren, 2015). Phenolics are capable of interacting with the cytoplasmic membrane, cell wall, nucleic acids, and/or energy transport, by altering or inhibiting their functions (Kumar & Pandey, 2013;Sanhueza et al., 2017;Xie et al., 2015). Furthermore, they have the ability to denature enzymes, or bind to vitamins, minerals, and carbohydrates making them inaccessible to microorganisms (Kumar & Pandey, 2013).
The aim of this study was to investigate the antibacterial activity of individual phenolic compounds from a group of phenolic acids, flavonols, flavones, and flavan-3-ols in relation to resveratrol against several food-borne pathogens. In order to investigate possible synergistic, additive or antagonistic interactions of these compounds, they were used in combination with resveratrol as binary phenolic mixtures.

| Pure phenolic compounds and binary phenolic mixtures
The present study included commercially available phenolic com-    Concerning the structure-antibacterial activity relationship, although containing the highest number of free OH groups in their structure, catechin and epicatechin showed a weak effect which was TA B L E 2 The antibacterial activity of mixtures of resveratrol with selected phenolic compounds at a molar ratio 1:1 (expressed as the MIC value in µM) does not play a significant role in their antibacterial activity. Instead, the dominant factor could be a difference in the structure of ring C, like the presence or absence of OH and/or keto groups (Kumar & Pandey, 2013;Taguri et al., 2006). Except structural differences, all these compounds possess different mechanism of action on microbial cell. It has been described that catechin induced cytoplasmic damage, that quercetin can cause an increase in the permeability of the cytoplasmic membrane, and that lueolin affected the cytoplasmic membrane stability and inhibited enzymes (Sanhueza et al., 2017).

| Antibacterial activity of individual phenolic compounds
The results from the antibacterial activities of the tested phenolic compounds against gram-positive and gram-negative bacterial species were analyzed by PCA. Figure 1a shows have four OH groups while catechin, epicatechin, and quercetin with five OH groups showed lower antibacterial activity. Consequently, we could not confirm that the catechol structure feature in ring B enhances the antibacterial activity of the compound. This is in line with the findings of Kumar and Pandey (2013) and Taguri et al. (2006) F I G U R E 2 (a) The effects of increasing doses of resveratrol in the binary phenolic mixtures on their antibacterial activity. The results are shown for mixtures of resveratrol with kaempferol and with rutin at 2:1 and 4:1 molar ratios, respectively (expressed as MIC values in µM); (b) The interaction between the compounds in the same binary phenolic mixtures described by the fractional inhibitory concentration index (FICI) who also could not establish a clear relationship between the total number of OH groups and antibacterial activity.

| Antibacterial activity of binary phenolic mixtures
Despite numerous studies examining the antibacterial effects of combined natural compounds such as essential oils (Palaniappan & Holley, 2010;Patrone, Campana, Vittoria, & Baffone, 2010;Periago & Moezelaar, 2001;Rivera Calo, Crandall, O'Bryan, & Ricke, 2014) or phenols and organic acids (Del Valle et al., 2016;Vasconcelos de Oliveira, Montenegro Stamford, Gomes Neto, & Leite de Souza, 2010), information on the interaction of individual phenolic compounds with resveratrol are rather scarce (Chan, 2002;Del Valle et al., 2016). In this study, the antibacterial activity of selected phenolic compounds with resveratrol was tested by the microdilution method, and their interaction as binary phenolic mixtures was described by the fractional inhibitory concentration index (FICI) (Mundy, Pendry, & Rahman, 2016;Palaniappan & Holley, 2010;Petersen, Labthavikul, Jones, & Bradford, 2006). The data presented in Table 2 indicate that mixing phenolic acids with resveratrol at an equimolar ratio generally improved their antibacterial activity. The FICI index above 0.5 and lower than 4 indicates that the interactions of phenolic acids with resveratrol (Table 2)  The results from the antibacterial activities of the tested phenolic mixtures were also analyzed by PCA. Figure 2b shows the position of polyphenolic mixtures with resveratrol in the multivariate space, specifically with mixtures of resveratrol with kaempferol, luteolin, and rutin grouped in the same quadrant. These mixtures containing seven OH groups showed better antimicrobial activity than the remaining three mixtures with eight OH groups. This again is indicative that antibacterial activity cannot simply be estimated by the number of OH groups, it is necessary to take into account other factors, such as position of OH groups, solubility, polarity, medium pH, and bacterial properties.

ACK N OWLED G M ENT
This study was partially supported by the Croatian Science Foundation (Project No. 8652).

CO N FLI C T O F I NTE R E S T
The authors declare that they have no conflict of interest.

E TH I C A L S TATEM ENT
The study did not involve any human or animal testing.