3.1.4. Flavones

Studies conducted on the antibacterial activity of flavones [59] suggested that at least one hydroxy group in the A ring (especially at C-7) is vital for antibacterial activity. Hydroxyl groups in other positions such as C-5 and C-6 can also increase the antibacterial action [60]. However, the substitution of OH with OMe at C-7 was seen to reduce the antibacterial activity. For instance, the compound 5,7-dihydroxy-flavone with two OH at positions 5 and 7 has been found to be more potent against *Ralstonia solanacearum* (i.e., MIC: 25 and 300 μg/mL) compared to 5-hydroxy-7-methoxy-flavone with OMe at position 7 and OH at position 5 (Figure 1E) [61]. The importance of the –OH group at position 5 of flavones for their antimicrobial activity against MRSA strains has also been reported [62]. One of the investigations on plant isoflavonones suggested that the hydroxyl group's C-5, 6 and 7 position is crucial for antimicrobial action [63]. The presence of the prenyl (C5) group at position 6 without cyclization of this substituent with the A ring has also been reported to improve antibacterial activity [52].

### *3.2. Number of Functional Groups Attached*

The number of functional groups attached has been found to have a significant influence on antimicrobial activity. The 2, 4- or 2, 6-dihydroxylation of the B ring and 5, 7-dihydroxylation of the A ring in the flavanone structure are essential for anti-MRSA activity [64]. Moreover, 5-hydroxyflavanones and 5-hydroxyisoflavanones with one, two

or three additional hydroxyl groups at the 7, 2, and 4 positions inhibited the growth of *Streptococcus mutans* and *S. sobrinus* [65]. Caffeic acid had higher antimicrobial activity than p-coumaric acid due to the additional –OH group on the phenolic ring of the former compound [66].

### *3.3. Presence of C2=C3 Double Bond*

It has been observed that flavanones with C2=C3 are more active than the corresponding flavones. For example, naringenin showed antibacterial effects on all the tested bacteria, whereas apigenin showed almost no effect [67]. In addition, the C2=C3 double bond was found to be responsible for the antifungal activity of 5,7-dihydroxyflavonoids, while hydrogenation of the C2=C3 bond reduced the antifungal effect [68]. The flavonoids apigenin luteolin, dinatin, and daidzein, C2=C3 had better anti-influenza virus activities, compared with catechin and epicatechin belonging to the flavanols class of compounds that lack the C2=C3 bond [69].

### *3.4. Type of Functional Group*

The hydrophobic substituents such as prenyl groups, alkylamino chains, alkyl chains, and nitrogen- or oxygen-containing heterocyclic moieties have been reported to enhance the activity of all the flavonoids [48]. Variation in the antimicrobial activity of polyphenols also depends on variation in the functional group they have [70]. The substitution of the phenyl moiety by a propyl or a methyl group has been found to be deleterious for the antibacterial effect against *S. aureus* and *B. subtilis* [71]. This negative antimicrobial effect was also observed against *L. monocytogenes,* when substituting the phenyl with the propyl moiety [71].

The naphthoquinone 5,8-dihydroxy-1,4-naphthoquinone without chlorine was very active against three Gram-positive (*S. aureus*, *B. subtilis*, and *L. monocytogenes*) and three Gram-negative (*E. coli*, *P. aeruginosa*, and *S. Enteritidis*) strains but in a lower extent against *P. aeruginosa*. The compound 2,3-dichloro-5,8-dihydroxy-1,4-naphthoquinone with chlorine was significantly less active against *E. coli* and *P. aeruginosa* [70]. The presence of gallic or galloyl moieties was found to promote the antibacterial activity of epigallocatechin gallate by inducing damage to the bacterial membrane [72]. The antibacterial action of caffeic acid and their alkyl esters against specific strains of *S. aureus* and *E. coli* showed that longer alkyl side chains were more effective against the Gram-positive bacterium, while caffeic acid esters with medium length alkyl side chain were more effective against the Gram-negative bacterium which was also far less susceptible to caffeic acid and its esters [73].

### **4. Enhancement of Antimicrobial Activity of Plant Derived Polyphenols by Biochemical Methods**

Higher potency of the antimicrobial or their use in low concentration is preferred in food application to avoid any changes in the organoleptic properties and minimize interaction with the complex food matrices [14,74]. The following biochemical means may be used to obtain high potent antimicrobials.

### *4.1. Enhancement Using Ascorbic Acid and Transition Metals*

One of the promising approaches to enhance antimicrobial activity by non-enzymatic means can be mild oxidation of plant phytochemicals, particularly polyphenols by reactive oxygen species (ROS), using ascorbic acid (AA) and transition metals (Cu (II), Fe (II), Fe (III) systems. However, the reaction mechanism of AA oxidation in the presence of transition metals is still unclear, and different mechanisms have been proposed.

In one study, the AA and Cu2+ reaction were projected to yield one mole of hydrogen peroxide (H2O2) and one mole of dehydro-ascorbic acid in the pH range of 2.6–9.3 [75]. Additionally, it has been assumed that H2O2 is formed when AA reacts with Cu(II) in the presence of O2 within a pH range of 2.5–4.0 [76]. At physiological pH (7.0), H2O2 production increased when the copper (Cu2+) concentration was deficient, compared with

AA [77]. It was further postulated that the ascorbate mono-anion would dominate when the pH is over 4.25. This form can further be oxidized with the concomitant reduction of copper II to copper I [78]. At pH higher than 4.25, rapid redox cycling of copper generates superoxide, peroxide, and hydroxyl radicals via a copper assisted Fenton reaction, and at a pH lower than 4.25, the level of superoxide in the solution decreases as superoxide anion reacts with hydrogen to form the hydroperoxyl radical (HO2 •) [78].

The reaction product of AA and Cu2+ leads to cleavage of viral and plasmid DNA, which could be withdrawn in the presence of metal chelators such as EDTA, stating that copper plays an essential role in the oxidation of AA [79]. The reaction was also withdrawn in the presence of the catalase enzyme, confirming the fact that H2O2 is mainly produced in the AA/Cu2+ reaction [79]. The hydroxyl radical generated by the AA/Cu2+ system is lesser than that generated by the ascorbate/H2O2 system [80]. It has also been reported that transition metal ions, such as Fe (III) and Cu (II), are reduced by ascorbate. Their lower oxidation states (e.g., Fe (II) and Cu (I), respectively) (Equations (1) and (2) below) may further give rise to Fenton reactions with H2O2, producing hydroxyl radicals [81].

$$\text{Cu}^+ + \text{H}\_2\text{O}\_2 \rightarrow \text{Cu}^{2+} + \text{OH}^\bullet + \text{OH}^- \tag{1}$$

$$\rm Fe^{2+} + H\_2O\_2 \rightarrow Fe^{3+} + OH^\bullet + OH^- \tag{2}$$

In the presence of limited oxygen, polyphenols can be oxidized non-enzymatically [82].The polyphenols containing a catechol ring are oxidized to semiquinone and benzoquinone radicals (Figure 2). At the same time, the oxygen is reduced to H2O2, and the whole process is mediated by the redox cycle of Fe3+/Fe2+ and Cu2+/Cu+ [83]. Recent reviews have illustrated the antimicrobial action of quinone and its derivatives [84–86]. It may be suggested that, in presence of ascorbic acid and transition metals, redox oxygen species may be generated, which may oxidize the polyphenols to corresponding quinones or benzoquinones, enhancing antimicrobial activity [87,88].

**Figure 2.** The figure illustrates the oxidation of the catechol ring containing polyphenols, which is oxidized to benzoquinone radical with an intermediate formation of semiquinone in presence of Cu2+ catalyst. The reaction starts with the initiation step of O2 being converted to O2 −, followed by a propagation step during which O2 − is converted to H2O2 [89].

Phenolics from plants have been combined with other substances, sometimes referred to as adjuncts, such as transition metal ions or vitamin C to enhance phenolic efficacy [90,91]. Enhancement of antimicrobial activity was observed in the case of pomegranate rind Ext by Cu (II) alone or with both Cu (II) and AA combinations against many bacterial strains [92,93]. The addition of copper (II) sulfate and AA ascertained the enhancement of antimicrobial activities of whole and sub-fractionated white tea against *S. aureus* [94]. Enhanced antimicrobial activity against *S. aureus* and *E. coli* was also observed by the addition of the AA to the (+)-catechin–copper (II) mixture [95]. A recent study revealed

the antibacterial effect of AA against *S. enterica* subsp. *Enterica* serovar Typhi and *Vibrio fluvialis* could be enhanced when applied in a combination with linalool and copper [96]. However, the mechanism behind the enhancement in antimicrobial activity due to the addition of transition metals and AA to the plant polyphenols has not been appropriately elucidated yet.

#### *4.2. Enhancement Using Degradation Products of Ascorbic Acid in an Ethanolic Solution*

In aqueous (AQ) systems, AA is very unstable and efficiently degraded both aerobic (AB) and anaerobically. The degradation process of AA is complex and involves many oxidation–reductions and intermolecular rearrangemen<sup>t</sup> reactions. The degradation of AA via AB and anaerobic (AAB) pathways depends upon oxygen, heat, light, storage temperature, and time [97,98]. However, degradation of AA mainly results in the formation of volatile and brown products via self-degradation and non-enzymatic browning. The most commonly reported terminal products resulting from the AB degradation of AA and dehydroascorbic acid in acidic AQ conditions (pH 1–3), were found to be 3-hydroxy-2-pyrone and 2-furoic acid [99]. These are also amongs<sup>t</sup> the highest yielding products depending on the conditions utilized. Heat-induced (60–100 ◦C) AB degradation of different solutions of AA and dehydroascorbic acid demonstrated that both 3-hydroxy-2-pyrone and 2-furoic acid were the main degradation products of AA (Figure 3A). This included decarboxylation of 2,3-diketogulonic acid with the formation of xylosone, a mechanism already reported by the authors of [100], followed by a multi-step conversion of xylosone to the terminal products via oxidation, dehydration, and/or ketoenol tautomerism [100]. Xylosone is a gateway to numerous degradation products, and its presence as an AA or dehydroascorbic acid degradation product has been confirmed by several studies [101,102].

**Figure 3.** Mechanism of aerobic and anaerobic degradation of ascorbic acid. (**a**) aerobic degradation of ascorbic acid with the primary terminal product of degradation as 3-hydroxy-2-pyrone; (**b**) anaerobic degradation of ascorbic acid with the primary terminal product as furfural [103].

The term "AAB degradation" of AA refers to ascorbic acid degradation to some terminal product via a mechanistic pathway that does not require an oxidation step. The AAB degradation of AA does not generally require the removal of O2 from a reaction system; however, the lowering of oxygen concentration has the advantage of limiting the competing AB reactions [103]. The degradation of AA by reaction with oxygen occurs faster than its AAB degradation [104]. However, the AAB degradation rate can increase considerably with higher temperatures [104]. The pH level is also known to influence the rate of AAB degradation of AA, which increases as the pH is raised from 2.3 to 4.0 [105].

Regardless, the most commonly reported terminal product for the AAB degradation of AA is furfural [99,106]. The general mechanism to describe the formation of furfural is shown in (Figure 3B) and involves initial ring cleavage and hydration of AA rather than oxidation [107]. The subsequent steps require decarboxylation, acid-catalyzed dehydration, and cyclization. Several studies have shown that the formation of furfural is favored at lower pH values [108,109].

Another mechanism has been reported for the acid-catalyzed AAB degradation of AA in methanol, forming a bicyclic structure similar to dehydroascorbic acid. It then undergoes dehydration and decarboxylation via dihydrofuran-type intermediates to afford furfural [110]. The authors proposed that the mechanism would be equally valid in AQ systems but did not provide evidence.

Not all scholars agree that furfural is exclusive to the AAB degradation of AA. Instead, it has been suggested that it can be formed via an oxidatively generated dehydroascorbic acid degradation pathway [111,112]. Another product unique to the AAB pathway is, 4,5-dihydroxy-2-ketopentanal (otherwise known as 3-deoxypentosulose) generated upon storage of AA at pH 3.5, although the storage temperature was not so relevant to wine conditions (120 ◦C for 2 h) [113]. This compound has been proposed as an intermediate in the formation of furfural during the AAB degradation of AA. The intermediate furan compounds which were generated during AA degradation have been found to inhibit the proliferation of *S. typhi* and *B. subtilis* to different extents [114]. The MIC values of furfural and furoic acid (terminal products of AA degradation) against *B. subtilis* and *S. typhi* were 0.027, 0.015, and 0.029, 0.009 μM, respectively [114]. Recent studies have reported antimicrobial action of furoic acid and furan compounds [115,116] The reported end product of AB and AAB degradation might have potency in enhancing the antimicrobial activity of plant Ext. However, to date, there is no evidence of the enhancement of the antimicrobial activity of plant Ext using the degradation product of AA.

### *4.3. Enhancement Using Maillard Reaction Products*

The Maillard reaction (MR) is a heat-induced browning reaction widely employed in various fields in the food industry that has been used as an effective method for protein modification and the production of remarkable changes in the structure and bioactivity of proteins [117,118]. In particular, MR products (MRPs) have been shown to have significant antibacterial activities against a wide range of bacteria, with lower toxicity than antibiotics [119,120]. The MRPs possess many intermediate products (Figure 4), such as aldehydes, ketones, and heterocyclic compounds, which can effectively inhibit the growth of some Gram-positive and Gram-negative bacteria [119].

MR products may have the potential to exhibit a synergistic antimicrobial effect in conjunction with phytochemicals from plant compounds, thus enhancing their potency ( i.e., lowering the MIC value of the plant-derived compounds) [121]. The plant-derived polyphenols have been found to have additive, synergistic antimicrobial effects with the intermediate products of the MR such as diacetyls, carbonyls, and furfural [122,123]. Methylglyoxal (one of the intermediate products of MR) and catechin have been reported to positively affect antibacterial activity [124]. However, the antimicrobial activity of plant Ext or plant phytochemicals using MRPs is not ye<sup>t</sup> reported, and further research is needed in this area.

**Figure 4.** Scheme of Maillard reaction in different pH levels consisting of early stage with the formation of the ARP products and Schiff base, followed by the advanced stage, consisting of fission reaction, Stecker degradation, resulting in formation of advanced glycated products with a final stage of oxidation, condensation, cyclization, and rearrangemen<sup>t</sup> resulting in melanoidin polymers.

### *4.4. Enhancement Using Laccase–Mediator System*

The oxidation of organic compounds to produce functionalized molecules is essential in organic synthesis [125]. Controlled enzymatic oxidation or hypo-oxidation can ye<sup>t</sup> be another approach toward enhancing the antimicrobial activities of phytocompounds. Increased bioactivities have been observed by the biochemical transformation of triterpenes using oxidative enzymes [126]. Oxidoreductive enzymes such as laccase and peroxidise can transform phenols through oxidative coupling reactions with the production of polymeric products by self-coupling or cross-coupling with other molecules.

Laccase (EC 1.10.3.2), is a multi-copper oxidase that couples the four-electron reduction of oxygen with the oxidation of a broad range of organic substrates, including phenols, methoxy-substituted phenols, anilines, aryl diamines, hydroxyindoles, benzenethiols and inorganic/organic metal compounds by a one-electron transfer mechanism, making this green enzyme useful for carrying out several types of oxidative reactions [127–132]. Laccases use O2 as the electron acceptor to remove protons from the phenolic hydroxyl groups. This reaction gives rise to radicals that can spontaneously rearrange, which can either lead to the fission of C–C or C–O bonds of the alkyl side chains or the cleavage of aromatic rings [128]. The oxidation of a reducing substrate by laccase involves losing an electron and forming a free radical [129]. This radical is, in general, unstable and may undergo further laccase-catalyzed oxidation (e.g., quinone from phenol) or non-enzymatic reactions (e.g., hydration, disproportion, or polymerization) [132]. The electron transfer from the substrate to copper is controlled by the redox potential difference. The rate of substrate oxidation by laccase, which has high redox potential, is higher if it has a lower redox potential.

Enzymatic polymerization of phenolic compounds (catechol, resorcinol, and hydroquinone) has been carried out using laccase [133–135]. Intermediates (quinones) formation in the first stage of oxidation with further oxidation reaction, forming colored polymers,

was observed while evaluating the polymerization and the structures of the polymers by UV–Vis and Fourier transform infrared spectroscopy [134]. Changes in the color of flavonoids due to oxidation by the laccase enzyme were due to the polymerization and linkage of the quinones (Figure 5) formed as an intermediate [133]. Laccase oxidation of caffeic acid and isoeugenol was shown to enhance their antimicrobial activity against *S. aureus* and *E. coli* in liquid media [135]. Some low molecular weight phenolic compounds are usually produced as a result of oxidative metabolism by C ring cleavage of catechin and epicatechins [136]. The antimicrobial properties of one of these low molecular weight polyphenols, 3,4 dihydroxy benzoic acid have been well established [137,138]. Moreover, dimers and polymers of flavonoids have also been found to have superior antimicrobial effects in comparison to the parent monomer [139–141]. It has been suggested that the toxicity of the laccase-treated olive Ext can be due to the presence of phenolic compounds such as ortho-benzoquinones, quinonoid, or oxidative coupling polymers, which results because of Lac treatment is more toxic than the parent compounds [142]. Many studies have suggested polymerization of the phenolics using the Lac enzyme [143,144]. Enhanced antimicrobial activity of the resulting oligomers and polymers has also been reported [145,146].

**Figure 5.** Mechanism of catechin oxidation by laccase where catechin is polymerized into polycatechin with the intermediate formation of semiquinone radicals and orthoquinone.

Although laccase can oxidize a wide range of substrates, some substrates of interest cannot be oxidized directly by laccases, either because they are too large to fit into the enzyme active site or because they have an exceptionally high redox potential. By mimicking nature, it is possible to overcome this limitation with the addition of so-called "chemical mediators", which are suitable compounds that act as intermediate substrates for the laccase, whose oxidized radical forms can interact with the bulky or high redox-potential substrate targets [131]. Laccase–mediator system (Figure 6) has found its immense application in the degradation of lignin. Small redox molecules such as 3-hydroxyanthranilic acid (HAA) might act as "electron shuttles" between the enzyme and lignin and cause polymer de-branching and degradation [131]. Some examples of laccase mediators extensively used are 2,2-azino-bis-(3-ethylbenzothiazoline-6-sulphonic acid) (ABTS), N-hydroxybenzotriazole (HBT), N-hydroxyphtalimide (HPI), violuric acid (VLA), N-hydroxyacetanilide (NHA), methyl ester of 4-hydroxy-3,5-dimethoxy-benzoic acid (syringic acid), and 2,2,6,6- tetramethyl piperidine-1-yloxy (TEMPO) [131].

**Figure 6.** Schematic representation of laccase-catalyzed redox cycles according to which laccase is oxydize in presence of oxygen, itself oxidizing the mediator, and the oxidized mediator further initiates the oxidation of the substrate, with high redox potential [131].

Laccase is capable of oxidizing unreactive iodide to reactive iodine [147]. Phenolic compounds such as vanillin, which resembles substructures of softwood lignin, can be directly iodinated by reacting with laccase and iodide, resulting in compounds with antifungal activity [147]. The addition of redox mediators "acetosyringone" in catalytic concentrations increased the rate of iodide oxidation by ten-fold and the yield of iodo-vanillin by 50% [147].

Functionalization is the process of adding new functions, features, capabilities, or properties to material by changing the surface chemistry of the material [148]. In one study, functionalization of chitosan with phenolic acids such as caffeic acid or gallic acid using laccase from *Trametes versicolor* formed a product with enhanced antimicrobial activity against *E. coli* and *L. monocytogenes* [149]. The authors proposed the functionalization of chitosan with the phenolic acids by laccase catalyzed oxidation of phenolic acids to electrophilic o-quinones, which undergo a new oligomer/polymer-forming structure originated by C–C coupling between the benzene rings and C–O–C coupling involved with the phenolic side chains.

### *4.5. Enhancement Using Peroxidase Enzyme*

The peroxidases (EC 1.11.1.7) are heme proteins and contain iron (III) protoporphyrin IX (ferriprotoporphyrin IX) as the prosthetic group. They come under the class of oxidoreductases that catalyze the oxidation of a wide range of molecules, using peroxide as an electron acceptor [150]. The reduction of peroxides at the expense of electron-donating substrates makes peroxidases useful in several industrial and analytical applications. The common overall reaction of the peroxidases can be written as in the following Equation (3), where RH is a suitable peroxidase substrate and R is a free-radical product derived from it as follows:

$$2\text{RH} + \text{H}\_2\text{O}\_2 \rightarrow 2\text{R}^\bullet + 2\text{H}\_2\text{O} \tag{3}$$

Oxidation reactions carried out by peroxidase may be one-electron or two-electron oxidation. A classic example of one-electron oxidation is guaiacol assay through which guaiacol is oxidized to a free radical that undergoes a subsequent radical–radical combination to give a colored dimeric product. The dimerization can occur between two ring carbon atoms or by adding the oxygen of one phenoxy radical to the ring carbon of the other [151]. Two-electron oxidation is rare for most peroxidase enzymes. The example under this type is the oxidation of halide and pseudo-halide ions, specifically I<sup>−</sup>, Br<sup>−</sup>, Cl<sup>−</sup>, and NCS<sup>−</sup>. The oxidation of I− and NCS− is common for the peroxidases [152]. Many reactions are known in which oxidation of a substrate by peroxidases produces a two-electron oxidized product. These reactions can be rationalized by forming a free radical metabolite, followed by the second oxidation of the free radical to the final observed product.

Lactoperoxidase, together with thiocyanate ions and hydrogen peroxide generates, hypothiocyanite ions and the oxidized product which is known as the lactoperoxidase system. The oxidized effect possesses a broad spectrum of antimicrobial activity. Hence, much attention has been paid to the lactoperoxidase system [153], e.g., lactoperoxidase systems mediated by oxidized β-carotene/SCN− cycling lead to enhanced antimicrobial effects [154].
