**8. Mechanisms of action of phenolic compounds**

**7. Phenolic profile of honey**

**Figure 4.** Structure of the major chemical types of flavonoids [51].

extracts, variating with the sample [54].

duced by the same bee species [40].

that found in honey from high sunlight incidence regions.

shady locations [53].

296 Honey Analysis

Regions characterized by a hot and humid climate with very high exposure to sunlight (as in northeast Brazil) are particularly known to exert a marked influence on the poly‐ phenolic content of plants. Sun‐exposed plants such as juazeiro (*Ziziphus joazeiro* Mart.) can contain much more total phenolics than the same varieties or other when grown in the

Assays made with honey collected in the central and southern region of Amazonas state in Brazil found that total phenolic content of methanolic extracts from the honey samples ranged from 17.0 to 66.0 mg galic acid equivalent (GAE)/g of extract and also high antioxidant pro‐ file. Gallic, 3,4‐dihydroxybenzoic, 4‐hydroxybenzoic, vanillic, salicylic, syringic, coumaric, trans,trans‐abscisic, cis,trans‐abscisic and cinnamic acids, catechol and flavonoids, taxifolin, naringenin and luteolin were identified. Concentrations ranged from 0.02 to 67.0 mg/mL of

Brazilian honeys from the semiarid region, which were composed of 24 monofloral hon‐ eys produced by *Meliponini*, native species of bee, were found to present strong antioxidant activity. The total phenolic content varied from 0.31 to 1.26 mg GAE/g with differences (p ≤ 0.05) among samples from distinct floral sources. The scavenging activity of DPPH radi‐ cals varied from 11.2 ± 1.3% to 46.9 ± 1.9%. Phenolic compounds p‐coumaric, ellagic and 3,4‐hydroxybenzoic acid and the flavonoids rutin, catechin, chrysin and naringenin were detected in higher amounts in *Ziziphus joazeiro* Mart. honeys than in the other honeys pro‐

Fifty eight polyfloral honey samples, from different regions in Serbia, were studied to deter‐ mine their phenolic profile, total phenolic content and antioxidant capacity. It was reported that the phenolic content ranged from 0.03 to 1.39 mg GAE/g and the radical scavenging activity of DPPH radicals ranged from 1.31 to 25.61% [44], an antioxidant capacity lower than

All these studies found strong correlation between total phenolic content or total flavonoid content and radical inhibition capacity, indicating that phenolics and flavonoids are the pri‐ mary factors responsible for the antioxidant properties of the studied honeys. Consequently, these results reinforce the influence of the botanical source on honey antioxidant properties.

Several mechanisms have been proposed to explain the observed antioxidant activity of phe‐ nolic compounds. The first is the direct removal of radicals through the formation of more stable compounds from radical supply of hydrogen (**Figure 5**). The various possible reso‐ nance hybrids in flavonoids and phenolic acids structure make them less reactive, limiting the deleterious power of other reactive species [55].

**Figure 5.** Radical stabilizing resonance structures by monoelectric oxidation of hydroxyl group in galangin [55].

Another mechanism of action of its antioxidant activity is their metal chelating propriety (**Figure 6**), which removes ions such as Fe2+, which catalyzes the formation of free radicals by Fenton and Waber‐Heiss reactions and which are propagators responsible by reactive oxygen species; decreasing, so the intracellular oxidative stress [56].

The *in vitro* activity of phenolic compounds depends on their structure. In flavonoids, the hydroxyl groups are in the ortho position (**Figure 7A**), especially in ring B; the presence of double bond to oxygen in the ring C (**Figure 7B**) and hydroxyl groups at positions 3 and 5 (**Figure 7C**) were found to increase the antioxidant capacity, since they contribute to stabi‐ lizing resonance structures [32, 57]. The presence of glycosides, however, reduces the anti‐ oxidant activity. The antioxidant activity of glycosidated conjugate rutin decrease about 50% when compared to quercetin [32].

**Figure 6.** Possible flavonoid coordinating points with metals [32].

**Figure 7.** (A) Hydroxyl ortho position; (B) the presence of double‐bonded oxygen in the 5‐position of ring C; (C) the presence of hydroxyl at positions 3 and 5 [32].

Phenolic acids have increased activity in the presence of hydroxyl groups in the ortho posi‐ tion (**Figure 8**) or carbonyl groups in the ortho hydroxyls, as with syringic acid [57]. Moreover, in general, the hydroxycinnamic acids have shown *in vitro* activities higher than the hydroxy‐ benzoic acids [58].

However, tests on biological models show that the flavonoids and other phenolic compounds act modulating the expression and activity of enzymes related to antioxidant defences [59, 60]. Phenolic compounds have the ability to induce phase II enzymes, such as quinone reductase NADPH and GST, as well as inhibiting enzymes related to carcinogenesis such as protein activation 1 (AP1), nuclear factor (NF)‐κB and MAP‐kinases [32, 60].

It is also important to emphasize that phenolic compounds also have pro‐oxidant activity, dependent on its concentration. The presence of hydroxyl groups in the ortho position can also produce radicals or hydrogen peroxide, in the presence of copper ions and oxygen molecules [61, 62]. The flavonoid rutin and morin at concentrations above 100 μg mL‐1 were able to produce hydrogen peroxide and damage DNA through comet assay in human lym‐ phocytes. However, this effect was not observed with naringenin, and hesperidin in the same concentration, which do not have hydroxyl groups in ortho position on ring B [63]. The generation mechanism of hydrogen peroxide or radicals can explain the antimicrobial action of flavonoids and their toxic effects at higher concentrations to microorganisms [32].

**Figure 8.** Radical sequestration mechanism of hydroxycinnamic acid including resonance stabilization radical by intramolecular hydrogen bonding [58].

*In vivo* testing confirms the antioxidant activity observed *in vitro*. Phenolic extracts from two monofloral Cuban honeys were able to inhibit erythrocytes oxidative damage. This study indicated that honey contains relevant antioxidant compounds responsible, at least in part, for its biological activity and that uptake of its flavonoids may provide defence and promote cell functions in erythrocytes [64]

A study was undertaken to determine whether replacing sucrose in the long‐term diet with honey, which has high antioxidant content, could decrease deterioration in brain function during ageing. Rats were fed *ad libitum* for 52 weeks on a powdered diet that was either sugar‐free or contained 7.9% sucrose or 10% honey. Apparently, long‐term feeding of honey, sucrose and a sugar‐free diet may have some effects on anxiety and spatial memory in rats, with honey‐fed rats exhibiting less reduction in spatial memory and decreased anxiety at the completion of the study [65].

Phenolic acids have increased activity in the presence of hydroxyl groups in the ortho posi‐ tion (**Figure 8**) or carbonyl groups in the ortho hydroxyls, as with syringic acid [57]. Moreover, in general, the hydroxycinnamic acids have shown *in vitro* activities higher than the hydroxy‐

**Figure 7.** (A) Hydroxyl ortho position; (B) the presence of double‐bonded oxygen in the 5‐position of ring C; (C) the

However, tests on biological models show that the flavonoids and other phenolic compounds act modulating the expression and activity of enzymes related to antioxidant defences [59, 60]. Phenolic compounds have the ability to induce phase II enzymes, such as quinone reductase NADPH and GST, as well as inhibiting enzymes related to carcinogenesis such as protein

It is also important to emphasize that phenolic compounds also have pro‐oxidant activity, dependent on its concentration. The presence of hydroxyl groups in the ortho position can also produce radicals or hydrogen peroxide, in the presence of copper ions and oxygen

activation 1 (AP1), nuclear factor (NF)‐κB and MAP‐kinases [32, 60].

**Figure 6.** Possible flavonoid coordinating points with metals [32].

benzoic acids [58].

298 Honey Analysis

presence of hydroxyl at positions 3 and 5 [32].

Manuka honey, derived from the *Leptospermum scoparium* tree, was investigated about its protection effect against oxidative damage and improvement of the process of skin wound healing, using human dermal fibroblast cells. Up to 16 compounds were identified in this honey, with leptosperin derivatives and methyl syringate as the major ones. It protected against apoptosis, intracellular ROS production and lipid and protein oxidative damage. Manuka honey also protected mitochondrial functionality, promoted cell proliferation and activated the AMPK/Nrf2 signaling pathway, associated with antioxidant defence, as well as the expression of the antioxidant enzymes such as SOD and CAT [37].
