**8. Antioxidant activity**

Over many years, honey from different parts of the world have been shown to be one of the highest potential natural products in which phenolics, flavonoids, ascorbic acids, and some enzymes serve as potent antioxidants [32]. The antioxidant properties of honey are derived from both enzymatic (e.g., catalase, glucose oxidase, and peroxidase) and nonenzymatic substances (e.g., phenolic compounds, ascorbic acid, α-tocopherol, carotenoids, amino acids, proteins, and Maillard reaction products). The quantity and kind of these antioxidants are mainly dependent on the floral source. The main functional components of honey are flavonoids. They contribute significantly to the total antioxidant activity of honey and they act by several mechanisms including direct trapping of reactive oxygen species, inhibition of enzymes responsible for producing superoxide anions, chelation of transition metals involved in processes forming radicals, and prevention of the peroxidation process by reducing alkoxyl and peroxyl radicals [5, 15, 29]. The antioxidant activity of flavonoids in the majority of cases relies on the number and position of hydroxyl groups, additional substituents, and the glycosylation of flavonoid molecules. The presence of specific hydroxyl groups in the flavonoid rings improves antioxidant activity. Substitution patterns in the A ring and B ring, and the 2,3-double bond (unsaturated) and 4-oxo group in the C ring affect the antioxidant action of flavonoids as well. The glycosylation of flavonoids reduces their antioxidant activity compared to the analogous aglycones [5, 33].

These antioxidants may help to protect cellular damages from oxidative stress and lower the risk of chronic diseases. Furthermore, in recent years, there has been an increase in new methods for the research of free radicals and antioxidants in relation with advances in human health. Various studies have demonstrated that neuronal and behavioral changes occur with ageing, including in the absence of degenerative disease. Current studies indicate that dietary intake of antioxidant nutrients and cognition is closely related. Evidence from epidemiological, experimental and clinical studies demonstrates that the consumption of foods with high levels of dietary antioxidants might prevent or lower the risk of cognitive deterioration [34]. Many research models have been established in chemical and/or biological systems for the studies of mechanisms of action of antioxidants. Generally, antioxidant ability was measured and presented as total antioxidant capacity (TAC) [35, 36], total antioxidant potentials (TRAP) [37, 38], Trolox equivalent antioxidant capacity (TEAC) [39], ferric reducing/antioxidant power (FRAP) [40], and oxygen radical absorption capacity (ORAC) [41]. Mechanistically, these methods are based on either a single-electron transfer reaction or a hydrogen atom transfer reaction from an antioxidant or oxidant to a free radical. The total antioxidant activity is related to the radical scavenging ability and reductive activity [42].

**7. Biological activities**

70 Honey Analysis

**8. Antioxidant activity** 

pared to the analogous aglycones [5, 33].

Clinical investigations of the therapeutic potential of honey are gradually growing and scientific evidence for the efficacy of honey in some conditions is beginning to emerge. The healing effect of honey could be classified by its antiinflammatory, antibacterial, and antioxidant properties of its components. Furthermore, honey has been reported to be effective in gastrointestinal disorders, in healing of wounds and burns, and in treating venous ulcers [31].

Over many years, honey from different parts of the world have been shown to be one of the highest potential natural products in which phenolics, flavonoids, ascorbic acids, and some enzymes serve as potent antioxidants [32]. The antioxidant properties of honey are derived from both enzymatic (e.g., catalase, glucose oxidase, and peroxidase) and nonenzymatic substances (e.g., phenolic compounds, ascorbic acid, α-tocopherol, carotenoids, amino acids, proteins, and Maillard reaction products). The quantity and kind of these antioxidants are mainly dependent on the floral source. The main functional components of honey are flavonoids. They contribute significantly to the total antioxidant activity of honey and they act by several mechanisms including direct trapping of reactive oxygen species, inhibition of enzymes responsible for producing superoxide anions, chelation of transition metals involved in processes forming radicals, and prevention of the peroxidation process by reducing alkoxyl and peroxyl radicals [5, 15, 29]. The antioxidant activity of flavonoids in the majority of cases relies on the number and position of hydroxyl groups, additional substituents, and the glycosylation of flavonoid molecules. The presence of specific hydroxyl groups in the flavonoid rings improves antioxidant activity. Substitution patterns in the A ring and B ring, and the 2,3-double bond (unsaturated) and 4-oxo group in the C ring affect the antioxidant action of flavonoids as well. The glycosylation of flavonoids reduces their antioxidant activity com-

These antioxidants may help to protect cellular damages from oxidative stress and lower the risk of chronic diseases. Furthermore, in recent years, there has been an increase in new methods for the research of free radicals and antioxidants in relation with advances in human health. Various studies have demonstrated that neuronal and behavioral changes occur with ageing, including in the absence of degenerative disease. Current studies indicate that dietary intake of antioxidant nutrients and cognition is closely related. Evidence from epidemiological, experimental and clinical studies demonstrates that the consumption of foods with high levels of dietary antioxidants might prevent or lower the risk of cognitive deterioration [34]. Many research models have been established in chemical and/or biological systems for the studies of mechanisms of action of antioxidants. Generally, antioxidant ability was measured and presented as total antioxidant capacity (TAC) [35, 36], total antioxidant potentials (TRAP) [37, 38], Trolox equivalent antioxidant capacity (TEAC) [39], ferric reducing/antioxidant power (FRAP) [40], and oxygen radical absorption capacity (ORAC) [41]. Mechanistically, these methods are based on either a single-electron transfer reaction or a hydrogen atom Montenegro et al. [11] studied the antioxidant activity of unifloral honeys (quillay, ulmo, avellana, tiaca) of native plants from Chile. In this study, was observed an important correlation between total phenolic content and antioxidant activity evaluated by ferric reducing activity power—FRAP method. The ferric reducing activity power assay directly measures antioxidants with a reduction potential below the reduction potential of the Fe3+/Fe2+ couple and the reaction is reproducible and linearly related to the molar concentration of the antioxidant(s) present in the sample. Furthermore, some variations in antioxidant activity between honey samples with the same botanical origin were observed. This variability could be explained by different accompanying species and geographical origin zone of the honey [11].

The scavenging activity towards peroxyl/alkoxyl radicals (ORAC method) is one of the most employed assays. In fact, in the last years databases of the ORAC index of foods have been built to emphasize the benefits of establishing the antioxidant capacity of polyphenol-rich foods [43]. The method is based on the ability of antioxidants to prevent the consumption of a target molecule mediated by free radicals generated during the aerobic thermal decomposition of AAPH (2,2'-azo-bis(2-amidinopropane). The target molecules are most commonly used are beta-phycoerythrin, fluorescein, and pyrogallol red. The use of the pyrogallol red (PGR) as probe is related to the amount and reactivity of a given phenolic compound towards the free radicals generated in the AAPH (2,2'-azo-bis(2-amidinopropane) dihydrochloride) thermolysis. The ORAC-PGR index can be considered as a measure of the capacity of the sample to remove peroxyl and alkoxyl radicals [44]. In complex mixtures, concentration, chemical nature, and possibly the interaction between the antioxidants present in the sample determine this index.

Recently obtained results from our research group demonstrated that quillay honey's ORAC-PGR index is not correlated to phenolic compound content present in samples but is highly correlated to flavonoid content. This is due to the fact that flavonoids are the polyphenolic species to which the highest reactivity is attributed [45]. In addition, the ORAC-PGR index in honey of *A. petiolaris*, named commonly as corcolen honey, collected from honeybee colonies of the central zone of Chile was evaluated. The value of this parameter for these samples is correlated to the percentage of corcolen pollen present, meaning that mostly the phenolic compounds and compounds with scavenger capacity belong to corcolen species. These correlations are quite interesting since just by means of the melissopalynological assay there could be evidence of the phenolics composition as well as the antioxidant capacity of monofloral *Azara sp*. honey [22].

Finally, more recently, our group has obtained results indicating that honey quillay compounds are reactive toward hypochlorite (HOCl). Hypochlorite has an important role in defense mechanisms that take part in the immune response toward microorganisms. However, it has also documented that hypochlorite, in certain pathophysiological conditions, can damage macromolecules including proteins, DNA, RNA, and cell membrane lipids, changing their biological function. The consumption of PGR-induced by hypochlorite is inhibited by compounds able to react with this reactive species. PGR-hypochlorite indexes obtained for quillay honey samples indicated high hypochlorite-mediated oxidation protection potential, these results being comparable to those obtained via Trolox—water-soluble vitamin E analogue (unpublished results).
