Antioxidant Activity

Anthocyanins are highly reactive radical scavengers in various in vitro environments. Anthocyanins not only scavenge radicals, but through their ability to bind heavy metals such as iron, zinc, and copper, also prevent the formation of radicals.47 Anthocyanins may also exert antioxidant abilities through the protection or enhancement of endogenous antioxidants (i.e., sparing effect), or through the induction of antioxidant enzymes such as glutathione-S-transferase (GST) and superoxide dismutase (SOD).48,49 Also, there appears to be a synergism between anthocyanins, vitamin C, and other flavonoids which is similar to the reported recycling effect of vitamin E by vitamin C. This effect was observed in an investigation by Rossetto et al.31 where the flavonoid catechin was observed to regenerate malvidin 3-glucosides thereby increasing their antioxidant capacity in a micellar system with induced linoleic acid peroxidation.

20.2.1 Structural Characteristics Effecting Antioxidant Activity

The structural characteristics responsible for the antioxidant effect of antho-cyanins are generally associated with the number of free hydroxyls around the pyrone ring (greater number of hydroxyls = greater antioxidant capacity; refer to Figure 20.2); this is, however, an oversimplification. The antioxidant capacity of a polyphenolic compound is dictated not only by the number of free hydroxyls, but also by the basic structural orientation of the compound. The ring orientation will determine the ease of which a hydrogen atom from a hydroxyl group can be donated to a free radical and the ability of the compound to support an unpaired electron. The conjugation of the anthocyanin ring structure is also important. The C2-C3 double bound of the C-ring is consistently associated with a higher antioxidant capacity, having a stabilizing effect on the phenoxy radical.15 50 The positioning of hydroxyls in relation to one another is also a very important determinant in the antioxidant capacity of anthocyanins. Hydroxyl groups in close proximity, such as the ortho-hydroxyls of the B-ring, appear to greatly enhance the antioxidant capacity of the compound50,51 in experimental (in vitro) models; however, the availability of the highly reactive ortho-hydroxyls in a biological system (in vivo) has yet to be established. Conceptually, this site on the B-ring could form bonds with many compounds within biological fluids thus inhibiting the ability of this reactive site to participate in oxidation, metal chelation, or protein binding in vivo.

20.2.2 Glycosylation and Antioxidant Capacity

Anthocyanins are found in plants in glycosylated forms. Glycosylation is reported to influence the antioxidant capacity of flavonoids/anthocyanins.32,52,53 It is generally stated in the literature that glycosylation decreases the antioxidant capacity of anthocyanins by reducing free hydroxyls and metal chelation sites; however, contradictory results have been reported.27,54,55 It is important to note that the effect of glycosylation on antioxidant capacity will depend on the environment in which oxidation is being assessed; i.e., aqueous-soluble or lipid-soluble phases.

Glycosylation diminishes the antioxidant capacity of the anthocyanin in an artificial membrane system by decreasing the number of free hydroxyls and metal chelation sites. More importantly, glycosylation will decrease the flavonoids accessibility to membranes as a result of the increased polarity (i.e., increased water solubility) associated with the sugar moiety. The physiological relevance of this effect has, however, not been sufficiently established in vivo. Aglycones are less water soluble and therefore have an increased partitioning into the lipid-soluble phase of the artificial membrane system. One would assume that the increased antioxidant capacity of anthocyanidins (aglycones) in this environment would therefore be partly as a result of the increased lipid solubility of the aglycones over the glycosides. Conversely, other assay systems such as the oxygen radical absorbance capacity (ORAC) assay,54 the ferric reducing assay,27 and certain lipid oxidation models55 have found some glycosides to have higher antioxidant capacities than their respective aglycones. Therefore, the in vitro effect of glycosylation on antioxidant capacity will depend on the environment in which oxidation is being assessed (aqueous-soluble or lipid-soluble phase). Additionally, because anthocyanin aglycones have not been identified in the blood or urine, the physiological relevance of the antioxidant capacity of aglycones is questionable. This being said, as anthocyanin glycosides are generally believed to be cleaved by colonic microflora, the aglycones theoretically could have physiological relevance within the colon with the glycosides having more systemic relevance. It is clear that the respective in vivo antioxidant capabilities of the antho-cyanin aglycones vs. their glycoside derivatives require further investigation.

20.2.3 Absorption, Metabolism, and Biological Antioxidant Activity

The biological implications of an anthocyanins antioxidant activity will ultimately depend on the extent of its absorption and metabolism; unfortunately, the absorption and metabolism of anthocyanins are poorly understood. Originally, their absorption was speculated to only occur post-hydrolysis of the glycosidic bond; however, this has been proved incorrect as numerous studies have characterized anthocyanin glycosides in biological fluids. The limited pharmacokinetic data available suggest that the maximum plasma concentrations of 1 to 150 nmol/L are generally reached between 1 and 4 hours post-consumption of doses ranging between 0.1 to 1.0 g. Additionally, less than 1% of the initial dose is generally reported to be recovered in the urine.21 4256-60 Although the bioavailability of the parent anthocyanins appears to be low, concentrations of bioactive metabolites could contribute significantly to the anthocyanins bioactivity.

Anthocyanins were previously not believed to be significantly metabolized in humans; however, methylated and glucuronidated metabolites of anthocyanins have recently been reported.60-62 Furthermore, a study by Felgines et al.62 suggests that the excretion of anthocyanin metabolites may be as high as 2% of the initial ingested dose. Recent investigations in our laboratory have confirmed that the excretion of metabolites is higher than the excretion of total parent compounds.62" Although metabolism (methylation, sulfation, and glucuronidation) will affect the antioxidant capacity of these compounds, they will likely retain much of their bioactivity. Researchers administering quercetin and (-)-catechin to rats have observed an increased antioxidant capacity of the plasma/serum, even though the compounds were identified in the biological fluids as glucuronide and sulfate derivatives.63 This suggests that conjugated metabolites of the parent compounds contribute to anthocyanin bioactivity.4960 Furthermore, researchers propose that the antioxidant activities of anthocyanins are maintained even after their degradation under physiological conditions. It is believed that a portion of absorbed anthocyanins are broken down into benzoic acid derivatives, either spontaneously or as a result of bacterial metabolism in the intestine. Protocatechuic acid is one of these breakdown products that have been characterized in human and animal models.64 In cell culture experiments, the protocatechuic acid formed from cya-nidin glycosides was observed to have antioxidant properties comparable to that of commercial antioxidants including BHT (butylated hydroxyanisole) and vitamin E.65 This suggests that phenolic acid derivatives of the parent compounds may also play a role in the antioxidant defenses of the blood (serum/plasma) after the consumption of anthocyanins.49

20.2.4 Effect of pH on Antioxidant Activity

Anthocyanins exist in equilibrium in a variety of protonated, deprotonated, and hydrated forms. These range from colored quinonoid forms, to the flavylium ion, and to colorless hemiacetal forms.55 The expression of the predominant form is generally pH dependent. There is little evidence regarding the effect of pH on the biological activity of these compounds. However, in spite of the loss of color of anthocyanins at physiological pH (i.e., pH 7), evidence presented by Narayan et al.66 suggests that anthocyanin glycosides retain their antioxidant activity.

20.2.5 COMPARTMENTALIZATION AND ANTIOXIDANT ACTIVITY

Results of trials aimed at determining the link between antioxidant consumption, antioxidant status, and cancer have been inconsistent.3 Although anthocyanins have shown promise in many in vitro antioxidant and anticancer models, it has not been established if these compounds can reach their target of suspected action and if high enough concentrations are reached to elicit an efficient response. Youdim et al.67 were among the first to show evidence of the incorporation of anthocyanins into cells and cell membranes. In a cell culture experiment, using human aortic endothelial cells, cyanidin glycosides from the elderberry were observed to be incorporated into both the plasma membrane and cytosol. The cells were determined to have significant protection against oxidation induced by reactive oxygen species. Subsequently, Bagchi et al.68 recently reported on the cellular uptake of berry anthocyanins by endothelial cells. Although absorption was indicated in these studies, the mechanism by which anthocyanins enter intracellular compartments, where genetic materials exist, has yet to be determined. If the antioxidant capacity exhibited by anthocyanins is associated with cancer prevention, their mechanisms likely extend beyond the prevention of DNA base oxidation and lesion formation alone.

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