**2. Oxidative process**

The process of oxidation has been studied for many years [60], because of the importance it has both for the organisms and the foodstuffs. In live organisms, the oxidative metabolism is essential for the survival of the cells. Oxidation is related to the production of energy associated with the degradation of glucans, lipids and proteins, to the detoxification of many xenobiotics and to the immune response through some of the free radicals (FR) generated [61].

Oxygen is associated with the conditions for aerobic life and is the motive force for the maintenance of the metabolism and cellular viability, but, at the same time, it is responsible for the formation of partially reduced mediators with high reactivity, known as reactive oxygen species (ROS). The majority of ROS are FR, that is, active molecular species with a separated electron at a higher energy level, which, therefore have paramagnetic properties, providing them with high reactivity [20, 62].

The systems of antioxidant protection have to act on the substrates susceptible to oxidation in a controlled way to maintain the physiological equilibrium of the organism. The protective effect of some enzymes, such as superoxide dismutase (SOD), catalase and glutation peroxidase, may start when an excess of FR is produced. If this excess cannot be neutralized, oxidation of the lipidic membrane, the low‐density lipoproteins (LDLs), the protein cellular components, DNA and enzymes can occur, thereby destroying them [63, 64].

Using the oxygen radical absorbance capacity (ORAC) method, Wang and Goodman [52] evaluated the antioxidant capacity of 14 anthocyanins and obtained values more than 3.5 times greater than those for trolox (a synthetic antioxidant similar to vitamin E). Kuskoski et al. [51], using the ABTS method in purified and isolated patterns of anthocyanins, found an activity twice that of trolox and also confirmed the influence of the structure or the com-

Sources rich in anthocyanins are very interesting options as functional foods [53–58]. Here, the oxidative process, the antioxidant effect and the biological properties of the anthocyanin pigments, described in last years, are reviewed. Furthermore, the most commonly used chemical methods to determine the antioxidant capacity of the anthocyanins are outlined. An overview of the bioavailability of anthocyanins, the metabolism after their intake and their presence and influence in red wine is also given. Finally, an overview of some potential uses in food

The fertility field of flavonoids antioxidants (e.g. anthocyanins) has grown exponentially in recent decades in such a way that a number of areas are involved such as nutrition, food processing, physiology, biochemistry, pharmacology and analytical chemistry affecting foods and health. Emphasis in this contribution is given in most recent reviews and references. Some 150 journals are cited from the fields of food science and technology, nutrition, chemistry (analytical) and biochemistry, engineering, agriculture, medicine, pharmacy, biology, physiology and clinic. Taking into account that thousands of references are available, the authors apologize for those they may have overlooked or inadvertently omitted. For older references please consult, for example, some reviews [4–7] published on 2012 and the excel-

The process of oxidation has been studied for many years [60], because of the importance it has both for the organisms and the foodstuffs. In live organisms, the oxidative metabolism is essential for the survival of the cells. Oxidation is related to the production of energy associated with the degradation of glucans, lipids and proteins, to the detoxification of many xenobiotics and to the immune response through some of the free radicals (FR) generated [61].

Oxygen is associated with the conditions for aerobic life and is the motive force for the maintenance of the metabolism and cellular viability, but, at the same time, it is responsible for the formation of partially reduced mediators with high reactivity, known as reactive oxygen species (ROS). The majority of ROS are FR, that is, active molecular species with a separated electron at a higher energy level, which, therefore have paramagnetic properties, providing

The systems of antioxidant protection have to act on the substrates susceptible to oxidation in a controlled way to maintain the physiological equilibrium of the organism. The protective effect of some enzymes, such as superoxide dismutase (SOD), catalase and glutation peroxidase, may start when an excess of FR is produced. If this excess cannot be neutralized,

industry is attempted mainly focusing in the anthocyanin encapsulation topic.

bination of anthocyanins on the antioxidant capacity.

208 Flavonoids - From Biosynthesis to Human Health

lent monograph of Andersen and Markhan [59].

**2. Oxidative process**

them with high reactivity [20, 62].

It is worth emphasizing that arteriosclerosis is currently defined as chronic inflammation of the vascular system, triggered by a specific inflammatory agent, the oxidized LDL. The LDLs are very small particles made up by lipids, cholesterol and proteins, with the function of transporting cholesterol and lipids from the blood to the adipose and muscular tissue and, in general, to all cells of the body [65, 66]. However, the LDLs can be oxidized by the FR, affecting, consequently, the molecules of cholesterol and fatty acids that constitute each LDL. The oxidized LDLs are involved in the pathogenesis of coronary heart diseases [67, 68].

Environmental, dietary or physiological factors can provoke an imbalance in favour of oxidation, causing what is known as oxidative stress [69–71]. Whether the oxidation or the oxidative stress, in particular, is either a primary cause or a side effect of many chronic diseases and of the phenomenon of ageing itself has been a scientific debate prompted over the last few decades. Therefore, many efforts and resources have been devoted to finding out the role oxidants play in hindering oxidation, thus resulting in either the prevention or the retardation of the oxidative stress [72].

An excessive production of ROS, particularly hydroxyl radicals, can easily initiate the process of oxidation of the LDLs. In turn, they contribute to a greater or lesser degree to the onset of coronary heart diseases, rheumatoid arthritis, inflammatory diseases, cancer, renal diseases, pancreatitis, multiple sclerosis, Parkinson's disease, cataracts, diabetes, pulmonary disorders and all diseases related to cellular ageing [73]. The intake of dietary antioxidants, that is exogenous antioxidants (**Figure 3**), is very important [74], and some compounds of this family, that is vitamin E, β‐carotene and phenolic compounds, are only synthesized by plants [27, 31, 34, 35]. Therefore, it is important to maintain a balance between oxidants and antioxidants. It is worth bearing in mind that over a lifetime, as the individual ages, this balance tilts in favour of the oxidants [75].

In foods, oxidation can be one of the main causes of alterations leading to rancidity, deterioration and loss of nutritional, commercial and organoleptic quality (colour, taste, smell and texture), besides being a possible health risk to the consumer. For this reason, the food industry, by improving the preparation of the products and by using antioxidants, is trying to prevent and slow down the process of deterioration, in order to offer the consumer a safer deadline for use, which guarantees the quality of the food product [76–80].

However, according to studies carried out in vivo during the last two decades, FR and ROS are no longer seen only as [71] destructive factors but also (and perhaps first of all) as messengers involved in intracellular signalling. So, there has been a substantial change [10] in the conception of these processes in both normal and pathological conditions. Ideas about the role of FR in the functioning of cells and organisms have been revised, resulting in a new concept of redox equilibrium. Oxidative stress is then viewed as [72] a modulation of thiol redox reactions, involved mainly in signalling pathways. On this way, nonradical oxidants (enzymatically generated hydrogen peroxide, other peroxides, quinones, etc.) play a basic role [10] in the oxidation of thiols for the sake of signalling, the formation of free radical intermediates being not necessary. The common conviction of the beneficial effect that the phenolic plants exert on the improvement of health is being revised [64].

**Figure 3.** Summary of antioxidant defence system [74].

The potential health benefits of natural antioxidants, while interesting, seem to escape our basic understanding of biological oxidation processes. Oxidation balances both very beneficial, even crucial, outcomes with decidedly negative impacts. This suggests that moderation in the use of some antioxidants may be advisable. Note that a large measure of the biological oxidation occurring in the body is essential for extracting energy from food and is highly adaptive, depending on health status. Apart from energetics, oxidation supports immunological integrity. While the bulk of epidemiological evidence supports the nutritional/health value of fruits and vegetables [4, 11, 12, 19, 22, 33, 39, 40, 50], the doses of individual components they contain, such as specific antioxidants that may contribute to improved health and reduced risk of certain diseases, remain uncertain.

### **3. Bioavailability and metabolism of the anthocyanins**

The existing knowledge concerning with the absorption, distribution, metabolism and excretion (ADME) of anthocyanin compounds (including their decomposition within the gastrointestinal lumen) has been the subjects of several recent reviews [81–95]. In general, few comparative studies have been undertaken about their metabolism, physiological availability or biotransformation after intake in comparison with the number of studied devoted to absorption and distribution. Little information is also available on the effects of food matrix on anthocyanin bioavailability, particularly food matrices of the usual diet [92].

In general, anthocyanins are considered to have a remarkably low bioavailability (relatively low as well in comparison with that of other flavonoids), on the basis of the levels detected in human blood after ingestion [81]. This fact contrasts with the health‐promoting properties [81, 83, 84, 90] of anthocyanins, suggesting bioavailability and their interaction with other components present. Anthocyanins appear to be rapidly absorbed in the stomach and small intestine [89] and removed, being in the plasma and urine where reach low maximal concentrations [90]. After oral administration, anthocyanins follow a particular pattern different from other flavonoids [84]. A 20–25% of intact anthocyanins were detected in plasma few minutes after intake [86]. Kinetic studies have shown that anthocyanins have a rapid distribution and appearance in blood that is compatible with a tricompartmental model. Elimination takes place mainly through bile. Anthocyanins could be absorbed from the stomach as well as intestines where they undergo decomposition catalysed by microbiota. Bacterial action is capable of hydrolysing anthocyanins or aglucons into simpler phenolic compounds, which can be absorbed and still maintain free phenolic groups, retaining part of the reducing capacity of the original molecule. Active transport may play a role in the absorption of anthocyanins from the stomach as well as in their transfer within the kidney or liver [84]. The metabolic destination of the anthocyanins can differ depending on their aglucon structure, as well as on the tissue where they are metabolized (intestine or liver).

However, the persistence of anthocyanin metabolites, phenolic acid breakdown products (which could be responsible for the health benefits associated with anthocyanins) suggests enterohepatic recycling, leading to prolonged residence time, and supports the notion that anthocyanins are far more bioavailability than previously suggested [81, 88, 92]. However, the compounds as well as the molecular mechanisms involving all those biological events [83] still remain under exploited. The ability to cross membranes, pH effect, digestive enzymes, microbiota, biliary acids and food matrix are critical factors, which may contribute to this apparent paradox [86]. There are many doubts if the effect is due to the native compounds or other forms, their mechanism or which factors have crucial impact on bioavailability [86]. To clear the access both native and metabolized forms in vivo and to distinguish their different biological roles have been a very challenging task. Accumulative evidence, which is emerging, suggests multiple roles [92] explaining the apparent incongruity (poor absorption). Compared with other flavonoids, much remains to be discovered [94, 95] about details and mechanisms of anthocyanin absorption and transport. The activity of anthocyanins could be associated with the ability to elicit cell adaptive responses involving the transcription factor Brf2 by affecting the "nucleophilic one" of the organism [89]. Recent studies on the bioavailability topic are summarized in **Table 1** [82, 96–108].

The potential health benefits of natural antioxidants, while interesting, seem to escape our basic understanding of biological oxidation processes. Oxidation balances both very beneficial, even crucial, outcomes with decidedly negative impacts. This suggests that moderation in the use of some antioxidants may be advisable. Note that a large measure of the biological oxidation occurring in the body is essential for extracting energy from food and is highly adaptive, depending on health status. Apart from energetics, oxidation supports immunological integrity. While the bulk of epidemiological evidence supports the nutritional/health value of fruits and vegetables [4, 11, 12, 19, 22, 33, 39, 40, 50], the doses of individual components they contain, such as specific antioxidants that may contribute to improved health and

The existing knowledge concerning with the absorption, distribution, metabolism and excretion (ADME) of anthocyanin compounds (including their decomposition within the gastrointestinal lumen) has been the subjects of several recent reviews [81–95]. In general, few

reduced risk of certain diseases, remain uncertain.

**Figure 3.** Summary of antioxidant defence system [74].

210 Flavonoids - From Biosynthesis to Human Health

**3. Bioavailability and metabolism of the anthocyanins**


**Table 1.** Bioavailability of anthocyanins.

### **4. Biological activity of the anthocyanins**

Establishing the biological activities of phytochemicals, flavonoids and polyphenol is dependent on the complete understanding of their intake, absorption, metabolism and excretion; however, to date, this had only realized for a limited few structures [109]. The increasing evidence of potential therapeutic effects that present anthocyanin compounds has boosted the interest in the knowledge of their biochemistry and biological effects during the last two decades [95, 110–112]. Biological properties of anthocyanins depend on their bioavailability. The chemical structure of anthocyanins [113] determines their rate and extent of intestinal absorption and nature of the metabolites in the plasma. The growing and current interest in the study of anthocyanin compounds [114, 115] stems from their wide applicability in the prevention and even in the treatment of various human diseases. They could also be used in the control of the viruses that cause immunodeficiency, such as the causal agent of AIDS, and they have a strong activity against the influence A and B viruses as well as against the herpes virus [116]. Though many articles have been devoted to varying biological effects of anthoxyanins, only a limited number of studies deal with their antimicrobial activity [117].

The favourable effects of anthocyanins on improvement of vision in humans (increase in visual acuity), one of the first reported, were described in 1966, which prompted their introduction into ophthalmology [56, 57]. It continues to be an interesting field of study due to the prevalence of myopia in today's society [118]. Although these effects are not completely understood [119], it has been confirmed that cyanidin helps regeneration of rhodopsin. Anthocyanins have been associated with substances that strengthen the capillaries, reinforce the action of vitamin C and favour the accumulation of this vitamin in the liver and in the suprarenal glands. Blackcurrant anthocyanins inhibit transient myopia, reduce eye fatigue, improve dark adaptation and enhance retinal blood flow with glaucoma [56]. Anthocyanin‐rich bilberry extract has a protective effect on visual function during retinal inflammation [116].

Anthocyanins have been shown to be effective in the prevention of arteriosclerosis and cardiovascular diseases [25, 40, 41, 72]. Commercial extracts of *Vaccinium myrtillus* (bilberry) [120, 121] contain glucosides of delphinidin and cyanidin and, since 1977, have been used to inhibit platelet aggregation [122] because of their preventive effect in the initial stage of the formation of thrombi, in the treatment of some diseases related to poor microcirculation resulting from capillary fragility, and also to prevent the oxidation of the LDLs [123–125].

Moreover, it has been demonstrated that these preparations accelerate the spontaneous process of cicatrization and that they have a preventive and curative activity against gastroduodenal ulcers induced in rats. These effects are probably due to their influence on the biosynthesis of mucopolysaccharides [126], which improves the efficacy of the gastric mucous layer and increases the base substance of the connective tissue and of the capillaries.

Another described effect is the inhibition in vitro that certain anthocyanins have on the porcine pancreatic elastase [127]. This enzyme attacks fibres and collagen, playing an important role in some pathologies, such as arteriosclerosis, emphysema and rheumatoid arthritis. Beneficial effects have also been described in experiments with diabetes, with a substantial reduction observed in the sugar concentration in urine and plasma of rats treated with the anthocyanin pigments of grapes [128]. It is suggested that anthocyanins act by reducing the biosynthesis of collagen, lipoproteins and glycoproteins, as well as reducing the activity of elastase and adenosine deaminase, which are both known to be high in diabetic patients.

**4. Biological activity of the anthocyanins**

Establishing the biological activities of phytochemicals, flavonoids and polyphenol is dependent on the complete understanding of their intake, absorption, metabolism and excretion; however, to date, this had only realized for a limited few structures [109]. The increasing evidence of potential therapeutic effects that present anthocyanin compounds has boosted the interest in the knowledge of their biochemistry and biological effects during the last two decades [95, 110–112]. Biological properties of anthocyanins depend on their bioavailability. The chemical structure of anthocyanins [113] determines their rate and extent of intestinal absorption and nature of the metabolites in the plasma. The growing and current interest in the study of anthocyanin compounds [114, 115] stems from their wide applicability in the prevention and even in the treatment of various human diseases. They could also be used in the control of the viruses that cause immunodeficiency, such as the causal agent of AIDS, and they have a strong activity against the influence A and B viruses as well as against the herpes virus [116]. Though many articles have been devoted to varying biological effects of anthoxyanins, only a limited number of studies deal with their antimicrobial activity [117].

**Comments References**

[96]

[98]

[99]

[100]

[105]

[106]

[108]

Pharmacokinetic trial to evaluate the bioavailability of anthocyanins and colonic polyphenol metabolites

Bioavailability studies and anticancer properties of malvidin‐based anthocyanins, pyranoanthocyanins

Effect of red cabbage fermentation on anthocyanin bioavailability and plasma antioxidant capacity in

Bioavailability and uptake of anthocyanins and their metabolites from grape/blueberry juice and

Application of the developed flavonoid‐poor menu meals to the study of the bioavailability of bilberry

Effects of processing sour cherry fresh fruit to the final juice product on the content of anthocyanins and

Anthocyanin stability, mucus binding, and uptake into epithelial cells in healthy individuals that

Pharmacokinetic characterization and bioavailability of strawberry anthocyanins relative to meal intake [97]

Bioavailability of red raspberry anthocyanins and ellagitannins: new insights [82]

Tissue bioavailability and intake of tart cherry anthocyanins [101] Confirmation and identification of tart cherry anthocyanins in several target tissues of healthy rats [102] Bioactive anthocyanins in 'Queen Garnet' plum: maturity and bioavailability [103] Use of anthocyanins as bioactive colourants in lipstick formulations [104]

Absorption and bioavailability of anthocyanins across the gastrointestinal mucosa [107]

after consumption of aronia berry extract in plasma and urine

and nonoxonium derivatives

212 Flavonoids - From Biosynthesis to Human Health

smoothie in vivo and in vitro

anthocyanins as model flavonoids

other related polyphenols

**Table 1.** Bioavailability of anthocyanins.

retained red grape or chokeberry juice in the mouth

humans

Anthocyanins are recognized for their various [67, 129] pharmacological and medicinal properties. They are antimutagenic, anti‐inflammatory and vasotonic [111, 112, 130, 131]. They protect against radiation, are chemoprotective against the toxicity of platinum, are used in therapy against cancer and are hepatoprotective against carbon tetrachloride. They also have other effects due to several actions of a variety of enzymes and metabolic processes.

There are various patented pharmaceutical preparations [132] containing flavylium salts and anthocyanins for the treatment of wounds, gastroduodenal ulcers, inflammation of the mouth and throat, vascular diseases and other diseases linked with the lipidic and the glyceric acid metabolisms. More recently, they have been used in the treatment of circulatory diseases.

Some studies specify the anticarcinogenic effect of anthocyanins [12, 13, 19, 111, 112, 133–135]. They inhibit the growth of carcinogenic cells that provoke colon cancer, induce the apoptosis effect, have the capacity to inhibit in vitro the growth of cells that cause tumours in humans and are even able to act as modulators of the macrophages in the immune response [89, 112].

Anthocyanins are effective against cytotoxicity, lipidic peroxidation, and as protectors of DNA, by forming co‐pigments of DNA‐anthocyanins. Moreover, anthocyanins have cellular antioxidant mechanisms comparable to or greater than other micronutrients, such as vitamin E. The capacity of the anthocyanins for stabilizing triple‐helical complexes of DNA [136] by forming complexes of anthocyanins‐DNA [137] is well established.

Pharmacokinetics of anthocyanins has recently reviewed [85, 113, 138, 139]. The most recent papers published on the subject are summarized in **Table 2** [96, 101, 140–151]. Anthocyanins are metabolized to a structurally diverse range of metabolites that exhibit dynamic kinetic profiles. A multicompartmental (theoretical physiologically based) pharmacokinetic (PBMK) model has been proposed [138] in order to describe the anthocyanins fate in vivo. Understanding the elimination kinetics of these metabolites is key to the design of future studies [152] concerning with their utility in dietary intervention or as therapeutics for disease risk reduction.


**Table 2.** Selected papers on pharmacokinetics of anthocyanins in the 2014–2016 period.

In words of Kay [152], 'These studies on (flavonoid) metabolism and biological activity of metabolites mark a new beginning in phytochemical research and, in this respect, this work is in its infancy'. Phenol‐Explorer web database gathers polyphenol metabolites [153] identified in human and animal biofluids, from 221 publications.
