State of the Art of Anthocyanins: Antioxidant Activity, Sources, Bioavailability, and Therapeutic Effect in Human Health

antioxidants Review State of the Art of Anthocyanins: Antioxidant Activity, Sources, Bioavailability, and Therapeutic Effect in Human Health Noelia Tena 1,* , Julia Martín2and Agustín G. Asuero 1 1 Departamento de Qu í mica Anal í tica, Facultad de Farmacia, Universidad de Sevilla, Prof. Garc í a Gonz á lez 2, E-41012 Sevilla, Spain; [email protected] 2Departamento de Química Analítica, Escuela Politécnica Superior, Universidad de Sevilla, C/Virgen de África 7, E-41011 Sevilla, Spain; [email protected] *Correspondence: [email protected] Received: 21 April 2020; Accepted: 19 May 2020; Published: 23 May 2020   Abstract: The antioxidant activity of anthocyanins in food is well known. Numerous antioxidant assays have been proposed to measure the capacity of anthocyanins to prevent the oxidation process that naturally occurs. Different solvents, temperatures, and pH levels are applied in each assay, and these factors should be taken into account in order to obtain useful and reproducible results. The concentration and the structure of these compounds are directly related to their antioxidant capacity and their environment. However, the effectiveness of the anthocyanin ingestion against diseases is also influenced by its bioavailability. Novel methodologies that simulate the digestion process have been developed in order to facilitate the current knowledge of anthocyanins bioavailability. Studies highlight the potential synergy effect between parent compounds and their derivatives (metabolites, conjugated products, and microbe-generated metabolites). The aim of this review is to provide an overview of advantages and disadvantages of the most common methods to determine the antioxidant activity of anthocyanins, chemical structure, and concentration of these compounds in different edible fruits, vegetables, and plants; their bioavailability after intake; as well as the main therapeutic effect described in the scientific literature. Keywords: anthocyanins; antioxidant activity; anthocyanin content; bioavailability; encapsulation; therapeutic effects 1. Introduction In recent years, the interest in plants and food containing antioxidant properties has increased. The chemical compounds present in vegetables and fruits with these capacities are: vitamins C and E, carotenoids, and flavonoids. The anthocyanins, which are the most important group of flavonoids in plants, are pigments with a flavylium cation (AH + ) structure that act as acid. This structure is directly related to its antioxidant activity. Most of the functional properties and the sensory quality of the anthocyanins can be explained by their chemical reactivity. The structures and properties of anthocyanins are dependent on different factors such as pH, temperature, and solvents which should be controlled to carry out antioxidant activity studies of these compounds [1–9]. Free radicals, reactive oxygen species (ROS), and/or reactive nitrogen species (RNS) are required for the proper performance of the human body and its organs. These radicals are on-balance by a redox homeostasis in our body. However, the body may be occasionally affected by an oxidative stress resulting from an off-balance state. This stress is important in the development of chronic degenerative diseases including coronary heart disease, cancer, and aging [ 2 ]. Anthocyanins have been described as compounds that prevent or inhibit, the oxidation by scavenging free radicals and Antioxidants 2020,9, 451; doi:10.3390/antiox9050451 www.mdpi.com/journal/antioxidants Antioxidants 2020,9, 451 2 of 28 reducing the oxidative stress. On a regular basis, anthocyanins act as H-atom donator or as single electron transfer. Different methods of analysis based on both mechanisms have been proposed to determine the antioxidant activity of anthocyanins. The antioxidant activity of these compounds depends on their total concentration, structure, and environment. A literature compilation about the concentrations of the most common anthocyanins in different foods is presented in this review in order to have an overview of the different sources of anthocyanins. The beneficial properties attributed to the dietary ingestion of anthocyanin-rich foods (eye health, cardiovascular diseases, antiobesity, antidiabetic, antimicrobial, anticancer or neuroprotective effect) have been deeply documented in studies carried out with experimental models. These health benefits contrast with the apparent small portion (<1–2%) of these compounds absorbed by our organism [ 3 – 5 ]. During the digestion process, anthocyanins undergo to an intense variation in pH that together with the enzymatic and bacterial action can cause the hydrolysis and transformation of anthocyanins into metabolites, conjugated products, or simpler phenolic compounds [ 7 , 10 – 12 ]. The question is: How can anthocyanins be so influential in health? Are anthocyanins the only responsible of their beneficial effects? Last scientific developments highlight the potential synergy effect between parent compounds, metabolites (phases I and II), conjugated products, and microbe-generated metabolites to explain those biological events [4,11–13]. Due to their particular physicochemical features, bioavailability of anthocyanins is very difficult to assess. The first studies were performed analyzing blood and urine to determine the anthocyanin concentration levels after the ingestion of foods rich in anthocyanins [ 14 – 16 ]. However, the low absorption percentage obtained led to in vitro assays (mostly using cell culture systems) in order to facilitate the knowledge of their biochemical and chemical changes as well as the influence of the digestion steps. Last studies have emphasized the key role of the microbiota in the transformation of anthocyanins, which is not considered in in vitro assays but it is still poorly considered in in vivo and ex vivo studies [14]. This review aims to highlight some aspects regarding the antioxidant activity of anthocyanins and their bioavailability after intake. The first part includes an exposition of the most common antioxidant bioassays used to determine in vitro the antioxidant activity of anthocyanins, being the advantages and disadvantages of each bioassay identified. Afterwards, the effect of the chemical structure and the environment in the ability of the anthocyanins to prevent oxidation is discussed and presented together with information about different sources and range of concentration of these compounds in food. The second part of the manuscript exposes the bioavailability and metabolism of anthocyanins as well as a summary including the main therapeutic effects of anthocyanins on different diseases. 2. Antioxidant Bioassays for Anthocyanins Numerous antioxidant assays have been proposed to measure the ability of anthocyanins to prevent the oxidation process that naturally occurs. Depending on the source of the anthocyanins and their nature, in most of the cases, an extraction step before carrying out the antioxidant bioassay is needed. The extraction process is a critical step in the determination of the antioxidant activity bioassay, presenting a challenge due to the low stability of anthocyanins after extraction and their tendency to remain bound to the matrix of the sample. Multiple alternatives have been proposed in literature for this procedure [ 17 ]. Results show that temperature, pH, solvent system, solvent-to-solid ratio, and number of extractions are factors that play an important role in the extraction efficiency and that should be optimized for each sample [ 18 , 19 ]. Once the anthocyanins are in liquid solution, the antioxidant activity could be determined by different bioassays. In general, two different mechanisms can be used to explain the antioxidant activity of anthocyanins: Hydrogen atom donator (HAT) and single-electron transfer (SET). In HAT mechanism, the free radical R • removes a hydrogen atom from the antioxidant (AH + ) converting the free radical to a more stable product. In the SET mechanism, the antioxidant (AH + ) donates an electron to the free radical reducing the oxidized intermediates into the stable form [ 20 ]. However, the difficulty in distinguishing between HAT and SET reactions is high. In most situations, Antioxidants 2020,9, 451 3 of 28 these two reactions take place simultaneously, and the mechanism of the reaction is determined by the antioxidant structure, solubility, the partition coefficient, and solvent polarity [ 21 ]. Different antioxidant bioassays have been commonly used in the literature to determine the antioxidant activity of anthocyanins. A summary of the most common antioxidant bioassays used for anthocyanins is shown in Table 1. Table 1. Commonly used methods for measurement in vitro of antioxidant activity Bioassay Reagents Involved in the Reaction Detection Method DPPH (Diphenyl-1-picrylhydrazyl) assay Free radical (DPPH•+) Decrease of Abs. at 515 nm Spectrophotometric or colorimetric ORAC Assay (Oxygen Radical Absorbance Capacity) 2,20-azobis(2-amidino-propane) dihydrochloride (AAPH) to produce free radical β-phycoerythrin or Fluorescein or Pyrogallol red Decrease of fluorescence Fluorescence spectroscopy TRAP assay (total peroxyl radical trapping antioxidant parameter) 2,20-azobis(2-amidopropane) hydrochloride (ABAP) to produce free radical Luminol Decrease of luminescence Chemiluminescence FCT (ferric thiocyanate) assay Ferrous chloride, formation of red ferric thiocyanate Increase of Abs. at 500 nm Spectrophotometric FRAP (Ferric Reducing Antioxidant Power) assay FeCl3·6H2O, formation of blue ferrous complexes Increase of Abs. at 593 nm Colorimetric CUPRAC, Cupric Ion Reducing Antioxidant Capacity Cupric neocuproine, formation of Cu(I)-neocuproine Increase of Abs. at 550 nm Spectrophotometric ABTS [2,20-azino-bis (3-ehtylbenzothiazoline-6-sulfonic acid) diamonium salt] assay Free radical (ABTS•+) Decrease of Abs. at 415 nm Colorimetric Methods of inhibited autoxidation Lipid molecules, azoinitiator O2consumption/ hydroperoxide formation Oxygen electrode, pressure gauge, detection of conjugated dienes Note: Abs, absorbance. Each one of these methods provides information about the antioxidant activity of the anthocyanins under specific conditions. In order to have more information about the reaction mechanism, a more comprehensive description of the procedure followed in each one of these bioassays are presented together with their main advantages and disadvantages. 2.1. DPPH (Diphenyl-1-Picrylhydrazyl) Assay DPPH assay is a spectrophotometric method that can be applied for both solid and liquid samples, not being specific for any particular antioxidant. Thus, it can be applied to determine the total antioxidant capacity of the sample. This assay is based on the ability of the free radical (DPPH • ) to react with hydrogen donor (AH + ). The free radical presents an intense abortion in the UV–vis spectral region at 515 nm, the absorbance at 515 nm decreases when the free radical is reduced. The antioxidant activity of anthocyanins for DPPH has been assessed by several authors [ 22 – 26 ]. Reliable results can be obtained with this method in a fast and simple manner. Currently, a colorimetric alternative has been proposed to extend the application of this assay where a spectrophotometer is not available [ 27 ]. Furthermore, as the free radical is stable its production is not necessary every time that an analysis is carried out. The main disadvantages could be the complexity of the analysis, as it turns more complex when other compounds present in the sample absorb also at 515 nm and many antioxidants are inert to DPPH. 2.2. ORAC Assay (Oxygen Radical Absorbance Capacity) ORAC assay is a fluorescence method that combines the sample (AH + ) with a fluorescent compound, protein phycoerthrin ( β -PE) and with a generator of free radicals, 2,2 0 -azobis(2-amidino-propane) dihydrochloride (AAPH). This assay is based on the loss of the fluorescent compound when it is oxidized by the generated free radicals. When antioxidants (AH + ) are involved in the reaction, the fluorescent compound is protected from oxidative degradation. Hence, Antioxidants 2020,9, 451 4 of 28 the fluorescence signal remains. The fluorescence signal is monitored during 1 h at λ em =565 nm and λ ex =540 nm respectively. The antioxidant activity of anthocyanins has been assessed by ORAC assay by several authors [ 26 , 28 – 30 ]. This is considered a good method to determine the antioxidant capacity of hydrophilic and hydrophobic samples, as it is easily adaptive to different samples changing the generator of free radicals. One disadvantage is the non-specificity of the fluorescence compounds that can react with the sample losing fluorescence even without the addition of a free radical generator. 2.3. TRAP Assay (Total Peroxyl Radical Trapping Antioxidant Parameter) TRAP assay is a chemiluminescence method that consists of the following components: (i) thermolabile azo-radical initiator (e.g., 2,2 0 -azobis(2-amidopropane) hydrochloride (ABAP)), which produces radicals (R • ) that react rapidly with O 2 to give a peroxyl radicals (ROO • ); (ii) oxidizable compounds with chemiluminescence properties to monitor the reaction progress (e.g., Luminol); and (iii) the sample with the antioxidant properties (AH+). The antioxidant activity of anthocyanins has been assessed by TRAP assay by several authors [ 31 – 34 ]. This assay is sensitive to all known chain-breaking antioxidants. However, an important disadvantage to highlight is the difficulty comparing the results between laboratories due to the amount of different end points that can be used. It is relatively complex method, time-consuming, and requires a high degree of experience. Furthermore, and such as ORAC assay, the oxidizable compounds are non-specific and other non-radical chain reaction could occur. 2.4. FCT (Ferric Thiocyanate) Assay The FTC assay is a spectrophotometric method that consists of the oxidation of ferrous chloride to ferric ion by reacting with peroxide. The peroxides are formed during the reaction that takes place when the sample is mixed with ethanol, water, phosphate buffer solution (pH =7), and linoleic acid. Then, ferrous chloride in hydrochloric acid is added to the reaction and the ferric ion formed is combined with ammonium thiocyanate producing ferric thiocyanate, which is red. The absorbance of the sample is measured at 500 nm until the maximum value is reached. Thisassayisusedtomeasuretheamountofperoxideproducedduringtheinitialstagesofoxidation. In the case of the anthocyanins this method has been applied to determine the antioxidant activity avoiding the peroxidation of polyunsaturated fatty acid [ 35 ]. This assay is simple and reproducible. Nevertheless, results are not reliable in the case that compounds within the sample absorb around 500 nm. This drawback is common not only in this method, but in other spectrophotometric assays. 2.5. FRAP (Ferric Reducing Antioxidant Power) Assay FRAP assay is a colorimetric method that can be employed for the determination of the total antioxidant activity of anthocyanins. It is based on the reduction of complexes of 2,4,6-tripyridyl-s-triazine(TPTZ) withferric chloridehexahydrate (FeCl 3· 6H 2 O)under acidicconditions. The solution turns slightly brownish, forming blue ferrous complexes once the reduction is completed. The absorbance is measured at 593 nm against the blank. This method has been extensively applied to determine the antioxidant activity of anthocyanins in different matrices: in elderberry [ 36 ]; in Roselle extract [ 37 ]; in raspberries, blackberries, red currants, gooseberries, and Cornelian cherries [ 38 ]; or in carrots, cabbage, cauliflower, potatoes, onions, asparagus and eggplant [ 39 ]. However, the results of this method are in the most of the cases compared to the results of other antioxidant assays [ 40 ]. Rapid and reproducible results are obtained with this method. However, some limitations related to FRAP assay should be considered. On the one hand, the samples must be aqueous and the pH value is critical. On the other hand, some compounds without antioxidant properties can reduce Fe 3+ to Fe 2+ inducing an overestimation of the antioxidant activity of the sample. Antioxidants 2020,9, 451 5 of 28 2.6. CUPRAC (Cupric Ion Reducing Antioxidant Capacity) Assay CUPRAC assay is a spectrophotometric method similar to FRAP. In this method the anthocyanins react with the CUPRAC reagent (cupric neocuproine) producing the Cu(I)-neocuproine which is a chromophore that absorbs at 450 nm. This method has been successfully applied to various food extracts [7]. ThemainadvantagesofthismethodisrelatedtothepositivecharacteristicsoftheCUPRACreagent: availability andeasy accessibility, rapidity, stability, low-cost, sensitivity towards thiol-typeantioxidants unlike FRAP, and responsiveness to both hydrophilic and lipophilic antioxidants. Nevertheless, the detection is based on the absorption at 450 nm which is non-specific enough and the presence of other compounds present in the sample could be interfered in the results. 2.7. ABTS (2,20-Azino-bis (3-ehtylbenzothiazoline-6-sulfonic acid) Diamonium Salt) Assay The most recent ABTS assay method is based on decolorization techniques. It consists of the production of a stable radical, blue/green ABTS chromophore, by the reaction of ABTS with potassium persulfate. This stable radical has a maximum absorbance at 415 nm. A drop in absorbance of this compound occurs when the radical reacts with the antioxidant. This method can determine the antioxidant activity of mixtures of substances, helping to distinguish between additive and synergistic effects. The antioxidant activity is calculated relatively to the reactivity of Trolox standard under similar conditions. This assay is frequently combined with DPPH assay for the determination of antioxidant activity of anthocyanins [ 41 – 43 ]. This method is simple and not a large sample volume and time of analysis are necessary. Nevertheless, a standard solution is required in order to obtain accurate results. This assay is also described in the literature as TEAC [6-hydroxy-2,5,7,8-tetramethylchroman- 2-carboxylic acid (Trolox)] equivalent antioxidant capacity. The same free radical is used both TEAC and ABTS assays. However, different reagents have been proposed for the generation of the green–blue ABTS •+ , resulting in different TEAC assays. This radical is produced by the oxidation of 2,2 0 -azinobis (3-ethylbenzothiazoline-6-sulfonic acid; ABTS). The oxidation can be reached in different ways: (i) in TEAC assay I, metmyoglobin reacted with H 2 O 2 generating the ferrylmyoglobin radical, which then reacted with ABTS. (ii) In TEAC assay II, the ABTS •+ is formed by the reaction with manganese dioxide. (iii) In TEAC assay III, enzymatic reaction using horseradish peroxidase is applied. Other proposals consist of applying electrochemical oxidation or using 2,2 0 -azobis-2-amidinopropane, dihydrochloride (AAPH) or potassium persulfate (K2S2O8) as oxidants. These alternatives are interchangeable when the appropriate solvent is selected. However, differences in assay conditions can be found, such as the reaction time or the wavelength used for the detection, for instance, sometimes in order to avoid interferences 734 nm is preferable to 415 nm. These assays have been used to determine the antioxidant capacity of the anthocyanins, as a consequence some results has been reported in wine [ 44 ]; in corn [ 45 ]; in pomegranate juice [ 46 ] or in blueberries [47]. The advantages of TEAC assay I are the simplicity, reproducibility, and flexibility to determine the antioxidant capacity in hydrophilic and lipophilic foods. However, the pre-addiction of the sample before the radical generation could result in an overestimation of the antioxidant capacity. Other possible disadvantages of this assay may be the fact that ABTS is not found naturally, and that any compound with a redox potential lower than ABTS•+may react with the radical. Other alternatives applied to determine the antioxidant activity of anthocyanins involve the use of enzymes [ 48 ] or the use of chromatographic techniques. The latter alternative can not only extract the anthocyanins to determine their antioxidant activity [ 49 ], but also determining the total anthocyanins concentration (TAC), and the identification and quantification of the presence of each anthocyanin presents in the sample [50,51]. The in vitro assays exposed in this manuscript provide information about the antioxidant activity of the anthocyanins determined by their capacity to neutralize the initiators of the oxidation process Antioxidants 2020,9, 451 6 of 28 (absorbing photons, neutralizing ROS or chelating metals ion) stopping the initiation steps of the autoxidation process. Thus, these assays are based on the reaction of the anthocyanins with: (i) some colored persistent radical such as the free radicals used in DPPH test and TEAC test; or (ii) other oxidizing agents like Fe 3+ ions used in FRAP test or Cu 2+ ions used in CUPRAC test. This neutralization capacity is measured under specific conditions of temperature, light, or the combination of both. Other assays to measure the antioxidant capacity of anthocyanins are based on a competitive probe reaction where the competitive reaction of radicals with the anthocyanins or with a probe, is monitored by fluorimetric techniques in the case of the ORAC assay, among others. All of these methods offer information on the actual antioxidant activity. They trap free radical and should be considered as chain-breaking antioxidants. This property can be measured properly only with methods based on inhibited autoxidation, while others (such as DPPH, ABTS) provide only a rough estimation. However, different specific studies have been carried out to determine the preventive antioxidant capacity [ 52 ]. Other specific assays to gather information about the inhibition of lipid substrate oxidation, where different factors and mechanism of oxidation are studied simultaneously, can be found in the literature [53]. Asithasbeenexplainedabove,theassayspresentedinthismanuscriptfollowdifferentmechanisms of reaction to measure the antioxidant capacity. Therefore, the information provided by them and the interpretation of their results should be carefully considered. For example, that is the case of the results provided by FRAP and CUPRAC assays versus ORAC assay which consist in a radical-trapping reaction and directly measures the capacity to neutralize initiators. Despite the fact that the methods mentioned above are the most commonly used to determine the antioxidant capacity of anthocyanins, there is currently a claim for the development of different alternatives in order to provide direct information about the capacity to prevent the autoxidation in the biological system. Diverse alternatives have been proposed to measure the inhibition of autoxidation. That is the case exposed by Matera et al. [ 54 ], where the antioxidant activity is measured by studying the inhibited autoxidation in aqueous micelles. For that purpose, linoleic acid and Triton-X100 were mixed under controlled pH together with an initiator and the extracted anthocyanins. The reaction was tracked by monitoring the oxygen consumption with a Clark-type electrode. Another alternative to measure the capacity of anthocyanins to inhibit autoxidation was proposed by Tisuda et al. [ 55 ], where the antioxidative activity was measured by using linoleic acid autoxidation in different systems; liposome, rabbit erythrocyte membrane, and rat liver microsomal. This type of study provides direct information about the relation between the results of antioxidants assays and their biological activity in the prevention of the autoxidation. Currently, the European Food Safety Authority (EFSA) has published a guidance for the scientific requirements for health claims related to antioxidants, oxidative damage, and cardiovascular health. In this guidance, the European Health Claims Regulation has given recommendation to determine the biological effect of food supplements with purported antioxidant activity. According to this document, the antioxidant properties of foods are based on scavenging free radicals that are in vitro studied in model systems and it is not established that this capability has a beneficial physiological effect in humans. Thus, the in vivo human studies in plasma using methods—such as TRAP, TEAC, FRAP, and ORAC—carried out to establish the antioxidant capacity of the plasma do not demonstrate that it has a beneficial physiological effect in humans. Consequently, the protection of cells from premature ageing to promote healthy ageing in relation to the antioxidant properties of food are not sufficiently defined because they are established by non-specific criteria. In order to provide information about the prevention of the autoxidation under specific criteria, the EFSA guidance recommend some assays for specific biological molecules. For example, it proposes: (i) the monitoring of F2-isoprostanes to determine the capacity for the protection of the lipids from oxidative damage; (ii) the analyses of 8-hydroxy-2-deoxy-guanosine in blood, tissue and urine to assess oxidative damage to DNA; and (iii) the monitoring of protein carbonyls by ELISA, that can be applied to determine the protection of protein from oxidation damage [56]. Antioxidants 2020,9, 451 7 of 28 All the assays explained in detail in this manuscript provide results that generally express the antioxidant activity as mmol Trolox Equivalent per kg of fresh weight. However, these results do not report exactly about the antioxidative activity of foods. For this purpose, information about the size portion of each food in the diet is also important to evaluate its antioxidant activity [ 57 ]. Due to the relevance of this type of results with regard to the antioxidant activity of anthocyanins, the USDA National Nutrient Database for Standard Reference has published a database where the total concentration of anthocyanins of different fruits and vegetables has been determined together with the service size that should be ingested from each fruit or vegetable [58]. 3. Classification and Natural Sources of Anthocyanins Health and therapeutic effects of anthocyanins are related to their chemical and biochemical reactivity, which are partially explained by their antioxidative activities [ 59 , 60 ]. However, the antioxidative activity of anthocyanins does not necessarily transfer to biological activity because any actions on the body depends both on bioavailability and cellular molecular targets [ 61 ]. Furthermore, not all the blue, red, and purple fruits, vegetables, and flowers have the same composition and concentration of anthocyanins and in consequence the same antioxidative activity. The fruits with the highest concentration of anthocyanins are berries, currants, grapes, and some tropical fruit. In the group of edible vegetables, leafy vegetables, grains, roots, and tubers show the highest concentration of anthocyanins as well [ 62 ]. Furthermore, the presence of anthocyanins can be detected in different parts of the plant such as stem, leaves, and storage organs. In nature, these pigments are commonly present as anthocyanin, which is in the form of glycoside, and as anthocyanidin also known as aglycone (one or more saccharide bonded with the aglycone). The base structure of anthocyanins is shown in Figure 1. Nowadays the number of anthocyanins identified in nature is higher than 600 [ 6 ]. Among these anthocyanins, glycoside forms of delphinidin, cyanidin, petunidin, peonidin, malvidin, and pelargonidin, are the most abundant [ 54 , 55 , 62 – 65 ]. Table 2shows the most common anthocyanins that have been identified in different fruits, vegetables, and edible flowers and the numerical code that has been assigned to each anthocyanin to refer them in the manuscript. Antioxidants 2020, 9, 451 7 of 27 All the assays explained in detail in this manuscript provide results that generally express the antioxidant activity as mmol Trolox Equivalent per kg of fresh weight. However, these results do not report exactly about the antioxidative activity of foods. For this purpose, information about the size portion of each food in the diet is also important to evaluate its antioxidant activity [57]. Due to the relevance of this type of results with regard to the antioxidant activity of anthocyanins, the USDA National Nutrient Database for Standard Reference has published a database where the total concentration of anthocyanins of different fruits and vegetables has been determined together with the service size that should be ingested from each fruit or vegetable [58]. 3. Classification and Natural Sources of Anthocyanins Health and therapeutic effects of anthocyanins are related to their chemical and biochemical reactivity, which are partially explained by their antioxidative activities [59,60]. However, the antioxidative activity of anthocyanins does not necessarily transfer to biological activity because any actions on the body depends both on bioavailability and cellular molecular targets [61]. Furthermore, not all the blue, red, and purple fruits, vegetables, and flowers have the same composition and concentration of anthocyanins and in consequence the same antioxidative activity. The fruits with the highest concentration of anthocyanins are berries, currants, grapes, and some tropical fruit. In the group of edible vegetables, leafy vegetables, grains, roots, and tubers show the highest concentration of anthocyanins as well [62]. Furthermore, the presence of anthocyanins can be detected in different parts of the plant such as stem, leaves, and storage organs. In nature, these pigments are commonly present as anthocyanin, which is in the form of glycoside, and as anthocyanidin also known as aglycone (one or more saccharide bonded with the aglycone). The base structure of anthocyanins is shown in Figure 1. Nowadays the number of anthocyanins identified in nature is higher than 600 [6]. Among these anthocyanins, glycoside forms of delphinidin, cyanidin, petunidin, peonidin, malvidin, and pelargonidin, are the most abundant [54,55,62–65]. Table 2 shows the most common anthocyanins that have been identified in different fruits, vegetables, and edible flowers and the numerical code that has been assigned to each anthocyanin to refer them in the manuscript. Figure 1. Structure of anthocyanins R3 = sugar, and anthocyanidins R3 = H. In supplementary information, Tables S1–S6 show the most common anthocyanins identified in different fruits, vegetables, and flowers, grouped according to their chemical structure: delphinidin and its derivatives (Table S1), cyanidin and its derivatives (Table S2), petunidin (Table S3), peonidin (Table S4), malvidin (Table S5) and pelargonidin and its derivatives (Table S6). Each table also indicates the natural source of the anthocyanins, the type of extraction that was applied, the chromatographic method used to identify and quantify them and the antioxidant assays applied to determine the antioxidant capacity. Table 2. Anthocyanins identified in different fruits, vegetables, and edible flowers and their codes. Code Anthocyanin Code Anthocyanin Figure 1. Structure of anthocyanins R3=sugar, and anthocyanidins R3=H. In supplementary information, Tables S1–S6 show the most common anthocyanins identified in different fruits, vegetables, and flowers, grouped according to their chemical structure: delphinidin and its derivatives (Table S1), cyanidin and its derivatives (Table S2), petunidin (Table S3), peonidin (Table S4), malvidin (Table S5) and pelargonidin and its derivatives (Table S6). Each table also indicates the natural source of the anthocyanins, the type of extraction that was applied, the chromatographic method used to identify and quantify them and the antioxidant assays applied to determine the antioxidant capacity. Antioxidants 2020,9, 451 8 of 28 Table 2. Anthocyanins identified in different fruits, vegetables, and edible flowers and their codes. Code Anthocyanin Code Anthocyanin 1 Delphinidin 30 Petunidin 3-arabinoside 2 Delphinidin 3-arabinoside 31 Petunidin 3-galactoside 3 Delphinidin 3-galactoside 32 Petunidin 3-glucoside 4 Delphinidin 3-glucoside 33 Petunidin 3-halactoside 5 Delphinidin 3,5-diglucoside 34 Petunidin 3-rutinoside 6 Delphinidin 3-rutinoside 35 Peonidin 7 Delphinidin 3-O-(6”-p-coumaroyl-glucoside) 36 Peonidin 3-galactoside 8 Cyanidin 37 Peonidin 3-glucoside 9 Cyanidin 3-arabidoside 38 Peonidin 3-rutinoside 10 Cyanidin 3-galactoside 39 Peonidin 3-(60-malonylglucoside) 11 Cyanidin 3-glucoside 40 Peonidin 3-(3”,6”-dimalonylglucoside) 12 Cyanidin 3,5-diglucoside 41 Peonidin 3-glucoside/malvidin 3-galactoside 13 Cyanidin 3-rutinoside 42 Peonidin 3-arabinoside/malvidin 3-glucoside 14 Cyanidin 3-(60-malonylglucoside) 43 Peonidin 3-O-sophoroside-5-O-glucoside 15 Cyanidin 3-(3”,6”-dimalonylglucoside) 44 Peonidin 3-p-hydroxybenzoylsophoroside-5-glucoside 16 Cyanidin 3-xyloside 45 Peonidin 3-caffeoylsophoroside-5-glucoside 17 Cyanidin 3-xylosylrutinoside 46 Peonidin 3-dicaffeoylsophoroside-5-glucoside 18 Cyanidin 3-dioxaloylglucoside 47 Peonidin 3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside 19 Cyanidin 3-halavtoside 48 Peonidin 3-caffeoy-feruloylsophoroside-5-glucoside 20 Cyanidin 3-O-sophoroside 49 Malvidin 21 Cyanidin 3-sophoroside-5-rhamnoside 50 Malvidin 3-arabinoside 22 Cyanidin 3-sambubioside 51 Malvidin 3-galactoside 23 Cyanidin 3-sambubioside-5-rhamnoside 52 Malvidin 3-glucoside 24 Cyanidin-3-p-hydroxybenzoylsophoroside-5-glucoside 53 Malvidin 3,5-diglucoside 25 Cyanidin-3-caffeoylsophoroside-5-glucoside 54 Pelargonidin 26 Cyanidin-3-caffeoyl-p-hydroxybenzoylsophoroside-5-glucoside 55 Pelargonidin 3-glucoside 27 Cyanidin 3-(p-coumaroyl)-diglucoside-5-glucoside 56 Pelargonidin 3-rutinoside 28 Cyanidin 3-(p-coumaroyl)-diglucoside-5-glucoside 57 Pelargonidin 3,5-diglucoside 29 Petunidin The main conclusion from Table S1 is that delphinidin 3-glucoside (code 4) is the most common and abundant delphinidin in fruits and edible flowers. However, this group of anthocyanins is not so frequent in vegetables, being delphinine the most common. The results showed in Table S2 pointed out that the most common cyanindin in fruit and edible flower is cyanidin 3-glucoside (code 11), being also abundant cyanidin 3-galactoside (code 10) in fruits. In vegetables, cyanidin (code 8) is the most common and abundant but cyanidin 3-glucoside (code 11) is abundant in some grains as well. Table S3 shows that the following anthocyanins can be identified in fruits: petunidin 3-arabinoside, petunidin 3-galactoside, and petunidin 3-glucoside (code 30, 31, 32) in almost all the studied fruits, with petunidin 3-glucoside (code 32) being the most abundant and the only petunidin identified in the flowers. Petunidin (code 29) is the only anthocyanin of this group identified in the studied vegetables. Table S4 reveals that no peonidin was identified in flowers. In fruits, peonidin 3-glucoside (code 37) was the most abundant but also peonidin 3-galactoside (code 36) was identified in the most of them. In vegetables, the most common anthocyanin of this group was peonidin (code 35). From the group of malvidin (Table S5) it can be concluded that the most abundant in fruits is malvidin 3-galactoside (code 51) followed by malvidin 3-glucoside (code 52). The last stage was also the most abundant in flowers followed by malvidin 3,5-diglucoside (code 53). In vegetables, the malvidin was the only anthocyanin quantified of this group. Table S6 shows that pelargonidin 3-glucoside (code 55) was the most abundant in fruits followed by pelargonidin 3-rutinoside (code 56). The pelargonidin 3,5-diglucoside (code 57) was the most abundant in flowers and the pelargonidin (code 54) was the only anthocyanin of this group identified in the studied vegetables. The protection of these pigments against oxidation process depends on their structures. Not all of them possess the same activities to scavenge diverse reactive oxygen or nitrogen species. Antioxidants 2020,9, 451 9 of 28 The antioxidant ability of anthocyanins depends on the ring orientation since it will determine the willingness to donate a proton and the capacity to transfer and electron. The number of free hydroxyls around the pyrone ring and their positions also play a key role in the antioxidant activity [ 2 ]. The presence of other types of radicals in the main structure has an important role in the antioxidant activity as well. Hence, anthocyanins chalcones and quinoidal bases with a double bond conjugated to the keto group are efficient antioxidants at scavenging free radicals. Also, the glycosylated B-ring structure of anthocyanins contributes to the high antioxidant activity, where orthohydroxylation and methoxylation substantially increase the antioxidant activity. Furthermore, anthocyanidins have higher antioxidant activity in comparison with anthocyanins, which has been reported in the literature. The reason may be the lower stability of the anthocyanidin compared to the anthocyanin due to its structure, what consequently makes anthocyanidin highly reactive [ 66 ]. Acylation of anthocyanin with one or more phenolic acids has a significant increase in antioxidant activity [54,67], but glycosylation leads to a reduction in the activity [66,68]. The efficacy of scavenging diverse free radicals differs from one anthocyanin to the other. Pelargonidin-3-glucoside, cyanidin-3-glucoside, and delphinidin-3-glucoside and their standard aglycones have strong antioxidative activity in a liposomal system and reduced formation of malondialdehydeby UVBirradiation[ 55 , 69 ]. Furthermore, theresults pointedout thehighest inhibitory effect on lipid peroxidation and O 2• scavenging activity of delphinidin and delphinidin-3-glucoside followed by cyanidin and pelargonidin [ 2 ]. On the contrary, pelargonidin had the highest inhibitory effect on hydroxyl radical scavenging activity [ 66 ]. Moreover, a study demonstrates the highest inhibitory effect on copper (II)-induced low-density lipoprotein (LDL) oxidation of cyanidin and cyanidin-3-glucoside compared with other phenolic acids, anthocyanins, and anthocyanin aglycones, whereas delphinidin has intermediate efficacy [ 70 ]. Another study analyzed the antioxidant activity of malvidin-3-glucoside and the result showed that the quinoidal-base and pseudo-base of malvidin-3-glucoside significantly inhibited peroxidation of linoleate compared with catechin, malvidin, and resveratrol [ 71 ]. However, the oxidation activity assigned to these anthocyanins is dependent on the type of reactive species and in consequence on the type of antioxidant assay carried out for the determination of the antioxidant activity. Thus, FRAP and TEAC assays have reported the significant reduction of the antioxidant activity by the metoxilation in the position 5 or 3 and 5 in petunidin and malvidin monoglucoside respectively [ 72 ]. Another factor implicated in the reactivity of the anthocyanins and also in their antioxidative activity is the pH. In the literature different studies have demonstrated the effect of the pH in the antioxidant capacity of the anthocyanins from different sources; Roselle [ 26 ], wine [ 71 , 72 ], black rice complexed with cycloamylose [ 73 , 74 ], palm juice [ 75 ], and Hibiscus acetosella [ 76 ]. The pH is an important factor that should be controlled in order to determine the reactivity of the anthocyanins. The acid nature of the anthocyanin structure is shown in Figure 2. This acid nature is due to the conjugation of the double bonds in the rings of the main structure and the hydroxyl groups at C4 0 , C5, and C7 respectively. The hydroxyl group at C7 is the strongest acid. The deprotonation can be produced at acid pH~4 yielding a neutral quinonoid base stabilized by tautomerization with the hydroxyl group at C5. The hydroxyl group at C4 is also susceptible to be deprotonated at higher pH~7 yielding the anionic base. If the pH level is still rising to basic pH, higher than 8, the deprotonation is produced in the C5 yielding the dianionic base which can lead to the chalcone anion [ 1 ]. Hence, the pH of the solution controls the proportions of protonated and deprotonated hydrated and isomeric form of anthocyanin, affecting its reactivity. Therefore, the pH should be controlled during the extractions process of anthocyanins and also during the antioxidative bioassays because their results are pH dependent [2,26]. Once the identification of the most common anthocyanins in different foods has been carried out and the influence of the structure and the environment in the capacity of anthocyanins to prevent the oxidation has been discussed, the next step was to determine the bioavailability of these anthocyanins and their implication, enhancing human health. Antioxidants 2020,9, 451 16 of 28 also suggested that some individuals are more susceptible to the protective effects of anthocyanin consumption: (i) overweight; (ii) over 50 years; and (iii) those with increased risk of cardiovascular disease. Another meta-analysis of 99 randomized controlled trials showed that the consumption of anthocyanin rich-products decreased significantly both systolic and diastolic blood pressure regardless of the health status of the participants [168]. In in vitro assays, anthocyanins have also shown inhibition of the porcine pancreatic elastase [ 169 ], an enzyme that plays a significant function in pathologies such as arteriosclerosis, emphysema, or rheumatoid arthritis, etc., by attacking fibers and collagen. Moreover, acceleration in the cicatrization process due to anthocyanin-rich extract has been demonstrated, showing preventive and curative activity against gastroduodenal ulcers induced in rats [ 7 ]. Their influence on the biosynthesis of mucopolysaccharides provably improves the efficacy of the gastric mucous layer, and increases the base substance of the connective tissue and of the capillaries [170]. Antiobesity and Antidiabetic effects: Anthocyanins have shown anti-obesity effects through multiple mechanisms such as inhibiting lipid absorption, regulating lipid metabolism, increasing energy expenditure, suppressing food intake and regulating gut microbiota, which suggests anthocyanins are promising candidates in anti-obesity therapies [ 171 ]. Kwon et al. [ 128 ] observed that anthocyanins-added diet from black soybean in rats decreases body weight gains, being significantly lowered in the rats fed with a high fat diet plus black soybean anthocyanins compared with the rats fed with high fat diet without black soybean. Anthocyanins also improved the lipid profile and suppressed the high fat diet-induced weight gain in liver intermediately and decreased the weights of epididymal and perirenal fat pads. In addition, type 2 diabetes is closely related to obesity [ 66 ]. Anthocyanins can alleviate complications in type 2 diabetes by inhibiting intestinal glucose absorption, inducing pancreatic insulin secretion, upregulating glucose transporter type 4, and suppressing hepatic gluconeogenesis [ 172 ]. After the supplementation of a high-fat diet during 13 weeks with different berries in mice, Heyman et al. [ 173 ] observed that those supplemented mice gained lesser body weight and presented lower fasting insulin levels than the control group as well as mediated positive effects on glucose homeostasis. Jankowski et al. [ 130 ] described a substantial decrease in the sugar concentration in urine and blood serum after streptozotocin injection in fed rats with grapes. The mechanisms of anthocyanins suggested by the authors were the reduction of the biosynthesis of collagen, lipoproteins, and glycoproteins, as well as the reduction of the activity of elastase and adenosine deaminase (both high in diabetic patients). Treatment with cherries in rats resulted in a significant reduction of blood glucose and urinary microalbumin and an increase of the creatinine secretion level in urea [ 174 ]. The pulp, seed and skin from “red chilto” (a red fruit from Argentina) had a hypoglycemic effect and acted increasing glucose absorption, decreasing glucose diffusion rate and promoting glucose transport across the cell membrane [ 175 ] in an in vitro simulated gastroduodenal digestion. Consumption of blueberries and apples/pears in humans was also associated to a lower risk of type 2 diabetes [176]. Antimicrobial effects: The antimicrobial activity of anthocyanins against a wide range of microorganisms is also well documented. Possible mechanisms induced cell damage by destroying the cell wall, membrane and intercellular matrix [ 66 , 140 , 177 ]. Blackberry extracts have antibacterial activity with the highest sensitivity to Aeromonas hydrophilia and Listeria innocua [ 141 ]. Cranberry extracts have antibacterial activity towards Enterococcus faecium resistant to vancomycin, Pseudomonas aeruginosa, Staphylococcus aureus, and Escherichia coli [ 142 ]. Different types of berry extracts inhibit Gram-negative bacteria but not Gram-positive bacteria [ 143 ] probably because Gram-negative bacteria acts as a preventive barrier against hydrophobic compounds but not against hydrophilic compounds [178]. Anticancer activity: Possible mechanisms of the anticancer activity of anthocyanins have been described by many authors: antimutagenic activity; inhibition of oxidative DNA damage and carcinogen activation; induction of phase II enzymes for detoxification; cell cycle arrest; inhibition of cyclooxygenase-2 enzymes; as well as induction of apoptosis and antiangiogenesis [179–184]. Antioxidants 2020,9, 451 17 of 28 In breast cancer, anthocyanins cause the inhibition of key modulators that promote its progression anddevelopment byacting directlyin the DNAfragmentation andpromotingthedeath ofMCF-7 cancer cells [ 185 , 186 ]. In addition, the studies indicate that anthocyanins exert extensive in vitro anti-invasive and in vivo anti-metastaticactivities. Forexample, delphinidincanactasapotentialantimetastaticagent that suppresses PMA-induced cancer cell invasion through the specific inhibition of NF- κ B-dependent MMP-9 gene expression [ 187 , 188 ]. In lung cancer, the treatment of cyanidin-3-glucoside and cyanidin 3-rutinoside, isolated from mulberry, inhibits the migration and invasion of A549 cells and also decreases MMP-2 and uPA and enhances TIMP-2 and PAI. Anthocyanins also inhibit the growth of carcinogenic cells that provoke colon cancer, induce the apoptosis effect, and are even able to act as modulators of the macrophages in the immune response [ 180 ]. Forester et al. [ 189 ] also reported the positive effect of anthocyanin metabolites decreasing cell viability and causing cell cycle arrest and apoptosis in colon cancer. In oral and cervical cancer, the invasion of SCC-4 cells and HeLa cells were diminished by the treatment of peonidin 3-glucoside and cyanidin-3-glucoside [190]. It is also important to note that the structures of anthocyanins have a considerable influence on their biological activities [ 191 – 193 ]. In this sense, the type of aglycones, sugars, and acylated acids, and the position and degree of glycosylation and acylation seem to be the main factors influencing the anticancer property [ 191 ]. Jing et al. [ 192 ] compared the anticancer properties of anthocyanin-rich extracts using human colon cancer HT29 cell line. Authors reported the following growth inhibitory activity rates: purple corn >chokeberry and bilberry >purple carrot and grape >radish and elderberry. Those non-acylated monoglycosylated anthocyanins had greater anticancer property than those with pelargonidin, triglycoside, and/or acylation with cinnamic acid. Neurodegenerative diseases: Anthocyanins are also uniquely suited for the treatment of neurodegenerative diseases such as Alzheimer’s, Parkinson’s, or amyotrophic lateral sclerosis. Their main mechanisms include antioxidant pathways, calcium homeostasis, inflammation, protein homeostasis, and the balance of pro-survival and pro-apoptotic signaling [194,195]. In a primary cell model of Parkinson’s disease, dopaminergic cell death elicited by rotenone was suppressed by extracts prepared from blueberries, grape seed, hibiscus, blackcurrant, and mulberry [ 154 ]. Moreover, Strathearn et al. [ 154 ] observed that those extracts rich in anthocyanins and proanthocyanidins exhibited greater neuroprotective activity than extracts rich in other polyphenols. The oral dispensation of anthocyanins (200 mg/kg) in rats was able to regulate cholinergic neurotransmission, to restore Na + , K + -ATPase and Ca 2+ -ATPase activities, and to prevent memory deficits caused by scopolamine dispensation [ 156 ]. Rehman et al. [ 157 ] showed the neuroprotective effect of anthocyanins based on an artificial ageing model using D-galactose to induce oxidative stress and inflammatory response. The potential mechanisms of their action included: decreased expression of the receptor for advance glycation end product, reduced level of ROS, and lipid peroxidation. Shih et al. [ 155 ] observed that mice fed with anthocyanin-rich mulberry extracts demonstrated significantly less amyloid β protein and showed improvement of learning and memory ability in avoidance response tests. The fed mice also showed a higher antioxidant enzyme activity and less lipid oxidation in both brain and liver, as compared to the control mice. Besides, the treatment with anthocyanin-rich mulberry extract has been proved to decrease the levels of serum aspartate aminotransferase, alanine aminotransferase, triglyceride, and total cholesterol that increase with ageing. Furthermore, the therapeutic profile of anthocyanins can be improved by encapsulation [ 158 – 160 ]. For instance, in Alzheimer’s disease Amin et al. [ 158 ] showed that encapsulated nanoparticles loaded with anthocyanins are rapidly taken up by cells enhancing their neuroprotective profile against amyloid beta toxicity above that of anthocyanins alone. Similar activity was also observed in in vivo studies in mice [158,160]. 6. Conclusions In order to establish the antioxidant activity of anthocyanins and how their intake affects the human health, many factors should be taken into account. Firstly, an evaluation of the antioxidant activity Antioxidants 2020,9, 451 18 of 28 from a multiparametric perspective is requires as the total concentration, the structure, the nature of the sample, the pH and the mechanism of the reaction play an important role in their effect. On a regular basis, more than one antioxidant assay—one for each mechanism—should be carried out. Besides identification and quantification of the anthocyanins is also highly recommended to establish an accurate value for the antioxidant activity of a sample. Secondly, it is required to know the bioavailability of these compounds after their intake. Many studies that have demonstrated the benefits of anthocyanin-rich extracts in the prevention of diseases. Nonetheless, it is important to note that their efficacy depends on their bioavailability. Along the digestion process, anthocyanins are metabolized into various conjugates, which then ultimately metabolize into phenolic acid degradation products as well. The accumulated evidence suggests the synergy effect between all possible forms to explain their attributed health-promoting properties. An inter- and intra-individual variability in anthocyanins absorption, metabolism, distribution, and excretion is also evident. Among the main factors that probably affect this variability are: food matrix and processing, enzymatic levels and microbiota functionality. Attention should be paid in different lines: (i) to perform well-designed standardized methods when evaluating bioavailability; (ii) to consider inter or intra-individual variability in anthocyanin metabolism; (iii) to assess the antioxidant activity of anthocyanin metabolites during gastrointestinal digestion; and, since anthocyanin might exert different biological activities, (iv) to draw a more accurate characterization profiles. Supplementary Materials: The following are available online at http://www.mdpi.com/2076-3921/9/5/451/s1, Table S1: Natural source, type of extraction, chromatographic method and antioxidant assay applied to identify and quantify delphinidin and its derivatives identified with codes, Table S2: Natural source, type of extraction, chromatographic method and antioxidant assay applied to identify and quantify cyanidin and its derivatives identified with codes, Table S3: Natural source, type of extraction, chromatographic method and antioxidant assay applied to identify and quantify petunidin and its derivatives identified with codes, Table S4: Natural source, type of extraction, chromatographic method and antioxidant assay applied to identify and quantify peonidin and its derivatives identified with codes, Table S5: Natural source, type of extraction, chromatographic method and antioxidant assay applied to identify and quantify malvidin and its derivatives identified with codes, Table S6: Natural source, type of extraction, chromatographic method and antioxidant assay applied to identify and quantify pelargonidin and its derivatives identified with codes. Author Contributions: Investigation, N.T., J.M., and A.G.A.; Project administration, A.G.A.; Supervision, A.G.A.; Writing—original draft, N.T. and J.M.; Writing—review and editing, N.T. and J.M. All authors have read and agreed to the published version of the manuscript. Funding: This research received no external funding. Acknowledgments: The authors would like to thank the Vicerrectorado de Investigaci ó n de la Universidad de Sevilla for its support. We would also like to thank the reviewers for their helpful comments that have greatly contributed to the improvement of the document. We are also grateful for the very helpful revision by Irene García Sáez. Conflicts of Interest: The authors declare no conflict of interest. References 1. Dangles, O.; Fenger, J.A. The Chemical Reactivity of Anthocyanins and Its Consequences in Food Science and Nutrition. Molecules 2018,23, 1970. [CrossRef] [PubMed] 2. Miguel, M.G. Anthocyanins: Antioxidant and/or anti-inflammatory activities. J. Appl. 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