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Anthocyanins are widespread and biologically active water-soluble phenolic pigments responsible for a wide range of vivid colours, from red (acidic conditions) to purplish blue (basic conditions), present in fruits, vegetables, and coloured grains. The pigments' stability and colours are influenced mainly by pH but also by structure, temperature, and light. The colour-stabilizing mechanisms of plants are determined by inter- and intramolecular co-pigmentation and metal complexation, driven by van der Waals, π-π stacking, hydrogen bonding, and metal-ligand interactions. This group of flavonoids is well-known to have potent anti-inflammatory and antioxidant effects, which explains the biological effects associated with them. Therefore, this review provides an overview of the role of anthocyanins as natural colorants, showing they are less harmful than conventional colorants, with several technological potential applications in different industrial fields, namely in the textile and food industries, as well as in the development of photosensitizers for dye-sensitized solar cells, as new photosensitizers in photodynamic therapy, pharmaceuticals, and in the cosmetic industry, mainly on the formulation of skin care formulations, sunscreen filters, nail colorants, skin & hair cleansing products, amongst others. In addition, we will unveil some of the latest studies about the health benefits of anthocyanins, mainly focusing on the protection against the most prevalent human diseases mediated by oxidative stress, namely cardiovascular and neurodegenerative diseases, cancer, and diabetes. The contribution of anthocyanins to visual health is also very relevant and will be briefly explored.

Over the years, anthocyanins have emerged as one of the most enthralling groups of natural phenolic compounds and more than 700 distinct structures have already been identified, illustrating the exceptional variety spread in nature. The interest raised around anthocyanins goes way beyond their visually appealing colors and their acknowledged structural and biological properties have fueled intensive research toward their application in different contexts. However, the high susceptibility of monoglycosylated anthocyanins to degradation under certain external conditions might compromise their application. In that regard, polyglycosylated anthocyanins (PGA) might offer an alternative to overcome this issue, owing to their peculiar structure and consequent less predisposition to degradation. The most recent scientific and technological findings concerning PGA and their food sources are thoroughly described and discussed in this comprehensive review. Different issues, including their physical-chemical characteristics, consumption, bioavailability, and biological relevance in the context of different pathologies, are covered in detail, along with the most relevant prospective technological applications. Due to their complex structure and acyl groups, most of the PGA exhibit an overall higher stability than the monoglycosylated ones. Their versatility allows them to act in a wide range of pathologies, either by acting directly in molecular pathways or by modulating the disease environment attributing an added value to their food sources. Their recent usage for technological applications has also been particularly successful in different industry fields including food and smart packaging or in solar energy production systems. Altogether, this review aims to put into perspective the current state and future research on PGA and their food sources., (© 2022 Institute of Food Technologists®.)

Bacterial quorum sensing (QS) is a cell-cell communication system that regulates several bacterial mechanisms, including the production of virulence factors and biofilm formation. Thus, targeting microbial QS is seen as a plausible alternative strategy to antibiotics, with potentiality to combat multidrug-resistant pathogens. Many phytochemicals with QS interference activity are currently being explored. Herein, an extract and a compound of bioinspired origin were tested for their ability to inhibit biofilm formation and interfere with the expression of QS-related genes in Pseudomonas aeruginosa and Staphylococcus aureus . The extract, a carboxypyranoanthocyanins red wine extract (carboxypyrano-ant extract), and the pure compound, carboxypyranocyanidin-3- O -glucoside (carboxypyCy-3-glc), did not cause a visible effect on the biofilm formation of the P. aeruginosa biofilms; however, both significantly affected the formation of biofilms by the S. aureus strains, as attested by the crystal violet assay and fluorescence microscopy. Both the extract and the pure compound significantly interfered with the expression of several QS-related genes in the P. aeruginosa and S. aureus biofilms, as per reverse transcription-quantitative polymerase chain reaction (RT-qPCR) results. Indeed, it was possible to conclude that these molecules interfere with QS at distinct stages and in a strain-specific manner. An extract with anti-QS properties could be advantageous because it is easily obtained and could have broad, antimicrobial therapeutic applications if included in topical formulations.

Tyrosinase is the central enzyme involved in the highly complex process of melanin formation, catalyzing the rate-limiting steps of this biosynthetic pathway. Due to such a preponderant role, it has become a major target in the treatment of undesired skin pigmentation conditions and also in the prevention of enzymatic food browning. Numerous phenolic-based structures from natural sources have been pointed out as potential tyrosinase inhibitors, including anthocyanins. The aim of the present study was to individually assess the tyrosinase inhibitory activity of eight purified compounds with a variable degree of structural complexity: native anthocyanins, deoxyanthocyanins, and pyranoanthocyanins. The latter two, the groups of anthocyanin-related compounds with enhanced stability, were tested for the first time. Compounds 1 to 4 (luteolinidin, deoxymalvidin, cyanidin-, and malvidin-3- O -glucoside) revealed to be the most effective inhibitors, and further kinetic studies suggested their inhibition mechanism to be of a competitive nature. Structure-activity relationships were proposed based on molecular docking studies conducted with mushroom tyrosinase (mTYR) and human tyrosinase-related protein 1 (hTYRP1) crystal structures, providing information about the binding affinity and the different types of interactions established with the enzyme's active center which corroborated the findings of the inhibition and kinetic studies. Overall, these results support the applicability of these compounds as pigmentation modulators.

Due to their physical and chemical characteristics, anthocyanins are amongst the most versatile groups of natural compounds. Such unique signature makes these compounds a focus in several different areas of research. Anthocyanins have well been reported as bioactive compounds in a myriad of health disorders such as cardiovascular diseases, cancer, and obesity, among others, due to their anti-inflammatory, antioxidant, anti-diabetic, anti-bacterial, and anti-proliferative capacities. Such a vast number of action mechanisms may be also due to the number of structurally different anthocyanins plus their related derivatives. In this review, we highlight the recent advances on the potential use of anthocyanins in biological systems with particular focus on their photoprotective properties. Topics such as skin aging and eye degenerative diseases, highly influenced by light, and the action of anthocyanins against such damages will be discussed. Photodynamic Therapy and the potential role of anthocyanins as novel photosensitizers will be also a central theme of this review.

Many efforts have been made in the past two decades into the search for novel natural and less-toxic anti-diabetic agents. Some clinical trials have assigned this ability to anthocyanins, although different factors like the food source, the amount ingested, the matrix effect and the time of consumption (before or after a meal) seem to result in contradictory conclusions. The possible mechanisms involved in these preventive or therapeutic effects will be discussed-giving emphasis to the latest in vitro and in silico approaches. Therapeutic strategies to counteract metabolic alterations related to hyperglycemia and Type 2 Diabetes Mellitus (T2DM) may include: (a) Inhibition of carbohydrate-metabolizing enzymes; (b) reduction of glucose transporters expression or activity; (c) inhibition of glycogenolysis and (d) modulation of gut microbiota by anthocyanin breakdown products. These strategies may be achieved through administration of individual anthocyanins or by functional foods containing complexes of anthocyanin:carbohydrate:protein.

Malvidin-3-O-glucoside, malvidin-3,5-O-diglucoside, malvidin-3-O-(6-O-coumaroyl)-glucoside-5-O-glucoside from Chinese Vitis davidii red wine were used to investigate the role of glucoside, diglucoside and coumaroylated glucoside moieties on their transport efficiency through MKN-28 gastric and Caco-2 intestinal cells. Due to the already described conversion of 3-O-glucosylated anthocyanins into 3-O-glucuronidated, the 3-O-glucuronidated metabolite of malvidin-3-O-glucoside was also tested. The antiproliferative activity was higher for the glucuronidated metabolite in both cell lines. All anthocyanins were transported through MKN-28 gastric cells and Caco-2 intestinal cells with transport efficiencies ranging from 4% to 9% in MKN-28 and from 3% to 5% in Caco-2. No significant differences on transport efficiencies were observed at 180 min among the different anthocyanins in MKN-28. The transport efficiency of malvidin-3-O-glucuronide at 180 min was about 3-4% in Caco-2 and MKN-28 cells. Computational studies were performed to evaluate the interaction between anthocyanins and glucose gastric transporters GLUT1 and GLUT3, which supported the experimental findings., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Two anthocyanins from purple-fleshed sweet potato were isolated and characterized by LC-MS and NMR analysis. They were identified as peonidin-3-(6'-hydroxybenzoyl)-sophoroside-5-glucoside and peonidin-3-(6'-hydroxybenzoyl-6″-caffeoyl)-sophoroside-5-glucoside. The acid-base dynamics of these acylated anthocyanins was evaluated by means of pH jump techniques. Equilibrium and kinetic constants were determined and, in general, these anthocyanins demonstrated a higher capacity in retaining the red and blue colors at acidic and basic pH values, suggesting a higher resistance to pH variations compared to the parent anthocyanin, peonidin-3-O-glucoside. The presence of acyl groups and additional glucoside moieties seems to determine this particular characteristic. The fluorescence properties of these anthocyanins were evaluated. Overall, the species present at higher pH values (7-9) showed higher fluorescence intensity for both anthocyanins, with an optimum λ ex /λ em pair at λ ex 610 nm/λ em 640 nm. The fluorescence characteristics of these anthocyanins were used to evaluate their location in gastric and intestinal cells by fluorescence microscopy., (Copyright © 2019 Elsevier Ltd. All rights reserved.)

Acylated anthocyanins from purple-fleshed sweet potato (PFSP) have been reported to have multiple benefits to human health. Although, the bioavailability of these anthocyanins remains unknown. In the present study, a characterization of the gastrointestinal bioavailability of PFSP anthocyanins was assayed and compared with the bioavailability of Red Wine anthocyanins. Acylated anthocyanins showed higher resistance to overall simulated digestions when compared to less complex anthocyanins from Red wine, with degradation at the intestinal level of about 30% and 45%, respectively. Transport rates of absorption of acylated anthocyanins were not dependent on the cell type. However, a higher transport efficiency was observed in gastric cells (8%) when compared to the intestinal cells (5%). Glucose and proteins, but not starch, decreased the transport efficiency of anthocyanins in about 3-7% in gastric cells and 2-3% in intestinal cells. These results bring new insights and lay the groundwork for further research on acylated anthocyanins bioavailability., (Copyright © 2018 Elsevier Ltd. All rights reserved.)

Anthocyanins may protect against a myriad of human diseases. However few studies have been conducted to evaluate their bioavailability so their absorption mechanism remains unclear. This study aimed to evaluate the role of two glucose transporters (GLUT1 and GLUT3) in anthocyanins absorption in the human gastric epithelial cells (MKN-28) by using gold nanoparticles to silence these transporters. Anthocyanins were purified from purple fleshed sweet potatoes and grape skin. Silencing of GLUT1 and/or GLUT3 mRNA was performed by adding AuNP@GLUT1 and/or AuNP@GLUT3 to MKN-28 cells. Downregulation of mRNA expression occurred concomitantly with the reduction in protein expression. Malvidin-3-O-glucoside (Mv3glc) transport was reduced in the presence of either AuNP@GLUT1 and AuNP@GLUT3, and when both transporters were blocked simultaneously. Peonidin-3-(6'-hydroxybenzoyl)-sophoroside-5-glucoside (Pn3HBsoph5glc) and Peonidin-3-(6'-hydroxybenzoyl-6″-caffeoyl)-sophoroside-5-glucoside (Pn3HBCsoph5glc) were assayed to verify the effect of the sugar moiety esterification at glucose B in transporter binding. Both pigments were transported with a lower transport efficiency compared to Mv3glc, probably due to steric hindrance of the more complex structures. Interestingly, for Pn3HBCsoph5glc although the only free glucose is at C5 and the inhibitory effect of the nanoparticles was also observed, reinforcing the importance of glucose on the transport regardless of its position or substitution pattern. The results support the involvement of GLUT1 and GLUT3 in the gastric absorption of anthocyanins.

High-fat (HF) diets are thought to disrupt the profile of the gut microbiota in a manner that may contribute to the neuroinflammation and neurobehavioral changes observed in obesity. Accordingly, we hypothesize that by preventing HF-diet induced dysbiosis it is possible to prevent neuroinflammation and the consequent neurological disorders. Anthocyanins are flavonoids found in berries that exhibit anti-neuroinflammatory properties in the context of obesity. Here, we demonstrate that the blackberry anthocyanin-rich extract (BE) can modulate gut microbiota composition and counteract some of the features of HF-diet induced dysbiosis. In addition, we show that the modifications in gut microbial environment are partially linked with the anti-neuroinflammatory properties of BE. Through fecal metabolome analysis, we unravel the mechanism by which BE participates in the bilateral communication between the gut and the brain. BE alters host tryptophan metabolism, increasing the production of the neuroprotective metabolite kynurenic acid. These findings strongly suggest that dietary manipulation of the gut microbiota with anthocyanins can attenuate the neurologic complications of obesity, thus expanding the classification of psychobiotics to anthocyanins.

There is a growing market demand for the incorporation of plant-derived ingredients into new products for the cosmetic industry. Anthocyanins are polyphenols arising from plant secondary metabolism that have been shown to possess many bioactive properties such as free radical scavenging, antimicrobial, and chemopreventive activities. In this work, the biological activities of red wine and blackberry anthocyanins were assessed by developing a new keratinocyte barrier model using the HaCat cell line and a microelectrode-based biosensor device, referred to as Electric Cell-Substrate Impedance Sensing (ECIS). Cells were seeded at the optimal cellular density of 1.6 × 10 6 cells per mL and the half-time was calculated to be 3.55 ± 0.67 hours. The compounds' cytotoxicity was assessed and anthocyanin pigments showed no cytotoxicity towards keratinocyte cells. Wound healing assays were also performed using ECIS and it was observed that the tested pigments enhanced the healing rate of keratinocyte cells by reducing the healing time more than 50%. Cyanidin-3-glucoside presented the best results recovering 50% of the injured area in 1.48(±0.15) hours, followed by the blackberry anthocyanins (2.01 ± 0.18 hours), malvidin-3-glucoside (2.03 ± 0.09 hours) and red wine anthocyanins (2.36 ± 0.76 hours). All presented significant differences from the control 4.91(±1.11) hours.

This study was designed to evaluate the pharmacokinetics of Port and table red wine anthocyanins in healthy men. Volunteers were recruited to drink 250 mL of a table red wine (221 mg of anthocyanins) and 150 mL of young Port red wine (49 mg of anthocyanins). Venous blood was collected from participants at 0, 15, 30, 60 and 120 min after wine ingestion. Urine samples were collected at baseline and at 120 min. Anthocyanins and anthocyanin metabolites in plasma and urine samples were quantified by HPLC-DAD and tentatively identified by LC-MS. Red wine anthocyanins were detected in their intact forms in both plasma and urine samples, but the glucuronylated metabolites of peonidin and malvidin (PnGlucr and MvGlucr) were the two main derivatives detected after both red wine consumptions. For the first time, and supported by the synthesis of Mv3Glucr, the main pathway followed by Mv3glc after absorption was described and involves anthocyanidin conjugation with glucuronic acid after glucose removal. Despite the lower total content of anthocyanins ingested when volunteers drank Port wine, no differences were observed in the plasma C max of MvGlucr and PnGlucr after table and Port red wine consumption. The relative bioavailability of anthocyanins in Port wine was 96.58 ± 5.74%, compared to the anthocyanins present in red wine. In conclusion, both Port and table red wines are good sources of bioavailable anthocyanins.

Scope: This study was designed to evaluate the influence of ethanol on the bioavailability of blackberry anthocyanins., Methods and Results: A total of 18 participants were recruited to consume 250 mL of a blackberry puree (650 mg of anthocyanins) without (BBP) or with 12% ethanol (BBP 12%). Venous blood was collected from participants at baseline and at 15, 30, 60, and 120 min after puree ingestion. Urine samples were collected at baseline and at 120 min. Plasma and urine concentration of anthocyanins and anthocyanin conjugates were quantified by HPLC-DAD. Methyl-cyanidin-glucuronide (Me-Cy-Glucr) and 3'-methyl-cyanidin-3-glucoside (3'-Me-Cy3glc) were the main anthocyanin conjugates detected in all plasma and urine samples. Urinary concentration of these anthocyanin conjugates were positively correlated with their plasma concentration. Ethanol increased plasma C max of Me-Cy-Glucr and 3'-Me-Cy3glc. Participants were then stratified according to their body mass index (BMI) and body fat mass. After BBP consumption, plasma C max of Me-Cy-Glucr and 3'-Me-Cy3glc tended to be decreased in overweight/obese participants, in comparison to normal weight participants. The increase on plasma C max of Me-Cy-Glucr and 3'-Me-Cy3glc induced by ethanol was more pronounced in the group of overweight/obese participants., Conclusions: Ethanol seems to enhance Cy3glc metabolism that appears to be compromised in overweight and obese individuals., (© 2016 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

The chemical modification of anthocyanins (water-soluble pigments) into more lipophilic compounds is very important to expand their application in the food, medical and cosmetic industries. In this work, the synthesis of a pure malvidin-3-glucoside-oleic acid ester derivative was achieved by enzymatic catalysis. This approach allowed us to synthesize a novel compound, malvidin-3-O-(6''-oleoyl)glucoside (Mv3glc-OA), which was structurally characterized by mass spectrometry and for the first time by NMR spectroscopy. The enzymatic reaction revealed to be regioselective giving only one ester product. Antioxidant features of the malvidin-3-glucoside lipophilic derivative by means of DPPH, FRAP and lipid peroxidation assays were assessed, which confirmed that the structural modification of the genuine malvidin-3-glucoside into a more lipophilic compound did not compromise its antioxidant potential and protected more effectively a lipidic substrate from oxidation, which is an important insight for future technological applications.

In this study, the gastric transport efficiency of malvidin-3-glucoside and several derivatives was assayed on the MKN-28 cell model. The transport efficiency was found to increase for all compounds with the incubation time. Pyranoanthocyanins may slightly impair transport efficiency levels in comparison with native anthocyanins. Among the pyranoanthocyanin derivatives the presence of the carbonyl group and the absence of charge were important for the transport efficiency percentage of oxovitisin and apparently compensated the negative effect associated with the additional ring. Moreover, the antiproliferative properties of these compounds in the MCF-7 cancer cell line were assayed, oxovitisin being the most effective compound in inhibiting the proliferation of MCF-7 cells. Also, a kinetic incorporation of oxovitisin was assayed revealing that this pyranoanthocyanin is quickly incorporated into cells. This study confirms the importance of the natural micro-oxidative processes that occur during the ageing of anthocyanin-containing food and their impact on their bioavailability and bioactivity properties.

Microglia mediate multiple aspects of neuroinflammation, including cytotoxicity, repair, regeneration, and immunosuppression due to their ability to acquire diverse activation states, or phenotypes. Modulation of microglial phenotype or microglia-neuron crosstalk can be an appealing neurotherapeutic strategy. Anthocyanins are a class of flavonoids found e.g., in berries that has been attracting interest due to its neuroprotective potential. However, there are no data clarifying the impact of anthocyanins on microglial phenotype or on microglia-neuron crosstalk (CX3CR1/CX3CL1). N9 microglia cell line was treated with 1μM cyanidin (Cy), cyanidin-3-glucose (Cy3glc) and a methylated form of cyanidin-3-glucose (Met-Cy3glc) in basal conditions and with LPS/IL-4 stimulation. SH-SY5Y cell line was treated with the conditioned medium of microglia and with the anthocyanins alone. At basal conditions, microglia treatment with anthocyanins for 24h induced a less pro-inflammatory profile. Decreased TNF-α mRNA expression was induced either by Cy and Met-Cy3glc. LPS markedly increase IL-6 mRNA expression, which was lowered by Cy3glc. IL-1β LPS-induced expression was reverted by Cy. Cy increased CX3CL1 mRNA expression in SH-SY5Y comparing either with control or LPS. Anthocyanins and metabolites were not able to shift microglia to an M2 strict phenotype however they did interact with microglia biology. There was an attenuation of M1 phenotype and increase of neuronal expression of CX3CL1 mRNA. Understanding how flavonoids modulate microglia-neuron crosstalk can open new directions for future nutritional interventions., (Copyright © 2016 Elsevier B.V. All rights reserved.)

The study of the antioxidant properties of six deoxyanthocyanidins (deoxypeonidin, deoxymalvidin, luteolinidin, apigeninidin, guaiacylcatechinpyrylium and syringylcatechinpyrylium) and an anthocyanin (cyanidin-3-glucoside) was carried out. The aim was to evaluate the relationship between the structure and the antioxidant properties of individual deoxyanthocyanidins, compared to a common anthocyanin derivative, cyanidin-3-glucoside. The ability of these compounds to inhibit lipid peroxidation in a liposome membrane system was examined by monitoring oxygen consumption and the antiradical and reducing capacities were determined using the DPPH and FRAP assay, respectively. The results showed that all the compounds tested presented antioxidant properties. Cyanidin-3-glucoside presented higher antiradical and reducing activities in the DPPH and FRAP assay, although in the liposome model, the guaiacylcatechinpyrylium was more effective inhibiting lipid peroxyl radicals. Additionally, the anti-proliferative effects of deoxyanthocyanidins, have been evaluated against two cancer cell lines from stomach (AGS, MKN-28) and one colon cancer cell (Caco-2), and compared with the effect of the respective anthocyanins. Considering the antiproliferative activity, all compounds were active against Caco-2 cell line, being the ones with glucose moiety and oaklin Scp the most active. Deoxyanthocyanidins, and in particular, guaiacylcatechinpyrylium may be regarded as potential food colorants., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

The gastric absorption of red wine anthocyanins was evaluated using a gastric MKN-28 cell barrier model. Anthocyanin transport was not affected by the presence of 4% ethanol and decreased with the increase of pH. Gastric cells pretreated with anthocyanins were found to increase anthocyanin transport. The presence of d-(+)-glucose was found to decrease anthocyanin uptake, suggesting the involvement of glucose transporters. RT-PCR assays revealed that GLUT1, GLUT3, and MCT1 transporters were expressed in MKN-28 cells. Computational studies were performed to provide a structural characterization of the binding site of hGLUT1 to glucose or different anthocyanins under different forms. Docking results demonstrated that anthocyanins can bind to glucose transporters from both intracellular and extracellular sides. Anthocyanins seem to enter into the transporter by two main conformations: B ring or glucose. From MD simulations, hGLUT1 was found to form complexes with all anthocyanins tested in the different protonation states.

Anthocyanins are naturally occurring compounds abundant in the human diet. Evidence has accumulated regarding the positive association of their intake with healthy biological effects. The microbiota has just been started to be considered as a metabolic organ, hence contributing to the metabolism of phenolic compounds and, consequently, to their bioavailability and the biological effects displayed by them. This review aimed to compile information regarding interaction of anthocyanins with the microbiota, from two perspectives: (i) identification of their colonic metabolites as potential bioactive molecules and (ii) their role as prebiotic agents. These perspectives are key points in anthocyanin metabolomics. Several metabolites have been identified after anthocyanin consumption with potential health benefits, in particular phenolic acids and simple phenols. On the other hand, microbiota modulation is closely related to several physiological impairments, and its modulation has been considered as a possible mechanism by which phenolic compounds may exert their effect.

Anthocyanins are major flavonoids in many plant foods and have been related to health promotion. In the human organism anthocyanins are metabolised to different metabolites. One of the most important phase II reactions of flavonoids is the methylation of the catechol group. This feature is expected to have an effect on the antioxidant and antiproliferative properties of flavonoids including anthocyanins. In this work, delphinidin-3-glucoside, cyanidin-3-glucoside and petunidin-3-glucoside methylated metabolites were obtained by enzymatic hemi-synthesis. The compounds were identified as monomethylated products by HPLC-MS and NMR. The methylated metabolites were found to still retain significant radical scavenging activity and reducing activity, suggesting that they could act as potential antioxidants in vivo. The antiproliferative activity of the metabolites in comparison with the parental anthocyanins was also evaluated in three cancer cell lines by sulforhodamine B assay. The conjugation with methyl groups decreased or did not alter the antiproliferative effect of the original anthocyanin., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

The bioavailability of flavanols, anthocyanins and anthocyanin-derived pigments like flavanol-anthocyanin dimers already reported to occur in food products is a major unsolved issue. The absorption of the flavanol-anthocyanin dimer (+)-catechin-(4,8)-malvidin-3-O-glucoside (Cat-Mv3glc) through Caco-2 cells was assessed by performing transepithelial transport assays. The ability of Cat-Mv3glc to cross Caco-2 cells was compared with that of malvidin-3-glucoside (Mv3glc), (+)-catechin (Cat) and procyanidin B3 (Cat-Cat), in order to evaluate the influence of some structural features on the transport efficiency. The flavanol-anthocyanin dimer was absorbed in this intestinal model although with a lower efficiency than the monomers Cat and Mv3glc. On the other hand, Cat-Mv3glc was found to cross the intestinal barrier model more significantly than Cat-Cat. This feature may be related to the presence of the glucose moiety in its structure. Overall, this study brings more insights into the bioavailability of anthocyanins and flavanols and represents the first report on the bioavailability of flavanol-anthocyanins., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

The bioavailability of anthocyanins is the most difficult one to assess amongst all flavonoid compounds as a result of their occurrence under different structures in equilibrium depending on pH. Due to their rapid appearance in plasma, the absorption of anthocyanins is likely to occur at the gastric level. Further investigations of the mechanisms by which anthocyanins are absorbed are limited by the lack of testable gastric epithelial cell models that form functional barriers. The methods available to evaluate the absorption of drugs at the gastric level make use of isolated gastric epithelial cells, which is both time and labour consuming. In the present study, a biologically relevant in vitro model of moderately differentiated adenocarcinoma stomach cells (MKN-28) was used as gastric barrier. The transepithelial electrical resistance (TEER) of MKN-28 cell monolayers was evaluated at pH values that cover the physiologic range of the stomach, ensuring the integrity of the cell monolayer . The immunofluorescence assay attested the localization of occludins at the cellular margins, which is associated with a non-disrupted membrane. Anthocyanins were found to cross MKN-28 cells in a time dependent manner and probably via a saturable transport mechanism.

Anthocyanins (cyanidin-3-glucoside (Cy-3-gluc) and delphinidin-3-glucoside (Dp-3-gluc)) and their respective vinylpyranoanthocyanin-catechins (portisins) were studied in order to evaluate the cytotoxicity effect on the estrogen responsive human breast cancer cell line (ER+) MCF-7 and their effect on estrogen receptor (ER-alpha and ER-beta) expression. Other flavonoid classes and phenolic molecules were also tested, aiming to study possible structural features related with these effects. Also, the antiproliferative effect of Cy-3-gluc and Dp-3-gluc was studied by an immunofluorescence assay. Generally, all the anthocyanin pigments studied inhibited, in a dose-dependent manner, the growth of the (ER+) MCF-7. The cytotoxicity effect was higher when cells were treated with Dp-3-gluc and its respective portisin. Altogether, the results point to the ortho trihydroxylated moiety in the phenolic ring as an important structural feature for more potent cytotoxicity effect on MCF-7 cells comparatively to the effect observed with the similar dihydroxylated compounds. In order to elucidate the molecular mechanism involved, expression of estrogen receptor was assayed by RT-PCR and real time RT-PCR. The higher antiproliferative effect observed after cell treatment with Dp-3-gluc was not followed by modification on ER expression. However, the anthocyanin Cy-3-gluc was able to induce a downregulation of ER levels although with no significant effect on MCF-7 proliferation.

This work aims to study the phase II metabolization of anthocyanins that is likely to occur in vivo. Anthocyanins (delphinidin, cyanidin, and malvidin-3-glucosides) were incubated with phase II enzymes in the presence of activated cofactors in order to obtain glutathionyl conjugates, methylated and glucuronydated compounds. Overall, the three anthocyanins tested were metabolized in vitro. Two compounds were detected by HPLC after incubation of human liver cytosolic fraction with cyanidin-3-glucoside and one compound with delphinidin-3-glucoside. These compounds were identified as monomethylated products. LC-MS analysis yielded mass data that fit with the anthocyanin structures bearing an additional methyl group in ring B. Several compounds were detected by HPLC after incubation of human liver microsomes with malvidin, cyanidin, and delphinidin-3-glucosides. These compounds were identified as monoglucuronides products after HPLC analysis. Conjugation with glutathione also occurred as proved by the mass data obtained. However, in this case, two anthocyanin equilibrium forms (flavylium and chalcone or water adducts) conjugated with glutathione were detected. Overall, the data of the present work shows the feasibility of the in vitro enzymatic hemisynthesis of metabolites and glutathione conjugates of anthocyanins. This first experimental approach may further allow the achievement of new purified forms of anthocyanins, some of which do not occur in nature, and also the determination of whether these compounds are the bioactive forms responsible for some of the biological activities reported for anthocyanins.