Iva Fernandes

Interplay between Anthocyanins and Gut Microbiota

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.

Influence of anthocyanins, derivative pigments and other catechol and pyrogallol-type phenolics on breast cancer cell proliferation.

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.

Mechanistic approach by which polysaccharides inhibit α-amylase/procyanidin aggregation.

The present work studies the inhibition of aggregation of α-amylase and procyanidin fractions by different polysaccharides (arabic gum, β-cyclodextrin, and pectins). Several analytical approaches, namely, fluorescence quenching, nephelometry, and dynamic light scattering (DLS), were used. In general, nephelometry showed that the presence of the polysaccharides in solution reduced the formation of insoluble aggregates. The fluorescence quenching measurements showed two effects: arabic gum and β-cyclodextrin reduce the quenching effect of procyanidin fractions on α-amylase fluorescence, whereas pectins do not affect the quenching of α-amylase fluorescence by procyanidin fractions. DLS measurements have revealed that the polysaccharides studied induce a decrease in aggregates size, which probably is due to the formation of smaller aggregates resulting from the disruption and reorganization of the procyanidin fractions/α-amylase aggregates. Overall, the results obtained for arabic gum and β-cyclodextrin strongly suggest that the main mechanism by which these two compounds inhibit protein/polyphenol aggregation is by molecular association between these polysaccharides and polyphenols, competing with protein aggregation. In the case of pectins, the results obtained provide evidence that the main mechanism by which they reduce protein/polyphenol aggregation is by forming a protein/polyphenol/polysaccharide complex, enhancing its solubility in aqueous medium.

Antioxidant and biological properties of bioactive phenolic compounds from Quercus suber L.

Phenolic compounds, namely, hydrolyzable tannins and low molecular weight phenolic compounds, were isolated and purified from Portuguese cork from Quercus suber L. Some of these compounds were studied to evaluate their antioxidant activity, including free-radical scavenging capacity (DPPH method) and reducing capacity (FRAP method). All compounds tested showed significant antioxidant activity, namely, antiradical and reducing properties. The antiradical capacity seemed to increase with the presence of galloyl groups. Regarding the reducing capacity, this structure-activity relationship was not so clear. These compounds were also studied to evaluate the growth inhibitory effect on the estrogen responsive human breast cancer cell line (ER+) MCF-7 and two other colon cancer cell lines (Caco-2 and HT-29). Generally, all the compounds tested exhibited, after a continuous exposure during a 48 h period, a dose-dependent growth inhibitory effect. Relative inhibitory activity was primarily related to the number of phenolic hydroxyl groups (galloyl and HHDP moieties) found in the active structures, with more groups generally conferring increased effects, except for HHDP-di-galloyl-glucose. Mongolicain B showed a greater potential to inhibit the growth of the three cell lines tested, identical to the effect observed with castalagin. Since these compounds are structurally related with each other, this activity might be based within the C-glycosidic ellagitannin moiety.

Flavonoid metabolites transport across a human BBB model

This study aimed to evaluate the transmembrane transport of different flavonoids (flavan-3-ols, anthocyanins and flavonols) and some of their metabolites (methylated and conjugated with glucuronic acid) across hCMEC/D3 cells (a blood-brain barrier (BBB) model). Further metabolism of the tested compounds was assayed and their transport modulated in an attempt to elucidate the mechanisms behind this process. The transport across hCMEC/D3 cells was monitored in basolateral media at 1, 3 and 18 h by HPLC-DAD/MS. All the flavonoids and their metabolites were transported across hCMEC/D3 cells in a time-dependent manner. In general, the metabolites showed higher transport efficiency than the native flavonoid. No further biotransformation of the metabolites was found as consequence of cellular metabolism. Anthocyanins and their metabolites crossed this BBB cell model in a lipophilicity-dependent way. Quercetin transport was influenced by phosphatase modulators, suggesting a phosphorylation/dephosphorylation regulation mechanism. Overall, this work suggests that flavonoids are capable of crossing the BBB and reaching the central nervous system.

Antioxidant and antiproliferative properties of methylated metabolites of anthocyanins.

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.

On the bioavailability of flavanols and anthocyanins: flavanol-anthocyanin dimers.

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.

Enzymatic Hemisynthesis of Metabolites and Conjugates of Anthocyanins

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.

Antioxidant and antiproliferative properties of 3-deoxyanthocyanidins.

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.

Experimental and Theoretical Data on the Mechanism by Which Red Wine Anthocyanins Are Transported through a Human MKN-28 Gastric Cell Model

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.