- Research Paper
- Open Access
Potential anti-inflammatory, anti-adhesive, anti/estrogenic, and angiotensin-converting enzyme inhibitory activities of anthocyanins and their gut metabolites
© Springer-Verlag 2011
- Received: 31 August 2011
- Accepted: 13 December 2011
- Published: 5 January 2012
Epidemiological studies have indicated a positive association between the intake of foods rich in anthocyanins and the protection against cardiovascular diseases. Some authors have shown that anthocyanins are degraded by the gut microflora giving rise to the formation of other breakdown metabolites, which could also contribute to anthocyanin health effects. The objective of this study was to evaluate the effects of anthocyanins and their breakdown metabolites, protocatechuic, syringic, gallic, and vanillic acids, on different parameters involved in atherosclerosis, including inflammation, cell adhesion, chemotaxis, endothelial function, estrogenic/anti-estrogenic activity, and angiotensin-converting enzyme (ACE) inhibitory activity. From the assayed metabolites, only protocatechuic acid exhibited a slight inhibitory effect on NO production and TNF-α secretion in LPS-INF-γ-induced macrophages. Gallic acid caused a decrease in the secretion of MCP-1, ICAM-1, and VCAM-1 in endothelial cells. All anthocyanins showed an ACE-inhibitory activity. Delphinidin-3-glucoside, pelargonidin-3-glucoside, and gallic acid showed affinity for ERβ and pelargonidin and peonidin-3-glucosides for ERα. The current data suggest that anthocyanins and their breakdown metabolites may partly provide a protective effect against atherosclerosis that is multi-causal and involves different biochemical pathways. However, the concentrations of anthocyanins and their metabolites, as used in the present cell culture and in vitro assays mediating anti-inflammatory, anti-adhesive, anti-estrogenic, and angiotensin-converting enzyme inhibitory activities, were often manifold higher than those physiologically achievable.
- Phenolic acids
- Estrogen receptor
Polyphenolic substances, such as anthocyanins, exert a great variety of physiological activities responsible for the health effects attributed to some foods, including a reduced risk of cardiovascular diseases. This is partly due to their anti-inflammatory properties (González-Gallego et al. 2010; Landberg et al. 2011; Rotelli et al. 2003), antioxidant and free radical scavenging activities (García-Alonso et al. 2009; Gray and DeWitt 1999; Kahkonen and Heinonen 2003; Matsumoto et al. 2002; Tsuda et al. 1994; Wang et al. 1997), peroxidation inhibition (Tsuda et al. 1996), and estrogenic/anti-estrogenic activity (Cassidy et al. 2003). It has been largely proven that the beneficial potential of polyphenols as part of a healthy diet cannot be only explained by their antioxidant characteristics (Virgili and Marino 2008).
One of the biological mechanisms by which flavonoids exhibit anti-inflammatory effects appears to be associated with the inhibition of nitric oxide (NO) production (Vallance and Leiper 2002). A critical step in both inflammation and atherosclerosis is the adhesion of circulating monocytes to vascular endothelial cells, which involves vascular cell adhesion molecule-1 (VCAM-1) and intercellular adhesion molecule-1 (ICAM-1). Studies in vitro suggest that flavonoids participate in the prevention and attenuation of inflammatory diseases by decreasing ICAM-1 and VCAM-1 levels (Kwon et al. 2005; Lotito and Frei 2006). In addition, dietary anthocyanins and hydroxycinnamic acids have been reported to reduce TNF-α-induced up-regulation of various inflammatory mediators such as ICAM-1 or MCP-1. Therefore, the ability of polyphenols to mediate inflammatory processes is likely to contribute to their antiatherogenic properties.
The effect of flavonoids on the arterial wall due to their estrogenic activity is well known. Flavonoids reduce the risk of cardiovascular diseases (Kris-Etherton et al. 2002), decrease serum cholesterol, low-density lipoproteins (LDL) and triglyceride levels (Ricketts et al. 2005), prevent osteoporosis (Dang and Lowit 2005), and improve menopausal symptoms (McCann et al. 2005). Notably, work has already shown that the estrogenic properties of wine flavonoids including anthocyanins are due, in part, to their ability to bind the estrogen receptor (Chalopin et al. 2010; Schmitt and Stopper 2001).
In addition, flavonoids have been suggested to decrease cardiovascular risk by reducing levels of angiotensin II, a well-known proinflammatory mediator (Naruszewicz et al. 2007). It has already been reported that some anthocyanin-containing foods as well as delphinidin inhibit ACE activity (Actis-Goretta et al. 2006; Lacaille-Dubois et al. 2001; Persson et al. 2009).
Anthocyanins intake in humans has been estimated to be between 3 and 215 mg/day (Chun et al. 2007; Frankel et al. 1995; Pérez-Jimenez et al. 2011; Wu et al. 2006). Most studies have shown very low bioavailability of anthocyanins based only on the measurement in plasma or urine of the original anthocyanins and their conjugated metabolites, glucuronidated, and sulfated anthocyanins (Manach et al. 2005). More recently, it has been established that the intestinal microflora plays a key role in the metabolism of anthocyanins. After ingestion, anthocyanins can be hydrolyzed by intestinal glucosidases and the resulting aglycones are further metabolised in the large intestine to other breakdown metabolites such as protocatechuic, gallic, syringic, and vanillic acids (Avila et al. 2009; Forester and Waterhouse 2008; Keppler and Humpf 2005; Vitaglione et al. 2007). Moreover, gallic acid has been determined in plasma after its ingestion at levels as high as 1.8 μmol/L in its original form and at 2.2 μmol/L as its derivative 4-O-methylgallic acid (Shahrzad and Bitsch 1998; Shahrzad et al. 2001). Therefore, metabolites produced by the intestinal microflora could account partly for the health benefits associated with anthocyanin consumption in humans.
The aim of this study was to further elucidate the potential mechanisms by which anthocyanins and their metabolites reduce the initial stages of atherosclerosis. Specifically, we have studied the effect of anthocyanins and their metabolites on (1) NO production and TNF-α secretion in macrophages, and (2) ICAM-1, VCAM-1, and MCP-1 secretion in endothelial cells. The ACE-inhibitory activity of anthocyanins and their metabolites was also measured. Finally, their ERα- and ERβ-binding ability was measured. Docking studies helped to rationalize selectivity on ERs.
Cyanidin-3-O-glucoside, pelargonidin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin-3-O-glucoside, and peonidin-3-O-glucoside were purchased from Extrasynthese (Lyon, France). Acetate buffer saline, neutral red, Griess reagent, crystal violet, LPS (lipopolysaccharide), human recombinant TNF-α, mouse recombinant IFN-γ, 17-β-estradiol, PBS, Tween-20, BSA, ACE (peptidyl-dipeptidase A, EC 184.108.40.206), and the phenolic acids: gallic, syringic, protocatechuic, vanillic, sinapic, homogentisic, 4-hidroxybenzoic, phloroglucinol, 3-(2′,5′-dimethoxybenzoil) propionic (DMB propionic), coumaric and caffeic acids were purchased from Sigma-Aldrich Química S.A. (Madrid, Spain). Ethanol 99%, glacial acetic acid, and dimethyl sulfoxide (DMSO) were obtained from Panreac (Barcelona, Spain). Sodium dodecyl sulfate (SDS) was acquired from Fisher (Madrid, Spain). Dulbecco’s modified Eagle’s medium (DMEM), fetal bovine serum (FBS), penicillin/streptomycin, trypsin, l-glutamine, and nonessential amino acids were purchased from Lonza (Barcelona, Spain). Estradiol [2,4,6,7,16,17-3H(N)] and scintillation counting liquid (Optifase HiSafe2) were obtained from Perkin-Elmer (Salem, MA, USA). Estrogen receptors α and β (human recombinant produced in insect cells) were purchased from Invitrogen (Barcelona, Spain).
RAW 264.7 cells, a murine monocyte macrophage cell line, were maintained at 37°C in 5% CO2 according to standard protocols (Rimbach et al. 2000). The medium consisted of DMEM with 4.5 g/L glucose and l-glutamine supplemented with 10% FBS and 1% penicillin/streptomycin (5,000 U/mL). For experiments, cells were harvested with trypsin–EDTA and macrophages were plated in 24-well plates at a density of 5 × 104 in 0.5 mL of medium for the cytotoxicity test or in 6-well plates at a density of 2 × 105 in 2 mL of medium, for nitrite and TNF-α measurement. Finally, cells were cultured for 72 h until they reached 80% confluence. Cells in 6-well plates were treated with gallic, vanillic, protocatechuic, and syringic acids in a range of concentrations between 0.01 and 500 μM or DMSO (<0.1%) as follows; four different treatments were performed. (A) Cells were pre-treated for 3 h with the different compounds, washed twice with PBS, and then stimulated with 1 μg/mL LPS for 24 h. (B) Cells were pre-treated with the different compounds for 3 h, washed twice with PBS, and then stimulated with 1 μg/mL LPS plus 1,000 U/mL IFN-γ for 24 h. (C) Cells were co-incubated with the different compounds at 200 μM together with 1 μg/mL LPS for 24 h. (D) Cells were co-incubated with the different compounds at 200 μM and with 1 μg/mL LPS plus 1,000 U/mL IFN-γ simultaneously for 24 h. For all these experiments, control cells were grown under identical conditions but were not exposed to the test compound or LPS/IFN-γ. For the cytotoxic assay, cells were treated with all compounds at the maximal concentration used in the NO production assay.
EA.hy 926 cells, a cell line derived from human umbilical vein endothelial cells (HUVEC), were a generous gift from Prof. C.-J. S. Edgell, University of North Carolina at Chapel Hill. EA.hy 926 cells were generated by fusion of human umbilical vein endothelial cells (HUVEC) with the human lung carcinoma cell line A549 and have been extensively used as a cell model for endothelial function (Fuchs et al. 2005). Cells were grown in DMEM medium containing 4.5 g/L glucose and l-glutamine and supplemented with 10% FBS and 1% penicillin/streptomycin (5,000 U/mL). Cells were harvested with trypsin–EDTA and seeded in 0.5 mL of medium in 24-well plates or 2 mL of medium in 6-well plates and cultured until they reached 80% confluence. After pre-treatment in 6-well plates with 10 and 100 μM of gallic, vanillic, protocatechuic and syringic acids for 16 h, cells were stimulated with 10 ng/mL TNF-α for 6 h.
Estrogen receptor (ER) competitive binding assay
The binding activity of anthocyanins and acids to human ER was determined using a radioactivity assay based on the ability of the different compounds to compete with 3H-labeled estradiol for the estrogen receptor. For the ERβ- and ERα-binding assay, the corresponding pure estrogen receptor was used at 2.4 nM together with 5 nM tritium-labeled estradiol ([2,4,6,7,16,17-3H]estradiol). Unlabeled estradiol, anthocyanins, and acids were prepared in DMSO (<0.5%) and diluted in PBS-T (PBS + 0.15% Tween20) including receptors and Estradiol-H3*. Briefly, the same volume of each compound, Estradiol-H3*, and receptor were mixed in a final volume of 150 μL, thus the different compounds were tested at a concentration ranging from 1 to 200 μM. The mixture was incubated for 4 h at 23°C to allow receptor binding. Afterward, 50 μL of charcoal (charcoal 0.1 g/mL and BSA 0.02 g/mL) was added and the samples kept on ice for 15 min before being centrifuged at 6,000×g for 5 min to remove the non-bound Estradiol-H3*. An aliquot of this supernatant (150 μL) was added to 4 mL of scintillation counting liquid. The bound [3H] estradiol was measured in a WinSpectral 1414 Liquid Scintillation Counter (Beckman, LS 6500). Three independent experiments containing three replicates were performed for each compound tested. Results are expressed as the percentage of specific binding of [3H] estradiol to ER versus log of competitor concentration. IC50 values represent the concentration of test compound required to displace 50% [3H] estradiol from the receptor. IC50 values were determined by non-linear regression fitting of experimental data to a sigmoid equation.
Geometries of compounds X–Y were first optimized using the ab initio quantum chemistry program Gaussian 03 (Frisch et al. 2004) and the B3LYP/3-21G* basis set. As macromolecules, the X-ray structures of estrogen receptor complexes with genistein were chosen (PDB codes: 1x7r for ERα and 1x7j for ERβ). Crystallographic water molecule close to Arg394 (ERβ Arg346) and Glu353 (ERβ Glu305) were kept as they were considered to be part of the binding site. Different conformers of the ligands were docked using the Lamarckian genetic algorithm implemented in AutoDock 3.1 (Morris et al. 1998) by randomly changing the torsion angles and overall orientation of the molecule. A volume for exploration was defined in the shape of a three-dimensional grid (80 × 80 × 90 Å3) with a spacing of 0.375 Å that enclosed the binding site and included the residues that are known to be crucial for activity. At each grid point, the receptor’s atomic affinity potentials for carbon, aromatic carbon, oxygen, nitrogen, sulfur, and hydrogen atoms were precalculated for rapid intra- and intermolecular energy evaluation of the docking solutions for each ligand. The original Lennard–Jonnes and hydrogen-bonding potentials provided by the program were used. The parameters for the docking using the LGA were identical for all docking jobs. After docking, the 100 solutions were clustered in groups with root mean square deviations less than 1.0 Å. The clusters were ranked by the lowest energy representative of each cluster.
The uptake of neutral red dye was used to assess cell viability as described previously (Valacchi et al. 2001). Macrophages and Ea.hy 926 cells were pre-treated in 24-well plates with the different test compounds for 24 h. After incubation, the culture medium was removed and replaced with fresh medium containing 50 μg/mL of neutral red. Following incubation for 2 h at 37°C, the medium was removed and the cells extracted using a solution comprising 50:49:1 (v/v/v) ethanol, water, and glacial acetic acid. Absorbance at 540 nm was recorded using a microplate reader (Power Wave XS, BIOTEK). For all cell culture experiments, compounds were dissolved in DMSO. The final DMSO concentration in the cell culture medium was 0.1% (v/v) or less. Pre-treatment for RAW 264.7 macrophages with up to 500 μM and treatment for Ea.hy 926 with up to 100 μM of any of the assayed compounds did not affect cell viability.
NO production was assessed by the measurement of nitrite concentration (NO2 −) in the medium using the Griess reaction (Wang and Mazza 2002b). Supernatants of cultured macrophages were collected and deproteinized with 0.3 M NaOH and 0.3 M ZnSO4. An equal volume of the Griess reagent (1% sulfanilamide/0.1% N-(1-naphthyl)-ethylenediamine dihydrochloride/2.5% H3PO4) and the deproteinized samples were incubated for 10 min at room temperature protected from light. The nitrite concentration was determined by measuring the absorbance at 548 nm against a standard curve for sodium nitrite (Park et al. 2000).
TNF-α secretion in RAW 264.7 macrophages
Supernatants from RAW 264.7 macrophages were collected for TNF-α secretion measurements as described above for NO production measurements. Upon collection, samples were centrifuged at 15,700×g for 10 min and the supernatants kept at −80°C until analysis. TNF-α secretion was measured using a commercially available enzyme-linked immunosorbent assay (ELISA) kit (Mouse TNF-α immunoassay, eBioscence).
Secretion of monocyte chemoattractant protein 1 (MCP-1), intercellular adhesion molecule 1 (ICAM-1), and vascular cell adhesion molecule 1 (VCAM-1) by EA.hy 926 cells
Upon collection of the supernatant from Ea.hy 926 cells, samples were centrifuged at 15,700×g for 10 min and the supernatants were kept at −80°C until analysis. The secretion of MCP-1, ICAM-1, and VCAM-1 by Ea.hy 926 cells was measured using commercially available ELISA kits (Diaclone, Bionova scientific).
Determination of ACE-inhibitory activity
ACE-inhibitory activity was measured by fluorescence using the method of Sentandreu and Toldrá (2006) with some modifications. Briefly, ACE (peptidyl-dipeptidase A, EC 220.127.116.11) working solution was diluted with 0.15 M Tris buffer (pH 8.3) containing 0.1 μM ZnCl2 with 0.04 U/mL of enzyme in the final reaction solution. A total of 40 μL of this working solution (or distilled water for the blank) was added to each microtiter plate well, with another 40 μL of distilled water for the blank (B) or 40 μL control (C) or 40 μL samples (S). The enzyme reaction was started by adding 160 μL of 0.45 mM o-Abz-Gly-p-Phe(NO2)-Pro-OH (Bachem Feinchemikalien, Bubendorf, Switzerland) dissolved in 150 mM Tris-base buffer (pH 8.3), containing 1.125 M NaCl, and the mixture was incubated at 37°C. The fluorescence generated was measured at 30 min using a multiscan microplate fluorimeter (FLUOstar optima, BMG Labtech, Offeuburg, Germany). 96-well microplates (Porvair, Leatherhead, UK) were used. Excitation and emission wavelengths were 350 and 420 nm, respectively. The software used to process the data was FLUOstar control (version 1.32 R2, BMG Labtech).
The ACE-inhibitory activity was evaluated in 11 phenolic acids: gallic, protocatechuic, syringic, vanillic, synapic, homogentisic, hydroxybenzoic, phloroglucinol, coumaric, caffeic, and DMB propionic acid and five anthocyanins: cyanidin-3-O-glucoside, malvidin-3-O-glucoside, delphinidin-3-O-glucoside, peonidin-3-O-glucoside, and pelargonidin-3-O-glucoside. All samples were diluted in distilled water or ethanol–water 30/70 (v/v).
The activity of each sample was tested in triplicate. Inhibitory activity was expressed as the concentration required to inhibit the original ACE activity by 50% (IC50). The formula applied to calculate the percentage of ACE-inhibitory activity was: 100 − (C − S)/(C − B), where C is the fluorescence of ACE with o-Abz-Gly-p-Phe(NO2)-Pro-OH (fluorescence substrate) and without inhibitor, S is the fluorescence of ACE with o-Abz-Gly-p-Phe(NO2)-Pro-OH and with sample as inhibitor, and B is the fluorescence of the fluorescent substrate o-Abz-Gly-p-Phe(NO2)-Pro-OH. This parameter was plotted versus sample concentration and non-linear logarithmic adjustment was performed as indicated by Quirós et al. (2007) to estimate IC50.
The results were reported as means ± standard deviation (SD) of at least three measurements or two in the case of ICAM-1, VCAM-1, and MCP-1, each performed in triplicate. One-way analysis of variance (ANOVA) was used to compare the means, and the least significant difference (LSD) test showed the values statistically different. Differences were considered significant at P < 0.05. All statistical analyses were performed with Statgraphics Plus 5.1 (Statistical Graphics Corporation, Inc., Rockville, MD, USA).
Effect of phenolic acids on NO production and TNF-α secretion in LPS and LPS/IFN-γ-stimulated macrophages
Effect of protocatechuic acid on the inhibition of nitric oxide production in RAW 264.7 macrophages
Inhibition of NO production (%)
LPS 1 μg/mL
LPS 1 μg/mL + IFN-γ 1,000 U/mL
16.2 ± 3.3*
4.3 ± 1.3*
21.3 ± 3.4*
6.1 ± 0.3*
27.4 ± 5.7*
12.7 ± 3.3*
Effect of protocatechuic acid on nitric oxide inhibition in RAW 264.7 macrophages using two different experimental conditions
Inhibition of NO production (%)
LPS 1 μg/mL
LPS 1 μg/mL + IFN-γ 1,000 U/mL
2.4 ± 16.9
3.7 ± 3.3
19.7 ± 7.5*
29.3 ± 4.1*
Earlier studies have shown that the anti-inflammatory action of flavonoids is mediated via their inhibition of iNOS protein and mRNA expression as well as their inhibition of nuclear factor kB (NF-kB) and STAT-1 activation, which are involved in the expression of several inflammatory genes (Chen et al. 2005; Hämäläinen et al. 1999).
We next assessed the effect of anthocyanin metabolites on TNF-α secretion. We found that exposure of macrophages to LPS led to TNF-α secretion (3.0 ± 0.1 ng TNF-α/mg protein), and that exposure to both LPS and IFN-γ together induced a synergistic effect on TNF-α secretion (11.4 ± 1.1 ng TNF-α/mg protein). Similar to the results obtained for NO production, we found that pre-treatment for LPS-stimulated cells with protocatechuic acid at concentrations higher than 10 μM caused a reduction in TNF-α secretion, with a significant inhibition at 100 μM protocatechuic acid of 39%. For cells treated with LPS + IFN-γ together, a low but significant inhibition of TNF-α secretion was observed only by protocatechuic acid at 50 and 100 μM (11.1 and 21.5%, respectively). Co-incubation with the other phenolic acids and stimulus did not affect TNF-α secretion. No effect on TNF-α was shown for any of the other polyphenols assayed, vanillic, protocatechuic, and syringic acids in our experimental conditions.
Monocyte-derived macrophages are the principal inflammatory cells in atheromas. Their activation is crucial to the progression of multiple inflammatory diseases such as septic shock, chronic inflammation, and atherosclerosis, via the release of inflammatory and cytotoxic mediators like cytokines or NO (Tamir and Tannenbaum 1996). In the present study, we have shown that exposing macrophages to LPS and IFN-γ simultaneously induces a synergistic effect in terms of NO production as well as TNF-α secretion, in accordance with other authors (Orlicek et al. 1996).
Studies in the literature investigating the effects of anthocyanins on NO production and TNF-α secretion are controversial. García-Alonso et al. (2004) did not find an effect of anthocyanins on NO production or TNF-α secretion when used to pre-treat RAW 264.7 macrophages 24 h prior incubation with LPS. In contrast, Hämäläinen et al. (1999) observed an inhibition of NO production as well as of iNOS protein and mRNA expression with pelargonidin treatment for macrophages exposed to an inflammatory stimulus (LPS). Also, Wang and Mazza (2002a) demonstrated an inhibitory effect of anthocyanins on LPS-induced NO production in macrophages.
Recently, Long et al. (2010) have shown that some flavonoids may increase the levels of hydrogen peroxide in the cell culture medium, thereby possibly affecting also some of the parameters that have been measured within the present. This increase in H2O2 in response to the flavonoid treatment may be due to a rapid degradation of some flavonoids at neutral pH and 37°C. This has been reported for anthocyanins such as delphinidin chloride, an extremely unstable compound, and may have also occurred in terms of its 3-glucoside (Avila et al. 2009). Furthermore, Long et al. (2010) demonstrated that different cell culture media may have different effects on H2O2 production for the same polyphenol test compound. Thus, compound instability and generation of H2O2 should be taken into account in interpreting effects of anthocyandins in cultured cells (Long et al. 2010).
Related to our findings, Yan et al. (2004) demonstrated that protocatechuic acid isopropyl ester reduced plasma TNF-α, NO, and hepatic malondialdehyde levels in a mouse model of septic shock induced by LPS and d-galactosamine. In our study, protocatechuic acid exhibited a protective effect in LPS/INF-γ-induced macrophages by inhibiting the overproduction of inflammatory mediators, namely NO and TNF-α.
In our work, and in accordance with other studies (Terra et al. 2007), we compared the ability of anthocyanin metabolites to inhibit NO production in macrophages using four different treatment protocols (pre-incubation with polyphenols and activation with LPS alone or LPS plus INF-γ, and co-incubation with polyphenols and LPS alone or LPS plus INF-γ). When macrophages were co-incubated simultaneously with the test compound and stimulus, protocatechuic acid was found to exhibit the strongest inhibition of NO secretion. In this case, we propose that protocatechuic acid acts by scavenging NO radicals or by a direct interaction with LPS or IFN-γ. However, the high level of inhibition of NO production and TNF-α secretion observed in cells pre-incubated with protocatechuic acid before LPS activation may be due to different mechanisms of action of flavonoids, as described above.
It needs to be considered that the concentrations of protocatechuic acid, which inhibited NO secretion in our cell culture experiments, were very high and rather not in the physiological range. Protocatechuic acid has been detected in plasma (human and rat) at concentrations that are around 200 nmol/L, which is 100-fold higher than the original anthocyanins concentration in plasma (between 1 and 10 nmol/L) (Caccetta et al. 2000) but still very much lower than the concentrations used in our cell culture experiments.
Our results suggest that foods rich in polyphenols, which may lead to elevated levels of protocatechuic acid in plasma, could be beneficial in the prevention of inflammatory diseases since they reduce the production of the cytotoxic oxidative stress mediator NO as well as the production of TNF-α, a crucial cytokine for the synergistic induction of NO synthesis.
Effect of phenolic acids on MCP-1, ICAM-1, and VCAM-1 secretion in endothelial cells
Effect of gallic acid on MCP-1, ICAM-1, and VCAM-1 secretion in Ea.hy 926
Gallic acid (μM)
96.2 ± 5.0
94.5 ± 4.4
88.0 ± 38.3
85.7 ± 3.1*
91.9 ± 0.6*
76.2 ± 13.7*
81.2 ± 2.4*
88.6 ± 5.6
50.8 ± 5.9*
78.9 ± 1.8*
79.9 ± 0.6*
41.2 ± 4.9*
Many epidemiological studies have reported that moderate wine consumption exerts a protective effect against cardiovascular diseases (Estruch 2000; Gronbaek et al. 2000). In addition, clinical studies have demonstrated that daily intake of wine reduces monocyte adhesion and circulating markers of inflammation (Badia et al. 2004; Estruch et al. 2004). Moreover, Sacanella et al. (2007) showed a more potent effect of red wine versus white wine, possibly due to its higher anthocyanin content. A suppression of NF-kB in white blood cells by red wine was suggested to play a key role in its anti-inflammatory effects (Blanco-Colio et al. 2000). In accordance with these studies, we observed a significant reduction in MCP-1, ICAM-1, and VCAM-1 levels when endothelial cells were pre-treated with gallic acid. This reduction was especially marked in the case of VCAM-1. Previous studies have reported that endothelial cells in human atherosclerotic lesions increase cell adhesion molecules (CAMs) and MCP-1. Therefore, gallic acid might be an effective protector against monocyte recruitment in inflammatory vessels and may prove useful in the prevention of atherosclerotic lesion development due, in part, to a decrease in MCP-1, which promotes monocytes infiltration into the arterial wall.
Determination of ACE-inhibitory activity
ACE-inhibitory activity of anthocyanins and phenolic acids
332.4 ± 40.1
157.3 ± 16.1
504.2 ± 31.5
83.9 ± 5.1
65.4 ± 4.0
70.8 ± 2.0
77.7 ± 2.3
104.6 ± 5.8
The in vitro ACE-inhibitory activity of flavonoids is due to the generation of chelate complexes with the zinc atom within the active center of ACE (García-Saura et al. 2005). Free hydroxyl groups of phenolic compounds are suggested to be important structural moieties to chelate the zinc ions, thus inactivating the ACE activity. It may therefore be the hydroxyl groups within the anthocyanins and phenolics acids that are responsible for their ACE-inhibitory activity. In fact, ACE-inhibitory activity has been already demonstrated in some compounds derived from plants such as flavonoids (Wille et al. 2001), terpenoids (Morigiwa et al. 1986), peptides (Kinoshita et al. 1993), and procyanidins (Wagner and Elbl 1992).
It is therefore likely that anthocyanins and their breakdown metabolites, phenolic acids, have hypotensive and protective effects on endothelial function due, at least in part, to their ACE-inhibitory effect since angiotensin II regulates arterial blood pressure, adhesion molecule expression, cytokines, chemokines, and growth factors within the arterial wall.
Relative affinity for ERα and ERβ
The first step to determine the estrogenic activity of a given compound is to measure the binding of this potential ligand to the estrogenic receptor. In general, the affinity of flavonoids to bind to ERα and β is lower than that of 17-β-estradiol. However, some studies have confirmed that genistein, daidzein, and equol have a good affinity for ER, especially for ERβ (Mueller et al. 2004).
Relative affinity of anthocyanins and phenolic acids for ERα and ERβ
ERα IC50 (μM)*
ERβ IC50 (μM)*
63.2 ± 0.8
61.3 ± 0.7
93.0 ± 0.8
64.4 ± 0.9
100.3 ± 0.9
Among the phenolic acid metabolites assayed, gallic acid showed affinity for ERß (100.3 μM ± 0.9) but did not show affinity for ERα.
It is interesting to note that delphinidin-3-glucoside and gallic acid, with similar structural features in the hydroxylation pattern of their B-ring, showed affinity for ERβ, but not for ERα. Moreover, delphinidin-3-glucoside demonstrated a binding affinity for ERβ that is approximately twofold higher than that of gallic acid. These results are in accordance with those obtained in the molecular modeling studies and could be explained by the hypotheses that two molecules of gallic acid are able to bind to the ER and display estrogenic/anti-estrogenic activities.
All other test compounds did not show any affinity for the ER at the concentrations tested.
Schmitt and Stopper (2001) have reported that anthocyanidins (the aglycones of anthocyanins) have high affinity toward ERα. They showed that pelargonidin (6.8 μM) had the highest affinity among the assayed anthocyanidins followed by delphinidin (10.4 μM) and cyanidin (12.2 μM) (Cornwell et al. 2004). In a similar way, Chalopin et al. (2010) showed that the endothelium-dependent vasorelaxation of delphinidin and a red wine extract is mediated via ERα. With respect to ERα, the presence of up to 2-OH groups in the B-ring of the molecular structure decreased the affinity of the anthocyanins to the ERα (Fang et al. 2001). In contrast, delphinidin-3-glucoside and gallic acid with 3-OH groups demonstrated the highest affinity for ERβ in our study.
Study of the binding mode on ERβ and ERα: docking studies
No binding poses were found for malvidin and cyanidin, in agreement with the absence of affinity, suggesting that these compounds are not suitable ER ligands.
Our results suggest that gallic acid could potentially be considered as an enterophytoestrogen; a gut microflora-derived metabolite that can exhibit higher estrogenic/anti-estrogenic activity than its corresponding precursor.
Nevertheless, the fact that these polyphenols demonstrate affinity for the estrogen receptors and display agonist/antagonist effects may suggest that these compounds could act in target genes and in tissues where they could collaborate in the health-promoting properties of anthocyanins.
The mechanisms underlying the antiatherogenic effect of anthocyanins consumption is probably multifactorial. Our study suggests that the protective health effects of anthocyanins might not only be due to anthocyanins themselves, but also to their metabolites produced by the action of the gut microflora. Both anthocyanins and their phenolic acid metabolites might play a role in decreasing vascular inflammatory markers, such as cytokines and adhesion and chemoattractant molecules. Despite observing a slight modulation of NO production by protocatechuic acid, the concentrations required to produce this effect do not fit with this being a potential mechanism of action for the antiatherogenic properties of anthocyanins. It might be the ability of anthocyanins and three of their metabolites to inhibit ACE activity, which could decrease the expression of inflammatory markers and therefore improve endothelial function. On the other hand, some of the assayed metabolites have shown a relatively important affinity for ER α and β, which regulate transcription of target genes, such as NF-kB. This affinity could also be implicated in atheromatosis. Overall, the concentrations of anthocyanins and their metabolites, as used in the present cell culture and in vitro assays mediating anti-inflammatory, anti-adhesive, anti-estrogenic, and angiotensin-converting enzyme inhibitory activities were often manifold higher than those physiologically achievable (Vitaglione et al. 2007). Further research, preferably in vivo, is necessary to determine whether, and to what extent, anthocyanin metabolites play a role in the prevention of atherosclerosis in humans.
This work was supported by the Spanish Ministry of Science and Innovation through projects AGL2006-05453, AGL2008-01713, AGL2009-07894, SAF2008-00945, and the Consolider-Ingenio 2010 Programme (FUN-C-FOOD, CSD2007-00063). M. Hidalgo wishes to thank CSIC, for a JAE predoctoral fellowship (JAEPre094).
- Actis-Goretta L, Ottaviani JI, Fraga CG (2006) Inhibition of angiotensin converting enzyme activity by flavanol-rich foods. J Agric Food Chem 54:229–234PubMedView ArticleGoogle Scholar
- Avila M, Hidalgo M, Sánchez-Moreno C, Pelaez C, Requena T, de Pascual-Teresa S (2009) Bioconversion of anthocyanin glycosides by Bifidobacteria and Lactobacillus. Food Res Int 42:1453–1461View ArticleGoogle Scholar
- Badia E, Sacanella E, Fernandez-Solá J, Nicolás JM et al (2004) Decreased tumor necrosis factor-induced adhesion of human monocytes to endothelial cell after moderate alcohol consumption. Am J Clin Nutr 80:225–230PubMedGoogle Scholar
- Blanco-Colio LM, Valderrama M, Alvarez-Sala LA, Bustos C et al (2000) Red wine intake prevents nuclear factor-kB activation in peripheral blood mononuclear cells of healthy volunteers during postprandial lipemia. Circulation 102:1020–1026PubMedGoogle Scholar
- Caccetta RA, Croft KD, Beilin LJ, Puddey IB (2000) Ingestion of red wine significantly increases plasma phenolic acid concentrations but does not acutely affect ex vivo lipoprotein oxidizability. Am J Clin Nutr 71:67–74PubMedGoogle Scholar
- Cassidy A, de Pascual-Teresa S, Rimbach G (2003) Molecular mechanisms by which dietary isoflavones potentially prevent atherosclerosis. Expert Rev Mol 30:1–15Google Scholar
- Chalopin M, Tesse A, Martínez MC, Rognan D, Arnal JF, Andriantsitohaina R (2010) Estrogen receptor alpha as a key target of red wine polyphenols action on the endothelium. PLoS One 5:e8554PubMedView ArticleGoogle Scholar
- Chen JC, Ho FM, Chao P-DL, Chen C-P et al (2005) Inhibition of iNOS gene expression by quercetin is mediated by the inhibition of IκB kinase, nuclear factor-κB and STAT1, and depends on heme oxygenase-1 induction in mouse BV-2 microglia. Eur J Pharmacol 521:9–20PubMedView ArticleGoogle Scholar
- Chun OK, Chung SJ, Song WO (2007) Estimated dietary flavonoid intake and major food sources of U.S. adults. J Nutr 37:1244–1252Google Scholar
- Cornwell T, Cohick W, Raskin I (2004) Dietary phytoestrogen and health. Rev Phytochem 65:995–1016View ArticleGoogle Scholar
- Dang ZC, Lowit C (2005) Dose-dependent effects of phytoestrogens on bone. Trends Endocrinol Metab 16:207–213PubMedView ArticleGoogle Scholar
- Estruch R (2000) Wine and cardiovascular disease. Food Res Int 33:219–226View ArticleGoogle Scholar
- Estruch R, Sacanella E, Badia E, Antúnez E et al (2004) Different effects of red wine and gin consumption on inflammatory biomarkers of atherosclerosis: a pro-spective randomized crossover trial. Effects of wine on inflammatory markers. Atherosclerosis 175:117–123PubMedView ArticleGoogle Scholar
- Fang H, Tong W, Leming MS, Blair R et al (2001) Structure-activity relationships for large diverse set of natural, synthetic, and environmental estrogens. Chem Res Toxicol 14:280–294PubMedView ArticleGoogle Scholar
- Forester SC, Waterhouse AL (2008) Identification of Cabernet Sauvignon anthocyanin gut microflora metabolites. J Agric Food Chem 56:9299–9304PubMedView ArticleGoogle Scholar
- Frankel EN, Waterhouse AL, Teissedre PL (1995) Principal phenolic phytochemicals in selected California wines and their antioxidant activity in inhibiting oxidation of human low-density lipoproteins. J Agric Food Chem 43:890–894View ArticleGoogle Scholar
- Frisch MJ, Trucks GW, Schlegel HB, Scuseria GE et al (2004) Gaussian 03, revision C.02. Gaussian Inc., WallingfordGoogle Scholar
- Fuchs D, Erhard P, Turner R, Rimbach G, Daniel H, Wenzel U (2005) Genistein reverses changes of the proteome induced by oxidized-LDL in EA.hy 926 human endothelial cells. J Proteome Res 4:369–376PubMedView ArticleGoogle Scholar
- García-Alonso M, Rimbach G, Rivas-Gonzalo JC, de Pascual-Teresa S (2004) Antioxidant and cellular activity of anthocyanidins and their corresponding vitisins A—studies in platelets, monocytes and human endothelial cells. J Agric Food Chem 52:3378–3384PubMedView ArticleGoogle Scholar
- García-Alonso M, Minihane AM, Rimbach G, Rivas-Gonzalo JC, de Pascual-Teresa S (2009) Red wine anthocyanins are rapidly absorbed in humans and affect monocyte chemoattractant protein 1 levels and antioxidant capacity of plasma. J Nutr Biochem 20:521–529PubMedView ArticleGoogle Scholar
- García-Saura MF, Galisteo M, Villar IC, Bermejo A et al (2005) Effects of chronic quercetin treatment in experimental renovascular hypertension. Mol Cell Biochem 270:147–155PubMedView ArticleGoogle Scholar
- González-Gallego J, García-Mediavilla MV, Sánchez-Campos S, Tuñón MJ (2010) Fruit polyphenols, immunity and inflammation. Br J Nutr 104:S15–S27PubMedView ArticleGoogle Scholar
- Gray JI, DeWitt DL (1999) Antioxidant and antiinflammatory activities of anthocyanins and their aglycon, cyaniding, from tart cherries. J Nat Prod 62:294–296PubMedView ArticleGoogle Scholar
- Gronbaek M, Becker U, Johansen D, Gottschau A et al (2000) Type of alcohol consumed and mortality from all causes, coronary heart disease and cancer. Ann Intern Med 133:411–419PubMedGoogle Scholar
- Hämäläinen M, Nieminen R, Vuorela P, Heinonen M, Moilanen E (1999) Anti-Inflammatory effects of flavonoids: genistein, kaempferol, quercetin, and daidzein inhibit STAT-1 and NF-κB activations, whereas flavone, isorhamnetin, naringenin, and pelargonidin inhibit only NF-κB activation along with their inhibitory effect on iNOS expression and NO production in activated macrophages. Med Inflamm 2007:1–10Google Scholar
- Kahkonen MP, Heinonen M (2003) Antioxidant activity of anthocyanins and their aglycons. J Agric Food Chem 51:628–633PubMedView ArticleGoogle Scholar
- Keppler K, Humpf HU (2005) Metabolism of anthocyanis and their phenolic degradation products by the intestinal microflora. Bioorg Med Chem 13:5195–5205PubMedView ArticleGoogle Scholar
- Kinoshita E, Yamakoshi J, Kikuchi M (1993) Purification and identification of an angiotensin converting enzyme from soy sauce. Biosci Biotechno Biochem 57:1107–1110View ArticleGoogle Scholar
- Kris-Etherton PM, Hecker KD, Bonanome A, Coval SM et al (2002) Bioactive compounds in foods: their role in the prevention of cardiovascular disease and cancer. Am J Med 113:71S–88SPubMedView ArticleGoogle Scholar
- Kwon HM, Choi YJ, Jeong YJ, Kang SW et al (2005) Anti-inflammatory inhibition of endothelial cell adhesion molecule expression by flavone derivatives. J Agric Food Chem 53:5150–5157PubMedView ArticleGoogle Scholar
- Lacaille-Dubois MA, Franck U, Wagner H (2001) Search for potential angiotensin converting enzyme (ACE)-inhibitors from plants. Phytomedicine 8:47–52PubMedView ArticleGoogle Scholar
- Landberg R, Sun Q, Rimm EB, Cassidy A et al (2011) Selected dietary flavonoids are associated with markers of inflammation and endothelial dysfunction in U.S. women1,2. J Nutr 141:618–625PubMedView ArticleGoogle Scholar
- Long LH, Hoi A, Halliwell B (2010) Instability of, and generation of hydrogen peroxide by, phenolic compounds in cell culture media. Arch Biochem Biophys 501:162–169PubMedView ArticleGoogle Scholar
- Lotito SB, Frei BD (2006) Dietary flavonoids attenuate tumor necrosis factor α-induced adhesion molecule expression in human aortic endothelial cells. J Biol Chem 281:37102–37110PubMedView ArticleGoogle Scholar
- Manach C, Williamson G, Morand C, Scalbert A, Remesy C (2005) Bioavailability and bioefficacy of polyphenols in humans. I. Review of 97 bioavailability studies. Am J Clin Nutr 81:230–242Google Scholar
- Matsumoto H, Nakamura Y, Hirayama M, Yoshiki Y, Okubo K (2002) Antioxidant activity of black currant anthocyanin aglycons and their glycosides measured by chemiluminescence in a neutral pH region and in human plasma. J Agric Food Chem 50:5034–5037PubMedView ArticleGoogle Scholar
- McCann MJ, Gill CI, McGlynn H, Rowland IR (2005) Role of the mammalian lignans in the prevention and treatment of prostate cancer. Nutr Cancer 52:1–14PubMedView ArticleGoogle Scholar
- Morigiwa A, Kitabatake K, Fujimoto Y, Ikekawa N (1986) Angiotensin converting enzyme inhibitory triterpenes from Ganoderma lucidum. Chem Pharm Bull 34:3025–3028PubMedView ArticleGoogle Scholar
- Morris GM, Goodsell DS, Halliday RS, Huey R et al (1998) Automated docking using a Lamarckian genetic algorithm and empirical binding free energy function. J Comput Chem 19:1639–1662View ArticleGoogle Scholar
- Mueller SO, Simon S, Chae K, Metzler M, Korach KS (2004) Phytoestrogens and their human metabolites show distinct agonistic and antagonistic properties on estrogen receptor α (ERα) and ERβ in human cells. Toxicol Sci 80:14–25PubMedView ArticleGoogle Scholar
- Naruszewicz M, Laniewska I, Millo B, Dluzniewski M (2007) Combination therapy of statin with flavonoids rich extract from chokeberry fruits enhanced reduction in cardiovascular risk markers in patients after myocardial infraction (MI). Atherosclerosis 194:179–184View ArticleGoogle Scholar
- Orlicek SL, Meals E, English BK (1996) Differential effects of tyrosine kinase inhibitors on tumor necrosis factor and nitric oxide production by murine macrophages. J Infect Dis 174:638–642PubMedView ArticleGoogle Scholar
- Park YC, Rimbach G, Saliou C, Valacchi G, Packer L (2000) Activity of monomeric, dimeric, and trimeric flavonoids on NO production, TNF-alpha secretion, and NF-kappa B-dependent gene expression in RAW 264.7 macrophages. FEBS Lett 465:93–97PubMedView ArticleGoogle Scholar
- Pérez-Jimenez J, Fezeu L, Touvier M, Amault N et al (2011) Dietary intake of 337 polyphenols in French adults. Am J Clin Nutr 93:1–9View ArticleGoogle Scholar
- Persson IA, Persson K, Andersson RG (2009) Effect of Vaccinium myrtillus and its polyphenols on angiotensin-converting enzyme activity in human endothelial cells. J Agric Food Chem 57:4626–4629PubMedView ArticleGoogle Scholar
- Quirós A, Ramos M, Muguerza B, Delgado MA et al (2007) Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis. Int Dairy J 17:33–41View ArticleGoogle Scholar
- Ricketts M-L, Moore DD, Banz WJ, Mezei O, Shay NF (2005) Molecular mechanism of action of the soy isoflavonas includes activation of promiscuous nuclear receptor. Rev J Nutr Biochem 16:321–330View ArticleGoogle Scholar
- Rimbach G, Valacchi G, Canali R, Virgili F (2000) Macrophages stimulated with INF-gamma activate NF-KappaB and induce MCP-1 gene expression in primary human endothelial cells. Mol Cell Biol Res Commun 3:238–242PubMedView ArticleGoogle Scholar
- Rotelli AE, Guardia T, Juárez AO, de la Rocha NE, Pelzer LE (2003) Comparative study of flavonoids in experimental models of inflammation. Pharmacol Res 48:601–606PubMedView ArticleGoogle Scholar
- Sacanella E, Vázquez-Agell M, Mena MP, Antúnez E et al (2007) Down-regulation of adhesion molecules and other inflammatory biomarkers after moderate wine consumption in healthy women: a randomized trial. Am J Clin Nutr 86:1463–1469PubMedGoogle Scholar
- Schmitt E, Stopper H (2001) Estrogenic activity of naturally occurring anthocyanidins. Nutr Cancer 41:145–149PubMedGoogle Scholar
- Sentandreu MA, Toldrá F (2006) A rapid, simple and sensitive fluorescence method for the assay of angiotensin-I converting enzyme. Food Chem 97:546–554View ArticleGoogle Scholar
- Shahrzad S, Bitsch I (1998) Pharmacokinetics of gallic acid and its relative bioavailability from tea in healthy humans. J Chromatogr B Biomed Sci 705:87–95View ArticleGoogle Scholar
- Shahrzad S, Aoyagi K, Winter A, Koyama A, Bitsch I (2001) Pharmacokinetics of gallic acid and its relative bioavailability from tea in healthy humans. J Nutr 131:1207–1210PubMedGoogle Scholar
- Tamir S, Tannenbaum SR (1996) The role of nitric oxide (NO) in the carcinogenic process. Biochim Biophys Acta 1288:F31–F36PubMedGoogle Scholar
- Terra X, Valls J, Vitrac X et al (2007) Grape-seed procyanidins act as antiinflammatory agents in endotoxin-stimulated RAW 264.7 Macrophages by inhibiting NFkB signaling pathway. J Agric Food Chem 55:4357–4365PubMedView ArticleGoogle Scholar
- Tsuda T, Watanabe M, Ohshima K, Norinobu S et al (1994) Antioxidative activity of the anthocyanin pigments cyanidin 3-O-d-glucoside and cyanidin. J Agric Food Chem 42:2407–2410View ArticleGoogle Scholar
- Tsuda T, Shiga K, Ohshima K, Kawakishi S, Osawa T (1996) Inhibition of lipid peroxidation and the active oxygen radical scavenging effect of anthocyanin pigments isolated from Phaseolus vulgaris L. Biochem Pharma 52:1033–1039View ArticleGoogle Scholar
- Valacchi G, Rimbach G, Saliou C, Weber SU, Packer L (2001) Effect of benzoyl peroxide on antioxidant status, NF-KappaB activity and interleukinalpha gene expression in human keratinocytes. Toxicology 165:225–234PubMedView ArticleGoogle Scholar
- Vallance P, Leiper J (2002) Blocking NO synthesis: how, where and why? Nat Rev Drug Discov 1:939–950PubMedView ArticleGoogle Scholar
- Virgili F, Marino M (2008) Regulation of cellular signals from nutritional molecules: a specific role for phytochemicals, beyond antioxidant activity. Free Radic Biol Med 45:1205–1216PubMedView ArticleGoogle Scholar
- Vitaglione P, Donnarumma G, Napolitano A, Galvano F et al (2007) Protocatechuic acid is the major human metabolite of cyanidin-glucosides. J Nutr 137:2043–2048PubMedGoogle Scholar
- Wagner H, Elbl G (1992) ACE-inhibitory procyanidins from Lespedeza capitata. Planta Med 58:297PubMedView ArticleGoogle Scholar
- Wang J, Mazza G (2002a) Inhibitory effects of anthocyanins and other phenolic compounds on nitric oxide production in LPS/IFN-α-activated RAW 264.7 macrophages. J Agric Food Chem 50:850–857PubMedView ArticleGoogle Scholar
- Wang J, Mazza G (2002b) Quantification of nitrite in the presence of anthocyanins using Griess and GC/Ms assays. J Food Biochem 26:339–354View ArticleGoogle Scholar
- Wang H, Cao G, Prior RL (1997) Oxygen radical absorbing capacity of anthocyanins. J Agric Food Chem 45:304–309View ArticleGoogle Scholar
- Wille PR, Ribeiro-do-Valle RM, Simoes CM, Gabilan NH, Nicolau M (2001) Effect of quercetin on tachykinin-induced plasma extravasation in rat urinary bladder. Phytother Res 15:444–446PubMedView ArticleGoogle Scholar
- Wu XL, Beecher GR, Holden JM, Haytowitz DB et al (2006) Concentrations of anthocyanins in common foods in the United States and estimation of normal consumption. J Agric Food Chem 54:4069–4075PubMedView ArticleGoogle Scholar
- Yan J-J, Jung J-S, Hong Y-J, Moon YS et al (2004) Protective effect of protocatechuic acid isopropyl ester against murine models of sepsis: inhibition of TNF-α and nitric oxide production and augmentation of IL-10. Biol Pharmacol Bull 27:2024–2027View ArticleGoogle Scholar