|Year : 2013 | Volume
| Issue : 1 | Page : 17-23
Red wine, white wine, rosé wine, and grape juice inhibit angiotensin-converting enzyme in human endothelial cells
Department of Medical and Health Sciences, Division of Drug Research/Pharmacology, Faculty of Health Sciences, Linköping University, Sweden
|Date of Submission||17-Apr-2012|
|Date of Acceptance||22-Jun-2012|
|Date of Web Publication||6-Feb-2013|
Department of Medical and Health Sciences, Division of Drug Research/Pharmacology, Faculty of Health Sciences, Linköping University, SE-581 85 Linköping
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Background: Beneficial effects of wine on cardiovascular disease have been shown previously, but the mechanism is still unknown. The renin-angiotensin system is an important mechanism in the body concerning regulation of blood pressure, fluid, and electrolyte balance, and the angiotensin-converting enzyme (ACE) is a key enzyme in this system. Aims: The aim of this study was to investigate the effect of red wine, white wine, rosι wine, and alcohol-free grape juice on somatic ACE-1 activity. The effects of the stilbene resveratrol and its glycoside, resveratrol-3-glycoside were also tested on ACE activity and concentration of nitric oxide (NO). Materials and Methods: Cultured endothelial cells from human umbilical veins (HUVEC) were incubated with wine, grape juice, resveratrol, or resveratrol-3-glycoside. Ethanol was used as control in the corresponding concentration (13%). Results: After incubation, a significant inhibition of ACE activity was seen with all the wines tested and the red grape juice. This inhibition was of a similar magnitude except for a lesser inhibition with the rosι wine. No significant inhibition was seen with the white grape juice, resveratrol, resveratrol-3-glycoside, or ethanol alone, and neither did resveratrol nor resveratrol-3-glycoside affect the concentration of NO. Conclusions: The effect of wine and grape juice on ACE activity in HUVEC is dependent on the amount of flavonoids and not on the content of alcohol or resveratrol.
Keywords: ACE, cardiovascular disease, ethanol, grape juice, resveratrol, wine
|How to cite this article:|
Persson I. Red wine, white wine, rosé wine, and grape juice inhibit angiotensin-converting enzyme in human endothelial cells. Int J Nutr Pharmacol Neurol Dis 2013;3:17-23
|How to cite this URL:|
Persson I. Red wine, white wine, rosé wine, and grape juice inhibit angiotensin-converting enzyme in human endothelial cells. Int J Nutr Pharmacol Neurol Dis [serial online] 2013 [cited 2021 Feb 27];3:17-23. Available from: https://www.ijnpnd.com/text.asp?2013/3/1/17/106975
| Background|| |
Numerous reports show the beneficial effects of wine on cardiovascular disease. Meta-analyses have come to the conclusion that a low to moderate intake of wine, 1-2 drinks per day, lowers the risk of cardiovascular disease by approximately 20-25%.  The cardiovascular protection by wine may be attributed to the polyphenol content present in grapes, for example, flavonoids, that is, catechins, flavonols, procyanidins and anthocyanidins, and the flavonoid-related substance stilbenes, that is, resveratrol and resveratrol-3-glycoside (piceid). , Beneficial effects of resveratrol on cardiovascular disease are inhibition of low-density lipoprotein (LDL) peroxidation, antioxidative effects, inhibition of platelet aggregation, anti-inflammatory activity, and vasorelaxing activity. 
Epidemiologic studies have indicated that the intake of alcohol has beneficial effects on cardiovascular disease. ,, However, it is important to bear in mind that large amounts of alcohol can also increase cardiovascular disease.
Vitis vinifera L. (Vitaceae), grapevine, is the most common species of the genus Vitis used for direct consumption of grapes and/or for producing grape juices, alcoholic beverages, and raisins. Other Vitis spp. are also used and also other related species such as the Muscadinia spp.
The color of the grape juice or wine depends on the specific pigments of the plant, that is, flavonoids of the grape used, green, red, or purple.
The flavonoid-related substances as stilbenes, the aglycone resveratrol and the resveratrol-3-glycoside are found primarily in the skin of grapes. Therefore, these substances are in higher amounts in wines and grape juices where the skin is present during manufacturing, that is, in red-coloured products. The amount of resveratrol and its glycosides also vary depending on the species of the grapes and the country of cultivation.  The beneficial properties of the flavonoids and resveratrol on cardiovascular disease are usually associated with their antioxidative effect. However, this is probably not the only mechanism. The renin-angiotensin system (RAS) is highly involved in cardiovascular disease, regulating blood pressure and water balance in the body. Angiotensin-converting enzyme (ACE) is synthesized by the endothelial cells and is a key enzyme in RAS, converting the inactive angiotensin I to form the active angiotensin II, which is the substance responsible for activating the angiotensin receptors involved in cardiovascular disease [Figure 1]. ACE inhibitors are the first line treatment of hypertension and heart disease. Previous studies have shown that a diversity of flavonoids inhibit ACE in vitro as well as in vivo. ,,,
|Figure 1: Schematic overview of the renin-angiotensin system showing endothelial cells synthesizing the angiotensin-converting enzyme (ACE) and nitric oxide (NO). Inhibition of ACE and upregulation of NO result in vasodilatation, inhibit proliferation of smooth muscle cells, reduce reactive oxygen species, and inhibit platelet aggregation|
Click here to view
RAS has a major influence on the circulation and there are several interactions known between RAS and nitric oxide (NO).  NO from endothelial cells relaxes the vascular smooth muscle and inhibits platelet aggregation, and a reduction of NO is believed to develop cardiovascular disease. As antioxidants may increase NO, it has been suggested that the beneficial effects of wine and resveratrol on cardiovascular disease is due to the increase in NO. ,
| Aim|| |
As the complete mechanism of wine and its cardiovascular benefits is not completely understood, the aim of this study was to investigate the effect on somatic ACE-1 activity. Red wine, white wine, and rosι wine were tested and compared to red and white grape juice without alcohol, to determine the importance of alcohol on ACE activity. Effects of resveratrol and the resveratrol-3-glycoside piceid on ACE activity and concentration of NO were also tested.
| Results|| |
ACE activity in HUVEC
After incubation with wine, all of the wines tested showed a significant inhibition of ACE activity in cultured endothelial cells from human umbilical veins (HUVEC); Bourgogne Chardonnay (wine no 1): P <0.001for the concentrations 5 and 10%, MasiCampofiorin (wine no. 2): P <0.001 for all concentrations tested, Dolce Vita Monica (wine no 3): P <0.01 for the concentrations 2 and 5% and P <0.001 for the concentrations 5 and 10%, Roodeberg (wine no 4): P <0.001 for the concentrations 5 and 10%, and Leonardo Rosato (wine no 5): P <0.01 for the concentration 10%, compared to 1.3% ethanol control [Figure 2].
|Figure 2: ACE activity in HUVEC after incubation for 10 minutes with wine. As control, 1.3% ethanol was used. The wine concentrations used were v/v % 2.5%, 5%, and 10%. All concentrations were final concentrations. The wines were Bourgogne Chardonnay (1), MasiCampofiorin (2), Dolce Vita Monica (3), Roodeberg (4) or Leonardo Rosato (5), n =6. **P < 0.01 and ***P < 0.001|
Click here to view
After incubating the cells with grape juice, a significant inhibition of ACE activity in HUVEC was seen with the Carbernet Sauvignon juice (no. 6) P <0.001 for the concentrations 5% and 10%, whereas no significant inhibition was seen with the Chardonnay juice (no 7) compared to the phosphate-buffered saline (PBS) control [Figure 3].
|Figure 3: ACE activity in HUVEC after incubation for 10 minutes with grape juice. The juices were Cabernet Sauvignon (6) or Chardonnay (7), n =6. ***P <0.001|
Click here to view
Compared to the dimethylsulfoxide (DMSO) control, resveratrol and resveratrol-3-glycoside did not show any significant effect on ACE activity in HUVEC [Figure 4] in any of the concentrations used.
|Figure 4: ACE activity in HUVEC after incubation for 10 minutes with resveratrol (R) or resveratrol-3-glycoside (R-3-G), n =6|
Click here to view
Concentration of NO in HUVEC
No significant change was seen in the concentration of NO after incubation for 24 hours with resveratrol or resveratrol-3-glycoside, compared to the DMSO control [Figure 5].
|Figure 5: NO concentration in HUVEC after incubation for 24 hours with resveratrol (R) or resveratrol-3-glycoside (R-3-G), n =6|
Click here to view
Cell viability/quality was performed with MTS [3-(4,5-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfonyl)-2H-tetrazolium] after exposure to wine at a concentration of 10%, grape juice 10%, resveratrol and resveratrol-3-glycoside 1 mg/mL, ethanol 1.3%, PBS, and DMSO [Table 1].
|Table 1: Cell viability/quality after exposure to the different wines (10%), grape juices (10%), resveratrol (1 mg/mL), resveratrol-3-glycoside (1 mg/mL), and controls (ethanol 1.3%, PBS, and DMSO)|
Click here to view
| Discussion|| |
This study shows that all the wines tested, irrespective of the color, red, white, or rosι [Figure 2], and the alcohol-free red grape juice [Figure 3] significantly inhibited ACE activity in HUVEC. The beneficial effect of wine on cardiovascular disease is often associated with red wine although there are reports on white wine having the same effect on cardiovascular disease. , These statements are confirmed by the results of this study.
Grapes are usually divided into red and white grapes. White grapes are green in color and derived evolutionarily from red grapes by mutations in two regulatory genes turning off the production of anthocyanins.  Anthocyanins are the main flavonoids found in red and purple grapes. , Flavan-3-ols, that is, catechin, epicatechin, epicatechin gallate, epigallocatechin gallate, and oligomeric and polymeric procyanidins are the most common flavonoids in the seeds of both white and red grapes. , Previous studies have shown that anthocyanidins, catechins, and procyanidins inhibit ACE activity in vitro as well as in vivo.,,, According to this, both red and white wine are expected to inhibit ACE activity, which is confirmed in the present study.
The flavonoid content in grapes varies according to the species, soil, climate, and geographic origin. The variation in ACE inhibition seen in this study is suggested to be due to these factors.
However, there also seems to be some variation in the inhibiting effect according to the type of wine, and the most conspicuous is the inferior inhibiting effect of rosι wine (wine no. 5). The color and, thereby, the content of flavonoids in rosι wine is dependent on the grapes, the wine, and the technique used when making the wine. The rosι wine tested is manufactured with red grapes and the color is dependent on how long the skin of the grapes is allowed to remain in contact with the juice. The amount of anthocyanidins/flavonoids in rosι wine, compared to red wine, is approximately 10% with variations depending on the type of grape and manufacturing technique.  Only red wine is fermented with skin during the entire manufacturing process, resulting in a higher amount of flavonoids compared to rosι and white wine.
The alcohol-free Cabernet Sauvignon grape juice (no. 6) inhibited ACE significantly, whereas the white alcohol-free alternative, Chardonnay grape juice (no. 7) showed no inhibiting effect on ACE. However, the Chardonnay wine (no. 1) showed a significant inhibition of ACE activity. The Chardonnay wine derives from France, whereas the Chardonnay grapes for producing the juice derive from Spain. The difference between the white wine and the white grape juice is probably due to the content of flavonoids and manufacturing technique.
Comparing the ethanol control and the PBS control, no significant effect was seen on ACE activity; neither of them significantly inhibited ACE activity. It is interesting to see that the Cabernet Sauvignon grape juice (no. 6) significantly inhibited ACE activity to the same degree as red wines (P <0.001) (nos. 2-4), and like the grape juice, the Roodeberg (wine no. 4) is made from Carbernet Sauvignon grapes. The conclusion of this study seems to be that the effects of wine and grape juice on ACE activity are in accordance with content of flavonoids, that is, anthocyanidins, procyanidins, and catechins, and not with the alcohol content. However, if alcohol extracts higher amounts of flavonoids from the grapes, an indirect effect of wine compared to grapes/grape juice may be seen. At the same time, it is important to note that alcohol in large quantities increases the risk of cardiovascular disease.
It could also be researched if the consumption of grapes as raisins or grapes can inhibit ACE activity and thereby prevent cardiovascular disease and also if the long-term storage of alcohol beverages containing flavonoids promotes forming of portisins, which are more potent ACE inhibitors. It has previously been shown that red wine vinegar beverage inhibits ACE activity in rats.
Resveratrol and its 3-glycoside, often called piceid are stilbenes, a group of flavonoid-related substances occurring in grapes and wines. The amount of resveratrol-3-glycoside in grapes is ten time higher than aglycone. ,
Although resveratrol and its glycoside were tested at high concentrations, 1 mg/mL compared to approximately 0.3 and 3 mg/L in wine, these substances did not inhibit ACE activity significantly in this study and this result is in accordance with Olszanecki et al, (2008).  Even if there seems to be a tendency to inhibit ACE activity with the highest dose of resveratrol and resveratrol-3-glycoside [Figure 4], this dose is high compared to the concentration in wine.  In addition, resveratrol is also metabolized by sulfation and glucuronidation, and very small amounts of free resveratrol are found in the blood plasma even after large oral doses. ,
The effect of resveratrol on cardiovascular disease is hypothesized to be antioxidative by increasing the concentrationof NO,, but this study showed no effect on NO concentration with resveratrol or resveratrol-3-glycoside.
| Conclusion|| |
In conclusion, red wine, white wine, rosι wine, and alcohol-free red grape juice inhibit ACE activity in HUVEC. No effect was seen with resveratrol or the resveratrol-3-glycoside piceid either on ACE activity or on the concentration of NO. It seems that the effect on ACE activity by wine and grape juice is dependent on the amount of flavonoids and not on the content of alcohol or resveratrol.
| Methodology|| |
The study on cultured endothelial cells from human umbilical veins (HUVEC) was approved by the Regional Ethics Committee at the Faculty of Health Sciences, Linkφping, Sweden (Dnr 03-602) and performed after informed consent from the mothers.
Cultured endothelial cells from HUVEC
The isolation and culture of endothelial cells from the vein of human umbilical cords have previously been described by Persson et al, (2006).  These cells express the somatic ACE-1 isoform. The cells from HUVEC were seeded in a 96-well microtiter plate, and at confluence, the medium was removed and replaced with medium without fetal calf serum (FCS) to avoid discrepancies in results due to ACE present in the serum. The cultured cells were treated with five different wines [Table 2], two different grape juices [Table 2], resveratrol, or resveratrol-3-glycoside. A corresponding volume of ethanol 13%, at a final concentration of 1.3% in the wells (for the wines), sterile PBS (for the juices), and DMSO (for resveratrol and resveratrol-3-glycoside) were used as controls. After incubation for 10 minutes with the different solutions at 1:1, 1:2, and 1:4 of wine or grape juice, final concentrations of 2.5, 5, and 10% in the wells, and 0.1, 0.5, and 1 mg/mL of resveratrol or resveratrol-3-glycoside, ACE activity was analyzed, as described below.
After incubation for 24 hours with resveratrol or resveratrol-3-glycoside, the medium was removed and stored at –20°C until nitrite analysis.
ACE activity in HUVEC
After incubating the cells with wine or grape juice at concentrations of 2.5, 5, and 10% and 0.1, 0.5, and 1 mg/mL of resveratrol or resveratrol-3-glycoside, ACE activity was analyzed with a commercial radioenzymatic assay (ACE-direct REA, Bόhlmann Laboratories, Allschwil, Switzerland) as previously described by Persson et al, (2006). Blank, standard, or tested substances were added to the wells with corresponding volumes of medium without FCS. After incubation for 10 minutes, the synthetic substrate 3 H-hippuryl-glycyl-glycine was added directly to all wells of the microtiter plate. Cells were incubated with the substrate for two hours at 37°C to allow for the ACE present to cleave the substrate to 3 H-hippuric acid. Then, 150 μl of medium plus substrate was transferred from each well into scintillation vials containing 50 μl 1 M HCl (to stop the enzymatic reaction). Scintillation liquid was added and the samples were counted in a scintillation counter (1217 Rack Beta Liquid Scintillation Counter from LKB Wallac, Turku, Finland). Inter and intra-assay coefficients of variation were below 5.6%.
Concentration of NO in HUVEC
Nitrite was analyzed as a marker of NO concentration as previously described by Persson et al, (2006).  Nitrite concentration was analyzed with a commercial nitrite/nitrate assay, nitric oxide (NO 2 /NO 3 ) assay kit obtained from R&D Systems, United Kingdom. The colorimetric detection of nitrite was determined with the Griess reaction; diazonium ions are produced when acidified nitrite reacts with sulfanilic acid and these diazonium ions form chromophore agents when reacting with N-(1-naphthyl) ethylenediamine. Optical density was determined with a spectramax reader at 540 nm (Molecular Devices, Wokingham, UK). Inter and intra-assay coefficients of variation were below 4.6%.
All chemicals for culturing cells were obtained from Life Technologies, Scotland, United Kingdom, except endothelial cell growth factor bought at Boehringer-Mannheim, Germany and heparin (Heparin LEO R ) from LEO Pharma AB, Malmφ, Sweden.
The wines [Table 2] were bought at Systembolaget, the Swedish Alcohol Retail Monopoly, Linkφping, Sweden.
The grape juice [Table 2] was manufactured and bought at Brunneby Musteri, Borensberg, Sweden.
Resveratrol (3,4',5-trihydroxystilbene) and resveratrol-3-glycoside (3,4',5-trihydroxystilbene-3-O-beta-D-glycopyranoside) were bought from Extrasynthese, Genay, France.
The CellTiter 96 AQ uesous One Solution Cell Proliferation Assay (Promega, Madison, WI, USA) and the colorimetric assay MTS were used to investigate viability/quality of the cells after incubating HUVEC with wine, grape juice, resveratrol, or resveratrol-3-glycoside.
One unit (U) of ACE activity was defined as the amount of ACE required to release 1 μmol hippuric acid min–1 and L–1 . Graph Pad Prism 5.0 was used for the statistical calculations. One-way analysis of variance (ANOVA) for repeated measures was performed followed by Dunnett's post test. The results are presented as mean ± s.e.m (standard error of the mean). Statistical significance is denoted as **P <0.01 and ***P <0.001.
| References|| |
|1.||Collins MA, Neafsey EJ, Mukamal KJ, Gray MO, Parks DA, Das DK, et al. Alcohol in moderation, cardioprotection and neuroprotection: Epidemiological considerations and mechanistic studies. Alcohol Clin Exp Res 2009;33:206-9. |
|2.||Das DK, Maulik N. Resveratrol in cardioprotection: A therapeutic promise of alternative medicine. Mol Interv 2006;6:36-47. |
|3.||Leifert WR, Abeywardena MY. Cardioprotective actions of grape polyphenols. Nutr Res 2008;28:729-37. |
|4.||Frémont L. Biological effects of resveratrol. Life Sci 2000;66:663-73. |
|5.||Alcohol and wine in health and disease. Proceedings of a conference. Palo Alto, California, USA. April 26-29, 2001. Ann N Y Acad Sci 2002;957:ix-xiii, 1-350. No abstract available. |
|6.||Korthuis RJ. Introduction to the special topics issue on alcohol and cardioprotection. Pathophysiology 2004;10:81-2. |
|7.||Shigematsu S, Ishida S, Hara M, Takahashi N, Yoshimatsu H, Sakata T, et al. Resveratrol, a red wine constituent polyphenol, prevents superoxide-dependent inflammatory responses induced by ischemia/reperfusion, platelet-activating factor, or oxidants. Free Radical Biology and Medicin 2003;34:810-7. |
|8.||Persson IA, Josefsson M, Persson K, Andersson RG. Tea flavanols inhibit angiotensin-converting enzyme activity and increase nitric oxide production in human endothelial cells. J Pharm Pharmacol 2006;58:1139-44. |
|9.||Persson IA, Persson K, Andersson RG. Effect of vaccinium myrtillus and its polyphenols on angiotensin-converting enzyme activity in human endothelial cells. J Agric Food Chem 2009;57:4626-9. |
|10.||Persson IA, Persson K, Hägg S, Andersson RG. Effects of green tea, black tea and Rooibos tea on angiotensin-converting enzyme activity and nitric oxide production in healthy volunteers. Public Health Nutr 2010;13:730-7. |
|11.||Persson IA, Persson K, Hägg S, Andersson RG. Effect of cocoa extract and dark chocolate on angiotensin-converting enzyme and nitric oxide in human endothelial cells and healthy volunteers-a nutragenomics perspective. J Cardiovasc Pharmacol 2011;57:44-50. |
|12.||Yan CS, Kim D, Aizawa T, Berk B. Functional interplay between angiotensin II and nitric oxide: Cyclic GMP as a key mediator. Arterioscler Thromb Vasc Biol 2003;23:26-36. |
|13.||Mannari C, Bertelli AA, Stiaccini G, Giovannini L. Wine, sirtuins and nephroprotection: Not only resveratrol. Med Hypotheses 2010;75:636-8. |
|14.||Schmitt CA, Heiss EH, Dirsch VM. Effect of resveratrol on endothelial cell function: Molecular mechanisms. Biofactors 2010;36:342-9. |
|15.||Klatsky AL, Friedman GD, Armstrong MA, Kipp H. Wine, liquor, beer, and mortality. Am J Epidemiol 2003;158:585-95. |
|16.||Dudley JI, Lekli I, Mukherjee S, Das M, Bertelli AA, Das DK. Does white wine qualify for french paradox? Comparison of the cardioprotective effects of red and white wines and their constituents: Resveratrol, tyrosol and hydroxytyrosol. J Agric Food Chem 2008;56:9362-73. |
|17.||Walker AR, Lee E, Bogs J, McDavid DA, Thomas MR, Robinson SP. White grapes arose through the mutation of two similar and adjacent regulatory genes. Plant J 2007;49:772-85. |
|18.||Monagas M, Gómez-Cordovés C, Bartolomé B, Laureano O, Ricardo da Silva JM. Monomeric, oligomeric and polymeric flavan-3-ol composition of wines and grapes from Vitisvinifera L. Cv. Graciano, Tempranillo, and Carbernet Sauvignon. J Agric Food Chem 2003;51:6475-81. |
|19.||Cantos E, Espin JC, Tomas-Barberan A. Varietal differences among the polyphenol profiles of seven table grape cultivars studied by LC-DAD-MS-MS. J Agric Food Chem 2002;50:5691-6. |
|20.||Belisario-Sanchez YY, Taboada-Rodriguez A, Marin-Iniesta F, Lopez-Gomez A. Dealcoholized wines by spinning cone column distillation: Phenolic compounds and antioxidant activity measured by the 1,1-diphenyl-2-picrylhydrazyl method. J Agric Food Chem 2009;57:6770-8. |
|21.||Mateus N, Oliveira J, Santos-Buelga C, Silva AMS, De Freitas V. NMR structure characterization of a new vinylpyranoanthocyanin-catechin pigment (a portisin). Tetrahedron Letters 2004;45:3455-7. |
|22.||Honsho S, Sugiyama A, Takahara A, Satoh Y, Nakamura Y, Hashimoto K. A red vinegar beverage can inhibit the renin-angiotensin system: Experimental evidence in vivo. Biol Pharm Bull 2005;28:1208-10. |
|23.||Li X, Wu B, Wang L, Li S. Extractable amounts of trans-resveratrol in seed and berry skin in Vitis evaluated at the germplasm level. J Agric Food Chem 2006;54:8804-11. |
|24.||Romero-Pérez AI, Ibern-Gómez M, Lamuela-Raventós RM, De la Torre-Boronat MC. Piceid, the major resveratrol derivative in grape juices. J Agric Food Chem 1999;47:1533-6. |
|25.||Olszanecki R, Bujak-Gizycka B, Madej J, Suski M, Wolkow PP, Jawien J, et al. Kaempferol but not resveratrol inhibits angiotensin converting enzyme. J Physiol Pharmacol 2008;59:387-92. |
|26.||Goldberg DM, Yan J, Soleas GJ. Absorption of three wine-related polyphenols in three different matrices by healthy subjects. Clin Biochem 2003;36:79-87. |
|27.||Walle T, Hsieh F, DeLegge MH, Oatis JE, Walle UK. High absorption but very low bioavailability of oral resveratrol in humans. Drug Metab Dispos 2004;32:1377-82. |
[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5]
[Table 1], [Table 2]