Flavonoid-Rich Grapeseed Extracts: A New Approach In High Cardiovascular Risk Patients?

P. Kar; D. Laight; K. M. Shaw; M. H. Cummings

Int J Clin Pract. 2006;60(11):1484-1492. 

Summary and Introduction

Summary

The management of traditional risk factors such as hypertension and dyslipidaemia has been successful in reducing the development of cardiovascular disease. However, this has not resulted in the amelioration of complications; prompting attention to be focused on novel markers of vascular risk such as endothelial dysfunction (a determinant of vascular tone), vascular inflammation, oxidative stress and insulin resistance. With an ever-growing interest in plant-derived products, agents that could have a beneficial effect on this complex web of pathophysiology have thus been a major area of research and interest. Flavonoids have been a major focus of attention since the days of the French paradox and the presence of high quantity of flavonoids in grapeseed extracts has prompted research looking at its effects on novel markers of vascular risk.

This review briefly summarises mechanisms implicated in the development of vascular disease and then focuses upon the potential role of the antioxidant properties of flavonoid-rich grapeseed extracts in the reversal of these processes.

Introduction

In the last few decades, a number of metabolic factors that are implicated in the pathogenesis of cardiovascular disease have been identified. They include dyslipidaemia, hypertension, altered glucose tolerance and hyperinsulinaemia.

However, while the management of these traditional vascular risk factors such as hypertension and dyslipidaemia has been successful in reducing the development or progression of cardiovascular disease, it has not totally ameliorated this complication.

Novel markers of vascular risk that have received much attention include endothelial dysfunction (a determinant of vascular tone), vascular inflammation, oxidative stress and insulin resistance. Recent reviews have focused upon the links between vascular tone and inflammation, oxidative stress, insulin sensitivity and the aetiology and pathogenesis of high cardiovascular risk-linked pathologies such as diabetes. With an ever-growing interest in plant-derived products, agents that could have a beneficial effect on this complex web of pathophysiology have thus been a major area of research and interest. This review briefly summarises mechanisms implicated in the development of vascular disease and then focuses upon the potential role of plant-derived flavonoids, especially flavonoid-rich grapeseed extracts, in the reversal of these processes.

The 'Complex' Linkage

Stern proposed the Common Soil hypothesis[1] in 1995 (linking atherosclerosis, inflammation and the metabolic syndrome) and there has been an abundance of evidence linking these conditions and the complex pathophysiology underlying the metabolic syndrome.

The vascular endothelium plays a key role in the regulation of vascular homeostasis and increasing evidence suggests that alterations in endothelial function contribute to the pathogenesis and clinical expression of cardiovascular disease.[2] Endothelial cells regulate vascular homeostasis by producing factors that act locally in the vessel wall and lumen, and a key endothelial product is nitric oxide (NO). Impaired NO production, increase in peroxynitrite formation, a pro-inflammatory milieu, and endothelial damage are some of the mechanisms leading to impaired vasodilatory capacity and endothelial dysfunction. In 1999, Duffy et al. revealed the importance of endothelial functional integrity in atherosclerosis and coronary ischaemia.[3] Atherosclerosis was seen to be dependent upon metabolic vasodilation via tonic release of endothelial NO and vasodilator prostanoids.[4]

Abnormal endothelial function is however often portrayed as a simple reduction in NO-mediated endothelium-dependent vasodilator responses, when this is just one feature of a more complex pattern of changes. Indeed, as vasodilator function decreases, there is increased vasoconstriction due to excess endothelin-1 (ET-1) synthesis[5,6] and other vasoconstricting agents. These alterations in vascular tone are coincident with a spectrum of pro-inflammatory and pro-thrombotic vascular changes. Endothelial dysfunction is now considered important both as a target and mediator in high cardiovascular risk patients such as type 2 diabetes.[7,8]

Oxidative stress has a deleterious effect upon endothelial function[9] and is a major culprit in the complex web of factors propagating the syndrome of insulin resistance and type 2 diabetes mellitus (DM).[7] Unsurprisingly, this has prompted a great deal of research and investigations looking at the effects of antioxidants on endothelial function, because of the therapeutic modality of reducing oxidative stress.

Atherosclerosis and the Role of Oxidative Stress upon Endothelial Function

Atherosclerosis is a chronic inflammatory disease that develops in lesion-prone regions of medium-sized arteries. As is well known, they may be present and clinically silent for decades before becoming active and producing clinical events such as cardiac death and cerebrovascular events.

Atherosclerosis can be taken as an example of a process for which there is substantial evidence of the effect of oxidative stress. Hypercholesterolaemia is universally accepted as a major risk factor for atherosclerosis. However, at any given concentration of plasma cholesterol, there is still great variability in the occurrence of cardiovascular events. One of the major breakthroughs in atherogenesis research has been the realisation that oxidative modification of low-density lipoprotein (LDL) might be a crucially important step in the development of the atherosclerotic plaque.[10,11] Native LDL causes the formation of foam cells from monocyte-derived macrophages in early atherosclerotic lesions only after the modification of LDL by various chemical reactions such as oxidation. As oxidation of LDL is primarily a free radical-mediated process that is inhibited by antioxidants, antioxidant depletion might be a risk factor for cardiovascular disease. Inactivation of endothelium-derived vasodilator NO is caused by vascular superoxide anion.[9,12] Nitric oxide combines with superoxide intravascularly to create peroxynitrite, a cytotoxic molecule, which then oxidises LDL.[13]

Evidence for LDL oxidation in vivo is now well established. In immunocytochemical studies, antibodies against oxidised LDL stain atherosclerotic lesions but not normal arterial tissue.[14] In young survivors of myocardial infarction (MI), an association has been demonstrated between increased susceptibility of LDL to oxidation and the degree of coronary atherosclerosis, whereas the presence of ceroid, a product of lipid peroxidation, has been shown in advanced atherosclerotic plaques.[15]

Apart from the atherogenic consequences of LDL oxidation, it is increasingly recognised that reactive oxygen and nitrogen species directly interact with signalling mechanisms in the arterial wall to regulate vascular function.[16]

The effects of antioxidants on these processes are complex but provide alternative mechanisms by which antioxidant supplementation might ameliorate vascular pathology, for instance by improving endothelial function.

Evidence suggests that the balance between pro-oxidant properties of peroxynitrite formed from NO and antioxidant properties of plasma[17] and NO[18] determines the progression of pathology in atherosclerosis.

The Role of Inflammation in the Metabolic Syndrome

In recent years inflammation has also been found to play a key role in the causation of atherosclerotic changes and C-reactive protein (CRP) has been deemed as an important biological marker of inflammation that has been linked to cardiovascular disease.[19] Profound interest has been generated in inflammation being a mediator in insulin resistance and furthermore in the pathogenesis and complications of type 2 diabetes.[20]

Current evidence suggests that oxidative stress, insulin resistance, endothelial dysfunction (in the peripheral arterial bed) and more recently inflammation might be acting synergistically, to promote the development of the metabolic syndrome and subsequent pathology.[21,22] Chronic subclinical inflammation is widely being recognised as a key component in the aetiology of macrovascular disease and possibly even the metabolic syndrome and type 2 diabetes [Insulin Resistance & Atherosclerosis (IRAS) study].[23] A rise in inflammatory markers (CRP, fibrinogen and white cell count) in conjunction with insulin resistance was demonstrated in the IRAS study. Similarly Ridker et al.[24] have shown a strong association between elevated CRP and atherosclerosis and also elevation in CRP and interleukin (IL)-6 levels being predictive of development of diabetes.[25] Clapp et al.[26] showed that inflammation can cause endothelial dysfunction, reduce vascular NO bioavailability and increase oxidative stress, which may alternatively suggest inflammation as the initiating factor. In this study, basal and stimulated endothelial NO bioavailability were measured, while systemic effects were determined by measuring cytokine response, total antioxidant status and urinary protein excretion.

The Importance of Nuclear Factor-κB

Nuclear factor-κB (NF-κB) is an inducible eukaryotic transcription factor of the rel family, which is critical in regulating transcription of specific genes, most of which, are involved in the immune, inflammatory responses and infection and induction of various agents including IL-1 and IL-6, and adhesion molecules. Research has shown an increase in NF-κB activation leads to a proinflammatory state (27) giving rise to suggestions that a decrease in this parameter might positively affect the pathophysiology of the metabolic syndrome. Examples of this are borne out by the effect of antioxidant and anti-inflammatory properties of glitazones,[28] while high-dose aspirin has also been said to have a similar effect on glucose metabolism in type 2 diabetes.[29] NF-κB acts as a transcription factor for various proteins including VCAM-1 and ELAM-1, which are responsible for monocyte adhesion to endothelium[30] - an initial trigger in the formation of atheroma.

Ochnoflavone, a naturally occurring bioflavonoid is thought to inhibit NF-κB due to its inhibition of lipopolysaccharide-induced NO formation - which may be the basis for its anti-inflammatory effects.[31] Recent research has also suggested flavonoid-rich products such as apple extracts[32] and green tea[33] to downregulate NK-κB signalling, thereby possibly reducing the pro-inflammatory state.

Flavonoids

In 1992, examination of WHO epidemiological data showed an intriguing anomaly in Toulouse, France where subjects, in spite of high saturated fat consumption, comparable cholesterol and similar risk factors showed considerably lower incidence of death from coronary heart disease compared with other countries such as the United States and the United Kingdom. This apparent discrepancy, also known as the 'French Paradox',[34] triggered a specific scrutiny for an explanation of this phenomenon. Review of the epidemiological data suggested that alcohol consumption, especially red wine, may have conferred superior protection compared with other beverages. This suggested that the beneficial effects of red wine were, at least in part, due to components other than alcohol. Although the specific mechanism behind the French paradox has not been identified, further studies, both in vivo and in vitro, showed flavonoid components in red wine to have antioxidant properties possibly contributing to cardiovascular benefits.[35-38]

While some studies suggest that flavonoid intake was not associated with reduced CHD,[39-41] two other prospective trials suggested a lower risk of MIs.[42,43] A total of eight cohort studies found lower CHD mortality with total or specific flavonoid intake[40,44-49] but one large cohort study of 38,445 women found a non- significant inverse association between flavonoid intake and CHD mortality.[41] However, a recent meta-analysis indicate a significant protective association between flavonoid intake and risk of CHD mortality, RR = 0.81 (CI 0.71-0.92).[50] One of the authors of the studies[51] that did not show any association described a high background consumption of milk consumption that might have contributed to the null finding, as milk intake can potentially prevent intestinal absorption of flavonoids.[52] Interestingly, most studies, apart from one[53] showed no association for stroke risks,[46,47] although it is likely that these studies did not have sufficient power to study strokes or indeed their various subtypes.

Chemistry of Flavonoids

Revival of interest in traditional medicine coupled with rapid advances in pharmacognosy has led to a greater understanding of the biochemistry and pharmacology of flavonoids.[54,55] Flavonoids are a subgroup of a class of compounds known as polyphenols and are derivatives of 2-phenyl-1-benzopyran-4-one with varied chemical structures present in fruits, vegetables, nuts and seeds. They are polyphenolic compounds possessing 15 carbon atoms; two benzene rings joined by a linear 3-carbon chain (Figure 1).

Figure 1.

 

Flavonoid structure.

Over 4000 flavonoids have been identified and they are divided into several groups according to their chemical structure. The six major subgroups are: chalcones, flavonols (quercetin and kaempherol), flavanone (the catechins), flavones (apigenin), anthocyanins and isoflavonoids (genistein).

Flavonoids, Oxidative Stress and Inflammation

Flavonoid intake has been inversely related with coronary heart disease in the Zutphen Elderly Study,[42] the Seven Countries Study[45] and a cohort study in Finland[48] (Figure 2). Subsequent research has shown the flavonoid component in wine to inhibit oxidation of human LDL[35] and also inhibit platelet aggregation and adhesion. Cishek et al. showed red wine to cause endothelium-dependent relaxation (EDR).[56] Subsequent experiments indicated that flavonoid monomers failed to show this effect. However, oligomeric procyanidins produced a dose-dependent EDR.

Figure 2.

 

Pathophysiological factors involved in the metabolic syndrome, atherosclerosis and type 2 diabetes and the potential action of flavonoid-rich grapeseed extracts (GSE).

Quercetin, a flavonoid prominent in onions and apples, has been epidemiologically linked with protection from coronary artery disease and cancer.[42,49] It has also been shown to inhibit monocyte adhesion to endothelial cells,[57] which is believed to be the first step in the process of atherosclerosis.

Chocolate derived from the plant Theobroma cacao, rich in flavonoids, have shown improved endothelium-dependent flow-mediated dilation (FMD).[58] Flavonoids in elderberry have been shown to confer significant protective effects against oxidative insult and thus have important implications upon preserving endothelial cell function and thereby preventing the initiation of endothelial cells associated with vascular disease.[59] In vitro studies have shown flavonoid-rich chocolates to inhibit lipoxygenase pathways, which give rise to proinflammatory leukotrienes.[60,61] In addition to this, some have shown chocolate procyanidins can modulate a variety of other cytokines such as IL-5, tumour necrosis factor-alpha, tumour growth factor-beta - reducing their inflammatory effects.[62-65] A recent review by Yoon and Back elucidates the anti-inflammatory effects of polyphenols highlighting the potential role for dietary polyphenols to confer health benefits in reducing inflammation.[66]

'Mechanism of Action' of Flavonoids

The mechanism underlying the effects of flavonoids/procyandins on the endothelium has yet to be defined. One of the suggested mechanisms of flavonoid action has been that it creates a 'pseudo-laminar shear stress response', which counters the endothelial dysfunction.[67] Laminar shear stress (LSS) is the frictional force generated by blood flowing over the endothelium and in the context of normal endothelial function, this is one of the important regulatory factors as it induces vasodilation through NO and prostacyclin synthesis while inhibiting vasoconstriction by suppressing endothelin-1 production.[68,69]

LSS also has been shown to alter expression of a spectrum of genes. Gene array studies have demonstrated that physiological levels of LSS suppress the mRNA levels of genes associated with vascular dysfunction and increase the expression of protective genes.[70,71]

Grapes and Grapeseed Extract

The medicinal and nutritional value of grapes (Vitis vinifera) has been heralded for thousands of years. Egyptians consumed this fruit at least 6000 years ago, and several ancient Greek philosophers praised the healing power of grapes - usually in the form of wine. European folk healers developed an ointment from the sap of grapevines to cure skin and eye diseases. Unripe grapes were used to treat sore throats and dried grapes (raisins) were used to heal consumption, constipation, and thirst. The ripe, sweet grapes were used to treat a range of health problems including cancer, cholera, smallpox, nausea, eye infections, and skin, kidney, and liver diseases.

A general composition of the grape ( ) consists of 2-6% stems, 5-12% skins, 80-90% juice and 0-5% seeds. Chemically, one of the important constituents are phenolic substances (frequently called polyphenols) which embrace many classes of compounds ranging from phenolic acids to simple and complex flavonoids. Grapeseeds, although they make up a small percentage of the weight of grapes, contain two-thirds of the extractable phenols.[72] The seeds are highest in phenol content and may contain up to 5-8% phenols by weight[73] and are essentially all flavonoids. They are also referred to as monomeric flavan-3-ols, which when joined together is known as oligomeric procyanidins. The procyanidins have been the subject of intensive research - mostly looking at its antioxidant role and its effect on the vascular endothelium.

Table 1.  General Composition of Grapes

Stems  2-6%
Skins  5-12%
Juice 80-90%
Seeds  0-5%

The rich presence of flavonoid components in grape juice/grapeseed extract (GSE) further prompted research in cardiovascular disease ( ). Initially, reduced platelet aggregation was noted with GSE in canine coronary arteries.[33] Suppression of lipid peroxidation has also been shown in neonatal rats. Rejuvenation of antioxidant system in central nervous system of rats, by GSE, has also been shown.

Table 2.  Examples of Research Undertaken with Grapes/Grapeseed Products

Year Product used Study outcome Reference
1995 Grape juice and red wine In vivo platelet activity inhibited and thrombosis in stenosed canine arteries Demrow et al.[36]
1997 Grape juice and red wine Reduced LDL oxidation in humans Miyagi et al.[35]
1997 Grapeseed extract More potent scavenger of oxygen-free radicals, cf. Vitamin C and E Bagchi et al.[87]
1999 Procyanidin-rich extract from grapeseed Attenuates development of atherosclerosis in rabbits Yamakoshi et al.[83]
1999 Purple grape juice Endothelial function improved; reduced LDL oxidation susceptibility Stein et al.[38]
2000 Grapejuice Human platelet aggregation inhibited Keevil et al.[76]
2001 Purple grape juice In vivo platelet aggregation decreased, enhanced nitric oxide release Freedman et al.[37]
2001 Grapeseed extract Plasma antioxidant capacity and lipid profile improved in hypercholesterolaemic subjects Vinson et al.[89]
2002 Grapeseed extract Post-prandial oxidative stress reduced; antioxidant levels increased in smokers Natella et al.[77]
2003 Grapeseed extract Significantly reduced oxidised LDL and inhibition of endothelial CD36 expression Bagchi et al.[74]
2004 Grapeseed extract Improved flow-mediated dilatation shown ultrasonically Clifton[78]
2005 Grapeseed polyphenols Decrease arterial pressure in hypertensive rats Peng et al.[85]
2005 Grapeseed extract Antioxidant status enhanced; decreased free radical-induced lipid peroxidation in CNS of aged rats Balu et al.[86]
2005 Grapeseed extract Reduction of pulmonary metastatic melanoma in mice Martinez Conesa et al.[94]
2005 Grapeseed extract Inhibits platelet function and release of reactive oxygen intermediates Vitseva et al.[75]

Further research showed reduced LDL oxidation in human high cardiovascular risk patients[35,38,74] reduced human platelet aggregation,[37,75,76] enhanced NO release[36] and improved human endothelial function.[39] Post-prandial oxidative stress was also seen to be reduced in models of oxidative stress (smoking).[77] Flow-mediated vasodilation has also seen to be improved after GSE administration.[78] In this context, it is important to remember that although there is no existing data relating impaired FMD to cardiac events in subjects without coronary disease, those patients with coronary disease who have very impaired FMD have more events.[79] Subjects with impaired FMD are also more likely to have coronary disease on angiography.[80] Statins improve mortality and one mechanism may be via their improvement of FMD.[81]

The University of California demonstrated that constituents present in GSE relaxed isolated blood vessels from rabbits by a pathway in which NO production is implicated,[82] while flavonoid-rich extract from GSE showed attenuation of development of aortic atherosclerosis in cholesterol-fed rabbits.[83]

Endothelium-dependent vasorelaxing activity in an aortic ring model is increased by incubation with grape products and these changes appear to be mediated by the NO-cGMP pathway.[84] Interestingly, in vitro tests using a cupric-ion-mediated LDL + VLDL oxidation model showed a synergistic effect of GSE with both Vitamin C and Vitamin E. Research by Peng et al. also indicated a decrease in arterial pressure in spontaneously hypertensive rats - probably via an antioxidant mechanism.[85]

Grapeseed extract has also been shown to be a more potent scavenger of oxygen-free radicals (an oxidative stressor) than other common antioxidants such as Vitamin C and E.[86,87] Uses of grapeseed-derived procyanidins (flavonoids with oligomeric structure) have also been shown to have an anti-hyperglycaemic effect on streptozotocin-induced diabetic rats. This was significantly increased if accompanied by a low insulin dose, which may partially have been due to the insulinomimetic activity of procyanidins on insulin-sensitive cell lines.[88] Research from Vinson et al. in 2001 used GSE in 17 subjects (nine normal and eight hypercholesterolaemic). Lipid profile and plasma antioxidant capacity were seen to be improved in the high-cholesterol subjects without a concomitant improvement in the healthy individuals.[89] Recent experimental trials have also suggested that proanthocyanidin-rich extract has protective effects against ischaemia-reperfusion-induced renal damage associated with oxidative stress.[90]

Other potential benefits of GSE have been centred on its effect on neoplasia and there have been several encouraging trials undertaken suggesting a beneficial effect.[90-94] Tebib et al.[95] reported an interesting finding in 1994 whereby faecal excretion of cholesterol by rats fed GSE was approximately twofold higher compared with the control group and could be attributed to a reduction or inhibition of intestinal cholesterol absorption.

Side-effects and Safety Profile of Grapeseed Extract

It is important to keep in mind with any potential treatment the possible side effects it brings with it. Fortunately, in vivo studies have not reported any major side effects and there are no known scientific reports of interactions between grapeseed and conventional medications either.

Grapeseed extract has been used as a dietary supplement for a number of years - both in Europe and the USA, and at present holds GRAS (Generally regarded as safe) status - as assessed by the FDA. 'Recommended dosage' has varied from 100 to 300 mg/day while pregnant and breast-feeding women are advised to avoid GSE.

Conclusion

Cardiovascular disease is one of the most important public health issues in the modern world - accountable for a sizeable portion of morbidity and mortality. An increasing population coupled with reduced exercise levels and dietary indiscretions have combined to increase the development of this pathology many fold. The recent past has seen an enormous advancement in the understanding of mechanisms underlying this condition. The links between oxidative stress, metabolic syndrome and inflammation are being increasingly unravelled and there is a great interest in therapies that influence the function of endothelial cells, which are key regulatory cells in the vessel wall. The identification of naturally occurring flavonoids that can modulate the production of NO in body cells is an exciting prospect.

The antioxidant and vascular protective aspects of flavonoid-rich products such as GSE, when combined with the potential hypolipidaemic and anti-platelet effects make a strong case for its potential in preventing and treating diseases associated with endothelial injury, oxidative damage and inflammation; chief among which are type 2 DM and atherosclerotic vascular disease. GSE are also an attractive proposition due to their wide availability and safety profile.

To date, there have been no studies examining the effects of GSE upon novel cardiovascular risk factors in high-risk patient groups. Given this observation and the emerging evidence that GSE may have an effect upon reducing oxidative stress, the use of GSE in such patients may demonstrate concomitant improvements in insulin resistance, endothelial function, inflammation in high-risk patient groups and ultimately cardiovascular outcome.

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