Effects of resveratrol and flavonols oncardiovascular function:Physiological mechanisms

Suwan Yap,1,2 Chengxue Qin,3 and Owen L. Woodman4*1School of Chemistry, University of Melbourne, Parkville, VIC, Australia2Bio21 Molecular Science and Biotechnology Institute, University of Melbourne, Parkville, VIC, Australia3Department of Pharmacology, University of Melbourne, Parkville, VIC, Australia4School of Medical Science and Health Innovations Research Institute, RMIT University, Bundoora, VIC, Australia

Abstract.Resveratrol and flavonols are commonly found together in

fruits and vegetables and, therefore, consumed in the diet.

These two polyphenols share both vasorelaxant and

antioxidant activity and may act together to improve

cardiovascular function. This review examines the

mechanisms by which resveratrol and flavonols influence

cardiovascular function and perhaps offer a new approach

for the development of therapeutic agents for the prevention

and/or treatment of coronary artery disease.

VC 2010 International Union of Biochemistry and Molecular Biology, Inc.Volume 36, Number 5, September/October 2010, Pages 350–359 �E-mail: owen.woodman@rmit.edu.au

Keywords: resveratrol, flavonol, cardioprotection, antioxidant,relaxation

1. Introduction

Epidemiological studies have indicated that the French popula-tion has a lower than predicted incidence of cardiovascular dis-ease (CVD), in particular coronary artery disease, given theircomparatively high-fat diet, an anomaly known as ‘‘the Frenchparadox’’ [1–3]. With risk factors for CVD, such as smoking andobesity, comparable with other Western populations, it hasbeen suggested that the regular consumption of red wine inthe French diet holds the key to cardioprotection [4,5]. Forexample, Gronbaek et al. examined the effect of different typesof alcoholic drinks on mortality for 10–12 years and demon-strated that there was a reduced risk of death with moderateconsumption of wine and not other alcoholic beverages [6].

Early studies demonstrated that in vitro incubation ofhuman coronary arteries and rat aortae with red winecaused endothelium-dependent vasodilation [7], which wasattributed to the inhibition of NO breakdown or endothelialnitric oxide synthase (eNOS) activation [8]. Later, it wasrevealed that it is the nonalcoholic component of wine inparticular, the polyphenols, such as resveratrol and flavo-nols, that are likely to contribute to the protective effects ofwine in the cardiovascular system [9,10].

2. Resveratrol

2.1. OverviewResveratrol (3,40,5-trihydroxystilbene) belongs to the stil-bene family of compounds that consist of two aromatic C-rings joined by an alkene bridge. It is considered a phytoa-lexin, a metabolite biosynthesized by plants in response toenvironmental stresses or microbial attacks [11]. Resveratrolexists as two isomers, the cis- and trans-diastereoisomers,but it is the trans isoform (Fig. 1A) that has been extensivelyinvestigated due to its greater natural abundance and bio-logical activity. Trans-resveratrol is found in high abundancein the skin of grapes and in the leaf epidermis as well as inmany fruits and vegetables that are components of thehuman diet such as mulberries, cranberries, blueberries,and peanuts [12]. Trans-resveratrol has also been isolatedfrom the dried roots of Japanese knotweed, Polygonumcuspidatum Sieb. et Zucc. (Polygonaceae), which has beenused in traditional Japanese and Chinese medicine forthe treatment of atherosclerosis and other inflammatorydiseases [13] amongst other disorders.

2.2. Vascular activitiesResveratrol has been demonstrated to exert both acute andchronic effects on the cardiovascular system [14–16]. Resver-atrol can acutely cause endothelium-dependent relaxation incarotid artery rings [17] but perhaps more importantly it hasbeen demonstrated to significantly improve endothelium-de-pendent relaxation to acetylcholine in rat aortae [18] aftersystemic administration for 21 days. In a similar study, it has

*Address for correspondence: Prof. Owen L. Woodman, School of Medical Sciences,RMIT University, P.O. Box 71, Bundoora, VIC 3083, Australia. Tel.: þ61-3-9925-7305;Fax: þ 61-3-9925 7063; E-mail: owen.woodman@rmit.edu.au.The first two authors contributed equally to this work.Received 23 May 2010; accepted 17 June 2010DOI: 10.1002/biof.111

Published online 27 August 2010 in Wiley Online Library(wileyonlinelibrary.com)

350

been shown that chronic treatment with resveratrol alsoresulted in a marked reduction of age-related deteriorationin endothelial function in mice demonstrated by improvedvascular responses to acetylcholine [19].

The ability of resveratrol to cause endothelium-depend-ent relaxation has been demonstrated to be mediated largelythrough nitric oxide as a nitric oxide synthase (NOS) inhibitor,NG-nitro-L-arginine methyl ester (L-NAME) abolished this effect[15]. Further evidence of a possible contribution of endothe-lium-derived NO to relaxation has been provided by Hsiehet al. who demonstrated an increase in eNOS expression in cul-tured bovine pulmonary artery endothelial (BPAE) cells treatedwith resveratrol [20]. It has also been shown that in resvera-trol-treated rats subjected to myocardial ischaemia/reperfusion(I/R), there is a significant increase in eNOS, neuronal nitric ox-ide synthase (nNOS) [21], and inducible nitric oxide synthase(iNOS) [22] proteins in the heart shown by western blot analy-sis. In addition, Wallerath et al. demonstrated a resveratrol-induced increase in eNOS mRNA and protein expression, result-ing in increased NO production [23]. Chronic resveratrol treat-ments also resulted in increased eNOS mRNA levels [19] andincreased nitric oxide production compared with controls [18].This was accompanied by improved endothelium-dependentrelaxation but the responses to an endothelium-independent di-lator were not tested. In a separate study, resveratrol treatmentfor 6 weeks failed to alter relaxation responses [24].

Resveratrol is also a known protein deacetylase Sirtuin 1(SIRT1) activator, and it is thought that the effects of resvera-trol on eNOS expression may be attributed to SIRT1 activation.SIRT1 activation results in deacetylation of eNOS at lysine res-idues, thereby stimulating eNOS activity [25]. A recent studyby Arunachalam et al. demonstrated that pretreatment of

human umbilical vein endothelial cells with resveratrol signifi-cantly reduced oxidant-induced decreases in SIRT1 levels andeNOS acetylation, thus enhancing NO production and endo-thelial function [26]. Knock down of endogenous SIRT1 byRNAi significantly attenuated resveratrol-induced increases ineNOS mRNA and protein expression [27] and reduced resvera-trol-induced increases in NO levels [25].

The structural similarity of resveratrol to a syntheticestrogen, diethylstilbestrol, led to studies which revealedthat it is also an agonist of estrogen receptor alpha (ERa)[28]. Estrogen is a known vasorelaxant [29], an effect relatedto stimulation of NO production [30]. It is thus thought thatthe vascular effects of resveratrol may be also in part medi-ated through ERa activation. The role of ERa in the endothe-lium has been investigated, where endothelium-dependentrelaxation is induced by polyphenol extracts in wild type butnot ERa knockout mice, providing evidence that the vasore-laxation properties are exerted through ERa activation [31]. Arecent study by Klinge et al. revealed that resveratrol, in asimilar fashion to 17b-estradiol, induces the interactionbetween ERa, caveolin-1, and Src, leading to phosphorylationof eNOS, and hence, NO production [32].

Taken together, the ability of resveratrol to improve NOproduction, and hence, endothelial function may contributein part to its protective effects in the cardiovascular systemgiven the well-established link between endothelial and vas-cular health.

2.3. Antioxidant activitiesResveratrol also possesses strong antioxidant activitiesin vitro. Its antioxidant activity was first demonstrated by

Fig. 1. Structures of trans-resveratrol (A), flavonol (B), quercetin (C), 30,40-dihydroxyflavonol (DiOHF) (D),40-hydroxy-30-methoxyflavonol (E), and DiOHF-6-succinamic acid (F).

Cardiovascular actions of resveratrol and flavonols 351

Frankel and Kanner where resveratrol inhibited low densitylipoprotein (LDL) oxidation by Cu2þ [9]. It was later shownthat resveratrol protects LDL against oxidation by both che-lating copper (but not iron) and by free radical scavengingmechanisms [33]. It has been demonstrated that trans-resveratrol scavenges superoxide [34], 2,2-diphenyl-1-picryl-hydrazyl radicals and inhibits production of thiobarbituricacid reactive substances (TBARS) in cultured human fibroblastcells or rat liver microsomes treated with an oxidant, tert-butylhydroperoxide (TBHP) [35]. In addition, it has beenshown that angiotensin II (AngII) and nicotinamide adeninedinucleotide phosphate (NADPH)-induced superoxide produc-tion in rat aorta was attenuated by resveratrol [18]. NADPHoxidase is considered the major source of reactive oxygenspecies in the vasculature [36,37]. Stivala et al. demonstratedthe importance of the three hydroxyl groups for antioxidantactivity of resveratrol as O-methylation of resveratrol virtuallyabolished any antioxidant capacity [35]. In addition, the pres-ence of the trans-olefinic double bond was shown to be im-portant for antioxidant activity where reduction of the doublebond or conversion to the cis geometry decreased antioxidantactivity [35]. Trans-resveratrol has also been shown to bemore effective than its cis counterpart in reducing Cu2þ-induced lipid peroxidation in human LDL [38,39]. Recent com-putational studies indicated that the 40-O radical of the cis-isoform was energetically less stable compared with the cor-responding trans-isomer radicals, providing an explanation forthe reduced antioxidant ability of cis-resveratrol [40].

Through its antioxidant ability, resveratrol reduces re-active oxygen species mediated cell death. For example, Sunet al. demonstrated that resveratrol was able to protectagainst lipid peroxidation and cell death, indicated byrelease of lactate dehydrogenase, in PC12 cells [41]. Inanother study, Goh et al. demonstrated that resveratroltreatment reduced hypoxia-induced superoxide levels in car-diomyocytes, and hence, prevented injury associated withhypoxia/reoxygenation [17].

Mechanistically, it is thought that resveratrol modu-lates antioxidant enzymes thereby reducing oxidative stress.It has been shown that the intracellular redox regulator thio-redoxin-1 and the cytoprotective enzyme hemeoxygenase-1were upregulated in a dose-dependent manner in culturedcells in response to resveratrol [42]. In another study, Span-ier et al. demonstrated a resveratrol-induced increase of glu-tathione peroxidase 1 (GPx1) and superoxide dismutase(SOD) mRNA expression, the major enzymes important inthe breakdown of H2O2 and superoxide [43]. In addition,resveratrol has also been shown to cause a concentration-dependent downregulation of NADPH oxidase subunits Nox-2 [44] and Nox-4 [43]. Because oxidative stress causes inac-tivation of NO and thus impairment of endothelial function,the ability of resveratrol to enhance NO production and pre-vent NO inactivation can improve vascular function.

2.4. Vascular and cardiac protectionChronic resveratrol treatment has also been shown to be car-dioprotective. In one study, hypercholesterolemic rats sub-

jected to permanent left anterior descending coronary artery(LAD) occlusion to induce myocardial infarction were foundto have impaired myocardial function, which was normalizedwith 2-week oral treatment with resveratrol. In addition, theauthors demonstrated that 2-week oral treatment withresveratrol normalized the effects caused by permanent LADocclusion such as decreased heme-oxygenase-1 expressionand increased caveolin-1/eNOS association and significantlyreduced cholesterol levels [45]. Miyazaki et al. demonstratedthat in mice fed with resveratrol for 1 week, the AngII type 1receptor expression in vivo was decreased significantly andAngII-induced hypertension was significantly attenuated inresveratrol-treated mice compared with controls [46]. Inanother study, steptozotocin (STZ)-induced diabetic ratstreated with resveratrol for 42 days showed significantlyreduced plasma malondialdehyde (MDA) levels comparedwith nontreated diabetic rats. In addition, resveratrol treat-ment also significantly reversed the decrease in relaxationresponse to acetylcholine (Ach) in the diabetic rat aorta [24].Similarly, in a study by Sulaiman et al., STZ-induced diabeticrats treated orally with resveratrol for 3-month reversed thereduction in SIRT1 expression in rat hearts as well as signifi-cantly improved cardiac function [47].

Isolated perfused hearts treated with resveratrol (10lM), either before global ischaemia or upon reperfusion,showed significant recovery of postischaemia myocardial con-tractile function [17,48,49]. Furthermore, MDA levels, an indica-tor of lipid peroxidation and oxidative stress, of the coronaryperfusate was shown to be significantly reduced in resveratrol-treated compared with vehicle-treated hearts, especially duringthe early reperfusion period [48]. Resveratrol has also beenshown to significantly improve cardiac function in an experi-mental model of myocardial I/R, where rats pretreated withresveratrol had significantly improved cardiac contractility dur-ing reperfusion [50]. Infarct size and MDA formation were alsoreduced significantly in resveratrol-treated rats compared withvehicle [50]. Using hearts from rats where resveratrol wasincluded in the diet, there was an improved recovery of coro-nary flow at the end of reperfusion and better recovery of con-tractile function compared with control hearts [51]. In addition,the work of Kaga et al. showed that oral administration ofresveratrol significantly reduced infarct size after myocardial in-farction compared with vehicle-treated rats [42]. Similar resultswere obtained by Xi et al. where resveratrol administration toisolated perfused rat hearts from 5 min before reperfusionuntil 30 min of reperfusion resulted in a significant reductionof infarct size compared with control hearts [52].

It is thought that resveratrol elicits cardioprotectionthrough targeting multiple mechanisms and sites of actions.First, it is thought that one possible mechanism of cardio-protection lies in its ability to stimulate NO formation whereHattori et al. showed that the protective effects of resvera-trol in rat isolated hearts were abolished by the addition ofa NO inhibitor, L-NAME or an iNOS inhibitor, aminoguanidine[49]. Furthermore, the inability of resveratrol to preventreperfusion injury in iNOS knockout mice supports evidencethat resveratrol mediated cardioprotection is in part relatedto NO formation [22].

352 BioFactors

Second, it is thought that resveratrol is able to upregu-late or activate certain survival kinases. Resveratrol hasbeen shown to mediate phosphorylation of Akt and CREB,which when blocked resulted in a lack of cardioprotection[17,53]. Recently, Brito et al. demonstrated the ability ofresveratrol to inhibit apoptosis by significantly increasinglevels of Bcl-2, an anti-apoptotic protein, as well as inhibi-ting peroxynitrite-induced caspase-3 activity [54].

Finally, a third proposed mechanism is that resveratrolmediates cardioprotection through Kþ channels. Cardiomyo-cytes subjected to hypoxia followed by reoxygenation withconcomitant treatment with resveratrol and selective mito-chondrial KATP and BKCa channel inhibitors were not pro-tected against cell death demonstrated by lactase dehydro-genase (LDH) release [17].

3. Flavonols

3.1. OverviewFlavonols (3-hydroxy-2-phenylchromen-4-one) are a class offlavonoids having a 3-hydroxyflavone backbone (Fig. 1B).They are present in a wide variety of fruits and vegetablesoften in combination with resveratrol. Hollman et al. [55,56]detected that the peak plasma concentrations of one of the

most abundant flavonols, quercetin, reach the micromolarrange in humans after ingestion of fruits and vegetablescontaining about 100 mg of quercetin. Furthermore, the dailyintake of flavonols exceeds that of vitamin E, a monophe-nolic antioxidant, and b-carotene on a milligram-per-day ba-sis. Thus, the amount of flavonol intake could provide phar-macologically significant concentrations in body fluids andtissues that are probably greater than that reached by vita-mins. Many studies have revealed two important biologicalactions of flavonols, like resveratrol, that may have benefi-cial effects on the cardiovascular system that is vasorelaxantand antioxidant activities.

3.2. Vasorelaxant activitySeveral studies have demonstrated that flavonols are effec-tive vasodilators [14,57,58]. They cause vasorelaxationpredominantly in an endothelium-independent manner[14,57,59], but endothelium-dependent relaxation also playsa small role. Chan et al. [57] demonstrated that removal ofthe endothelium reduced sensitivity to flavonols, withoutaffecting the maximum response.

The mechanism by which flavonols cause endothelium-independent relaxation remains unclear. In general, the pro-cess of contraction by vascular smooth muscle is initiated bycalcium-regulated phosphorylation of myosin light chain(MLC). Thus, relaxation requires a decreased intracellularcalcium concentration or increased MLC phosphatase activity[60] (Fig. 2). Based on these principles, it has been sug-gested that flavonols could interfere with calcium utilizationin several ways. Ko et al. [61] measured the influx of radioac-tive calcium into the aortic smooth muscle caused by nor-adrenaline or high potassium and showed that flavonols in-hibit the influx of extracellular calcium. Flavonols were foundto cause a concentration-dependent inhibition of calciumuptake compared with untreated aorta (mechanism 1, Fig. 2).

Flavonols might also affect the mobilization of intracel-lular calcium from the sarcoplasmic reticulum (SR) [72](mechanism 2, Fig. 2). Previous work from our laboratorydemonstrated that flavonols impaired the contraction causedby phenylephrine-induced calcium release from intracellularstores in rat isolated aorta [57]. Furthermore, Shoshan et al.[62] demonstrated that quercetin inhibited the influx of cal-cium via the Ca2þ-ATPase at the SR store in smooth muscle.

Third, flavonols could inhibit protein kinase C (PKC) ina concentration-dependent manner, preventing phosphoryla-tion of L-type calcium channels or other proteins that regulatecross-bridge cycling [60] (mechanism 3, Fig. 2). Herrera et al.[73] demonstrated that flavonols inhibited the tonic contrac-tion induced by the PKC activator, phorbol 12-myristate-13-ac-etate. In another study, quercetin inhibited PKC in cell-freeextracts from mouse brain [63] as well as intact cells [64].

A fourth mechanism for flavonol action is thought tobe the NO-cyclic guanylyl monophosphate (cGMP) pathway(mechanism 4, Fig. 2). The precise mechanisms by whichcGMP relaxes vascular smooth muscle is unclear, however, itis proposed that cGMP can activate a cGMP-dependent pro-tein kinase, inhibit calcium entry into the vascular smoothmuscle, activate potassium channels and decrease inositol

Fig. 2. Proposed mechanisms of flavonol-inducedvasorelaxation. (1) Inhibition of Ca21—entry throughvoltage-gated calcium channel [61], (2) inhibition ofmobilization of intracellular calcium from thesarcoplasmic reticulum [57,62], (3) inhibition of PKCactivity [63,64], (4) increase in NO and cGMP [65–67],(5) increase in cAMP content [68–70], and (6) inhibitionof Rho kinase [71]. See text for details and fulldiscussion. PLC, phospholipase C; DG, diacylglycerol;PKC, protein kinase C; IP3, inositol 1,4,5-triphosphate;SR, sarcoplasmic reticulum; AC, adenyl cyclase; MLC,myosin light chain; cAMP, cyclic adenosinemonophosphate; cGMP, cyclic guanylyl monophosphate.[Color figure can be viewed in the online issue, which isavailable at wileyonlinelibrary.com.]

Cardiovascular actions of resveratrol and flavonols 353

1,4,5-triphosphate (IP3) and PKC. The eventual low-intracellu-lar Ca2þ concentration in smooth muscle cells reduces cellu-lar contractility and yields relaxation. Lemos et al. [65] dem-onstrated that flavonol-induced relaxation was inhibited by aNOS inhibitor, L-NAME, suggesting the relaxation is associ-ated with the production of cGMP. Furthermore, the vasore-laxation could also result from elevated NO levels in thesmooth muscle cells. NO is generally produced by eNOSattached to the endothelium membrane and diffused tosmooth muscle cells and regulate the vascular tone. Theincreased NO level could result from increased synthesis ofNO by eNOS [66,67] or decreased degradation of NO byscavenging superoxide [74]. This pathway might be the maincontributor to the endothelium-dependent relaxation inducedby flavonols.

Apart from interfering with calcium utilization, flavonolsmay also increase with MLC phosphatase activity. MLC phos-phatase removes phosphate from MLC to promote smoothmuscle relaxation [60]. Increases in cyclic adenosine mono-phosphate (cAMP) led to a reduction in MLC phosphoryla-tion, which decreases the interactions between actin andmyosin and thus causes vasorelaxation (mechanism 5, Fig.2). It has been demonstrated that quercetin can increaseaortic cAMP content [70] and thus lead to vascular dilatation.The increased cAMP level could also be due to the inhibitionof cAMP phosphodiesterase [68,69].

More recently, a synthetic flavonol, 30,40-dihydroxyfla-vonol (DiOHF) was shown to attenuate vascular contractionthrough inhibition of the RhoA/Rho-kinase signaling pathwayin endothelium-denuded rat aorta [71]. Rho kinase, a serine/threonine kinase, phosphorylates MLC phosphatase, inhibi-ting its activity and thus promoting the phosphorylated stateof MLC for contraction. By inhibiting this pathway, flavonolscould improve vasorelaxation (mechanism 6, Fig. 2).

3.3. Antioxidant activityIn addition to their vasodilator activity, flavonols are effectiveantioxidants. The predominant mechanism is probably bythe donation of a single electron to the radical resulting inthe formation of a semiquinone radical, which can donate afurther electron to form the orthoquinone as shown inFig. 3. It has been demonstrated by electron paramagneticresonance (EPR) spectroscopy that the spin distribution dur-ing oxidation of quercetin remains entirely on the 40-OH ofthe B-ring favoring the donation of two electrons leading tothe formation of an orthoquinone [75].

Flavonols have low-redox potentials [0.23 < E7 (redoxpotential at pH ¼ 7) < 0.75 V], thus they are able to reducehighly oxidizing free radicals thermodynamically with redoxpotentials in the range (2.31 > E7 > 1.0 V), such as superox-ide (E7, 0.940 V), peroxyl (E7, 1.00 V), alkoxyl (E7, 1.600 V),and hydroxyl radicals (E7, 2.310 V) by hydrogen atom dona-tion [76]. By scavenging such reactive species, flavonols mayreduce further formation of ROS and interrupt oxidative reac-tions. Furthermore, by scavenging superoxide radicals, thebioavailability of NO increases [67,77,78] which may contrib-ute to improved endothelial function in the vasculature afterischaemia reperfusion. By limiting the formation of peroxyni-trite, flavonols could also improve endothelial function byreducing nitration of the NOS cofactor, BH4. This may in turnreduce the uncoupling of eNOS and production of more ROSfrom eNOS monomer [79]. Thus, flavonols, by scavengingsuperoxide and reducing peroxynitrite formation, may helpto prevent endothelial dysfunction during reperfusion. Thereare several reports that flavonols can reduce injury causedby I/R, where there is an extensive production of ROS[77,80–83].

These biological activities have generated great inter-est in searching for and designing flavonols for potential useas therapeutic agents in the prevention and treatment ofCVD. To facilitate the development of flavonols as therapeu-tic agents, it is desirable to better understand the structuralcharacteristics that influence their biological activity.

3.4. Structure-activity relationships of flavonolsHerrera et al. [72], Rice-Evan et al. [76], Dugas et al. [84],Chan et al. [57], and Woodman et al. [85] have revealed sev-eral important features of flavonols that are required for vas-orelaxant and antioxidant activity. Most structural featuresfavoring vasodilator activity parallel those that favor antioxi-dant activity [57]. The basic structure of flavonols with anaccepted ring notation system is shown in Fig. 1.

The number and the orientation of hydroxyl groups onthe B-ring appear to be the most significant determinant oftheir vasorelaxant activity. Flavonols with three contiguousOH groups (a pyrogallol group) enhance contraction inresponse to a variety of stimuli, whereas the presence ofone or two hydroxyl groups favors relaxation [73]. Flavonolswith a 20,40-dihydroxy group are weak vasodilators, whereasa catechol group, comprising hydroxyl groups at C30 and C40,is associated with strong vasorelaxant activity [73]. Chanet al. have shown that the catechol moiety on the B-ring

Fig. 3. Oxidation of flavonols via donation of a single electron.

354 BioFactors

also improves antioxidant activity [57,86]. Qin et al. [87]studied a series of synthetic DiOHF derivatives bearing vari-ous substituents (Me, OMe, Cl, CF3, and t-Bu) at the 30-posi-tion and a common 40-OH. It was demonstrated that the anti-oxidant activity was lost with all of these substitutions,supporting the importance of the catechol moiety on theB-ring for antioxidant activity [87]. Oxidation of flavonolsoccurs on the B-ring when the catechol is present, yielding arelatively stable ortho-semiquinone radical [88] by facilitat-ing electron delocalization [89] as shown in Fig. 3, whereasflavonols lacking catechol systems form relatively unstableradicals and are weak scavengers [90,91]. Antioxidant capa-bility is impaired when there is obstruction of the 30,40-cate-chol structure, for example, by 40-O-methylation [83]. More-over, flavonols with more than two hydroxyl groups on theB-ring, such as a pyrogallol group, have been reported tohave pro-oxidant properties. They could promote hydrogenperoxide production [92] from which the Fenton reactionmay generate the highly reactive hydroxyl radical [93].

Furthermore, the co-planar conformation given by thedouble bond linkage between C2 and C3 and the 4-carbonylgroup on the C-ring are required for the vasodilator activity[73], and the free 3-OH group on the C-ring is essentialfor the endothelium-dependent component of relaxation[57,70,85]. Flavonols with a 3-OH and 30,40-catechol arereported to be 10-fold more potent than ebselen, a knownreactive nitrogen species (RNS) scavenger, against peroxy-nitrite [94]. It has been suggested that the conjugation andplanar system of the C-ring permits a resonance effect thatlends stability to the flavonol radical and is, therefore, criti-cal in optimizing antioxidant activity [95]. Removal of the3-OH group abrogates coplanarity and conjugation, therebycompromising scavenging ability [75,95,96]. It is also postu-lated that the B-ring hydroxyl groups form hydrogen bondswith the 3-OH, aligning the B-ring with the heterocycle andthe A-ring. Eliminating this hydrogen bond leads to a minortwist of the B-ring, compromising the ability to delocalizeelectrons [75]. Because of this intramolecular hydrogenbonding, the influence of a 3-OH group is potentiated by thepresence of a 30,40-catechol [76].

When compared with the B-ring hydroxylation pattern,the impact of A-ring substitution on biological activity is ofquestionable significance compared with antioxidant activity.The work of Heim et al. [97] indicated that substitutions onthe A-ring correlate less with antioxidant activity but the ab-sence of substitution on this ring might enhance vasorelaxa-tion [57]. Another study from our laboratory reported thatwhen a large substitution, succinamic acid, is introduced tothe A-ring, the vasorelaxant activity is abolished [98] butantioxidant activity was preserved.

3.5. QuercetinQuercetin (3,30,40,5,7-pentahydroxyflavone, Fig. 1C) is a natu-rally occurring flavonol found in many foods and beveragessuch as berries, citrus, fruits, vegetables, herbs, and tea.Quercetin can cause both endothelium-dependant and endo-thelium-independent relaxation in isolated arteries [57,99].

In addition, quercetin possesses antioxidant activity enablingit to inhibit lipid peroxide formation in human LDL in vitro[100]. Sanchez et al. [101] demonstrated that quercetin pre-vents AngII-induced increases in the expression of thep47phox subunit of NADPH oxidase, one of the major sourcesof O�

2 in blood vessels [36,37]. Furthermore, rats treatedwith quercetin showed improved post-I/R cardiac function,improved mitochondrial function and increased Mn-SOD ac-tivity [102]. Chronic treatment with quercetin has also beenshown to improve endothelium-dependent relaxation aortaefrom spontaneously hypertensive rats [103].

3.6. 30,40-DihydroxyflavonolDiOHF (Fig. 1D) is a synthetic flavonol that is as potent asquercetin in inducing vessel relaxation and scavengingsuperoxide in rat isolated aorta [57]. DiOHF has been dem-onstrated to cause coronary and renal blood flow after intra-venous infusion in conscious sheep [83]. In addition, DiOHFwas able to preserve vasodilator reserve after I/R of rathindquarters [77] indicating improved vascular function. Inanother study, DiOHF significantly reduced myocardial infarctsize after I/R in anesthetized sheep, with the level of protec-tion being similar to that of ischaemic preconditioning (IPC),currently regarded as the most effective protection againstreperfusion injury [83]. More recently, Wang et al. demon-strated that daily treatment of goats with DiOHF during the4-week reperfusion period after myocardial ischaemia signifi-cantly reduced infarct size and prevented left ventricularremodeling, as well as inhibiting myocyte apoptosis in thenoninfarcted myocardium [104]. Levels of caspase-3, Bax andcytochrome c were also significantly reduced in the DiOHF-treated group compared with vehicle, indicating a decreasein apoptosis [104]. In addition, DiOHF treatment significantlyreduced the extent of myocyte apoptosis identified by termi-nal deoxynucleotidyl transferase dUTP nick end labeling(TUNEL) in the noninfarcted zone of the myocardium [104].Although these results demonstrated the potential of DiOHFin cardioprotection, its mechanism of action is still unknown.DiOHF possess both vascular and antioxidant activities, andit is not known whether either or both of these activities areimportant for cardioprotection. We have successfully synthe-sized a flavonol with the vasorelaxant activity that does notpossess antioxidant activity, 40-hydroxy-30-methoxyflavonol[87] (Fig. 1E) and a water-soluble flavonol with the selectiveantioxidant activity, DiOHF-6-succinamic acid [98] (Fig. 1F).These two flavonols will enable studies to cast light on thespecific biological activity required for cardioprotection.

3.7. Mechanisms of vascular andcardiac protectionIt is thought that flavonols elicits cardioprotection by target-ing multiple mechanisms and sites of actions. First, the vaso-relaxant activity of flavonols may help to dilate blood ves-sels and thus better re-establish perfusion in post-ischaemichearts. The postulated mechanism of action is describedabove and may be exerted in an endothelium-dependent

Cardiovascular actions of resveratrol and flavonols 355

and/or endothelium-independent manner. The endothelium-dependant relaxation effect of flavonols is mainly mediatedby the NO-cGMP pathway.

It has been demonstrated that vasorelaxant activity offlavonols is closely related to their calcium inhibition activityin rat isolated aorta [57], and in our laboratory, we havefound that DiOHF has a slight negative chronotropicresponse in isolated right atrium (Chan and Woodman,unpublished observations). Thus, flavonols may be able toreduce intracellular calcium overload in many pathologicalconditions, such myocardial ischaemia reperfusion. Further-more, unlike classical calcium channel blockers, no negativeinotropic effect was observed when DiOHF was administeredto conscious sheep [105].

Third, the antioxidant activity of flavonols could scav-enge various ROS, such as superoxide, peroxyl radical, andperoxynitrite [84]. By scavenging such reactive species, fla-vonols prevent the formation of highly reactive species andlimit the perpetuation of oxidative damage to the myocar-dium and blood vessels. Furthermore, ROS also play a majorphysiological role in several aspects of intracellular signalingand regulation [106]. Therefore, reduction in the levels ofROS by flavonols may also have profound effect on cellularsignaling pathways indirectly.

Lastly, chronic administration of flavonols has shownsome beneficial effect in animal models. DiOHF significantlyreduced oxidant stress and preserved endothelial-dependantrelaxation in aorta from diabetic rats when given for a week[107]. Furthermore, quercetin has been shown to induce aprogressive, dose-dependant, and sustained reduction inblood pressure in several rat models of hypertension[103,108–110] and metabolic syndrome when given chroni-cally [111,112].

4. Conclusions

In summary, polyphenolic compounds, such as resveratroland flavonols are effective vasodilators and antioxidants.Vasodilatation could help to establish better blood flow andthe antioxidant activity could reduce oxidative damage medi-ated by various ROS. These results indicate that polyphe-nolic compounds could be cardioprotective and have poten-tial as an adjunctive therapeutic agent for many CVDs, suchas diabetes and atherosclerosis and for the treatment ofmyocardial ischaemia reperfusion. These effects may alsohave effect on several redox-sensitive proteins and cellularsignaling pathways. A greater understanding of the cellularmechanisms of polyphenolic compounds will aid the devel-opment of these compounds as therapeutic agents for theprevention and the treatment of CVD.

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