antihypertensive, vasodilator and antioxidant effects of a vinifera grape skin extract

Journal of

JPP 2002 54 1515ndash1520 2002 The AuthorsReceived May 13 2002Accepted July 24 2002DOI 101211002235702153ISSN 0022-3573

Antihypertensive vasodilator and antioxidant effectsof a vinifera grape skin extract

R Soares de Moura F S Costa Viana M A V Souza K Kovary

D C Guedes E P B Oliveira L M S Rubenich L C R M Carvalho

R M Oliveira T Tano and M L Gusma4 o Correia

Abstract

Cumulative evidence suggests that moderate wine consumption exerts a cardioprotective effect We

investigated the occurrence of an antihypertensive effect of an alcohol-free hydroalcoholic grape skin

extract (GSE) obtained from skins of a vinifera grape (Vitis labrusca) in experimental rodent

hypertension models The vasodilator effect of GSE (polyphenols concentration 555 mg gshy 1) was also

assessed in the isolated mesenteric vascular bed of Wistar rats and the antioxidant effect was studied

on lipid peroxidation of hepatic microsomes Oral administration of GSE signi cantly reduced systolic

mean and diastolic arterial pressure in Wistar rats with desoxycorticosterone acetate-salt and NG-

nitro-L-arginine methyl ester (L-NAME) induced experimental hypertension In the rat isolated

mesenteric vascular bed pre-contracted with norepinephrine bolus injections of GSE induced

endothelium-dependent vasodilatation that was substantially inhibited by L-NAME but not by

indometacin tetraethylammonium or glibenclamide Lipid peroxidation of hepatic microsomes

estimated as malondialdehyde production was concentration-dependently inhibited by GSE In

conclusion the antihypertensive effect of GSE might be owing to a combination of vasodilator and

antioxidant actions of GSE These ndings also suggest that the bene cial effect of moderate red

wine consumption could be owing to an antihypertensive action induced by compounds occurring in

the skin of vinifera grapes

Introduction

Epidemiological studies have shown that red wine consumption may have a protectiveeŒect against coronary heart disease (St Leger et al 1979) This aspect is observed mainlyin some regions of France where a high intake of saturated fats is not accompanied byincreased risk of cardiovascular disease a condition termed the ` French paradox rsquo rsquo(Renaud amp de Lorgeril 1992) This paradox has been attributed in part to the moderateconsumption of red wine The mechanism of the cardioprotective eŒect of wine is notcompletely established but experimental evidence suggests that the benereg cial eŒects ofwine are probably owing to coronary vasodilatation (Flesch et al 1998) inhibition ofplatelet aggregation (Demrow et al 1995) inhibition of oxidation of low-densitylipoprotein (Frankel et al 1993) and reduction of endothelin synthesis (Corder et al2001) Biochemical studies have shown that wine is rich in polyphenols (Scalbert ampWilliamson 2000) compounds that have several pharmacological properties includingvasodilator (Andriambeloson et al 1997) hypotensive (Diebolt et al 2001) andantioxidant eŒects (Frankel et al 1993) As hypertension is also a very important riskfactor for coronary heart disease the present study addressed the possibility that anantihypertensive eŒect of grape skin compounds could also participate in thecardioprotective eŒect of wine We tested the hypothesis that an alcohol-freehydroalcoholic grape skin extract (GSE) obtained from skins of Vitis labrusca avinifera grape largely used in Brazil to produce red wine might possess antihypertensiveaction Antioxidant and vasodilator eŒects of the extract were also assessed in the lipidperoxidation of microsomal protein and in the rodent isolated mesenteric vascular bedrespectively

Department of PharmacologyIBRAG-CB State University of Riode Janeiro Rio de Janeiro Brazil

R Soares de Moura F S CostaViana M A V Souza D CGuedes E P B Oliveira L M SRubenich L C R M CarvalhoR M Oliveira T Tano M LGusma4 o Correia

Department of BiochemistryIBRAG-CB State University of Riode Janeiro Rio de Janeiro Brazil

K Kovary

Correspondence R Soares deMoura Departamento deFarmacologia IBRAG-CBUniversidade do Estado do Riode Janeiro Av 28 de setembro87 Rio de Janeiro CEP 20551-030 Brazil E-mail demourauerjbr

Funding Supported in part byConselho Nacional de Pesquisase Fundac a4 o de Amparo Pesquisado Estado Rio de Janeiro

1515

1516 R Soares de Moura et al

Materials and Methods

Preparation of GSE

V labrusca (Isabel varietal) red grapes were obtained fromselected vineyards located it the State of Rio Grande doSul Brazil The grapes were washed in tap water and theskins were separated from the pulp Approximately 100 gof grape skin was boiled in 400 mL of distilled water for5 min and then minced Ethanol (400 mL)was added to thedecoction which was then shaken for 4 h and kept in darkbottles inside a refrigerator (4 deg C) for 20 days The hydro-alcoholic extract of V labrusca skins was reg ltered throughWhatman no 1 reg lter paper and the ethanol was evaporatedunder low pressure at 55 deg C The extract was thenlyophilized and frozen at reg 20 deg C until use Typically 100 gof wet skins yields about 89 g of lyophilized extract

Drugs

Norepinephrine (noradrenaline) bitartrate acetylcholinechloride NG-nitro- l -arginine methyl ester (L-NAME)tetraethylammonium (TEA) indometacin deoxycholicacid deoxycorticosterone acetate and chemical reagentswere purchased from Sigma (St Louis MO USA) Nitro-glycerin and glibenclamide were gifts from CristaliaProdutos Quimicos Farmaceuticos Ltd and Hoechst doBrazil respectively All drug solutions were freshly pre-pared before each experiment Norepinephrine acetyl-choline nitroglycerin L-NAME TEA and deoxycholicacid were dissolved in water or physiological salt solution(PSS) deoxycorticosterone was dissolved in corn oil indo-metacin was dissolved in ethanol and glibenclamide wasdissolved in 50 ethanol50 dimethylsulfoxide Theextract was dissolved in PSS drinking water or in saline

Animals

All experiments were reviewed and approved by the EthicsCommittee of Animal Experiments of State University ofRio de Janeiro The experiments were performed withadult male Wistar rats (250plusmn 350 g) obtained from theAnimal Care Facility of the Department of PharmacologyState University of Rio de Janeiro The animals werehoused in plastic cages (three rats per cage) Bodyweightheart rate systolic mean and diastolic arterial pressurewere measured three times a week Cardiovascular par-ameters (systolic mean and diastolic arterial pressure andheart rate) were measured by the tail cuŒmethod using aLetica LE 5000 device For arterial pressure measurementsanimals were trained for at least 2 weeks to stay still undera piece of cloth until the arterial pressure was steadilyrecorded with minimal restraint and stress The reg rstmeasurement of cardiovascular parameters was discardedand the mean of two or three subsequent measurementswas recorded each day

L-NAME hypertension

The animals were treated orally with 50 mg kgshy 1 L-Name(L-NAME group n macr 10) or with 50 mg kgshy 1 L-NAMEplus 100 mg kgshy 1 GSE (L-NAME shy GSE group n macr 10)

dissolved in drinking water daily for 28 days Bodyweightand cardiovascular parameters were measured three timesa week

Desoxycorticosterone acetate (DOCA)-salthypertension

Twelve rats were uninephrectomized under ether anaes-thesia After recovery from surgery the rats were trainedfor measurements of systolic mean and diastolic arterialpressure After the period of adaptation the animals weretreated subcutaneously with deoxycorticosterone acetate(125 mg kgshy 1 per week)and were given a 09 NaCl02KCl drinking solution ad libitum for 30 days Sixteen daysafter the beginning of DOCA-salt treatment when theanimals became hypertensive a group of rats was treatedorally with 100 mg kgshy 1 GSE (DOCA-salt shy GSE groupn macr 6) daily from Day 16 until Day 28 Another group ofrats was continuously treated with DOCA and 09NaCl02 KCl drinking solution (n macr 6) until the end ofthe experiments

Isolated mesenteric vascular bed

The rat superior mesenteric vascular bed was isolatedaccording to McGregor (1965) Briemacr y male Wistar rats(250plusmn 350 g) were killed with inhaled CO2 Then the mes-enteric vascular bed was cannulated and perfused with PSSat macr ow rate of 4 mL minshy 1 generated by a peristaltic pump(Lifecare Model 4 AbbottShaw) The PSS had the fol-lowing composition (mm ) NaCl 118 KCl 47 CaCl2 25MgSO4 12 KH2PO4 12 NaHCO3 25 EDTA 002 andglucose 11 The PSS (37 deg C) was bubbled with 95 O2

5CO2 Perfusion pressure was measured with a transducerconnected to a preamplireg er and chart recorded Drugswere either dissolved in PSS and perfused at the desiredconcentration or were administered as bolus injectionsdirectly into the perfusion stream (volume 100 L) Per-fusion pressure was elevated (80plusmn 110 mmHg) with norepi-nephrine (1plusmn 01 m ) added to the perfusion macr uid Thevasodilator eŒect of drugs was expressed as percentagedecrease in relation to the pressor eŒect of norepinephrineWhen the pressor eŒect of norepinephrine reached aplateau the dose response to bolus injections of GSEwas obtained Acetylcholine (10 pmol) and nitroglycerin(1 mol) were administered as bolus injections to assessendothelial function The vasodilator eŒect of GSE wasstudied in vessels pre-treated with deoxycholic acid(25 mm ) dissolved in PSS for 4 min to chemically removethe endothelium L-NAME (300 m ) TEA (1 mm ) gliben-clamide (1 m ) or indometacin (01 m ) As the GSEinduced a long-lasting inhibitory eŒect on the norepine-phrine constrictor eŒect only one doseplusmn response curve toGSE was obtained in each vascular bed preparation

Lipid peroxidation of hepatic microsomes andestimation of polyphenols

Hepatic microsomes were prepared from livers of Wistarrats by tissue homogenization in 5 vols of ice-cold 025 mm

sucrose 50 m m Tris-HCl pH 74 using a Temacr on Potter-

1517Cardiovascular effects of Vitis labrusca skin extract

Elvehjem homogenizer The homogenate was centrifugedfor 20 min at 10000 g and the resulting supernatant wascentrifuged for 60 min at 100 000 g The microsomal pelletwas suspended in 20 glycerol 50 mm Tris-HCl pH 74and stored at reg 80 deg C Microsomal protein was determinedby the method described by Petersen (1977) with bovineserum albumin as standard For induction of lipid per-oxidation 1 mg of microsomal protein was treated with50 m ferrous iron (stock solution 100 m Fe(NH4)2 in005 m HCl) and 500 m H2O2 in 500 L (reg nal volume) of50 mm Tris-HCl pH 74 in the absence or presence ofdiŒerent concentrations of GSE After incubation for40 min at 37 deg C the samples were treated with 500 L of20 trichloroacetic acid (wv) and 10 L of 2 butylatedhydroxytoluene (wv) in ethanol After centrifugation at3000 g for 5 min supernatants were reacted with an equalvolume of 067 thiobarbituric acid (TBA) (wv) andincubated for 1 h in a boiling water bath The malondi-aldehyde (MDA)TBA complexes were detected on aShimadzu high-performance liquid chromatography sys-tem using the method described by Young amp Trimble(1991) with minor modireg cations

The concentration of polyphenols in lyophilized GSEmeasured by analysing for total phenol by the Folin-Ciocalteau procedure (Singleton amp Rossi 1965) was555 mg gshy 1

Statistical analysis

All results are presented as mean sup3 sem for the number ofrats One-way analysis of variance and the Studentrsquo s un-paired t-test were used for statistical analysis Values ofP 005 were considered statistically signireg cant

Results

Antihypertensive effect of GSE in L-NAMEhypertensive rats

The baseline systolic mean and diastolic arterial pressuresin L-NAME and L-NAME shy GSE groups were not signireg -cantly diŒerent (L-NAME group 157 sup3 3 96 sup3 2 and

L-NAMEL-NAME + GSE

140

120

100

80

60 L-NAMEL-NAMEL-NAME

L-NAME L-NAMEL-NAME + GSEL-NAME + GSE

160

140

120

100

80

200

180

160

ndash10 ndash5 0 5 10 15 20 25 30ndash10 ndash5 0 5 10 15 20 25 30ndash10 ndash5 0 5 10 15 20 25 30 Dia

sto

lic a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Mea

n a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Syst

olic

art

eria

l blo

od

pre

ssu

re

(m

mH

g)

Time (days)Time (days)Time (days)

Figure 1 EŒects of grape skin extract (GSE) on systolic mean and diastolic arterial pressure in rats treated with NG-nitro-l -arginine methyl

ester (L-NAME) From Day 0 (arrow) the animals were treated daily with oral L-NAME (50 mg kgshy 1 dayshy 1) or with oral L-NAME

(50 mg kgshy 1 dayshy 1) plus GSE (100 mg kgshy 1 dayshy 1) in drinking water P 005 (analysis of variance)

76sup3 4 mmHg L-NAME shy GSE group 160 sup3 2 100 sup3 3and 72 sup3 4 mmHg respectively) Four weeks of L-NAMEadministration increased systolic mean and diastolic ar-terial pressure to 198 sup3 2 154 sup3 3 and 131 sup3 3 mmHg in theL-NAME group and to 170 sup3 3 119 sup3 3 and 103 sup3 3 mmHgin the L-NAME shy GSE group respectively (P 005 be-tween L-NAME and L-NAME shy GSE groups) Figure 1shows the time course of changes in arterial pressure inboth groups Heart rate decreased in both groups in thereg rst week of treatment At the end of treatment the heartrate of the L-NAME group (256 sup3 4 beats minshy 1) wassignireg cantly lower then that of the L-NAME shy GSE group(281 sup3 5 beats minshy 1) The bodyweight of the L-NAMEgroup was not diŒerent compared with the L-NAME shy GSE group at the beginning of L-NAME treat-ment but at the end of the study the bodyweight of theL-NAME shy GSE group (342 sup3 8 g) was signireg cantlyhigher than that of the L-NAME group (306 sup3 8 g)

Antihypertensive effect of GSE in DOCA-salthypertensive rats

The baseline systolic mean and diastolic arterial pressurein DOCA-salt and DOCA-salt shy GSE groups were notsignireg cantly diŒerent (DOCA-salt group 193 sup3 3 113 sup3 8and 73sup3 3 mmHg DOCA-salt shy GSE group 194 sup3 5120 sup3 3 and 83 sup3 2 mmHg respectively) Administration ofDOCA in both groups induced a continuous increase inarterial pressure By Day 16 of DOCA-salt treatment thesystolic mean and diastolic arterial pressures were 223 sup3 9165 sup3 10 and 137 sup3 11 mmHg in the DOCA-salt group and212 sup3 4 156 sup3 6 and 130 sup3 8 mmHg in the DOCA-salt shy GSE group respectively The oral administration ofGSE to the DOCA-salt shy GSE group from Day 16 to Day28 reduced the increases in arterial pressure By Day 28 thesystolic mean and diastolic arterial pressures in the DOCA-salt shy GSE group were signireg cantly lower than the arterialpressures observed in the DOCA-salt group (DOCA-saltgroup 246 sup3 5 192 sup3 9 and 164 sup3 12 mmHg DOCA-salt shy GSE group 221 sup3 3 146 sup3 3 and 107 sup3 4 mmHgrespectively) Figure 2 shows the time course changes insystolic mean and diastolic arterial pressure in both groups


Dia

sto

lic a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Mea

n a

rter

ial b

loo

d p

ress

ure

(

mm

Hg

)

Syst

olic

art

eria

l blo

od

pre

ssu

re

(m

mH

g)

260

240

220

200

180

200 180

160

140

120

10080

60

DOCA-saltDOCA-salt + GSE

180

160

140

120

100

80

Time (days)ndash5 0 5 10 15 20 25 30 ndash5 0 5 10 15 20 25 30 ndash5 0 5 10 15 20 25 303535

Time (days)Time (days)

DOCA-saltDOCA-saltGSE GSE GSE



DOCA-salt

Figure 2 EŒects of grape skin extract (GSE) on systolic mean and diastolic arterial pressure in uninephrectomized rats treated with

deoxycorticosterone acetate (DOCA) and salt solution From Day 0 (arrow) rats were treated subcutaneously with DOCA (125 mg kgshy 1 per

week) and 09 NaCl02 KCl solution for drinking From Day 16 six rats were treated with DOCA-salt plus oral GSE (100 mg kgshy 1 dayshy 1)

until Day 28 The other six rats were treated only with DOCA-salt from Day 0 (arrow) until Day 30 P 005 (analysis of variance)

Vasodilator effect of GSE in the isolatedvascular mesenteric bed

The basal perfusion pressure was 29 sup3 3 mmHg Theincrease in perfusion pressure induced by norepineph-rine in control deoxycholic acid- L-NAME- TEA-indometacin- and glibenclamide-treated groups were85sup3 12 83 sup3 10 119 sup3 9 113 sup3 6 80 sup3 6 and 88 sup3 11 mmHgrespectively In vessels pre-contracted with norepinephrinebolus injections of GSE induced a dose-dependent vaso-dilator response The vasodilator eŒect of GSE was notaltered by TEA glibenclamide or indometacin but wassignireg cantly reduced by L-NAME (Figure 3) The per-centage vasodilator eŒects of acetylcholine and GSE (30

0

20

40

60

80

100

CONTROL

L - NAME

INDOMETACIN

TEA

GLIBENCLAMIDE

1 10 100Grape skin extract (mg)

Rel

axat

ion

(

)

Figure 3 Doseplusmn response curves for grape skin extract in nor-

epinephrine pre-contracted rat vascular mesenteric bed in the absence

(control) or presence of NG-nitro-l -arginine methyl ester (L-NAME)

indometacin tetraethylammonium (TEA) or glibenclamide P 005 (analysis of variance)

60 and 100 g)before deoxycholic acid were 40 sup3 3 31 sup3 1061sup3 9 and 76 sup3 5 respectively The percentage vasodilatoreŒects of acetylcholine and GSE were signireg cantly reducedafter endothelium removal (0sup3 0 0 sup3 0 1 sup3 1 and 3 sup3 2respectively) However the vasodilator eŒect of nitro-glycerin obtained before deoxycholic acid (65 sup3 8 )was not signireg cantly changed by endothelium removal(63 sup3 7 )

Antioxidant effect and polyphenolconcentration

As shown in Figure 4 GSE induced a concentration-dependent inhibition of iron-induced lipid peroxidation inrat liver microsomes as measured by the decrease in MDAformation

Discussion

The aim of the present study was to investigate the anti-hypertensive eŒect of an alcohol-free hydroalcoholic ex-tract prepared from skins of V labrusca a vinifera redgrape largely used in Brazil for wine production Approxi-mately 220 million litres of wine obtained from V labrusca

are consumed in Brazil each year The present study showsthat oral administration of 100 mg kgshy 1 dayshy 1 V labrusca

GSE which corresponds to 55 mg kgshy 1 dayshy 1 of poly-phenols (approximately the amount occurring in about275 mL kgshy 1 dayshy 1 of red wine Lo pez et al 2001) inducesa substantial antihypertensive eŒect in DOCA-salt hy-pertensive rats and prevents L-NAME-induced hyper-tension in rats Similarly the ethanol-free hydroalcoholicextract obtained from Cabernet sauvignon grape skin hasbeen shown to produce an antihypertensive eŒect (Soaresde Moura 2001) We have also observed that hydro-alcoholic extract obtained from the pulp of V labrusca

grapes did not reduce the hypertension induced by L-NAME in Wistar rats (R Soares de Moura unpublishedresults) It is possible that the antihypertensive eŒect of the


10

8

6

4

0

2

00 02 04 06 08 10Grape skin (mg mLndash1)

Mal

on

dia

ldeh

yde

pro

du

ctio

n

(n

mo

l (m

g p

rote

in)ndash1

)

Figure 4 EŒect of grape skin extract on the iron-induced lipid

peroxidation of rat liver microsomes

compounds of the hydroalcoholic extract of vinifera grapeskins which may also occur in wine could potentially playan important role in the cardioprotective eŒect of moderatered wine consumption (St Leger et al 1979)

The mechanism of the antihypertensive eŒect of GSE isnot known Given that our extract was obtained from avinifera grape the extract may share some eŒects similar tothose encountered in wine and other grape products Wineand grape products have been shown to induce manyimportant cardiovascular eŒects The eŒects of wine onvascular smooth muscle are controversial Red wine hasbeen shown to induce either vasodilatation (Fitzpatrick etal 1993 Flesch et al 1998) or no vascular eŒect (Rending etal 2001) in isolated vessels These controversial reg ndingsmay be owing to diŒerent wine varieties or even diversevessels studied A direct endothelium-dependent vaso-dilator eŒect of wine produced in oak barrels but not insteel tanks has been demonstrated in isolated vessels(Flesch et al 1998) Products obtained from red winesuch as polyphenol compounds induce vasodilatation(Andriambeloson et al 1997 1999) and reduce arterialpressure in normotensive rats (Diebolt et al 2001)

The present results demonstrate that GSE also has asubstantial direct vasodilator eŒect in the isolated mes-enteric vascular bed of the rat This inhibitory eŒect on thevascular smooth muscle is not inhibited by indometacinTEA or glibenclamide suggesting that the release of vaso-dilator prostanoids activation of Ca+2- or ATP-dependentpotassium channels do not play an important role in thevasodilator eŒect of GSE The vasodilator eŒect of GSE isendothelium-dependent because in the isolated mesentericvascular bed pre-treated with deoxycholic acid the vaso-dilatation induced by GSE and acetylcholine but notnitroglycerin was signireg cantly reduced Nitric oxide animportant modulator of vascular function (Vanhoutte ampMombouli 1996) participates in the vasodilator eŒect ofwine (Fitzpatrick et al 1993 Flesch et al 1998) and wine-

derived polyphenols (Andriambeloson et al 1999 Stocletet al 1999) Importantly nitric oxide is also released afterred wine ingestion in humans (Matsuo et al 2001) There-fore nitric oxide probably plays a very important role inthe vasodilator eŒect of GSE given that L-NAME aninhibitor of nitric oxide synthase signireg cantly reduced thevasodilator eŒect of GSE in the isolated mesenteric vascularbed of rats We speculate that the antihypertensive eŒect ofGSE may also be owing to its vasodilator eŒect

The antihypertensive eŒect of GSE probably does notinvolve modulation of the reninplusmn angiotensin system sincethe antihypertensive eŒect was observed in DOCA-salthypertensive rats a low-renin model of experimental hy-pertension An anti-adrenergic action of GSE also appearsto be unlikely because the vasodilator eŒect of the extractwas signireg cantly reduced in vessels without endotheliumand contracted with norepinephrine We could speculatethat the antioxidant eŒect of the extract demonstrated inour study could play an important role in the antihyper-tensive eŒect since GSE reduced the MDA formation in-vitro Previous experimental results have shown that oxi-dative stress increases in hypertension induced by inhibitionof NO synthase (Usui et al 1999) or by salt supplementationin uninephrectomized rats treated with DOCA (Wu et al2001) However our in-vitro results are apparently not inagreement with the reg ndings of Young et al (2000) whodemonstrated that oral ingestion of GSE in humans didnot change the MDA plasma levels This diŒerence may beowing to the distinct grape skin preparation or dose usedRecently it has been demonstrated that polyphenols fromred wine decrease endothelin-1 synthesis in cultured bovineaortic endothelial cells (Corder et al 2001) Consideringthat endothelin-1 plays a very important role in DOCA-salt (de Carvalho et al 1990) and in L-NAME hypertension(Naruse et al 2000) we also speculate that the reduction ofarterial pressure induced by GSE might involve the re-duction of endothelin-1 synthesis

Increases in arterial pressure induced by L-NAMEproduced a decrease in heart rate probably owing tobaroreceptor activation However in rats treated with L-NAME plus GSE the bradycardia was smaller probablydue to lesser increases in arterial pressure

Rats treated with L-NAME showed a reduction inbodyweight as the pressure increased However in animalstreated with L-NAME plus GSE the decrease in body-weight was signireg cantly smaller The mechanism of weightreduction induced by chronic L-NAME treatment is notknown As NO seems to modulate food intake in rodents(Morley amp Flood 1991) cerebral reduction on NO for-mation by L-NAME could participate in this eŒect At themoment we cannot propose a mechanism for the GSE-dependent inhibition of L-NAME-induced weight loss

The quality and chemical composition of wine variesaccording to many factors such as grape species cultivarvineyard terroir time of fermentation and ageing Thepercentage of wine polyphenols compounds that inducevasodilatation in isolated vessels varies among wines(Vin4 as et al 2000) and in-vitro studies have shown thatonly red wine produced in oak barrels induces vaso-dilatation in isolated rat aorta (Flesch et al 1998) However


wine ageing in oak barrels does not seem to be importantfor the vasodilator eŒect of vinifera grapes as previouslysuggested (Flesch et al 1998) since we demonstrated thatGSE which contains 555 mg gshy 1 polyphenols a concen-tration similar to that obtained in the lyophilized residue ofthe red wine Cabernet sauvignon (952 mg gshy 1 L M SRubenich unpublished observation) has substantial vaso-dilator antihypertensive and antioxidant eŒects eventhough the extract was not submitted to the ageing processNevertheless augmentation of some pharmacologicalproperties of compounds present in grape skin during theprocess of wine ageing in oak barrels might occur

In conclusion the present results provide experimentalevidence that an ethanol-free extract obtained from viniferagrape skin exhibits signireg cant antihypertensive vasodilatorand antioxidant eŒects The combination of these actionspresent in the vinifera grape skin may help explain whymoderate red wine consumption has a protective eŒectagainst cardiovascular diseases Although wine consump-tion might protect against coronary heart disease assuggested by many studies the harmful eŒects of ethanolsuch as pancreatitis liver dysfunction and addiction couldlimit the medical indication of chronic wine ingestionTherefore an alcohol-free product obtained from viniferagrapes with similar pharmacological actions as wine mightbe a potential medicine for prevention and treatment ofcoronary heart diseases and hypertension

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J Chromatogr 872 85plusmn 93

Wu R Millette E Wu L de Champlain J (2001) Enhanced

superoxide anion formation in vascular tissues from spontaneously

hypertensive and desoxycorticosterone acetate-salt hypertensive

rats J Hypertens 19 741plusmn 748

Young I S Trimble E R (1991)Measurementof malondialdehyde

in plasma by high performance liquid chromatography with macr uori-

metric detection Ann Clin Biochem 28 504plusmn 508

Young J F Dragsted L O Daneshvar B Lauridsen S T

Hansen M Sandstrom B (2000) The eŒect of grape-skin extract

on oxidative status Br J Nutr 84 505plusmn 513


Materials and Methods

Preparation of GSE

V labrusca (Isabel varietal) red grapes were obtained fromselected vineyards located it the State of Rio Grande doSul Brazil The grapes were washed in tap water and theskins were separated from the pulp Approximately 100 gof grape skin was boiled in 400 mL of distilled water for5 min and then minced Ethanol (400 mL)was added to thedecoction which was then shaken for 4 h and kept in darkbottles inside a refrigerator (4 deg C) for 20 days The hydro-alcoholic extract of V labrusca skins was reg ltered throughWhatman no 1 reg lter paper and the ethanol was evaporatedunder low pressure at 55 deg C The extract was thenlyophilized and frozen at reg 20 deg C until use Typically 100 gof wet skins yields about 89 g of lyophilized extract

Drugs

Norepinephrine (noradrenaline) bitartrate acetylcholinechloride NG-nitro- l -arginine methyl ester (L-NAME)tetraethylammonium (TEA) indometacin deoxycholicacid deoxycorticosterone acetate and chemical reagentswere purchased from Sigma (St Louis MO USA) Nitro-glycerin and glibenclamide were gifts from CristaliaProdutos Quimicos Farmaceuticos Ltd and Hoechst doBrazil respectively All drug solutions were freshly pre-pared before each experiment Norepinephrine acetyl-choline nitroglycerin L-NAME TEA and deoxycholicacid were dissolved in water or physiological salt solution(PSS) deoxycorticosterone was dissolved in corn oil indo-metacin was dissolved in ethanol and glibenclamide wasdissolved in 50 ethanol50 dimethylsulfoxide Theextract was dissolved in PSS drinking water or in saline

Animals

All experiments were reviewed and approved by the EthicsCommittee of Animal Experiments of State University ofRio de Janeiro The experiments were performed withadult male Wistar rats (250plusmn 350 g) obtained from theAnimal Care Facility of the Department of PharmacologyState University of Rio de Janeiro The animals werehoused in plastic cages (three rats per cage) Bodyweightheart rate systolic mean and diastolic arterial pressurewere measured three times a week Cardiovascular par-ameters (systolic mean and diastolic arterial pressure andheart rate) were measured by the tail cuŒmethod using aLetica LE 5000 device For arterial pressure measurementsanimals were trained for at least 2 weeks to stay still undera piece of cloth until the arterial pressure was steadilyrecorded with minimal restraint and stress The reg rstmeasurement of cardiovascular parameters was discardedand the mean of two or three subsequent measurementswas recorded each day

L-NAME hypertension

The animals were treated orally with 50 mg kgshy 1 L-Name(L-NAME group n macr 10) or with 50 mg kgshy 1 L-NAMEplus 100 mg kgshy 1 GSE (L-NAME shy GSE group n macr 10)

dissolved in drinking water daily for 28 days Bodyweightand cardiovascular parameters were measured three timesa week

Desoxycorticosterone acetate (DOCA)-salthypertension

Twelve rats were uninephrectomized under ether anaes-thesia After recovery from surgery the rats were trainedfor measurements of systolic mean and diastolic arterialpressure After the period of adaptation the animals weretreated subcutaneously with deoxycorticosterone acetate(125 mg kgshy 1 per week)and were given a 09 NaCl02KCl drinking solution ad libitum for 30 days Sixteen daysafter the beginning of DOCA-salt treatment when theanimals became hypertensive a group of rats was treatedorally with 100 mg kgshy 1 GSE (DOCA-salt shy GSE groupn macr 6) daily from Day 16 until Day 28 Another group ofrats was continuously treated with DOCA and 09NaCl02 KCl drinking solution (n macr 6) until the end ofthe experiments

Isolated mesenteric vascular bed

The rat superior mesenteric vascular bed was isolatedaccording to McGregor (1965) Briemacr y male Wistar rats(250plusmn 350 g) were killed with inhaled CO2 Then the mes-enteric vascular bed was cannulated and perfused with PSSat macr ow rate of 4 mL minshy 1 generated by a peristaltic pump(Lifecare Model 4 AbbottShaw) The PSS had the fol-lowing composition (mm ) NaCl 118 KCl 47 CaCl2 25MgSO4 12 KH2PO4 12 NaHCO3 25 EDTA 002 andglucose 11 The PSS (37 deg C) was bubbled with 95 O2

5CO2 Perfusion pressure was measured with a transducerconnected to a preamplireg er and chart recorded Drugswere either dissolved in PSS and perfused at the desiredconcentration or were administered as bolus injectionsdirectly into the perfusion stream (volume 100 L) Per-fusion pressure was elevated (80plusmn 110 mmHg) with norepi-nephrine (1plusmn 01 m ) added to the perfusion macr uid Thevasodilator eŒect of drugs was expressed as percentagedecrease in relation to the pressor eŒect of norepinephrineWhen the pressor eŒect of norepinephrine reached aplateau the dose response to bolus injections of GSEwas obtained Acetylcholine (10 pmol) and nitroglycerin(1 mol) were administered as bolus injections to assessendothelial function The vasodilator eŒect of GSE wasstudied in vessels pre-treated with deoxycholic acid(25 mm ) dissolved in PSS for 4 min to chemically removethe endothelium L-NAME (300 m ) TEA (1 mm ) gliben-clamide (1 m ) or indometacin (01 m ) As the GSEinduced a long-lasting inhibitory eŒect on the norepine-phrine constrictor eŒect only one doseplusmn response curve toGSE was obtained in each vascular bed preparation

Lipid peroxidation of hepatic microsomes andestimation of polyphenols

Hepatic microsomes were prepared from livers of Wistarrats by tissue homogenization in 5 vols of ice-cold 025 mm

sucrose 50 m m Tris-HCl pH 74 using a Temacr on Potter-






Results



L-NAMEL-NAME + GSE

140

120

100

80



160

140

120

100

80

200

180

160


sto

lic a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Mea

n a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Syst

olic

art

eria

l blo

od

pre

ssu

re

(m

mH

g)









Dia

sto

lic a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Mea

n a

rter

ial b

loo

d p

ress

ure

(

mm

Hg

)

Syst

olic

art

eria

l blo

od

pre

ssu

re

(m

mH

g)

260

240

220

200

180

200 180

160

140

120

10080

60


180

160

140

120

100

80






DOCA-salt







0

20

40

60

80

100

CONTROL

L - NAME

INDOMETACIN

TEA

GLIBENCLAMIDE


Rel

axat

ion

(

)








Discussion






10

8

6

4

0

2


Mal

on

dia

ldeh

yde

pro

du

ctio

n

(n

mo

l (m

g p

rote

in)ndash1

)














References



































359plusmn 363




1029plusmn 1031

















219plusmn 227









































Results



L-NAMEL-NAME + GSE

140

120

100

80



160

140

120

100

80

200

180

160


sto

lic a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Mea

n a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Syst

olic

art

eria

l blo

od

pre

ssu

re

(m

mH

g)









Dia

sto

lic a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Mea

n a

rter

ial b

loo

d p

ress

ure

(

mm

Hg

)

Syst

olic

art

eria

l blo

od

pre

ssu

re

(m

mH

g)

260

240

220

200

180

200 180

160

140

120

10080

60


180

160

140

120

100

80






DOCA-salt







0

20

40

60

80

100

CONTROL

L - NAME

INDOMETACIN

TEA

GLIBENCLAMIDE


Rel

axat

ion

(

)








Discussion






10

8

6

4

0

2


Mal

on

dia

ldeh

yde

pro

du

ctio

n

(n

mo

l (m

g p

rote

in)ndash1

)














References



































359plusmn 363




1029plusmn 1031

















219plusmn 227





































Dia

sto

lic a

rter

ial b

loo

d p

ress

ure

(mm

Hg

)

Mea

n a

rter

ial b

loo

d p

ress

ure

(

mm

Hg

)

Syst

olic

art

eria

l blo

od

pre

ssu

re

(m

mH

g)

260

240

220

200

180

200 180

160

140

120

10080

60


180

160

140

120

100

80






DOCA-salt







0

20

40

60

80

100

CONTROL

L - NAME

INDOMETACIN

TEA

GLIBENCLAMIDE


Rel

axat

ion

(

)








Discussion






10

8

6

4

0

2


Mal

on

dia

ldeh

yde

pro

du

ctio

n

(n

mo

l (m

g p

rote

in)ndash1

)














References



































359plusmn 363




1029plusmn 1031

















219plusmn 227





































10

8

6

4

0

2


Mal

on

dia

ldeh

yde

pro

du

ctio

n

(n

mo

l (m

g p

rote

in)ndash1

)














References



































359plusmn 363




1029plusmn 1031

















219plusmn 227







































References



































359plusmn 363




1029plusmn 1031

















219plusmn 227




































antihypertensive, vasodilator and antioxidant effects of a vinifera grape skin extract

Documents