Biosci. Biotechnol. Biochem., 76 (5), 954–960, 2012"-Viniferin Is More Eﬀective Than Its Monomer Resveratrol in Improving
the Functions of Vascular Endothelial Cells and the Heart
Nahla Z GHONDA,1 Shigeki Y OSHIDA,1 Shoji E ZAKI,1 Yoshie O TAKE,1 Chie M URAKAMI,1
Ahmed M LIKI,2 Abdelwahed G HORBEL,2 and Hitoshi M IYAZAKI1; y1
2Graduate School of Life and Environmental Sciences, Tsukuba University, Ibaraki 305-8572, Japan
Laboratory of Molecular Physiology of Grapevine, CBBC, B.P. 174, 1164 Hammam-Chatt, Tunisia

Received December 21, 2011; Accepted January 25, 2012; Online Publication, May 7, 2012
[doi:10.1271/bbb.110975]

The present study compared the eﬀects of resveratrol
and its dimer "-viniferin on vascular endothelial cells(VECs) functions, and on the blood pressure andcardiac mass of spontaneously hypertensive rats
(SHRs). Treatment of VECs with these compounds
enhanced cell proliferation via nitric oxide generationand protected the cells from oxidative stress by suppressing increases in intracellular oxygen species. "Viniferin was more potent than resveratrol in most ofthese eﬀects. "-Viniferin, but not resveratrol inhibitedangiotensin-converting enzyme activity in vitro. Three
weeks of "-viniferin treatment (5 mg/kg) reduced thesystolic blood pressure and improved the whole cardiac
mass and left ventricle mass indexes in SHRs. In
contrast, resveratrol administration (2.5 mg/kg) failed
to lower the blood pressure and signiﬁcantly improvethese mass indexes. These data suggest that "-viniferin
as well as resveratrol may be involved in protecting thefunctions of VECs and the heart.
Key words:

angiotensin-converting enzyme; "-viniferin;
resveratrol; spontaneously hypertensive rat;
vascular endothelial cell

Hypertension is the major risk factor for coronary
artery disease and a leading cause of mortality worldwide. The renin-angiotensin system (RAS) plays animportant role in the pathogenesis of hypertension andcardiac hypertrophy.1) These phenomena are attributablein part to excessive production of reactive oxygen
species (ROS), particularly superoxide and hydrogen
peroxide. ROS formation impairs nitric oxide (NO)generation and contributes to hypertension.2)
Polyphenols from grapes and wines are known todecrease the incidence of coronary heart disease. Diebolt
et al.3) found that short-term oral administration of red
wine polyphenolic compounds decreased blood pressure
in normotensive rats and enhanced endothelium-dependent relaxation. Mizutani et al.4) also indicated thatextracts of wine phenolics attenuated the elevation ofblood pressure in stroke-prone spontaneously hypertensive rats (SHRs). Stilbenes are the most importantpolyphenolic subclass occurring naturally in grapes andrelated products. Among them, resveratrol (3,40 ,5-trihydroxystilbene) has been the most widely studied for its
role in human health (Fig. 1A). The cardioprotectioneﬀect of resveratrol has been documented in diﬀerent
experimental settings of pressure overload.5,6) In SHRs,
a well-established genetic model of hypertension andsubsequent cardiac hypertrophy, resveratrol attenuatedthe pathological cardiac hypertrophy without lowering
blood pressure.7,8) In contrast, it reduced hypertension inmale Sprague-Dawely rats9) and obese Zucker rats.10) In
contrast to many reports describing the beneﬁcial eﬀects
of resveratrol on the vasculature and heart, there arevery few reports concerning the beneﬁcial functions of"-viniferin, a natural dehydrodimer of resveratrol. "Viniferin content is comparable to or greater thanresveratrol content, depending on the type of red wine
and the stage of noble rot development of grapes.11,12)Tran et al.13) indicated that "-viniferin has enhanced
antioxidant activity as compared to resveratrol. Mi
et al.14) demonstrated that "-viniferin was more potent
than resveratrol in inducing the vasorelaxation ofisolated thoracic aortas. We recently found that "viniferin is more eﬀective than resveratrol in inhibiting
platelet-derived growth factor (PDGF)-dependent cellproliferation and migration of cultured vascular smoothmuscle cells by reducing the increased intracellular
ROS levels induced by PDGF.15) These data suggest that"-viniferin as well as resveratrol may contribute topreventing the pathogenesis of atherosclerosis.
Angiotensin-converting enzyme (ACE) in RAS catalyzes the formation of angiotensin II from angiotensin I,
and also hydrolyses and inactivates the vasodilator
peptide bradykinin. ACE inhibition is an importanttherapeutic approach in the treatment of hypertension.
ACE inhibitors not only lower the blood pressure but
also ameliorate heart failure.16,17) Indeed, a number ofplant-derived compounds have been found to posses
in vitro ACE inhibitory activity, including procyanidins,
ﬂavanols,18,19) tannins,20) and peptides.21,22)
The present study was designed to compare the eﬀects
of resveratrol with those of "-viniferin on vascularendothelial cells (VECs) and ACE in vitro, and on bloodpressure and cardiac mass in SHRs.

y
To whom correspondence should be addressed. Fax: +81-29-853-7723; E-mail: hitomy1@sakura.cc.tsukuba.ac.jp
Abbreviations: ACE, angitonsin-converting enzyme; DAF-2DA, diaminoﬂuorescein-2 diacetate; DBP, diastolic blood pressure; DCF-DA,
0 0
2 ,7 -dichloroﬂuorescein diacetate; EGC, epigallocatechin; GPx, glutathione peroxidase; L-NAME, NG-nitro-L-arginine methyl ester; LPA,
lysophosphatidic acid; LV, left ventricle; NO, nitric oxide; ROS, reactive oxygen species; SBP, systolic blood pressure; SHR, spontaneously
hypertensive rat; VEC, vascular endothelial cell; WKY, Wistar-Kyoto rat

Eﬀects of "-Viniferin and Resveratrol on VECs and SHR

Materials and Methods
Materials. Trans-resveratrol and lysophosphatidic acid (LPA) werepurchased from Cayman Chemical (Tokyo). Epigallocatechin (EGC)
was obtained from Mitsui Norin (Tokyo). Dulbecco’s Modiﬁed Eagle’s
Medium (DMEM), fetal bovine serum (FBS), penicillin and streptomycin, Hanks’ balanced salt solution (HBSS), 20 ,70 -dichloroﬂuoresceindiacetate (DCF-DA), L-arginine, glutathione reductase (GR), trypsinEDTA, and corn oil were purchased from Sigma Chemical (St. Louis,
MO). Diaminoﬂuorescein-2 diacetate (DAF-2DA) was from Daiichi
Pure Chemicals (Tokyo). Glutathione reduced (GSH) was purchasedfrom MP Biomedicals (Illkrich). NG-nitro-L-arginine methyl ester(L-NAME) was obtained from Wako Pure Chemical Industries (Osaka,
Japan). Anti-eNOS antibody was from Cell Signaling Technology(Beverly, MA).
Puriﬁcation of "-viniferin. Polyphenolic compounds containing
resveratrol and "-viniferin derived from grapes were kindly provided
by Sun Bright (Tokyo), and "-viniferin was puriﬁed by the Gilson
Auto-Preparative HPLC System (Gilson, Middleton, WI) with a purityof about 98%. Separation was achieved on a Mightysil RP-18 column(10 mm i.d.  250 mm, Kanto Chemical, Tokyo).
Cell culture. VECs were isolated from porcine pulmonary arteries,
obtained at a local abattoir (Tsuchiura, Japan). The cells were cultured
in DMEM containing low amounts of glucose supplemented with 10%
FBS, 100 units/mL penicillin, and 100 mg/mL streptomycin at 37  C ina humidiﬁed 5% CO2 atmosphere.23) The responses of VECs to thecompounds were analyzed between passages 8 and 10 after 16-h
incubation in DMEM containing 1% FBS. Resveratrol and "-viniferindissolved in dimethyl sulfoxide (DMSO) were added to the medium at
a ﬁnal DMSO concentration of 1%. The same ﬁnal concentration of
DMSO was used in place of these reagents in control treatments.
Wound repair. Serum-starved VECs (3  105 cells/well) plated
onto 35-mm dishes were pretreated with various concentrations ofresveratrol or "-viniferin for 30 min. Then VEC monolayers weremanually displaced by scratching using a sterile 200 mL pipette tip to
generate a clean area of constant diameter.23) The cells were incubatedwith LPA, resveratrol, or "-viniferin at 37  C for 24 h. Immediately
after injury (0 h) and at 8 h and 24 h, the wounds were photographedwith an inverted microscope attached to a digital camera (Leica,
Wetzlar) and the cells that had migrated into the cell-free area werecounted. The number of these cells was determined in three randomly
selected microscopic ﬁelds.
Cell proliferation assay. Serum-starved VECs (2  105 cells/well)plated onto 35-mm dishes were treated with and without L-NAME for
1 h before adding various concentrations of resveratrol or "-viniferin
for 24 h. Cell proliferation was then determined by counting trypan
blue negative cells (viable cells) by trypan blue dye exclusion using ahemacytometer after trypsinization.23)
Cell viability. Hydrogen peroxide (H2 O2 )-induced cytotoxicity as to
VECs was evaluated in preliminary experiments by incubating thecells with increasing concentrations (0–700 mM) of H2 O2 for 24 h.
Because about 50% of cell viability was lost at 500 mM, thisconcentration was used in all subsequent cell viability investigations.
Serum-starved VECs (2  105 cells/well) plated onto 35-mm dishes
were pretreated with various concentrations of resveratrol or "viniferin for 24 h and then H2 O2 was added for another 24 h. Cell
viability responses were determined by counting the number of viable
cells per total cells by trypan blue dye exclusion.23)
Measurement of NO production. The ﬂuorescence reagent DAF2DA was used to measure NO production.24) Brieﬂy, VECs were
loaded with 10 mM DAF-2DA (Daiichi Pure Chemicals, Tokyo) for
30 min. Thereafter, the cells were co-incubated with 100 mM L-arginine(Sigma, St. Louis, MO) and various concentrations of resveratrol or"-viniferin for 2, 4, 6, and 8 h. Production of NO was estimated byrecording the ﬂuorescence at 485 nm as excitation and 530 nm as
emission using a luminescence spectrophotometer (PerkinElmer,
Waltham, MA).

955

Western blotting. After incubation with reagents, the cells werelysed with ice-cold lysis buﬀer consisting of 20 mM Tris–HCl pH 7.5,
150 mM NaCl, 1 mM EDTA, 10% glycerol (vol/vol), 100 mM NaF,
10 mM sodium pyrophosphate, 1% TritonX-100, 1 mM Na3 VO4 , 1 mM
PMSF, 10 mg/mL antipain, 10 mg/mL leupeptin, 10 mg/mL aprotinin,and centrifuged at 14,000 rpm at 4  C for 15 min. The supernatant wascollected and used for subsequent analyses. The protein concentrationof the cell lysates was determined using BCA reagents (Pierce,
Rockford, IL). After SDS–PAGE, the proteins were transferred topolyvinylidene diﬂuoride membranes. After blocking, the membranes
were incubated with an anti-phospho-eNOS antibody and then withperoxidase-conjugated secondary antibody. Blots were developed witha chemiluminescence kit according to the manufacturer’s instructions(Nacalai Tesque, Kyoto, Japan).
Determination of intracellular ROS. The generation of intracellular
ROS was monitored spectroﬂuorometrically using the oxidant-sensitive probe DCF-DA (Sigma) as described previously.25) Fluorescence
was monitored by confocal laser scanning microscopy (TCPSP2,
Leica) at an excitation wavelength of 488 nm and an emission
wavelength of 500–630 nm. Data were expressed as changes relative toinitial ﬂuorescence.
Catalase activity assay. VECs were treated with resveratrol or"-viniferin for 12, 24, and 48 h. After these time periods, the cells were
harvested in RIPA buﬀer and lysed by sonication. The sonicated cells
were centrifuged and the total protein concentration in the supernatant
was determined using BCA reagents (Pierce). H2 O2 at 10 mM (80 mL)
was incubated with 10 mL of 10 mM Tris–HCl (pH 7.4)/1 mM EDTA at
37  C for 2 min, and mixed with 10 mL of cell lysates. The absorbancewas measured at 240 nm at 25  C for 5 min; the changes in absorbanceover time are proportional to the H2 O2 decomposition. Catalase
activity was expressed as micromoles of H2 O2 consumed per min per
mg protein.
Glutathione peroxidase (GPx) activity assay. VECs and lysates
were prepared as described for the catalase activity assay. Thereafter,
50 mL of lysates was added to a reaction mixture containing 50 mL of
1  TE, 290 mL H2 O, 10 mL of 100 mM GSH, 50 mL of 10 U/mL
glutathione reductase, and 50 mL of 2 mM NADPH, and incubated at
37  C for 2 min. Then 10 mL of 7 mM tert-butyl hydroperoxide wasadded and NADPH oxidation was measured at 340 nm. Nonenzymaticoxidation of NADPH at 340 nm was measured by adding RIPA buﬀer
instead of the lysates. Enzymatic consumption of NADPH was
calculated by subtracting the nonenzymatic consumption of NADPH
from the total. GPx activity was expressed as nmol of NADPH
consumed per min per mg of protein.
In vitro assay for inhibition of ACE activity. ACE inhibitory activity
was measured spectroﬂuorometrycally using ACE Kit-WST Detection (Dojindo, Kumamoto, Japan) according to the manufacturer’s
instructions.
Animal experiments. Fourteen-week-old male SHRs and normotensive Wistar-Kyoto rats (WKYs) (Charles River Laboratories, Japan)
were maintained in accordance with the Guidelines for the Proper
Conduct of Animal Experiments, Science Council of Japan, and theprotocol approved by the Animal Care and Use Committee of the
University of Tsukuba. They were housed under an animal facility witha 12-h light/dark cycle and were fed the usual rat chow and tap water.
An adaptation period of one week for vehicle administration and bloodpressure measurements was allowed before the initiation of experimental protocols. Six rats were randomly assigned to each group andbody weight was measured every week. The SHRs were administered
2.5 mg/kg body weight resveratrol or 5 mg/kg body weight "-viniferin
daily by oral gavage for three weeks. The dose of resveratrol waschosen according to previous experimental models using hypertension
in SHRs.7,8) In order to give the same dose of the molecules, the doseof "-viniferin used was the double of that of the monomer. The
untreated WKYs and SHRs were administered vehicle orally by
gavage (corn oil, Sigma) for the same period.
Measurement of blood pressure. Systolic and diastolic bloodpressure (SBP and DBP) was measured once a week by tail-cuﬀ

956

N. Z GHONDA et al.

A(MW=228.24)(MW=454.47)

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Fig. 1. "-Viniferin as Well as Resveratrol Promotes Wound Repair and Proliferation of VECs.
A, Chemical structures of resveratrol and "-viniferin. B and C, Serum-starved VECs monolayers were wounded with a pipette tip, then treatedwith 5 mM LPA or the indicated concentrations of resveratrol or "-viniferin for 8 h (B) or 24 h (C). The number of cells that migrated into the cellfree area represents wound repair as described in ‘‘Materials and Methods.’’ D, Serum-starved VECs were treated with 200 nM CNP or theindicated concentrations of resveratrol or "-viniferin for 24 h. Cell proliferation was assessed by trypan blue dye as described in ‘‘Materials andMethods.’’ Data are mean  SE of three separate experiments for B and C, and four separate experiments for D.  p < 0:05 vs. untreated cells.
#
p < 0:05.

plethysmography (BP-98A; Softron, Tokyo) just prior to administration of the compounds. The ﬁnal SBP and DBP are presented as meansof ﬁve measurements.
Cardiac weight. At the end of the experiment the rats were scariﬁed,
and the hearts were excised immediately, cleaned, and weighed. The
atria were discarded and the free wall of the right ventricle wascarefully removed, leaving the left ventricle (LV) free wall and theintraventricular septum as total LV. They were weighed separately.
The heart and ventricular weights were normalized to body weight andwere expressed as heart and LV mass index (mg/g).
Statistical analysis. Data represent mean  SE. ANOVA was used
to assess signiﬁcant diﬀerences between treatment groups. Whentreatment was found to have a signiﬁcant eﬀect, Duncan’s multiple
range test was used to compare multiple group means. p < 0:05 wasconsidered statistically signiﬁcant.

Results"-Viniferin stimulates the wound repair of VECs more
eﬀectively than resveratrol by inducing cell proliferation
Initially we investigated the wound repair eﬀects of "viniferin and resveratrol on cultured VECs by incubating

the cells for 8 and 24 h. Treatment with each of thecompounds at 10, 20, and 30 mM for 24 h signiﬁcantly
enhanced the wound healing except for 30 mM "-viniferin(Fig. 1C) as LPA, a stimulator of wound repair in
VECs.26) In contrast, an 8-h incubation had no eﬀect(Fig. 1B). These data suggest that both compounds
enhance the wound repair by inducing cell proliferation,
but not cell migration. In addition, it should be notedthat "-viniferin at 10 and 20 mM more eﬃciently
promoted the healing than resveratrol at 30 mM. We
conﬁrmed the cell proliferation eﬀect of the compounds
by trypan blue exclusion assay (Fig. 1D). Again, "viniferin at 10 and 20 mM was more eﬀective thanresveratrol at 30 mM. Therefore, we concluded that "viniferin is more potent than resveratrol for theprotection of VECs.
NO production is involved in the proliferation eﬀectof "-viniferin and resveratrol on VECs
Because NO plays an important role in promoting
VEC proliferation and migration,27) we examined toEﬀects of "-Viniferin and Resveratrol on VECs and SHR

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α-Tubulin

α-Tubulin

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Fig. 2. "-Viniferin and Resveratrol Promote NO Production in VECs, Leading to Cell Proliferation.
A, "-Viniferin induces NO production more eﬀectively than resveratrol. Serum-starved cells were treated with the indicated concentrations ofresveratrol or "-viniferin for 2, 4, 6, and 8 h, and then NO production was measured using DAF-2DA as described in ‘‘Materials and Methods.’’
B, L-NAME, an NOS inhibitor, suppresses the proliferative eﬀect of resveratrol and "-viniferin on VECs. Serum-starved cells were treated with
30 mM resveratrol or 10 mM "-viniferin in the presence or absence of 1 mM L-NAME, and the proliferation was assessed using trypan blue dye as
described in ‘‘Materials and Methods.’’ Data are mean  SE of four separate experiments for A and B. C, Resveratrol and "-viniferin induce thephosphorylation of eNOS. Serum-starved cells were treated with 30 mM resveratrol and 10 mM "-viniferin, respectively, for 4, 8, 12, and 24 h.
Thereafter, cells were lysed and western blotted using anti-phospho-eNOS antibody. Representative data are shown from three independent
experiments.  p < 0:05 vs. untreated cells. # p < 0:05.

determine whether "-viniferin and resveratrol produce
NO. As we expected, both compounds signiﬁcantly
increased NO generation in a time-dependent manner(Fig. 2A). The NO production rate of "-viniferin at eachconcentration was signiﬁcantly higher than that ofresveratrol at the same concentration, which agrees wellwith the data on the wound repair and cell proliferation(Fig. 1C and D). We used 10 mM "-viniferin and 30 mM
resveratrol in all subsequent experiments. The proliferative eﬀects of "-viniferin and resveratrol were inhibitedby the addition of L-NAME, a general inhibitor of NO
synthase (NOS) (Fig. 2B). These data suggest that "viniferin and resveratrol induce VEC proliferation
through NO production.
We investigated to determine whether both compounds phosphorylate eNOS since eNOS is activated byphosphorylation.28) As Fig. 2C indicates, "-viniferin andresveratrol induced sustained phosphorylation of the
enzyme, which is consistent with the sustained NO
generation induced by these compounds.
"-Viniferin and resveratrol protect VECs from oxidative stress
Since oxidative stress is known to be one of the maincauses of endothelial injury and increasing risk ofhypertension,29) we examined the cytoprotective properties of "-viniferin and resveratrol against H2 O2 induced cell damage. As Fig. 3A indicates, "-viniferin
as well as resveratrol signiﬁcantly protected the cells
from the cytotoxic eﬀect of H2 O2 . Both compounds

reduced intracellular ROS levels in VECs exposed to
H2 O2 (Fig. 3B). That is, the increased intracellular
ROS was signiﬁcantly decreased by the 24-h pretreatment of the cells with "-viniferin and resveratrol.
Because phase II detoxifying enzymes are essential
components of the cellular antioxidant defense system
detoxifying ROS, we examined the activity of catalase
and GPx enzymes in the presence and the absence ofeach compound. Both compounds upregulated catalase
and GPx activities in a time-dependent manner(Fig. 3C). Therefore, we concluded that "-viniferin as
well as resveratrol attenuates intracellular ROS generation in VECs through the increased activities ofantioxidant enzymes, which confers resistance to oxidative stress.
"-Viniferin, but not resveratrol, inhibits ACE
Because ACE inhibitors are extremely eﬀective in
reducing blood pressure, we investigated the ACE
inhibitory eﬀect of "-viniferin and resveratrol in vitro.
ACE was incubated in the presence of "-viniferin,
resveratrol, epigallocatechin (EGC), or EDTA (Fig. 4).
"-Viniferin exhibited a potent ACE inhibitory activity
whereas resveratrol did not. Notably, the inhibitory
eﬀect of "-viniferin at 100 mM was more pronounced
than that of EGC, an established food-derived ACE
inhibitor.18) Together with the data on NO generation
shown in Fig. 2A, these data suggest the possibility
that "-viniferin, but not resveratrol, reduces bloodpressure.

958

N. Z GHONDA et al.
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Percentage of ACE inhibition

Fig. 3. "-Viniferin and Resveratrol Protect VECs from Oxidative Stress and Increase the Activities of Catalase and GPx.
A, "-Viniferin and resveratrol protect VECs from H2 O2 -induced cell death. Serum-starved cells were treated with the indicated concentrationsof resveratrol or "-viniferin for 24 h, washed, and incubated with 500 mM H2 O2 for another 24 h. Cell viability was assessed using trypan blue dye
as described in ‘‘Materials and Methods.’’ B, "-Viniferin and resveratrol suppress increased intracellular ROS levels in VECs exposed to H2 O2 .
Serum-starved cells were treated with 30 mM resveratrol or 10 mM "-viniferin for 24 h followed by incubation with 500 mM H2 O2 for 15 min.
Intracellular ROS accumulation was determined by measuring DCF-derived ﬂuorescence as described in ‘‘Materials and Methods.’’ Data aremean  SE of four separate experiments for A and B.  p < 0:05 vs. untreated cells. # p < 0:05 vs. H2 O2 -treated cells without stimuli. C, "Viniferin and resveratrol upregulate catalase and GPx activities. Serum-starved cells were incubated with 30 mM resveratrol or 10 mM "-viniferin
for 12, 24 or 48 h, and enzymatic activities were assessed as described in ‘‘Materials and Methods.’’ Data are mean  SE of four separate
experiments.  p < 0:05 vs. untreated cells.
100

EDTA
EGC
ε-Viniferin
Resveratrol

50

0
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0.1

1

10

Compound concentration (mM)

Fig. 4. "-Viniferin, but Not Resveratrol, Inhibits ACE Activity in Vitro.
The percentage of ACE inhibition was assessed using ACE KitWST Detection according to the manufacturer’s instructions. Data
are mean of a single experiment performed in triplicate.

"-Viniferin, but not resveratrol, loweres blood pressure in SHRs
We investigated the eﬀects of "-viniferin and resveratrol administration on blood pressure in SHRs (Fig. 5).
As expected, SBP in the "-viniferin-treated SHRs was
signiﬁcantly decreased at all time points as compared toage-matched vehicle-treated SHRs. In contrast, DBP
was unaltered by "-viniferin administration. Three-weekresveratrol treatment did not lower SBP or DBP. Rather,resveratrol tended to increase the blood pressure.
"-Viniferin is more eﬀective than resveratrol in
improving cardiac mass in SHRs
Because a reduction in cardiac mass with antihypertensive agents has been reported in experimental as wellas human hypertension,30,31) we examined the eﬀects of
the administration of each of the compounds on cardiac
mass (Table 1). The heart and LV mass indexes in thevehicle-treated SHRs were signiﬁcantly higher thanthose in the WKYs. These parameters were signiﬁcantly
reduced in the "-viniferin-treated SHRs as compared to
the vehicle-treated SHRs. In contrast, there was no
signiﬁcant diﬀerence in heart and LV mass indexes
between the vehicle-treated SHRs and the resveratroltreated SHRs (2.5 mg/kg). Therefore, the reduction in
cardiac mass indexes in the SHRs treated with "viniferin correlated with the observed reduction in SBP.
These data strongly indicate that "-viniferin more
eﬀectively improves cardiac mass than resveratrol in
hypertensive rats.

Discussion
The present study is the ﬁrst to describe the direct
eﬀect of "-viniferin on cultured VECs and on SHRs. Our
major ﬁndings are as follows: First, "-viniferin is more
eﬀective than resveratrol in inducing the proliferation
and wound repair in VECs via NO production and inprotecting VECs from oxidative stress-induced cell
death. Second, "-viniferin, but not resveratrol, inhibits
ACE activity in vitro. Third, "-viniferin, but notresveratrol, reduces blood pressure and improves cardiac
mass in SHRs.

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WKY,
Vehicle-treated SHR
ε-Viniferin-treated SHR
Resveratrol-treated SHR

15

16

17

DBP mmHg

SBP mmHg

Eﬀects of "-Viniferin and Resveratrol on VECs and SHR

210
200
190
180
170
160
150
140
130
120
110
100

18

*

WKY,
Vehicle-treated SHR
ε-Viniferin-treated SHR
Resveratrol-treated SHR

15

Age (weeks)

*

16

17

18

Age (weeks)

Fig. 5. "-Viniferin, but Not Resveratrol Attenuates the Elevation of Blood Pressure in SHRs.
Systolic blood pressure (SBP) and diastolic blood pressure (DBP) were measured by tail-cuﬀ plethysmography as described in ‘‘Materials andMethods.’’ Data are mean  SE of six rats per treatment.  p < 0:05 vs. vehicle-treated SHRs.

Table 1. Body Weight and Cardiac Mass Indexes after 3 Weeks of Treatment with "-Viniferin or Resveratrol in SHRs
HMI, heart mass index; LVMI, left ventricular mass index. Data are means  SE of six rats per treatment.  p < 0:05 vs. WKYs, # p < 0:05 vs.
vehicle-treated SHRs.

Heart weight (g)Body weight (g)HMI (mg/g)
LVMI (mg/g)

WKYs

Vehicle-treated
SHRs"-Viniferin-treated
SHRs

Resveratrol-treated
SHRs

1:26  0:03
364:33  1:4
3:45  0:07
2:55  0:05

1:42  0:03
357:12  33:98  0:12
3:01  0:05

1:23  0:02#
342:83  5:2#
3:60  0:04#
2:85  0:02#

1:37  0:06
358:60  7:2
3:82  0:11
2:97  0:06

Our data indicate that "-viniferin, but not resveratrol,
reduces blood pressure in SHRs. This distinct eﬀect isprobably due to the two points of diﬀerence between thetwo compounds. The ﬁrst is that "-viniferin generates NO
more eﬀectively than resveratrol. NO is a strong vasodilator that lowers the blood pressure. The second is that"-viniferin, but not resveratrol, inhibits ACE activity.
Our data are in good agreement with a recent report
indicating that "-viniferin has a more potent vasorelaxant
eﬀect than resveratrol in the isolated rat thoracic aorta.14)
The fact of no ACE inhibitory eﬀect of resveratrol in thepresent study is also consistent with past reports.19,32) Itshould be noted that ACE inhibition promotes the releaseof NO.31) ACE not only produces the vasoconstrictor
angiotensin II, but also degrades the vasodilator bradykinin. Bradykinin is known to stimulate endothelial NO
production. Thus, there is a possibility that "-viniferin
enhances NO generation by inhibiting ACE activity
which is followed by a blockade of bradykinin inactivation. There has been no report indicating the plasma
concentrations of "-viniferin after oral administration of
it to rats. Marier et al.33) found that oral administration of
resveratrol to rats at a dose of 50 mg/kg body weight
exhibited over 100 mM of total plasma concentration of
the aglycone and glucuronide forms of resveratrol. Also,
Abd El-Mohsen et al.34) identiﬁed the presence ofresveratrol metabolites not only in the plasma, but also
in the kidney, heart, and brain that are closely associatedwith the blood pressure. Although further investigation isnecessary for the concentrations of "-viniferin absorbed
in vivo, our data together with past reports suggest thepossibility that the ACE inhibitory eﬀect of "-viniferin is
involved in the "-viniferin-dependent reduction in bloodpressure.
We found that "-viniferin and resveratrol protect
VECs from H2 O2 -induced cell death by suppressing
intracellular ROS accumulation. Our ﬁndings support
the view that both compounds themselves exert anti-oxidant eﬀects due to the redox properties of theirphenolic hydroxyl groups.18) Direct scavenging of H2 O2
may reﬂect the ability of the two compounds to have thecytoprotective eﬀects against H2 O2 . In addition to this,"-viniferin and resveratrol may promote the vascular
expression of enzymes involved in scavenging H2 O2 ,such as catalase and GPx. Our thought is supported by
Ungvari et al.35) who demonstrated that in vitro treatment with resveratrol increased oxidative stress resistance in VECs through both direct scavenging of H2 O2and upregulation of antioxidant enzymes. In our experiments, 30 mM "-viniferin inhibited the wound healing
and exhibited a weaker eﬀect on the cell proliferation
than 10 and 20 mM "-viniferin. We cannot exclude thepossibility that high concentrations of "-viniferin are
toxic to VECs; however, these toxic eﬀects observed in
our in vitro experiments may have been due to theserum-starved condition (1% FBS) to clearly show thefunctions of the compound. Further study is necessary
concerning this issue.
The three-week resveratrol treatment failed to alter
the blood pressure in SHRs, consistent with previous
studies.7,8) Although the compound tended to improve
the whole cardiac and LV mass indexes, the eﬀects were
not signiﬁcant. In contrast, "-viniferin signiﬁcantly
reduced SBP and cardiac mass in SHRs. Caristrom
et al.36) demonstrated that dietary inorganic nitrate,
which is converted in vivo to NO and other bioactive
nitrogen oxides, prevented cardiac hypertrophy in rats
subjected to the unilateral nephrectomy and a chronically high-salt diet. Matsuoka et al.37) also indicated thatchronic L-arginine administration, which induces NO
generation, attenuated cardiac hypertrophy in SHRs.
Similar mechanism may mediate "-viniferin-dependent
cardioprotective eﬀect because this compound produces
NO in VECs.
To the best of our knowledge this is the ﬁrst report on
ACE inhibitory, antihypertensive, and cardioprotective

960

N. Z GHONDA et al.

eﬀects of "-viniferin. Further studies, including identiﬁcation of target molecules for "-viniferin and resveratrol in the cardiovascular system, are necessary to
clarify the mechanisms underlying these beneﬁcial
eﬀects.

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