Grape extract improves antioxidant status and physical performance in elite male athletes

©Journal of Sports Science and Medicine (2009) 8, 468-480

http://www.jssm.org

fa Research article



Grape extract improves antioxidant status and physical performance in elite
male athletes

Sophie Lafay 1, Caroline Jan 1, Karine Nardon 1, Benoit Lemaire 1, Alvin Ibarra 2
, Marc Roller 1,
Marc Houvenaeghel 3, Christine Juhel 4 and Louis Cara 4
1 Naturex SA, Site d’Agroparc, Avignon Cedex 9, France, 2 NAT’Life division, Naturex Inc, South Hackensack, NJ,
USA, 3 Salvator Hospital, Centre Régional de Médecine du Sport, Marseille Cedex 9, France, 4 Avantage Nutrition,
Luminy Entreprises, Grand Luminy, Marseille, France

cal performance, sportsmen, training, competition.

Abstract

Excessive physical exercise overproduces reactive oxygen spe-

cies. Even if elite sportsmen increase their antioxidant status by
Introduction
regular physical training, during the competition period, this
improvement is not sufficient to limit free radical production
Young athletes under a regular training program exhibit a
which could be detrimental to the body. The aim of this random-
substantial increase in antioxidant status when compared
ized, double-blind, placebo controlled, and crossover study on
to healthy sedentary people (Brites et al., 1999). Such an
20 elite sportsmen (handball = 10, basketball = 5, sprint = 4, and
volleyball = 1) during the competition period was to determine
effect results from an adaptative response to controlled
if the consumption of a grape extract (GE; Vitis vinifera L.) was
physical activity, inasmuch as exercise represents a form
able to improve the parameters related to (i) anti-oxidative status
of oxidative stress which may be defined as an increase in
and oxidative stress and (ii) physical performance. Specific intracellular steady state concentration of oxidants over
biomarkers of antioxidant capacity, oxidative stress, skeletal cell
physiological values (Cazzola et al., 2003; Evelson et al.,
muscle damage, and other general biomarkers were determined
2002; Radak et al., 2008). During a competition period
in plasma and urine before (D0) and after one month (D30) of
and/or overload training, this adaptative response does not
placebo or GE supplementation (400mg·d-1). Effort tests were
suffice to limit free radical production which could prove
conducted using the Optojump® system, which allows determin-
detrimental to the body (Klapcinska et al., 2005; Pa-
ing the total physical performance (EnRJ45), explosive power
(RJ110), and fatigue (RJL5). The plasma ORAC value was not
lazzetti et al., 2003; Pincemail et al., 2000). Antioxidants
modified in the placebo group; however, GE increased the may be administrated before competition, when exercise
ORAC value compared to the placebo at D30 (14 966+/-335 vs
is likely to be exhaustive and results in the generation of
14 242+/-339 µmol Teq·L-1; p < 0.05). The plasma FRAP value
free radicals that overwhelm the defensive mechanisms,
was significantly reduced in the placebo group, but not in the
causing oxidative stress (Gomez-Cabrera et al., 2008).
GE group. Therefore, GE limited the reduction of FRAP com-
Different types of supplementation such as selenium,
pared to the placebo at D30 (1 053.7+/-31.5 vs 993.7+/-26.7
vitamin E, vitamin C or polyphenols revealed that it was
µmol Teq·L-1; p < 0.05). Urinary isoprostane values were in-
possible to increase the already adapted antioxidant status
creased in the placebo group, but were not modified in the GE
(Margaritis et al., 2003; Morillas-Ruiz et al., 2005; 2006;
group. Consequently, GE limited the production of isoprostanes
compared to the placebo at D30 (1.24+/-0.12 vs 1.26+/-0.13 Rokitzki et al., 1994a; 1994b). Some studies further
ng·mg-1 creatinine; p < 0.05). GE administration, compared to
evaluated the impact of an antioxidant effect on perform-
the placebo at D30, reduced the plasmatic creatine phos-
ance or endurance in athletes; the results, however, dif-
phokinase concentration (CPK, 695.7+/-177.0 vs 480.0+/-81.1
fered largely depending on the type of supplementation,
IU·L-1, p = 0.1) and increased hemoglobin levels (Hb, 14.5+/-0.2
subjects and protocol (Clarkson et al., 2000).
vs 14.8+/-0.2 vs g·dL-1, p < 0.05), suggesting that GE admini-
Oligomeric proanthocyanidins (OPCs) are poly-
stration might protect cell damage during exercise. The high phenols and more specifically, polymers of flavanols
variability between sport disciplines did not permit to observe
(Manach et al., 2004). Their main dietary sources are to
the differences in the effort test. Analyzing each individual be found in grapes, cocoa, and apples (Santos-Buelga and
group, handball players increased their physical performance by
24% (p < 0.05) and explosive power by 6.4% (p = 0.1) after GE
Scalbert, 2000). OPCs are famed for their potent antioxi-
supplementation compared to the placebo. Further analyses dant capacity in vitro (da Silva et al., 1991; Mazur et al.,
showed that CPK and Hb were the only biomarkers correlated
1999; Shafiee et al., 2003; Teissedre et al., 1996; Vitseva
with the increase in performance. In conclusion, GE ameliorates
et al., 2005; Yilmaz et al., 2004). As regards human con-
the oxidative stress/antioxidant status balance in elite athletes in
sumption, there is substantial evidence that OPC intake
the competition period, and enhances performance in one cate-
from grapes or cocoa increase the antioxidant status
gory of sportsmen (handball). Our results suggest that the en-
among hypercholesterolemic, hyperlipidemic, hemodialy-
hancement in performance might be caused by the protective
sis patients, smokers or healthy volunteers (Castilla et al.,
action of GE during physical exercise. These findings encourage
2008; Gorinstein et al., 2006; Preuss et al., 2000; Serafini
conducting further studies to confirm the efficacy and mecha-
nisms of action of GE on elite and occasional athletes.
et al., 2003; Simonetti et al., 2002; Vigna et al., 2003).

Few studies have been released with respect to flavanols
Key words: Botanical extract, oxidative stress, exercise, physic-
in athletes (Morillas-Ruiz et al., 2006; Pavlovic, 1999).

Received: 17 December 2008 / Accepted: 17 July 2009 / Published (online): 01 September 2009

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Lafay et al.
469


To date, only a single study has been carried out on poly-
vitamin and mineral use over the 8 weeks prior to launch-
phenol consumption-related impact over performance ing our study; 4) fewer than 2 cigarettes per day; 5) under
(Pavlovic, 1999).
20g per day alcohol intake; 6) no vegetarian, vegetalian
Thus, the aim of the randomized, double-blind, nor deviated diet behavior; 7) no recent surgery; 8) no
cross-over clinical study presented herein is to ascertain blood transfusion over the three months prior to the study;
the effects of grape extract consumption (GE) titrated in and 9) no involvement in another clinical study. More-
flavanols on (i) anti-oxidative status and oxidative stress, over, as suggested by the Ethical Committee, they were
and on (ii) the physical performance among elite athletes asked not to consume any drugs which may enhance
under regular training conditions and in competition.
physical activity.


Table 1. Subject (n =20) baseline physical, hematologic and
Study design
lipemic characteristics. Values are means (± SEM).
This study was a randomized, double-blind, placebo con-
Age (y)
21.6 (2.0)
trolled, and crossover design. It was approved by the
Height (m)
1.87 (.02)
Ethical Committee from Salvator Hospital, Marseille,
Weight (kg)
84.7 (3.2)
Body Mass Index (kg·m-2)
23.9 (.55)
France (project # 206014). The promoter for the study
Hb (g.dL-1)
14.6 (.2)
was Naturex SA, Avignon, France. Dr Houvenaeghel,
Blood formula
Normal
M.D., from Salvator Hospital, Marseille, France, ensured
Cholesterol (g.L-1)
1.82 (.06)
medical investigations. Avantage Nutrition, Luminy
Triglycerides (g.L-1)
.56 (.06)
Technopole, Marseille, France, enforced scientific coor-

dination for our study.
Methods
The subjects were randomly divided into 2 groups:

each group was supplied with either 400mg GE supple-
Subjects
mentation, or 400mg of placebo (maltodextrin) under
Twenty elite male athletes were recruited after having capsule conditioning (2 capsules) to be taken along with
submitted their written consent. All subjects practiced their breakfast over a one month period. After one month
explosive sports in keeping with top elite standards (i.e. of supplementation and two weeks of a wash out period,
national leagues), namely: 10 handball players, 5 basket-
the treatments were reversed (Figure 1). Placebo and GE
ball players, 4 sprinters, and 1 volleyball player. The capsules were indiscernible. Supplementation was given
baseline characteristics of the latter are presented in Table
with breakfast over a one month period. The study was
1. Participants proved to be in good health as assessed by
double-blind while neither the M.D. nor the coordinator,
way of a medical history questionnaire, physical examina-
nor the participants knew the randomization codes. The
tion, and clinical laboratory tests. All subjects fulfilled the
promoter,--namely Dr Lafay--, was the sole member to
following eligibility criteria: 1) Practice of high level have knowledge thereof. The subjects were allowed to
national intensive sports under non-stop training condi-
maintain their standard eating patterns. They were asked
tions over a one-month period, including competition; 2) to complete a questionnaire on food intake frequency.
no metabolic disorders (type 1 or 2 diabetes, cardiovascu-
Moreover, the intensity of training had to be equivalent
lar, hepatic, gastrointestinal or renal diseases); 3) no among all participants: hence, they were further requested
pharmacological treatment, antibiotic nor supplemental to complete a questionnaire focusing on their training


n=20; 400mg Powergrape®/day
T
?
OU
Training and competition period
S
H
WA
n=20; 400mg Placebo /day
Weekly overtraining scores booklets
D0
D30
D0
D30
Booklet (Alimentary consumption, Overtraining scores)
Blood samples
Urinary samples
Effort test



Figure 1. Study design. The subjects, randomly divided into 2 groups, were assigned to supplementation either
400mg of GE or 400mg of placebo (maltodextrin) under capsule forms (2 capsules) with their breakfast during
one month. After one month of supplementation and two weeks of wash out period, the treatments were re-
versed. Effort test, blood and urine samples were done at day 0 (D0) and day 30 (D30) of each period.

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470
Grape extract improves antioxidant status on sportsmen






Figure 2. High-pressure liquid chromatography chemical fingerprint for Powergrape®: (1) gallic acid (5.9min); (2) B1 dimer
(11.5min) ; (3) B3 dimer (12.9); (4) catechins (14.4min); (5) B2 dimer (18.9min); (6) epicatechin (19.3min); (7) C1 trimer (21.5min); (8) B2-3-O-
gallate dimer (22.8min); (9) digallate trimer (27.3min), (10) digallate dimer (29.3min) ; (11) epicatechine-3-O-gallate (29.4min); (12) gallate trimer
(30.6min).

levels (over-training scores). At the beginning of the
Microbiology, heavy metals, pesticides and my-
study (D0), and after 30 days of supplementation of GE or
cotoxins were controlled for compliance.
placebo (D30), blood and urine were sampled from the

subjects after they had fasted overnight for > 10h. More-
Table 2. Mobile phase gradient used to obtain high-pressure
over, an effort test was conducted using a validated sys-
liquid chromatography chemical fingerprint of the grape
tem Optojump® (Microgate SRL, Bolzano, Italy) (Le-
extract.
Minutes
Phase A1
Phase B1
hance et al., 2005; Wilmot et al., 2004) associated with a
0 85
15
cardiofrequence-meter. Strict compliance with terms and
30
65
35
conditions was assessed as follows: a cross-analysis of
32
0
100
information contained in a diet consumption frequency
40
0
100
questionnaire together with the counting of the capsules
41
85
15
brought back to the laboratory at D30 of each period.
45
85
15

1Phase A: distilled water/trifluoroacetic acid 5.10-3%;
Grape extract
phase B: acetonitrile 65%/trifluoroactic acid 5.10-3%.
The GE used in this trial was obtained from whole grapes,
HPLC was achieved using column SBC18 (250 X
4.6mm) at 25°C.
Vitis vinifera L. (Vitaceae). This extract, registered as

Powergrape® (batch 4990), was manufactured by Naturex
Sample collection and analyses of endogenous parame-
SA, France. The extract is obtained using a hydro-
ters
alcoholic solvent (30% ethanol) and the plant:extract ratio

is between 10:1 and 15:1, calculated on dry basis. This Sample collection
extract is spray dried with maltodextrin (0 to 8% w/w) Blood samples were collected in dry, EDTA/K3-coated
and silica (0 to 2% w/w). It is standardized in total poly-
and lithium/heparin-coated tubes. One EDTA/K3-coated
phenols (>90%) by the Folin method (Folin and Denis, tube was used for hemoglobin (Hb) analysis conducted
1915), in total flavanols (>50%) by the vanillin method less than two hours after sampling. Serum (blood in dry
(Broadhurst et al., 1978) and in flavanols monomers and
tubes) and plasma (blood in EDTA/K3-coated tubes) were
gallic acid (>12%) by HPLC-DAD.
separated by centrifugation at 4°C and 2000g for 10min.
The high-pressure liquid chromatography chemical
Serum was immediately treated with 10µL·mL-1 of inhibi-
fingerprint for the whole grape extract is presented in tor cocktail (EDTA, antibiotic and anti-protease). Suitable
Figure 2. The method for performing this analysis was as
portions for the biochemical analysis described below
follows: HPLC was achieved using column SBC18, 250 were stored at -20°C.
X 4.6mm at 25°C. Mobile phase A involved the use of
One portion of plasma coming from lith-
distilled water/trifluoroacetic acid 5.10-3%, whereas mo-
ium/heparin-coated tubes was treated with pyrogallol 12%
bile phase B consisted in acetonitrile 65%/trifluoroactic (20µL·mL-1) for vitamin E analysis. Another portion was
acid 5.10-3%. The gradient used is presented in Table 2. treated with metaphosphoric acid 5% (4µL·mL-1) for
The flow rate amounted to 0.7mL.min-1, and the elution vitamin C analysis. The remaining plasma was directly
was monitored at 280 nm. Analyses were performed by stored at -80°C for antioxidant capacity determination.
Naturex SA.
Plasma coming from EDTA/K3-coated tubes was stored

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Lafay et al.
471


at -80°C for oxidized-LDL analysis. Red blood cells com-
360nm and the emission wavelength was 440nm. The
ing from EDTA/K3-coated tubes were washed with 5 method has been previously validated, and we imple-
mmol·L-1 phosphate-buffered saline, pH 7.4, hemolized at mented quality control checks by spiking plasma samples.
4°C with bi-distilled water and stored at -80°C for endo-
Urinary isoprostanes: The level of isoprostanes in
gen antioxidant enzyme analysis. Urine samples were urine samples was determined using an enzyme immuno-
collected in sterile tubes for creatinine and isoprostane assay kit (Cayman Chemical, Ann Arbor, MI and Oxford
analysis.
Biomedical Research, Oxford, MI). The amounts of iso-

prostanes were expressed in nanograms and corrected
Analyses
with creatinine values.
Biomarkers of antioxidant status and oxidative stress

Oxygen radical absorbance capacity (ORAC): ORAC of Biomarker of skeletal muscle damage
plasma samples was determined. This fluorescence Plasma creatin phosphokinase (CPK) was assessed using
method is widely used for assessing antioxidant capacity specific enzymatic and colorimetric kit methods
in biological samples. It is based on the inhibition of a (Biomérieux, Craponne, France).
peroxy-radical-induced oxidation initiated by the thermal-

based decomposition of azo compounds such as AAPH General plasmatic biomarkers: Triglycerides, choles-
using fluorescein as a fluorescent probe and Trolox as a terol, serum ferritin, urea, and red blood cell hemoglo-
standard substrate (Huang et al., 2002; Ou et al., 2001).
bin
Ferric reducing ability of plasma (FRAP): FRAP
Plasma triglycerides and cholesterol levels were deter-
was also determined on 100µL plasma samples diluted mined using commercial kits (Biotrol, Paris, France and
1:4 and the tripyridyltriazine complex formed with the Biomérieux, Charbonnière-les-bains, France, respec-
reduced ferrous ions was measured with spectrofluorome-
tively). A polyvalent control serum (33-plus Biotrol,
try (LS 5, Perkin Elmer, Norwalk, CT) (Benzie et al., Paris, France) was treated in parallel to samples and
1996).
served as control in order to appreciate result accuracy in
LDL oxidation: Anti-oxLDL autoantibodies were the triglycerides and cholesterol analyses. Serum ferritin
determined using a commercially available ELISA kit was assessed using a specific enzymatic kit (Roche Diag-
(Biomedica, Vienna, Austria).
nosctics, Meylan, France). Serum urea was assessed using
Antioxidant enzymes: Superoxide dismutase specific enzymatic and colorimetric kit methods
(SOD), gluthation peroxidase (GPx), and catalase: SOD
(Biomérieux, Craponne, France). Red blood cell Hb was
(Peskin and Winterbourn, 2000), GPx (Ozdemir et al., measured using a haematology blood cell counter (Sys-
2005; Prasad et al., 2005) and catalase (Bai et al., 1999; mex XT2000i, Roche Diagnostics, Meylan, France).
Deisseroth and Dounce, 1970) were determined on red

blood cells with three different commercial kits (FLUKA,
Physical performance measurements (Effort tests)
Buchs, Switzerland; TREVIGEN Inc, Gaithersburg, MD, For measuring physical performance, the Optojump®
USA; SIGMA, Saint Louis, MO, USA, respectively).
method was used (Microgate, Bolzano, Italy). This
Plasma vitamin E: To determine plasma vitamin E method calculated the energy in Joules (J) produced by
concentration, the used HPLC equipment included a pho-
each sportsman by measuring the flight time during sev-
todiode array detector on-line with a Waters Millennium. eral single vertical rebound jumps. The Optojump® sys-
The separation was carried out using a Nucleosil column tem is able to measure with 1/1000th precision all flying
(150mm × 4.6mm, 5 µm, Interchim, Montluçon, France). times. This method consists of 2 bars (100 x 4 x 3 cm),
Elution was performed with an isocratic mobile phase of one containing the reception and control unit, the other
pure methanol at a flow rate of 2mL.min?1. Alpha-
embedding the transmission electronics.
tocopherol acetate (Sigma Aldrich) was added to plasma The Optojump® test was conducted at D0 and D30 of
samples as an internal standard. Samples were extracted each period. Figure 3 summarizes the Optojump® test for
twice with hexane after precipitation of the proteins. The each sportsman, which consisted of: (i) 3 successive sin-
extract was evaporated to dryness under N2, dissolved in gle vertical Counter Movement Jumps (CMJ), starting
ethanol-methylene chloride (65:35, v:v) and injected into from a resting standing position, from which the subjects
the HPLC.
were instructed to jump as high as possible, keeping their
Ascorbic acid: Ascorbic acid was determined ac-
hands on their hips throughout the jump, starting with
cording to the method previously described by Tessier et knees bent at 90°. For the CMJ test, they had to jump
al. (1996). Ascorbic acid from 500µL of supernatant was starting with a preliminary counter movement until a 90°
oxidized with 200µL of 2g·L?1 of potassium ferricyanide angle. The flight time was recorded which allowed the
(Sigma) and 3.68 mol·L?1 of sodium acetate buffer (pH determination of the energy (J) reached during the jump.
6.9) and processed into the following derivative, i.e. qui-
Each single CMJ was separated from the other by a 1
noxaline complex with 200µL of 10 mg·mL?1 of o-
minute rest. This first series of CMJ was considered as the
phenylenediamine (Sigma). Dehydroascorbic acid was warm-up period necessary for the adaptation to the test.
determined using a Waters HPLC/fluorescence detector. (ii) A 2 minutes rest period. (iii) Sportsmen performed a
The sample (40µL) was injected into a Hypersil BDS-C18
series of vertical Rebound Jumps (RJ) during 45 seconds,
column (5µm, 25cm) at 30ºC and eluted with a mobile from which they were instructed to jump as high and as
phase consisting of 80mmol·L-1 Na2HPO4, 80mmol.L-1 fast as possible, keeping their hands on their hips
NaH2PO4 and methanol (30:30:40 v/v/v) at an throughout the jump, starting with knees bent at 90°. The
0.8mL.min?1 flow rate. The excitation wavelength was Total Rebound Jump Power (EnRJ45) is the total

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472
Grape extract improves antioxidant status on sportsmen




3 SINGLE VERTICAL COUNTER
MAXIMAL REPETITIVE
MAINTAINED REBOUND
MOVEMENT JUMPS
REBOUND JUMP POWER
JUMP POWER
(CMJ)
(RJ110)
(RJL5)
Separated by
=
=
1 minute rest
10 FIRST RJ
5 LAST RJ
3 minutes
2 minutes
45 seconds
WARM UP
REST
TOTAL REBOUND JUMP POWER
(EnRJ45)



Figure 3. Optojump® methodology. Each sportsmen performed the test at day 0 (D0) and after 30 days (D30) of each period. The test included
3 steps: (i) a warm up phase of adaptation where they had to perform three single vertical Counter Movement Jumps (CMJ) separated by 1 minute
rest, (ii) a 2 minutes of rest, and (iii) to perform a continuum Rebound Jumps (RJ) during 45 seconds, namely Total Rebound Jumps Power (EnRJ45),
which represents the total physical performance. The Maximal Repetitive Rebound Jump Power (RJ110), which represents the explosive power, is the
average energy (J) calculated from the 10 first RJ of this 45 seconds sequence; and the Maintained Rebound Jump Power (RJL5), which represents
the fatigue, is the average energy (J) calculated from the last 5 RJ of this session.

accumulated energy (J), calculated from the successive RJ
placebo capsules (Table 3). With the ORAC method,
developed during 45 seconds. The Maximal Repetitive plasma antioxidant capacity increased from 13 885 ± 360
Rebound Jump Power (RJ110) is the average energy (J) to 14 996 ± 335µmol Trolox eq·L-1 (p < 0.05) after one
calculated from the 10 first RJ of this 45 second sequence,
month of GE supplementation; whereas, this parameter
and the Maintained Rebound Jump Power (RJL5) is the remained unaltered after one month of placebo supple-
average energy (J) calculated from the last 5 RJ of this mentation (14 488 ± 380 vs 14 242 ± 339µmol Trolox
session.
eq·L-1 at D0 and D30 respectively; p = 0.7).
EnRJ45 represents the total physical performance;
With the FRAP method, a significant decrease in
RJ110 gives the average explosive power at the beginning
plasma antioxidant capacity is exhibited following pla-
of a repeated resistance exercise; and RJL5 represents the
cebo capsule consumption (1 084 ± 43 vs 994 ± 27 µmol
capacity to maintain a maximal explosive force in the Fe2+·L-1; p < 0.05) whereas no significant modification
ultimate phase of a resistance exercise, which is a meas-
was observed after the GE period (1 071 ± 32 vs 1 054 ±
ure of fatigue.
32 µmol Fe2+·L-1; p = 0.3).

No significant variation of oxidized LDL concen-
Statistical analyses
tration, catalase, and SOD activities was disclosed;
Data are presented as means +/- SEM. All observed re-
whereas, such was not the case as regards glutathione
sults at the start of the second phase were compared with peroxidase (GPx) (Table 3). This enzyme activity de-
those at the start of the first phase by a paired t test. Be-
creased during the placebo period (p < 0.05) while re-
cause baseline data of the 2 phases did not differ, data maining unaltered throughout the whole grape extract
from both groups were pooled by placebo or grape extract
intake period.
treatments (n = 20).
Analyses of the vitamins C and E revealed that
Comparisons of after-treatment values and within ascorbic acid concentration was not modified during the
treatments were made by the paired t test. Comparisons of
study, whereas plasma vitamin E concentration was sig-
relative values were performed by using paired z tests. nificantly higher after GE supplementation as compared
Pearson’s correlation analysis was used to probe the cor-
to placebo supplementation (p < 0.05; Table 3).
relation between different biomarkers determined in the
During placebo supplementation, urinary excretion
blood, and the results obtained with the effort test. Values
of isoprostanes was markedly increased, disclosing
of p < 0.05 were considered significant. Statistical analy-
thereby, an increase in oxidative injury and inflammation
ses were carried out using XLStat software (Version (1.18 ± 0.11 vs 1.64 ± 0.18ng·mg-1 creatinine at D0 and
2008.1.03, Addinsoft, France).
D30, respectively; p < 0.05). Such an increase did not

appear during the GE supplementation (1.24 ± 0.12 vs
Results
1.26 ± 0.13ng·mg-1 at D0 and D30, respectively).


Biomarkers of antioxidant status and oxidative stress
Skeletal muscle damage
Regardless of the method used to determine the plasma CPK activity reached 696 ± 177 and 480 ± 81 IU·L-1 after
antioxidant capacity, a significant increase of the plasma placebo and GE periods, respectively (p = 0.1) (Table 3).
antioxidant capacity is showed when volunteers con-
General biomarkers: No significant difference on
sumed whole grape extract capsules compared to the cholesterol and triglycerides was evidenced whatever

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Lafay et al.
473





Table 3. Body weight, plasma and urinary parameters, and relative values of effort test, before (D0) and after (D30) sup-
plementation of GE or placebo in elite sportsmen. Values are means (± SEM) of 20 determinations performed in duplicate
on samples from different subjects; n = 20 elite sportsmen.

Placebo n=20
Grape extract n=20

D0
D30
D0
D30
Weight (kg)
84.4 (3.1)
83.7 (2.9)
84.8 (3.1)
84.0 ( 3.1)
Biomarkers of antioxidant status and oxidative stress
ORAC (µmol·L-1)
14 488 (380)
14 242 (339)
13 885 (360)
14 966 (335) *#
FRAP (µmol·L-1 Fe2+)
1 084 (43)
994 (27) *
1 071 (32)
1 054 (32) #
LDLox (mU·mL-1)
481 (98)
473 (106)
556 (119)
576 (128)
SOD (U·g-1 Hb)
2 368 (85)
2 438 (77)
2 523 (105)
2 541 (92)
GPx (µmol·min-1·g -1 Hb)
17.1 (1.0)
15.1 (.9) *
18.9 (1.1)
17.4 (.9) #
Catalase (mol·min-1.g -1 Hb)
.625 (.039)
.560 (.051)
.653 (.030)
.673 (.057)
Vitamin E (µg·mL-1)
12.49 (.36)
12.50 (.46)
12.79 (.49)
13.15 (.41) #
Vit E/cholesterol ratio (µg·mg-1)
6.74 (.20)
6.97 (.20)
6.99 (.21)
7.24 (.25) #
Vit C (µmol·L-1)
62.8 (4.9)
57.6 (4.6)
55.8 (3.2)
54.5 (4.3)
Isoprostanes (ng·mg-1 creatinine)
1.18 (.11)
1.64 (.18) *
1.24 (.12)
1.26 (.13) #
Biomarker of skeletal muscle damage
Creatine phosphokinase (UI·L-1)
529 (122)
696 (177)
605 (158)
480 (81)
General plasmatic biomarkers
Triglycerides (g·L-1)
.54 (.05)
.58 (.06)
.59 (.07)
.68 (.10)
Cholesterol (g·L-1)
1.87 (.05)
1.81 (.05)
1.84 (.06)
1.85 (.05)
Ferritin (µg·L-1)
76.9 (9.7)
69.2 (9.3) *
79.0 (10.9)
69.8 (10.8) *
Urea (g·L-1)
.287 (.012)
.279 (.013)
.301 (.013)
.291 (.019)
Hemoglobin (g·dL-1)
14.7 (.1)
14.5 (.2)
14.5 (.2)
14.8 (.2) *#
Effort test
Performance (EnRJ45, %)
-.47 (3.77)
4.59 (5.99)
Explosive power (RJ110, %)
-1.53 (1.92)
.73 (2.65)
Fatigue (RJ5, %)
1.64 (3.22)
9.55 (5.34)
* Significantly different from the pre-treatment (D0); p < 0.05 (paired t test). # Significantly different from the placebo post-treatment
(D30); p < 0.05 (paired t test).

the period (placebo or GE) (Table 3). Regardless of the the placebo supplementation (19.5 ± 9.7% vs -4.4 ± 6.1%
capsule content the athletes had, no modification of urea respectively; p < 0.05). There was also significant evi-
concentration was observed; whereas, ferritin concentra-
dence of an effective trend towards explosive power
tion was significantly reduced in both cases (Table 3). Hb
(RJ110) (-3.6 ± 2.5% vs 2.8 ± 4.3% for placebo supple-
concentration was not modified in the placebo group; mentation vs GE supplementation respectively; p = 0.1).
however, GE supplementation increased Hb concentra-
First, it is noteworthy that results on general parameters
tion, as opposed to placebo intake, respectively (14.8 ± for the handball category (Table 4) are equivalent to those
0.2 vs 14.5 ± 0.2 g·dL-1, p < 0.05; Table 3).
found in the entire subject pool (Table 3). Different corre-

lation tests were performed among handball players: re-
Effort tests
sults evidenced unequivocal cross-relation between GE-
All sport categories
induced Hb concentration increase and performance (r =
No significant effect was found regarding total physical 0.717; p = 0.02) and CPK results (r = -0.67; p = 0.034).
performance (EnRJ45), explosive power (RJ110), and
fatigue (RJL5) (Table 3). More pointedly, inter-subject Discussion
variations observed in the present study reduced the like-

lihood of establishing a significant result. Even if the In the present study, we hypothesized that a grape extract
intensity of each volunteer’s training program was con-
(GE) supplementation at physiological doses would par-
trolled and remained consistent throughout the course of tially avoid antioxidant system down-regulation and con-
our study, subjects (i.e. selected athletes) practiced a vari-
sequently lower chronic and/or acute exercise-induced
ety of elite sports: hence, training protocols had to be oxidative damage in elite athletes while in competition.
adjusted accordingly. In order to reduce the variability Moreover, oxidative stress generated under such condi-
component, we decided to analyze the effort test results as
tions is likely to trigger oxidative skeletal muscle fatigue
a function of the following criteria: the type of training, and damage (Barclay et al., 1991; Powers et al., 1999)
and the sportsmanship category involved therefore. We which can affect exercise performance. Thus, the second
considered that only the handball category comprised a objective of this study was to cross-evaluate the effect of
sufficient number of volunteers to allow for statistical the GE supplementation on these parameters, along with
analysis (n = 10).
oxidative stress and the antioxidant status.
Sub-group: the handball category (n = 10): Con-
We demonstrated that ingestion of 400mg of GE
cerning the effort test for the handball sub-group (Table per day during one month resulted in a 9.48% increase in
4), performance (EnRJ45) is increased significantly dur-
antioxidant capacity determined by ORAC method com-
ing the grape extract supplementation phase compared to pared to placebo ingestion. In vitro, ORAC values of the

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474
Grape extract improves antioxidant status on sportsmen



Table 4. Body weight, plasma and urinary parameters, and relative values of effort test, before (D0) and after (D30) supple-
mentation of GE or placebo in handball players. Values are means (±SEM) of 10 determinations performed in duplicate on
samples from different subjects; n = 10 handball players.

Placebo n=10
Grape extract n=10

D0
D30
D0
D30
Weight (kg)
88.5 (5.7)
86.5 (5.4)
88.4 (5.7)
87.1 (5.7)
Biomarkers of antioxidant status and oxidative stress
ORAC (µmol·L-1)
13 678 (487)
13 946 (482)
13 336 (384)
15 132 (536) ***
FRAP (µmol·L-1 Fe2+)
1 168 (57)
988 (39) ***
1 110 (45)
1 060 (55) #
LDLox (mU·mL-1)
537 (174)
562 (189)
622 (202)
693 (229)
SOD (U·g-1 Hb)
2 381 (116)
2 549 (122) *
2 710 (157) ‡
2 679 (113) #
GPx (µmol·min-1·g -1 Hb)
17.0 (1.4)
14.2 (1.5) *
18.8 (1.7)
18.0 (1.6) #
Catalase (mol·min-1.g -1 Hb)
.580 (.056)
.545 (.090)
.640 (.044)
.681 (.088)
Vitamin E (µg·mL-1)
12.0 (.5)
12.0 (.7)
12.2 (.6)
12.9 (.7) ##
Vit E/cholesterol ratio (µg·mg-1)
6.7 (.3)
6.9 (.3)
6.9 (.4)
7.5 (.5) *#
Vit C (µmol·L-1)
65.8 (9.1)
55.5 (9.1)
59.3 (5.1)
54.7 (5.8)
Isoprostanes (ng·mg-1 creatinine)
1.3 (.1)
1.7 (.2) *
1.3 (.2)
1.3 (.2) #
Biomarker of skeletal muscle damage
Creatine phosphokinase (UI·L-1)
704 (223)
725 (326)
790 (300)
464 (120)
General plasmatic biomarkers
Triglycerides (g·L-1)
.47 (.04)
.58 (.07)
.53 (.05)
.66 (.10)
Cholesterol (g·L-1)
1.81 (.1)
1.76 (.10)
1.78 (.10)
1.78 (.10)
Ferritin (µg·L-1)
69.1 (14.7)
57.8 (13.3) *
68.8 (15.5)
56.8 (13.0) *
Urea (g·L-1)
.29 (.02)
.28 (.01)
.30 (.05)
.27 (.06)
Triglycerides (g·L-1)
14.6 (.20)
14.56 (.19)
14.52 (.21)
14.87 (.30) *
Effort test
Performance (EnRJ45, %)
-4.4 (6.1)
19.5 (9.7) †
Explosive power (RJ110, %)
-3.57 (2.50)
2.82 (4.25)
Fatigue (RJ5, %)
2.95 (3.47)
10.85 (6.88)
*, ** and *** denote p < 0.05 , p < 0.01 and p < 0.001, respectively, from the pre-treatment (D0) by paired t test. # and ## denote p < 0.05 and
p < 0.01, respectively, from the placebo post-treatment (D30) by paired t test. † significantly (p < 0.05) different from the placebo relative val-
ues by paired z test. ‡ significantly (p < 0.05) different from the placebo pre-treatment (D0).

GE used in the present study approximates 14 000µmol sorption of different antioxidant blends. Palazzetti and co-
Trolox eq·g-1 (External analysis, Lareal, France); hence, workers (2004) also demonstrated the significant increase
ingestion of 400mg of GE enables to increase the plasma of GPx activity after antioxidant supplementation com-
ORAC value, matching a consumption of 5 600µmol pared to placebo. As regards amateur male athletes while
Trolox equivalent per day. This result is congruent with under competition conditions, the antioxidant supplemen-
the work of Cao et al. (1998), to the effect that a diet tation maintained the glutathione reductase activity in the
involving around 3 300µmol Trolox per day increases treated group after three months; whereas, it decreased
significantly the plasma ORAC value of the participants. significantly in the placebo group (Tauler et al., 2006).
Additionally, Prior et al. (2007) recently demonstrated This decrease was also exhibited in soccer players having
that consumption of mixed grape powder increases dra-
volunteered to participate in an incremental treadmill
matically post-prandial antioxidant capacity. Interestingly,
running exercise (Klapcinska et al., 2005). This enzyme is
the variation of the plasma antioxidant capacity deter-
clearly linked with GPx because it regenerates the oxi-
mined by the FRAP value differs substantially from our dized glutathione, cofactor of GPx; it is noteworthy there-
findings obtained through the ORAC method. While the fore, that there was solid evidence of enzyme activity
ORAC method allows to highlight an increase of the preservation, akin to our GPx related findings. Even if the
antioxidant capacity with GE, the FRAP method, -- con-
type and the dose of antioxidant supplementation differ in
sidered more so as an indirect method (Prior and Cao, these studies, results are congruent with the present study.
1999) --, revealed that GE limited the decrease of the The optimization of plasma ORAC and FRAP values
antioxidant capacity compared to placebo. These results could also be explained by the free scavenging activity of
evidenced that GE consumption increases the athletes’ absorbed metabolites from grapes. Plasma flavanol me-
antioxidant status; they further revealed, however, that the
tabolites were not determined in our study hereby; how-
interpretation of the changes in plasma antioxidant capac-
ever, numerous studies to date (Baba et al., 2002; Dono-
ity relies for the most part on the method used, a case in van et al., 2000; Donovan et al., 2002; Gonthier et al.,
point formerly demonstrated by Prior and Cao (1999).
2003; Holt et al., 2002; Sano et al., 2003) substantiated
The increase of the plasma vitamin E concentration
the bio-availability of grape or cocoa flavanols under
and the preservation of the GPx levels could be explained
different bio-active forms (Spencer et al., 2001; Tomas-
by the increase of the plasma ORAC value and the limita-
Barberan et al., 2007).
tion of the FRAP value reduction induced by the con-
Other blood antioxidant determined biomarkers,
sumption of GE polyphenols. The same result on vitamin enzymatic such as superoxide dismutase, catalase or non
E was shown in endurance athletes (Vasankari et al., enzymatic such as vitamin C, did not differ significantly
1997b) or in triathletes (Palazzetti et al., 2004) after ab-
whatever the period. These results are in agreement with

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Lafay et al.
475


those of Palazzetti et al. (2004) and Margaritis et al.
Numerous studies have reported that exogenous
(2003); the latter revealed no variation in vitamin C and antioxidants protect against skeletal muscle fatigue
SOD in supplemented triathletes compared to placebo. (Barclay and Hansel, 1991; Diaz et al., 1994; Khawli and
Concerning catalase activity, Tauler et al. (2003) studied Reid, 1994; Novelli et al., 1990; Reid and Moody, 1994;
its variation on athletes supplemented with vitamin C, or Shindoh et al., 1990; Travaline et al., 1997). Most studies,
with a blend of vitamin E, vitamin C and beta-carotene however, have applied synthetic or enzymatic antioxi-
(Tauler et al., 2006). In both cases they exhibited an in-
dants and resorted to in vitro muscle preparations to quan-
crease in catalase activity that was not disclosed in our tify muscular contractions (Barclay and Hansel, 1991;
study.
Diaz et al., 1994; Khawli and Reid, 1994; Reid and
One of the biomarkers most commonly investi-
Moody, 1994; Shindoh et al., 1990).
gated within biological systems remains the degree of
The determination of serum creatine phos-
lipid peroxidation; to such an end, several methods may phokinase (CPK) is used as an indirect index of exercise-
be implemented (Slater, 1985; Vasankari et al., 1995). In induced muscle damage (Chevion et al., 2003) and is
the present study, lipid peroxidation was determined in associated indirectly with the increment of the permeabil-
blood, using specific autoantibodies against oxidized LDL
ity in the muscle cell membrane (Janssen et al., 1989).
and in urine using the ELISA method to determine iso-
This permeability is produced by the oxidative stress
prostanes. No effect was shown on oxidized LDL what-
induced by physical exercise (Child et al., 1999; Pa-
ever the period, whereas significant results were obtained lazzetti et al., 2003; Van der Meulen et al., 1991). Moril-
for isoprostane urinary excretion. Urinary isoprostane las-Ruiz et al. (2006) showed that consumption of a natu-
values were significantly increased in the placebo group, ral antioxidant beverage containing 1.2g per liter of poly-
but were not modified in the GE group. Therefore, com-
phenols by cyclists increased the serum CPK concentra-
pared to the placebo, GE limited the production of iso-
tion similarly to the placebo beverage suggesting that the
prostanes significantly after the administration period. antioxidant capacity of polyphenols has no repercussion
Oxidized LDL results are in agreement with results re-
on cellular muscle damage. The same result was obtained
leased by Vasankari et al. (1997b; 1997c); however, oxi-
by Helgheim et al. (1979) with a vitamin E supplementa-
dized LDL results are not consistent with some studies tion and by Margaritis and coworkers (2003), on triath-
showing an increase of this parameter in soccer and bas-
letes supplemented with an antioxidant blend. Contrari-
ket-ball players (Pincemail et al., 2000), in weekend war-
wise, while using the same sportsmen and supplementa-
riors (Chang et al., 2002) or a decrease in sedentary vol-
tion the latter revealed a significant reduction of the mag-
unteers (Vasankari et al., 1998a) after intense exercise. To
nitude in duathlon-induced creatine kinase isoenzyme
our knowledge, no existing study has evidenced as yet, a mass increase during normal and overload training (Pa-
decrease of oxidized lipoproteins following antioxidant lazzetti et al., 2004). Branched-chain amino acids
supplementation in elite athletes.
(Coombes and McNaughton, 2000), vitamin C (Jakeman
Isoprostanes are specific end-products of non
and Maxwell, 1993), allicin (Su et al., 2008) or CoQ10
enzymatic free radical catalyzed oxidation of arachidonic (Kon et al., 2008) supplementations on different volun-
acid. It is believed that isoprostanes are formed while still
teers generated the same result: a lower CPK activity as
esterified to phospholipids in cellular membranes and are compared to the control group. Because of the supplement
released through the action of phospholipase to circulate antioxidant effects, muscle membrane integrity should be
freely in the body (Mastaloudis et al., 2001; Morrow et maintained, preventing the release of enzymes into blood
al., 1992; Watson et al., 2005). Quantification of iso-
circulation. All these results are in agreement with the
prostanes has been referred to as the ultimate criterion for
findings presented here, hereby exhibiting a trend towards
in vivo lipid peroxidation and oxidative stress, notably in decreasing the serum CPK concentration among elite
acute or chronic inflammatory conditions (Basu, 2008; athletes involved in competition while absorbing the
Pratico et al., 1998). An increase in isoprostane concen-
grape extract, as compared to placebo intake. Such a trend
tration has been reported in athletes during exercise in (p = 0.09) results from a very high variability already
several studies (Mastaloudis et al., 2001; Steensberg et al.,
reported by Hartman and Mester (2000): authors observed
2002) showing induced-exercise oxidative stress. The that athletes with chronically low CPK exhibited mainly
effect of antioxidant supplementation on this biomarker low variability; those with chronically higher values ex-
has been probed and widely documented as regards