00:00, 01 марта 2008, Научные статьи

Medicina Sportiva

Effects of supplemetation with red grape skin polyphenolic extract and interval swimming test on the blood antioxidant status in healthy men

Авторы:
Sadowska-Krępa E., Kłapcińska B., Kimsa E.
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1 () 2008, 01 марта 2008
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1-7
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Общеспортивная тематика
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Аннотация

Исследование было направлено на оценку воздействия безалкогольного экстракта полифенола красного винограда на уровень антиоксидантов в крови здоровых мужчин после плавательного теста.

Effects of supplemetation with red grape skin polyphenolic extract and interval swimming test on the blood antioxidant status in healthy men

Abstract

Introduction: Grapes Vitis vinifera and red wine are rich sources of polyphenolic substances which may potentially enhance the antioxidant capacity of plasma in humans.

The aim of the study: The study was aimed at the assessment of the effects of the alcohol-free red grape skin polyphenolic extract on the blood antioxidant status in healthy male subjects subjected to interval swimming test.

Materials and methods: Fourteen physical education students randomized to two groups: the control group (n=5) and the experimental group (n=9) supplemented with an alcohol-free polyphenolic extract from the skins of Vitis vinifera red wine grapes. One capsule containing 390 mg of red grape skin extract was administered three times daily for 6 weeks. The students participated in the interval-type swimming test (free-style with moderate to high intensity) at two occasions i.e. before the onset of the study (1st trial) and then once more after 6 weeks (2nd trial). In venous blood samples taken at rest, immediately after completion of interval swimming test and after 1h recovery the activities were measured of antioxidant enzymes: glutathione peroxidase (GSH-Px), superoxide dismutase (SOD), catalase (CAT), glutathione reductase (GR), level of glutathione (GSH). Creatine kinase (CK) activity and concentrations of uric acid (UA), lactate (LA) and total antioxidant status (TAS) were measured in plasma.

Results: A marked tendency was observed toward lower, as compared to values recorded in the 1st trial, pre- and post-exercise CK activities after a 6-wk -long supplementation with red grape skin extract (F=4.01; P=0.056 by 3-way ANOVA). A 6 wk-long supplementation had an insignificant modification of antioxidant enzyme (SOD, CAT, GSH-Px, GR) activities, concentrations of non-enzymatic antioxidants (GSH, UA) and total antioxidant status (TAS) were observed. The beneficial effect of an alcohol-free red wine grape extract on hemodynamic parameters. A comparison of swimming speeds and heart rates (HR) attained by subjects during interval swimming tests has evidenced that during the 2nd trial the participants from the supplemented group were able to swim faster at the final 50 m repeat (improvement from 1.18±0.03 to 1.31±0.11 ms-1) at HR's slightly lower from those recorded during the first trial. Mean swimming speed attained by participants from the control group at the final 50 m distance during the 2nd trial was also higher (improvement from 1.22 to 1.29 ms-1), however this was accompanied with an increase in HR from 184.6 to 187.6 beatsmin-1.

Conclusions: The above presented results indicate that the supplementation with the alcohol free red wine grape polyphenolic extract may influence on attenuation of the post-exercise release creatine kinase (CK) into the circulation. Supplementation with this extract seems to be promising for the improvement of the hemodynamic status of subjects subjected to physical loads.

Key words: red wine grape polyphenolic substances, antioxidant enzymes, total antioxidant status (TAS), creatine kinase (CK), physical exercise

Introduction

Oxygen consumption during intense physical work may increase as much as 20-fold which is associated with a markedly increased production of oxygen radicals (1). Reactive oxygen species (ROS), including hydroxyl radicals, superoxide radicals, hydroperoxides or aldehydes are known to be toxic, mutagenic and carcinogenic to cells. It has been suggested that an increased production of ROS plays an important role in exercise-induced muscle damage (1-3). In spite of the intervention of the cell's antioxidant defense system, free radical-mediated lipid peroxidation can lead to the loss of integrity of cell membranes and to tissue damage.

Inactivation and removal of reactive oxygen species (ROS) depend on the capacity of the antioxidant defense system determined by a dynamic interaction between antioxidant enzymes (superoxide dismutase - SOD, EC 1.15.1.1, glutathione peroxidase - GSH-Px, EC 1.11.1.9), catalase - CAT, EC 1.11.1.6, glutathione reductase -GR, EC 1.6.4.2), and the non-enzymatic antioxidants which include low-molecular weight molecules such as tocopherol (vitamin E), ascorbic acid (vitamin C), beta-carotene, glutathione - GSH, and uric acid.

It has been suggested that exercise-induced muscle damage could be prevented through the supplementation of dietary antioxidants (4). Antioxidant properties have also been evidenced in red wine grapes (Vitis vinifera) and red wines. Many studies have confirmed that grapes Vitis vinifera and red wine are rich sources of polyphenolic substances, that are usually subdivided into two groups, the flavonoids (quercetin, catechins, flavonols, anthocyanidins) and nonflavonoids (phenolic acids and resveratrol). These substances may potentially enhance the antioxidant capacity of plasma in humans as they behave as scavengers of ROS, as well as metal chelators and enzyme modulators (57). Resveratrol (3,5,4'-trihydrostilbene), a prominent representative of polyphenols found in the skins of red grapes and in red wine, has a variety of bioactivities associated with health promotion (8). Other human dietary sources of resveratrol are peanuts and peanut butter. The polyphenolic structure of this stilbene polyphenol confers antioxidant activity and may prevent oxidative stress-induced cellular damage (9,10). Resveratrol is also known as a potent antiarrhythmic agent with cardioprotective properties associated with its antioxidant activity and upregulation of nitric oxide (NO) production (11). This natural polyphenolic phytochemical as a chemoprevention agent has been shown to inhibit tumor initiation and the growth of cancerous cells through increased apoptosis, to reduce inflammatory processes by inhibition of prostaglandin production, cyclooxygenase-2 activity and to prevent the progression of fungal infection (9).

Numerous previous studies have been dedicated to the assessment of the benefits from antioxidant supplementation in physically active individuals and professional athletes (4,12). To our knowledge, there are no published reports on the combined effect of the exercise and supplementation with polyphenols derived from the skins of red wine grapes on the antioxidant status. The objective of the present study is to examine the effects of the alcohol-free red grape skin polyphenolic extract on the blood antioxidant status in healthy male subjects subjected to interval swimming test.

Methods

Subjects

Fourteen nonsmoking male physical education students aged 21.0±0.6 yr volunteered to take part in this study. All subjects were informed of the purpose and the nature of the study before giving their written consent to participate in the experiment, which had been approved by the Ethics Committee of the Academy of Physical Education. The basic characteristics of the subjects is presented in Table 1. None of the participants of the study was following a special diet or taking any drug known to affect antioxidant status of the blood.

Exercise protocol

The participants were randomly assigned into two groups: 1- the control group (n=5) and 2- the experimental group (n=9) supplemented with an alcohol-free polyphenolic extract from the skins of Vitis vinifera red wine grapes (Andean Medicine Centre Ltd. UK). The supplement presented in soft gelatinous capsules was administered at a dose of 1 capsule containing 390 mg of red grape skin extract three times daily for 6 weeks. The phenolic composition of the extract, as declared by the producer, was 188 mg-g-1 of polyphenols (catechin, gallic acid, quercetin, trans-resveratrol, cis-resveratrol) and 35 mg-g-1 of anthocyanidins (malvidin, peomidin, petunidin, delphinidin, cyanidin). The students participated in the interval-type swimming test (free-style with moderate to high intensity) at two occasions i.e. before the onset of the study (1st trial) and then once more after 6 weeks (2nd trial).

The interval freestyle (front crawl) swimming test consisted of six repeats of 50m with the first distance at 70-75% effort and subsequent 50m repeats with progressively increasing velocity with 10-20 seconds rest between swims and the last swim as a maximal effort. The heart rate (HR) was monitored before the start of the test and at the end of each distance by using Sport Tester Polar S610i (Polar Electro Oy, Finland) and the times on each repeat were measured by a stopwatch.

Venous blood samples were drawn into heparinized test tubes from the antecubital vein at rest, immediately after completion of interval swimming test and after 1h of passive recovery.

Analytical procedures

Fresh whole blood samples were immediately assayed for reduced glutathione (GSH) by a colorimetric method (13) with 5,5'-dithiobis-2-nitrobenzoic acid and the activity of GSH-Px by using commercial kit (RANSEL RS505, Randox,UK). The remaining blood was centrifuged for 10 min at 1,000 g at 4°C to separate plasma and erythrocytes. The erythrocytes were washed three-times with cold (4°C) saline and kept frozen at - 20°C, for not longer than 2 weeks, until being assayed for activities of antioxidant enzymes, i.e. SOD using a commercially available RANSOD SD125 kit (Randox, UK), CAT by the method of Aebi (14) and GR according to Glatzle et al. (15). The activity of CAT was expressed as the rate constant (k) of a first order reaction of hydrogen peroxide decomposition related to the hemoglobin (Hb) content (k-gHb-1). One GR activity unit was defined as the reduction of 1^mol of oxidized glutathione (GSSG) per minute, monitored by a decrease in absorbance at 340 nm due to oxidation of NADPH consumed for reduction of GSSG. The activities of all antioxidant enzymes were measured at 37°C and expressed per 1 g of hemoglobin as assesed by a standard cyanmethemoglobin method using a diagnostic kit (HG980; Randox, UK).

Table 1. Basic characteristics of the subjects (n=14)

 

First trial

Second trial

Variable

Control Group (n=5)

Supplemented group (n=9)

Control Group (n=5)

Supplemented group (n=9)

Age (years)

21.0 (0.5)

21.6 (0.7)

21.0 (0.5)

21.6 (0.7)

Height (m)

180.3 (6.3)

182.3 (8.2)

180.3 (6.3)

182.3 (8.2)

Body mass (kg)

78.5 (8.9)

77.3 (8.5)

78.3 (8.3)

77.1 (8.8)

BMI (kg-m-2)

23.8 (0.6)

23.7 (0.9)

23.5 (0.5)

23.4 (0.9)

 

Fresh plasma samples were assayed for activities of creatine kinase (CK, EC 2.7.3.2) at 37°C using commercial diagnostic kits (Randox, UK). The plasma concentration of lactate (LA) was assessed at 30°C by commercially available enzymatic, colorimetric kit (Randox, UK). The level of the Total Antioxidant Status (TAS) in fresh plasma samples was assayed using commercial diagnostic kits (Randox, UK).

Statistical analysis

Mean and (±SD) were calculated for all variables. All data were tested for homogeneity of variances using the Levene's test and then analyzed using a 3-way analysis of variance (ANOVA) for repeated measures, followed by the Bonferroni test. All statistical analyzes were performed using STATISTICA 5.0 (StatSoft, Inc. 1995) software. The level of significance was set at p<0.05.

Results

The obtained results are presented in Tables 1 to 3. Basic characteristics of participants is presented in Table 1. No between-group differences were found in somatic parameters recorded in both trials.

The results of biochemical analyses are presented in Tables 2 and 3. Supplementation with red grape skin extract appeared not to affect significantly the activity of SOD. There was only a tendency towards slightly higher SOD activities to occur immediately post-exercise. Despite the lack of significant differences between the pre- and post exercise values a repeated measures 3-way ANOVA revealed a significant (F = 4.47; p<0.05) -effect of exercise.

Similarly, no significant, exercise-induced changes in CAT and GSH-Px activities were found in both the control and supplemented groups in each trial. There was only a tendency toward slightly higher CAT and GSH-Px activities immediately post-test. Interestingly, the contrary trends in GSH-Px activities were observed between 1st and 2nd trial, i.e. an increase (significant in the case of resting values; p<0.05) in the control group and a decrease - in the experimental group receiving the supplement. In both groups of subjects the post-exercise changes in GR activities recorded in both trials were small and insignificant.

No significant effect was found of the supplementation and exercise on concentrations of GSH in erythrocytes.

There was a tendency toward delayed increase in UA concentration in plasma during the recovery period from the interval swimming test, as reflected by a significant exercise- effect (F=24.04; p<0.05 by 3-way ANOVA). Ingestion of the red grape skin extract led to a significant (p<0.05; by 3-way ANOVA) decrease in the resting plasma concentrations of UA in subjects from the supplemented group.

Plasma concentrations of TAS were slightly higher after interval swimming test, the effect of exercise was significant (F=27.05; p<0.001 by 3-way ANOVA), but the effect of the supplementation did not reach the significance level.

The interval swimming test induced changes in plasma activity of CK, the results are presented in Table 3. A significant increase in CK activities was observed immediately after completion of the swimming test, and a significant exercise-effect (F=27.19; p<0.0001) was revealed by 3-way ANOVA. Moreover, a marked tendency was observed toward lower, as compared to values recorded in the 1st trial, pre- and post-exercise CK activities after a 6-wk -long supplementation with red grape skin extract, and the effect appeared to be close to the significance level (F=4.01; p=0.056 by 3 -way ANOVA). It is noteworthy that all CK activities were within the reference range for men (24-195 U-l-1) (16).

The results of plasma lactate (LA) determinations recorded during interval swimming test are presented in Table 3. The post-test lactate concentrations were found to be significantly (p<0.001) higher than the pre-test values. A significant main effect of exercise (F=2048.3; p<0.001) on LA level was found by 3-way ANOVA.

The swimming speeds and post-test heart rates (HR) (Table 4) recorded during all repeats of the interval swimming test are presented in Table 4. With the aim of revealing whether supplementation with the red grape extract could affect performance, we have calculated the differences between swimming speeds achieved at individual repeats of the interval swimming test during the 2nd and the 1st trials. Mean results calculated separately for the control and the supplemented groups are presented in Fig. 1. The significant differences (p<0.05) in swimming speeds between the control group and the experimental group supplemented with the red grape skin extract were measured at the five distance of individual 50m repeats of the interval freestyle swimming.

Table 2. Mean (±SD) pre- and post-test activities of the antioxidant enzymes: superoxide dismutase (SOD), catalase (CAT), glutathione peroxidase (GSH-Px), glutathione reductase (GR) and the plasma Total Antioxidant Status (TAS) in the control and the experimental group supplemented with red grape skin extract

 

Control group

Supplemented group

Variable

First trial

Second trial

First trial

Second trial

rest

after test

1h

post

Rest

after test

1h

post

Rest

after test

1h

post

rest

after test

1h

post

SOD

1043.4

1185.1

907.1

1182.7

1375.4

1467.1

1199.8

1240.1

1257.9

1176.2

1359.4

1478.3

(U-gHb-1)

(691.9)

(683.0)

(420.2)

(140.9)

(158.8)

(197.2)

(386.1)

(395.9)

(527.6)

(128.5)

(219.9)

(364.7)

CAT

177.2

185.2

196.0

207.9

198.2

200.8

207.9

209.9

216.5

187.2

214.6

230.9

(s-1gHb-1)

(33.5)

(37.3)

(46.6)

(24.6)

(28.8)

(19.2)

(32.8)

(29.4)

(39.2)

(33.5)

(42.7)

(51.7)

GSH-Px

27.2

30.9

34.9

43.1*

49.3

44.3

35.9

42.1

38.1

27.9

31.0

34.9

(U-gHb-1)

(4.13)

(6.6)

(7.2)

(5.6)

(12.7)

(5.8)

(3.9)

(9.8)

(6.6)

(8.7)

(10.8)

(9.5)

GR

23.0

21.9

24.3

23.3

26.0

23.6

23.7

23.0

24.2

25.8

26.7

25.1

(U-gHb-1)

(4.3)

(2.7)

(4.3)

(7.9)

(3.2)

(9.2)

(2.7)

(2.8)

(4.6)

(6.3)

(4.7)

(7.1)

TAS

798.1

891.8

959.3

814.7

972.5

053.6

773.9

835.2

908.8

811.4

878.7

1029.9

(|imol/L)

(166.1)

(97.9)

(139.5)

(132.0)

(131.2)

(100.7)

(55.3)

(64.0)

(132.4)

(114.9)

(141.8)

(213.9)

 

Note: Significantly (*p<0.05) different from the corresponding value in 1st trial

 

Table 3. Mean (±SD) pre- and post-test concentrations of blood glutathione (GSH), plasma lactate (LA) and uric acid and activities of plasma creatine kinase (CK) and lactate dehydrogenase (LD) in subjects from the control and the experimental group supplemented with red grape skin extract

Variable

Control group

Supplemented group

First trial

Second trial

First trial

Second trial

Rest

after test

1h

post

Rest

after test

1h

post

Rest

After test

1h

post

Rest

after test

1h

post

GSH (re-gHb-1)

1.8 (0.2)

1.7 (0.4)

1.8 (0.4)

2.2 (0.3)

1.9 (0.5)

2.3 (0.7)

2.3 (0.2)

1.8 (0.2)

1.5 (0.2)

2.4 (0.3)

1.8 (0.1)

2.3 (0.4)

Uric acid (mg-dl-1)

2.8 (0.7)

3.2 (0.7)

3.9* (0.7)

4.7 (1.1)

5.4 (0.8)

6.5* (0.5)

4.0* (0.9)

4.9 (1.2)

5.9 (1.8)

3.5* (0.7)

3.9 (0.8)

4.4 (0.5)

CK (U-l-1)

142.3 (52.4)

186.0** (57.2)

167.2* (40.8)

172.7* (59.6)

208.3* (79.2)

180.5 (60.2)

135.2 (59.7)

168.2* (75.1)

151.1 (74.8)

101.2* (28.3)

127.3* (31.9)

116.2 (33.8)

LA (mmol-l-1)

2.1 (0.4)

14.5** (0.9)

2.8 (0.6)

3.4 (0.6)

12.9** (0.7)

2.8 (0.6)

3.4 (0.6)

13.8** (0.7)

3.2 (0.9)

3.2 (0.5)

13.7** (1.2)

2.9 (0.6)

 

Note: Significantly (*p<0.05, ** p<0.001) different from the resting value; Significantly (*p<0.05) different from the corresponding value in 1st trial.

 

Table 4. Swimming speeds and mean HRs recorded at each of six 50 m repeats of interval freestyle swimming test in the control and the experimental group supplemented with red grape skin extract

 

Control group

Supplemented group

Repeat No.

1th trial

2nd trial

1th trial

2nd trial

 

V (m-s-1)

HR

V (m-s-1)

HR

V (m-s-1)

HR

V (m-s-1)

HR

1

1.08

147.2

1.10

155.0

1.07

142.0

1.12

152.0

(0.03)

(15.8)

(0.02)

(11.7)

(0.09)

(10.8)

(0.05)

(13.8)

2

1.07

157.4

0.88

164.2

1.12

158.6

1.10

161.1

(0.03)

(16.0)

(0.48)

(12.1)

(0.07)

(9.3)

(0.07)

(9.6)

3

1.14

167.0

1.11

165.6

1.15

168.8

1.15

171.2

(0.04)

(14.1)

(0.08)

(19.2)

(0.09)

(8.6)

(0.07)

(5.3)

4

1.20

170.0

1.13

173.0

1.19

170.8

1.16

170.0

(0.07)

(14.1)

(0.11)

(9.8)

(0.06)

(8.6)

(0.09)

(7.4)

5

1.22

180.0

0.99

179.0

1.20

175.7

1.19

173.8

(0.12)

(9.7)

(0.48)

(8.8)

(0.15)

(10.0)

(0.08)

(8.9)

6

1.22

184.6

1.29

187.6

1.18

186.7

1.31

185.3

(0.02)

(9.5)

(0.18)

(9.1)

(0.03)

(12.4)

(0.11)

(8.6)

 

Discussion

Numerous previous studies have evidenced that the efficiency of the antioxidant defense system in humans depends on endogenous production of antioxidants and on adequate dietary vitamin and micronutrient intake (17). It is well established that several naturally occurring foods, such as skins and seeds of red wine grapes contain a very high concentration of flavonoid and nonbioflavonoid polyphenols, which are potent antioxidants. The antioxidant capacity of red wine and alcohol-free red grape skin extracts has been shown in different in vitro and in vivo systems (18). Polyphenols act as antioxidants by the hydrogen-donating property of their hydroxyl groups (6,19,20). Apart from their capacity to scavenge free radicals, polyphenols may prevent hydroxyl radical formation through the Haber-Weiss/Fenton reactions, due to their metal-chelating properties (21). It was evidenced that consumption of red wine increases the antioxidant capacity of serum (7). Much less is known about the potentially beneficial effects of the extract prepared from the grape Vitis vinifera on the antioxidant status of physically active individuals, both men and women, which has motivated us to undertake our investigation.

The most interesting finding of the present study was that a 6 week-long consumption of the red grape skin extract resulted in a significant decrease (main effect) in CK activities and a significant decline in resting CK activity in the plasma (Table 3). The release of creatine kinase (CK), an integral part of the ATP/creatine phosphate energy system of muscle, into circulation, as evidenced by an increase in activity of this enzyme in the serum or plasma, is considered the most sensitive indicator of muscle injury (22). There is a substantial evidence that exercise-induced oxidative stress may play a pivotal role in both the initiation and progression of muscle cell membrane disruption (23). One of the major factors responsible for the increase in the permeability of plasma membranes, which results in a "leakage" of intracellular proteins into the circulation, is peroxidation of membrane lipids, markedly enhanced during strenuous exercise (24). In the study reported here, post-exercise CK activities recorded in both groups during both trials were significantly higher from the resting values. It is worth noting that CK activities recorded during the 1st trial in both groups of participants were very close to each other. Interestingly, as compared to the 1st trial, CK activities recorded during the 2nd interval swimming test, were higher in the control group, while the contrary trend toward lower values was observed in subjects receiving the red grape skin extract. Most likely, the antioxidant capacity of this extract played an important role in maintaining the integrity of the cell membrane post-exercise and limiting CK release from skeletal muscle. In this regard, our results are in accordance with those reported by Morillas-Ruiz et al. (25) for the polyphenol-supplemented individuals subjected to intense exercise.

It should be stressed, however, that although the antioxidant properties of most of the phenolic components of red wine grapes have been shown in vitro, much less is known about the antioxidant potential of individual components in vivo (18,26,27). Moreover, in most studies the antioxidant effects of the specific red wine constituents appeared not to be secondary to changes in plasma concentrations of antioxidant vitamins (28,29).

Although the results of our study have indirectly evidenced the antioxidant potential of the red grape skin extract in attenuating signs and symptoms of exercise-induced skeletal muscle injury in young physically active men, little direct influence on their blood antioxidant status, as mostly insignificant modification of antioxidant enzyme (SOD, CAT, GSH-Px, GR) activities, concentrations of non-enzymatic antioxidants (GSH, UA) and total antioxidant status (TAS) were observed. Post-exercise increases in antioxidant enzyme activities appeared also to be relatively small and mostly insignificant, although the main-effect of exercise appeared to be significant, but only in the case of SOD.

The discrepancies in findings among studies suggest that type and intensity of exercise may affect the response of the blood antioxidant defense system. In a recent study of Nikolaidis et al. (30) with adolescent swimmers aged 9-11 y subjected to an interval swimming test (12 bouts of 50 m at a pace of 70-75 % of the participant's maximum 50 m speed) significant increases in CAT activity, TAS as well as significant decreases in GSH were reported. Although directions of post-exercise changes recorded in our study were similar, i.e. the red blood cell activities of SOD, CAT and GSH-Px also tended to increase in response to the interval swimming test; the magnitude of changes was much less (Table 2). Similarly, the post-exercise decline in GSH concentration observed in both groups of participants has not reached the level of significance. As expected, the plasma concentrations of urate tended toward delayed increase during the recovery period, which appeared to be significant only in the control group (Table 3). Interestingly, in subjects supplemented with the red wine grape extract the resting plasma concentrations of uric acid, recorded during the 2nd trial, were significantly lower than the baseline values. These observations suggests that interval swimming test, as applied in our study, resulted only in subtle changes in the capacity of the antioxidant defense system in the blood.

Among the most promising health implications related to consumption of flavonoids and polyphenolic components derived from red wine grapes are their antiatherogenic and cardioprotective effects (8,11,31,32). Such a suggestion originated from epidemiologic observations among inhabitants of Southern France who have a very low mortality rate due to coronary heart disease (CHD) despite a high-fat diet, little exercise, and a wide-spread cigarette smoking. This phenomenon, known as French Paradox, is attributed to a relatively high intake of red wine.

Several experiments performed on animal models have evidenced a strong cardioprotective effect of red wine polyphenols. Mokni et al. (10), who studied selected hemodynamic parameters, such as heart rate (HR) and developed pressure (DP) in isolated hearts from control and resveratrol treated rats, observed a marked improvement in post- ischemic indices of myocardial function after 7 day-long intra-peritoneal administration of this polyphenol (but not after an acute treatment), which could be only partly explained by its antioxidant properties. Another group (33) who examined the effect of resveratrol on ventricular function of rat hearts after ischemia-reperfusion (I/R)-induced injury observed also a dramatic improvement in post-ischemic ventricular functional recovery. Moreover, these cardioprotective effects were correlated with both the antioxidant activity of resveratrol and up-regula-tion of nitric oxide (NO) production. Previous studies (34) with the use of human umbilical vein endothelial cells exposed to an alcohol-free red wine grape extract evidenced augmented release of NO from endothelial cells in a concentration-dependent manner, which may be attributed to an over expression of endothelial NO synthase (eNOS) mediated by red wine polyphenols.

Direct effects of red wine or alcohol-free wine extracts on endothelial cells have been reported by several authors, who concluded that NO release induced by direct action of some anthocyanins and other polyphenols present in red wine may be involved in the increase in coronary flow vascular reserve, as observed after consumption of red wine in both healthy human volunteers and patients with CAD (8,35,36).

The results of our study seem to support indirectly these observations on the beneficial effect of an alcohol-free red wine grape extract on hemodynamic parameters. A comparison of swimming speeds and heart rates (HR) attained by our subjects during interval swimming tests (Table 4, Fig.1) has evidenced that during the 2nd trial the participants from the group supplemented with the red grape skin extract were able to swim faster at the final 50 m repeat (improvement from 1.18±0.03 to 1.31±0.11 m-s-1) at HR's slightly lower from those recorded during the first trial. Mean swimming speed attained by participants from the control group at the final 50 m distance during the 2nd trial was also higher (improvement from 1.22 to 1.29 m-s-1), however this was accompanied with an increase in HR from 184.6 to 187.6 beats/min. Moreover, although all students enrolled for the experiment were from the same study year, which implies that their weekly activity profile was similar, those from the control group had generally worse results in swimming speeds (Fig. 1) recorded during the 2nd trial (except for the final 50 m distance). Swimming speeds attained during the 2nd trial by individuals supplemented with the red grape skin extract were not worse than those recorded before starting supplementation (1st trial), and a marked improvement was observed at the last 50 m distance. Of importance is the fact that there was a marked tendency toward lower HR values over the last three 50 m repeats despite unchanged (on 4th and 5th repeats) or visibly higher swimming speed attained at the last 50 m distance during the 2nd interval swimming test.

Taking together, the differences in the response to physical loads, as those applied in the interval swimming test, seem to support the hypothesis of the beneficial effects of supplementation with the alcohol free red wine grape extract on maintenance of integrity of the muscle cell membranes and attenuation of the post-exercise release of the muscle cell enzyme (CK) into the circulation, despite only a minor impact on the capacity of the blood antioxidant defense system. Supplementation with red wine grape polyphenolic extract seems to be promising for the improvement of the hemodynamic status of subjects subjected to physical loads.

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