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JOURNAL OF MEDICINAL FOOD
J Med Food 11 (1) 2008, 000–000
© Mary Ann Liebert, Inc. and Korean Society of Food Science and Nutrition
DOI: 10.1089/jmf.2007.558
Intake of Purple Grape Juice as a Hepatoprotective Agent in Wistar Rats
Caroline Dani,
1
Lívia S. Oliboni,
1
Matheus A.B. Pasquali,
2
Marcos R. Oliveira,
2
Fernanda M. Umezu,
1
Mirian Salvador,
1
José C.F. Moreira,
2
and João A.P. Henriques
1
1
Instituto de Biotecnologia, Universidade de Caxias do Sul, Caxias do Sul;
and
2
Centro de Estudo em Estresse Oxidativo, Departamento de Bioquímica,
Universidade Federal do Rio Grande do Sul, Porto Alegre, Rio Grande do Sul, Brazil
ABSTRACT Grape juice is a source of polyphenols, as catechin, anthocyanidins, resveratrol, and others. Some health ben-
efits have been attributed to these compounds (e.g., antioxidant and antitumorigenic properties). In this study, we investigated
the possible antioxidant activity of two different grape juices: organic purple grape juice and conventional purple grape juice.
The antioxidant activity of both grape juices was evaluated by an animal model of three groups: control and organic and con-
ventional juices. After 30 days, all animals were sacrificed, and blood and liver were collected to evaluate lipid peroxidation
level (thiobarbituric acid-reactive substances [TBARS] assay), protein oxidative level (carbonyl assay), and catalase (CAT)
and superoxide dismutase (SOD) activities. The group treated with organic grape juice showed the highest SOD and CAT ac-
tivities in both plasma and liver when compared with the conventional and control groups (P .05). In plasma, we observed
a positive correlation among SOD and CAT activities, resveratrol, and all anthocyanin contents, suggesting that these polyphe-
nols may be, at least in part, responsible for this increased antioxidant defense. The grape juices were capable of reducing
carbonyl and lipid peroxidation levels in plasma and liver. However, in plasma, the organic group showed lower carbonyl and
TBARS levels when compared to the conventional grape juice group (P .05). Our findings suggest that the intake of pur-
ple grape juice, especially of organic juice, induces a better antioxidant capacity when compared to conventional juice and
that this may be an important issue for further investigations in the area of biochemical functional foods.
KEY WORDS:
•
antioxidant
•
hepatoprotective
•
oxidative stress
•
phenolic content
127
INTRODUCTION
T
HE LIVER REGULATES
many important metabolic func-
tions. Hepatic injury is associated with distortion of
these metabolic functions.
1
Additionally, the liver is the
key organ of metabolism and excretion, and it is continu-
ously and variedly exposed to xenobiotics because of its
strategic placement in the body. Thus, liver diseases re-
main one of the more serious health problems. The CCl
4
-
induced hepatotoxicity model is frequently used for in-
vestigating hepatoprotective effects of drugs and plant
extracts.
2,3
CCl
4
-induced toxicity is a well-characterized murine
model for the study of oxidative damage in vivo. The toxi-
city of CCl
4
results from its reductive dehalogenation by the
liver cytochrome P450 enzyme system into the
trichloromethyl free radical, which readily interacts with
molecular oxygen to form trichloromethyl peroxyl radicals.
4
Both radicals are able to attack proteins and lipids or still
abstract hydrogen atoms from an unsaturated lipid, leading
to membrane lipid peroxidation, cellular dysfunction, and,
finally, to cell necrosis.
5
In a recent study, grape leaf extracts were able to reduce
the damage caused by CCl
4
.
6
The possible hepatoprotective
activity of purple grape juices, either organic or conven-
tional, has not been reported so far.
Grape juice is a very rich source of polyphenols, such as
flavonoids, tannins, and resveratrol.
7
Although there are
studies reporting that Vitis vinifera grape juices show an-
tioxidant activity,
8–10
there is no reference in literature about
this on Vitis labrusca cultivars. Presently, there is an in-
creasing interest in a healthier and more environmentally
friendly production method for fruits. Organic production is
a cultivation method characterized by restrictions against the
use of synthetic pesticides and fertilizers, as well as of ge-
netic engineering.
11
Given these considerations, the aim of the present study
was to investigate the beneficial effects of two different pur-
ple grape juices—organic and conventional—in reducing
the damage to liver and the oxidative stress in plasma and
liver, using the well-established murine model.
Manuscript received 18 August 2007. Revision accepted 3 October 2007.
Address reprint requests to: Caroline Dani, Instituto de Biotecnologia da Universidade
de Caxias do Sul, Universidade de Caxias do Sul, Rua Francisco Getúlio Vargas, 1130,
Caxias do Sul, RS, Brazil, 95070-560, E-mail: carolinedani@yahoo.com.br
MATERIALS AND METHODS
Grape juices
The grape juice samples used in this study were produced
from V. labrusca, variety Bordo, vintage 2005. The organic
juices were produced with organically cultivated grapes (no
pesticides) and were obtained from Cooperativa Aecia (An-
tonio Prado, RS, Brazil), which received the ECOVIDA cer-
tificate, a guarantee of organic food production. The con-
ventional juices were obtained from Vinhos Monte Reale
(Flores da Cunha, RS, Brazil). Both grape juices were kindly
donated by these wineries.
Phenolic compounds
Total phenolic content was measured by using the mod-
ification of Singleton et al.
12
of the Folin-Ciocalteu col-
orimetric method. High-performance liquid chromatogra-
phy (HPLC) analysis was used in order to quantify the
presence of individual phenolic compounds. Prior to
HPLC analysis, 5 mL of each sample was filtered through
a cellulose membrane with a 0.20-mm diameter. The
equipment used in the analysis consisted of a chromato-
graphic system of liquid gradient, LC-DAD Series 1100
chromatograph (Hewlett-Packard, CA, USA), with a de-
tector system of diode array. A Zorbax 300 SB C18 pre-
column (12 mm 4.6 mm, 5 m particle size) and C18-
ODS column (150 mm 4 mm, 5 m particle size) were
used in the equipment.
In order to quantify the resveratrol compound, we used a
mobile phase of ultrapure water and acetonitrile (75:25
vol/vol) (pH 3.0), at a constant flow of 1.0 mL/minute for
20 minutes, in a controlled-temperature room at 20°C. The
peak was detected at 306 nm, and the amount of sample in-
jected was 20 L.
13
In order to determine cyanidin-3-glucoside, delphidin-3-
glucoside, peonidin-3-glucoside, and malvidin-3-glucoside,
a mobile phase with solvents A (ultrapure water, formic acid,
and acetonitrile [87:10:3 by volume]) and B (ultrapure wa-
ter, formic acid, and acetonitrile [40:10:50 by volume]), at
a constant flow of 0.8 mL/minute, in a controlled-tempera-
ture room at 25°C, was applied. The peak was detected at
518 nm, and the amount of sample injected was 50 L. The
elution conditions were 50–60% (30 minutes), 60–100% (30
minutes), and 100–50% (10 minutes).
14
Animals
Twenty-four male Wistar rats (60 days old, weighing
200 50 g) from our breeding colony were used in the ex-
periments. The animals were handled under standard labo-
ratory conditions of a 12-hour light/dark cycle and fixed
temperature (25 2°C). Food and water were available ad
libitum. All experimental procedures were performed in ac-
cordance with the U.S. National Institutes of Health’s Guide
for the Care and Use of Laboratory Animals with the ap-
proval of the local ethics committee.
Treatment
The animals were randomly allocated into one of the three
experimental groups (n 8): group 1 served as the control
and received vehicle saline, and conventional or organic pur-
ple grape juices were given to groups 2 and 3, respectively.
The doses of purple grape juice were determined by calcu-
lating the amount of juice that would be consumed daily in
average by a 70-kg male human.
15
Juices were administered
to the rats (7 L of grape juice/g of body weight) twice a
day. During the experiment, the amount was adjusted ac-
cording to the animals’ weight. Before sacrifice, the ani-
mals’ blood was colleted and kept in heparin-coated tubes.
On day 30, half of the animals received a single intraperi-
toneal CCl
4
(3 mL/kg) dose. The animals that received CCl
4
or only vehicle (mineral oil [control]) were killed 6 hours
later by decapitation. Liver samples were isolated and stored
at 70°C until analysis.
Oxidative stress analyses
We used the thiobarbituric acid-reactive species (TBARS)
output during an acid-heating reaction as an index of lipid
peroxidation, which is widely adopted as a sensitive method
for the measurement of lipid peroxidation, as previously de-
128 DANI ET AL.
T
ABLE
1. T
OTAL
P
HENOLIC
C
ONTENT AND
L
EVELS OF
R
ESVERATROL AND
A
NTHOCYANINS
(C
YANIDIN
,
D
ELPHINIDIN
, P
EONIDIN
,
AND
M
ALVIDIN
)
IN
O
RGANIC AND
C
ONVENTIONAL
G
RAPE
J
UICES
Grape juice
Organic Conventional
Total phenolic compounds (mg of catechin/mL) 262.50 0.70* 119.59 3.53
Resveratrol amount (ppm) 0.213 0.005* 0.075 0.010
Cyanidin (ppm) 11.79 0.42* 0.76 0.04
Delphinidin (ppm) 26.30 1.15* 4.10 0.40
Peonidin (ppm) 19.21 1.43* 8.59 0.82
Malvidin (ppm) 232.46 4.25* 95.26 1.95
Data are mean SD values.
*Statistically different between the two grape juices (P .05).
scribed.
16
In brief, the samples were mixed with 10%
trichloroacetic acid and 0.67% thiobarbituric acid and then
heated in a boiling water bath for 15 minutes. TBARS were
determined by absorbance at 535 nm.
The oxidative damage to proteins was assessed by deter-
mining carbonyl groups based on the reaction with dinitro-
phenylhydrazine, as previously described.
17
In brief, pro-
teins were precipitated by addition of 20% trichloroacetic
acid and redissolved in dinitrophenylhydrazine, and the ab-
sorbance was read at 370 nm.
Antioxidant enzyme assays were performed in tissue ho-
mogenates, as previously described. Catalase (CAT) activ-
ity was assayed by measuring the rate of decrease in H
2
O
2
absorbance at 240 nm.
18
Superoxide dismutase (SOD) ac-
tivity was assayed by measuring the inhibition of adrenaline
autooxidation at 480 nm, as previously described.
19
Statistical analyses
Biochemical data are expressed as mean SEM values,
and analysis of variance and Tukey’s test were performed
using the SPSS (Chicago, IL) version 12.0 package. All tests
were performed in duplicate. Pearson’s correlation coeffi-
cient was used to test the correlation between polyphenol
content and the assays.
RESULTS
Table 1 shows the content of phenolic compounds in the
two types of purple grape juice used in this study. The two
types present a statistical difference in the content of total
phenolic compounds (P .05), especially in resveratrol
amount; the organic purple grape juice had higher amounts
in both parameters. Important differences could be observed
between both grape juices with regard to content of antho-
cyanins (malvidin, cyanidin, delphinidin, and peonidin); we
also observed that the organic juice is richer in the amount
of all phenolics than the conventional juice (Table 1).
In this study, we have demonstrated that grape juice, es-
pecially the organic one, was capable of altering oxidative
parameters in plasma. It was observed that the animals that
received organic grape juice showed lower plasma lipid per-
oxidation levels when compared to conventional grape juice
and control groups (P .05) (Fig. 1A). We found a nega-
tive correlation between lipid peroxidation (TBARS) and to-
tal phenolic content (r 0.511) and resveratrol (r
0.546), cyanidin (r 0.604), peonidin (r 0.512),
delphinidin (r 0.593), and malvidin (r 0.526) con-
HEPATOPROTECTION BY PURPLE GRAPE JUICES 129
*
A
0.00
Control
1.75
TBARS (nmol MDA/mg protein)
Control Organic Conventional
0.25
0.50
0.75
1.00
1.25
1.50
Organic
Conventional
**
*
*
B
0
4
nmol carbonyl/mg protein
Control Organic Conventional
1
2
3
C
0
50
Sod (USod/mg protein) Cat (UCat/mg protein) Sod/Cat
20
10
30
40
*
**
*
**
*
**
FIG. 1. (A) TBARS level, (B), carbonyl levels (protein oxidation
assay), and (C) antioxidant enzyme activities and their ratio in the
plasma of rats treated with different grape juices. Control rats received
saline. Data are mean SD values. *P .05 compared to control;
**P .05 compared to conventional grape juice treatment. MDA,
malondialdehyde.
*
0.00
1.25
TBARS (nmol MDA/mg protein)
Control Organic Conventional
0.25
0.50
0.75
1.00
Vehicle
CCl4
#
*
#
*
FIG. 2. TBARS content in liver of chronic grape juice-treated Wis-
tar rats. Data are mean SD values. *P .05 compared to control
vehicle;
#
P .05 compared to control CCl
4
. MDA, malondialde-
hyde.
tents (P .01 for all comparisons). Indeed, the protein ox-
idative damage decreased after treatment with both grape
juices, showing a significant difference in relation to the
control (P .05) (Fig. 1B). In this assay, the polyphenol
content showed an important correlation; we also observed
a negative correlation between carbonyl content and
polyphenol total amount (r 0.799) and resveratrol (r
0.679), cyanidin (r 0.604), peonidin (r 698), del-
phinidin (r 0.629), and malvidin (r 0.692) contents
(P .05 for all comparisons). When analyzing the activi-
ties of antioxidant enzymes, we observed that the group
treated with organic grape juice had higher SOD and CAT
activities as compared to the conventional grape juice and
control groups. We observed a positive correlation between
the SOD and CAT activities (r 0.707; P .01). We ob-
served a positive correlation between SOD and CAT activ-
ities and total phenolic content (r 0.773 and 0.578, re-
spectively) and also between resveratrol (r 0.775 and
0.602), cyanidin (r 0.775 and 0.632), peonidin (r
0.687 and 0.578), delphinidin (r 0.766 and 0.629), and
malvidin (r 0.721 and 0.588) contents (P .01 for all
comparisons). This suggests that these polyphenols may be
responsible for this increased antioxidant defense. The
SOD/CAT ratio of the organic group presented the lowest
level when compared to conventional juice and control
groups (P .05) (Fig. 1C). This ratio showed a negative
correlation with the content of phenolic compounds; we ob-
served this correlation with resveratrol (r 0.621), cyani-
din (r 0.608), peonidin (r 0.609), delphinidin (r
0.619), and malvidin (r 0.615) (P .05 for all com-
parison).
CCl
4
damage was quantified through the lipid peroxida-
tion detection assay, and the level of lipid peroxides was
significantly increased in the liver of rats after the CCl
4
in-
jection (P .05) (Fig. 2). However, after treatment with or-
ganic grape juice these levels decreased significantly (P
.05) when compared to conventional grape juice and con-
trol treatments (Fig. 2). This could be explained by the phe-
nolic content; we observed a negative correlation between
liver lipid peroxidation and total phenolic content (r
0.511) and resveratrol (r 0.546), cyanidin (r
0.604), peonidin (r 0.512), delphinidin (r 0.593),
and malvidin (r 0.526) contents (P .05 for all com-
parisons).
Figure 3 shows the capacity of CCl
4
to induce protein ox-
idative damage in liver when compared to control (P .05).
A significant attenuation of the oxidative damage induced
by the CCl
4
injection can be observed in the groups that
were given grape juice (P .05), but the group that received
conventional grape juice showed lower values (higher pro-
tection against damage) when compared to the group that
130 DANI ET AL.
*
0.0
1.5
nmol carbonyl/mg protein
Control Organic Conventional
0.5
1.0
Vehicle
CCl4
#
*
*
(
**
)
(
**
)
#
*
*
Vehicle
CCl4
*
(
**
)
*
0
60
UCAT/mg protein
Control Organic Conventional
20
10
30
40
50
Vehicle
CCl4
A
*
(
**
)
*
*
0
80
USOD/mg protein
Control Organic Conventional
20
10
30
40
50
60
70
Vehicle
CCl4
B
*
0.0
3.5
Sod/Cat Ratio
Control Organic Conventional
1.5
1.0
0.5
2.0
2.5
3.0
C
#
FIG. 3. Liver protein peroxidation (carbonyl) levels in liver. Data
are mean SD values. *P .05 as compared to control vehicle;
#
P .05 compared to control CCl
4
; **P .05 compared to con-
ventional grape juice treatments.
FIG. 4. Levels of enzyme activity—(A) CAT, (B) SOD, and (C)
SOD/CAT ratio—in liver in rats treated with different grape juices
and that received CCl
4
or vehicle injection. *P .05 compared to ve-
hicle;
#
P .05 compared to CCl
4
; **P .05 compared to organic
and conventional grape juice treatments.
received organic grape juice (P .05) (Fig. 3). However,
in the groups that received vehicle in addition to grape juice,
the organic grape juice group showed a more significant de-
crease when compared to the conventional grape juice
group, but both grape juices provided protection when com-
pared to the vehicle-only group (P .05) (Fig. 3).
Figure 4 shows the effects of grape juice treatment on
CAT and SOD and on the ratio of both enzymes’ activities
in liver. Modifications in CAT activity in the liver were ob-
served between the control and CCl
4
-treated groups, with
the CCl
4
group showing higher values (P .05) (Fig. 4A).
However, a significant increase in CAT activity was also
observed in the organic grape juice group when compared
to the conventional grape juice and control groups that had
both received vehicle in addition (P .05). We observed a
correlation between CAT activity and total phenolic content
(r 0.372) and resveratrol (r 0.380), cyanidin (r
0.376), delphinidin (r 0.381), peonidin (r 0.373), and
malvidin (r 0.376) contents (P .01 for all comparisons).
SOD activity was reduced in CCl
4
-treated rat liver (P .05)
(Fig. 4B). We observed that both juices reduced SOD ac-
tivity in liver when compared to the control group that re-
ceived vehicle only (P .05) (Fig. 4B). The liver SOD/CAT
ratio decreased significantly when compared to the control
and conventional groups (Fig. 4C).
DISCUSSION
In our study we observed that chronic treatment with
grape juice was able to reduce the lipid peroxidation level
in liver and plasma after CCl
4
injection, whereas the organic
juice induced a more significant reduction than the conven-
tional juice. These protection activities could indicate a he-
patoprotective action of grape juice since the CCl
4
damage
was smaller after grape juice intake.
Orhan et al.
6
showed that the ethanol extract of V.
vinifera—despite not having measured its active compounds
in their study—was capable of inducing a possible hepato-
protective action. They believed that it could be due to (1)
inhibiting the cytochrome P450-dependent oxygenase ac-
tivity, (2) preventing lipid peroxidation, and (3) stabilizing
the hepatocyte membrane, induced by polyphenolic com-
pounds.
We also observed that CCl
4
injection increased lipid per-
oxidation level 6 hours after a single intraperitoneal injec-
tion, suggesting that this agent makes a good topic for in-
vestigating the antioxidant effect of chronic treatment with
grape juice. The phenolic content of the juices could be a
possible explanation for this effect. Organic grape juice
showed higher contents of total phenolic compounds and
other phenolics, such as resveratrol and anthocyanins, asso-
ciated with antioxidant activities.
9,20–23
Phenolic compounds are secondary metabolites produced
and accumulated in plant tissues. Depending on the pres-
ence of biotic and abiotic factors (e.g., phytopathogenesis,
water availability), different amounts of these compounds
in plant organs would result. Actually, organic farming is a
small-scale practice, in which there is no use of chemical
protective substances like pesticides or artificial fertilizers
to promote plant growth. Since pesticides are not used, the
plants are more susceptible to the action of phytopathogenic
organisms, resulting in the production of larger amounts of
phenolic compounds.
11
This study shows that the choice of
agricultural practice used for grapes (organic vs. conven-
tional) results in different amounts of total phenolic com-
pounds, resveratrol, and anthocyanins. The correlations ob-
served between amount of resveratrol and anthocyanins with
antioxidant enzyme activity could help explain why some
important studies attributed health benefits
9,20–23
to grape
juice intake, as we have shown our study.
Our study further showed that especially after treatment
with organic grape juice the liver SOD/CAT ratio was lower
than that in the control group. This parameter is very im-
portant because as a result of an imbalance between these
two enzymes oxidative stress may be induced, and it par-
ticipates in some diseases.
24,25
SOD activity leads to the
production of hydrogen peroxide, which can react with iron
via the Fenton reaction to generate hydroxyl radicals, which
are thought to be the most toxic oxygen molecules in vivo.
26
CAT could scavenge an excess of hydrogen peroxide, avoid-
ing its potential role as an oxidative stress-facilitating mol-
ecule. Our results showed that grape juice treatment induced
CAT activity in a different way than SOD, reducing the
SOD/CAT ratio and suggesting a better antioxidant protec-
tive status once there would be no extra hydrogen peroxide
to overcome Fenton chemistry.
In conclusion, based on earlier reports
27–29
providing ev-
idence of antiplatelet and antioxidant benefits from grape
consumption and on our results showing reduced oxidative
stress in liver and plasma, it seems reasonable to recommend
that moderate quantities of purple grape juice be regularly
included in daily servings of fruits and vegetables in order
to help maintain a healthy life by attenuating oxidative dam-
age and providing hepatoprotective action, at least in some
in the organs studied.
ACKNOWLEDGMENTS
We thank the Universidade de Caxias do Sul (Caxias do
Sul, RS, Brazil), CAPES, IBRAVIN, CNPq, and FAPERGS
for their help and financing during the research.
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