Effect of Grape Seed Extract on Lead Induced Hypertension and Heart Rate in Rat
Fatemeh Zarea Mehrgerdi,
Mohammad Kazem Gharib Naseri
The main objective of this study was to evaluate the
potential protective effect of red Grape Seed Extract (GSE) on lead induced
hypertension (HTN) and Heart Rate (HR) in male Wistar rats. The rats were
randomly assigned to one of 4 groups: Each group received lead acetate
(100 ppm in drinking water), GSE (100 mg kg-1, orally) or Lead
+ GSE for 45 days. Another group assigned as control group provided with
tap water and regular pellet food. The Systolic Blood Pressure (SBP) and
heart rate were determined by tail plethysmography coupled to a computer
system. There was a sustained elevation of SBP in lead exposed rats that
significantly increased at day 18 (lead treated, 112.7 ±2.7 mmHg,
vs. control, 105.6±2.6 mmHg, n = 10, p<0.05) and reached a maximum
level at day 36 (lead treated, 124.9 ±2.3 mmHg, vs. control, 103.6 ±3.1
mmHg, n = 10, p<0.001). However, the other three groups; showed no
significant changes in SBP. Furthermore, the heart rate was increased
sustainly in lead exposed animals that was statistically significant at
days 36 and 45 (lead treated group, 404.5±9.4 vs. control group,
381.7 ±6.7, n = 10, p<0.05). The blood lead level in both lead
and lead + GSE treated groups was increased significantly compared with
control and GSE treated groups (p<0.001). However, GSE administration
had no effect on the blood lead level in lead treated group. According
to the result of this study, it may be concluded that GSE could have beneficial
effect in protecting the cardiovascular system through its antioxidant
activity against oxidative stress.
Lead is a bluish-gray metal and cumulative poison that exists
in combination with organic and inorganic compounds. Chronic exposure
to low levels of lead causes hypertension (HTN) in humans and animals
(Gonick et al., 1997; Ni et al., 2004; Vaziri et al.,
1997, 1999b; Vaziri and Sica, 2004). Although different considerations
have been raised to explain the pathogenesis of lead-induced hypertension
but the mechanism is not defined clearly. Several studies have suggested
the primary involvement of the increased production of Reactive Oxygen
Species (ROS) observed in lead-exposed animals (Gonick et al.,
1997; Vaziri et al., 1997). Other studies revealed strong evidence
for increased hydroxyl radical (OH) activity in rats with lead-induced
HTN and lead-treated cultured endothelial cells (Ding et al., 2000,
2001). It has been previously shown that increased ROS leads to enhanced
NO inactivation, depressed NO bioavailability and compensatory up regulation
of NO synthases (NOSs) in rats with lead-induced HTN (Gonick et al.,
1997; Ni et al., 2004; Vaziri et al., 1997, 1999b; Vaziri
and Sica, 2004). In addition, lead induced hypertension is accompanied
by a marked increase in plasma and tissue lipid peroxidation products,
an increase in malodialdehyde (MDA) and substantial reduction in urinary
excretion of stable NO metabolites (NOX) (Ding et al., 2001; Gonick
et al., 1997; Vaziri et al., 1997). In many studies it has
been demonstrated marked amelioration of hypertension together with normalization
of plasma MDA concentration and urinary NOX excretion with a variety of
antioxidants including: lazaroid (Vaziri et al., 1997) dimethylthiourea
(Ding et al., 2001), vitamin E (Vaziri et al., 1999b) and
vitamin C (Marques et al., 2001).
In animals exposed to lead in tap water, lead exposure affects
the renin-angiotensin system, inducing sympathetic hyperactivity and increasing
sensitivity to stimulation of cardiac and vascular ß receptors and dopaminergic
receptors (Boscolo and Carmignani, 1988; Victery, 1988). The involvements
of sympathetic nervous system (SPNS) and circulating catecholamines have
been implicated in lead-induced hypertension. In vitro electrophysiological
study showed that superfusion of a low concentration (5 µM) of PbCl2
enhanced excitatory postsynaptic potentials (EPSPs) in some of the SPNS
examined but reduced inhibitory postsynaptic potentials (IPSPs) in other
SPNS tested. On the other hand in vivo study showed that intrathecal
injection of PbCl2 (10 and 100 nmol) increased both the heart
rate and mean arterial pressure (Lai et al., 2002).
Grape seed is a waste product of the winery and grape juice
industries. The composition and properties of grape seed have been extensively
investigated and reported to have many favorable effects on human health
such as lowering of Low-Density Lipoprotein (LDL), reduction of cardiovascular
disease and cancer (Kim, 2005; Nomoto et al., 2004). In addition,
the seed extract of Vitis vinifera (GSE) are reported to have antimicrobial
and free radical scavenging properties and to be a good source of proanthocyanidins
(Roychowdhury et al., 2001; Shi et al., 2003). Proanthocyanidins
are potent natural antioxidants of various polyphenolic components (Shi
et al., 2003). These compounds posses a broad spectrum of antioxidative
properties with greater potency than vitamin E and C, that protects the
organs against free radicals and oxidative stress, both in vitro
and in vivo (Aldini et al., 2003; Roychowdhury et al.,
2001). However, up to now there was no investigation carried out on the
effect of GSE on the lead-induced hypertension. We hypothesized that grape
seed extract could play an important role in the scavenging of free radicals
and could thereby reduce the lead induced HTN. Therefore, the main objective
of this study was to determine the possible changes in arterial blood
pressure and heart rate in sub-chronic lead-exposed rats and to evaluate
the potential protective effect of red grape seed hydro-alcoholic extract
in these changes.
MATERIALS AND METHODS
Animals and treatments: The study protocols were approved by the
Physiology Research Center Ethics Committee for animals and were performed
according to the international conventions on animal experimentation.
Experiments were carried out during April to June 2007 with the use of
40 male Wistar rats at 3 months of age, at the beginning of the experiment.
The animals were housed in a climate controlled, light-regulated space
with 12 h light and dark cycles. They had free access to a regular rat
chow food. The rats were randomly assigned to one of 4 groups (10 rats
each): Group A (control) received tap water; Group B, provided with tap
water contained 100 ppm lead acetate for 45 days; Group C, provided with
tap water and received GSE (100 mg kg-1, orally, once a day)
via gavage for 45 days. Group D, provided with tap water contained 100
ppm lead acetate and GSE (100 mg kg-1, orally, once a day)
via gavage for 45 days. All groups received normal chow pellet food.
Grape seed extract preparation: Grape, as large clusters with
red berries, was bought from a local super market in Ahwaz, Iran and identified
by botanist as Vitis vinifera L. Grape seeds were separated from
the grapes manually, air-dried (in shade, 25-30°C) for one week and milled
to fine powder (a particle size of < 0.4 mm). The grape seed powder was
macerated in 70% ethanol (25% w/v) for 72 h at room temperature and was
stirred three times a day. The mixture filtered with cheese cloth and
the filtrate dried at room temperature (25-30°C) to remove ethanol and
grape seed extract was obtained as a powder (yield: 25-30%).
Heart rate and blood pressure recording: At the beginning and
day 9, 18, 27, 36 and 45 of the experiments conscious rat were placed
in a restrainer, prewarmed and allowed to rest for about 20 min prior
to blood pressure and heart rate measurements, that were determined by
tail plethysmography coupled to a computer system (Power Lab, AD Instruments,
Australia) (Gonick et al., 1997). Three consecutive recordings
(5 min apart) were performed and the averages of recordings were calculated
for each rat.
Blood lead measurement: At the end of experiments (day 45) blood
samples were collected in EDTA contained tubes through cardio-puncture
and the lead content of the samples was measured by graphite atomic absorption
spectrometry (Carl Ziess, Germany) following digestion of the blood in
a solution of nitric acid and perchloric acid (Parsons et al.,
Statistical analyses: Results are expressed as mean±SEM. Each
of the above mentioned studies were performed in a group of ten rats.
Comparisons were performed by repeated measurement ANOVA followed by LSD
test. The level of statistical significance was defined as p<0.05.
RESULTS AND DISCUSSION
As expected lead exposure resulted in a marked increase
in arterial blood pressure. There was a sustained elevation of Systolic
Blood Pressure (SBP) although it did not reach statistical significance
before day 18 (Fig. 1). The SBP in lead exposed rats
significantly increased at day 18 (lead treated, 112.7±2.7 mmHg, vs. control,
105.6±2.6 mmHg, n = 10, p<0.05, repeated measurement ANOVA followed by
LSD test) and reached a maximum level at day 36 (lead treated, 124.9±2.3
mmHg, vs. control, 103.6±3.1 mmHg, n = 10, p<0.001, repeated measurement
ANOVA followed by LSD test) and remained constant through out the experiment
Systolic blood pressure
n = 10) in different groups of rats during 45 day period of
experiment. *p<0.05, **p<0.01 and ***p<0.001, significant difference
between lead treated and other groups (repeated measurement
ANOVA followed by LSD test)
period Fig. 1. However, administration
of GSE along with lead resulted in a significant prevention of the blood
pressure to increase in the GSE+lead treated group. In contrast to the
lead-exposed animals, the other three groups; control, GSE and lead+GSE
treated animals, showed no significant changes in SBP.
There was a sustained increase in heart rate in lead exposed
animals that reached its maximum at day 36 and it was statistically different
from control group (lead treated group, 404.5±9.4 vs. control group, 381.7±6.7,
n = 10, p<0.05, repeated measurement followed with LSD test, Fig.
2). Nevertheless, in those animals that received GSE+lead, the heart
rate was not different from control or GSE treated groups and remained
The blood lead level in both groups, lead and lead+GSE treated
groups was increased significantly compared with control and GSE treated
groups (lead exposed group, 259±12 µg dL-1, vs. control group,
65±8 µg dL-1, n = 10, p<0.001, One way ANOVA, followed by LSD
test). However, GSE administration had no effect on the blood lead level
in lead treated group (Fig. 3).
The present study investigated for the first time whether
GSE had antihypertensive effects on the lead-induced hypertension. In
this study, we used rats, which were treated with 100 ppm lead acetate
in their drinking water, for 45 days. This amount of lead is considered
Heart rate (Mean±SEM, n =
10) in different groups of rats during 45 day period of experiment.
# p<0.05, lead treated group vs. control group and *p<0.05,
lead treated group vs. GSE treated group (repeated measurement
ANOVA followed by LSD test)
Blood lead level (Mean±SEM,
n = 10) in control animals and in different groups of rats after
45 days treatment with Lead (100 ppm), GSE (100 mg kg-1,
orally) or lead + GSE. ***p<0.001 significantly differ from
control or GSE treated rats (one-way ANOVA followed by LSD test)
low level of exposure, similar to the level seen in the
environment, although the result values are not directly comparable to
those of humans. Previous studies have shown that exposure for longer
duration (three and more months) to low level (100 ppm) of lead, not high
level (5000 ppm), results in hypertension in rats (Khalil-Manesh et
al., 1993; Purdy et al., 1997). Present results show that sub-chronic
(45 day) exposure will also results in increased systolic blood pressure
and heart rate. These effects were prevented when the GSE was simultaneously
administered with lead, but it has no effect on blood pressure and heart
rate when administered alone.
Lead-induced hypertension is the element of chronic lead
intoxication syndrome that has received more attention in the past decade
and different considerations have been raised to explain the pathogenesis
of lead-induced hypertension. Several studies have focused on the involvement
of endothelium dysfunction to generate NO in this model of hypertension.
These studies demonstrated the existence of an altered NO synthesis, probably
because of a diminished eNOS activity and/or increased NO catabolism by
oxygen free radicals. In vitro studies have demonstrated that NOS
activity is inhibited by lead (Mittal et al., 1995) and previous
study from Vaziri et al. (1999a) reported that a rise in vascular
eNOS expression in lead treated rats was accompanied by inhibition of
eNOS activity. In this regard, Vaziri et al. (2003) and
Ding et al. (2000) have demonstrated an increased lipid peroxidation
and enhanced hydroxyl radical generation in rats and cultured endothelial
cells after exposure to lead, respectively. In several earlier studies,
some evidence was found that oxidative stress and increased ROS activity
lead to enhance NO oxidation and depressed NO bioavailability in rats
with lead-induced HTN (Ding et al., 2001; Gonick et al.,
1997; Vaziri et al., 1997, 1999a). They have further shown that
lead-induced oxidative stress is primarily due to increased hydroxyl radical
generation in both intact animals and cultured endothelial cells (Ding
et al., 2000, 2001).
In another study, it has shown that lead may exert a stimulatory
effect on sympathetic preganglionic neurons (SPNs). This effect may result
mainly from the reduction of inhibitory postsynaptic potentials (IPSPs)
and to a lesser extent, enhancement of excitatory postsynaptic potentials
(EPSPs) in SPNs by low concentration of lead. The activation of SPNs may
cause an enhancement of sympathetic outflow resulting in an increase of
blood pressure and heart rate (Lai et al., 2002). In addition,
it has shown that chronic exposure to lead is able to strongly increase
plasma levels of adrenaline and, mostly, noradrenaline well agrees with
other data showing lead to increase sympathetic nerve activity (Carmignani
et al., 2000).
Grapes (Vitis vinifera) are one of the most widely
consumed fruits worldwide and are rich in polyphenols. Grape seeds are
byproducts formed during the industrial production of grape juice and
wine. They are a potent source of proanthocyanidins, which are mainly
composed of dimers, trimers and oligomers of monomeric catechins (Agarwal
et al., 2007; Veluri et al., 2006). Although the mechanism
of the beneficial health effects of grape seed polyphenols is not well
understood, several lines of evidence strongly suggest that they are powerful
antioxidants and are able to serve as free radical scavengers (Joshi et
al., 2001; Mittal et al., 1995). In addition, it has shown
that the polyphenols of grape seed could protect against cardiac cell
apoptosis via the induction of endogenous cellular antioxidant enzymes
(Du et al., 2007). The antioxidant activity of grape seed`s polyphenols
is more potent than vitamin C and vitamin E (Aldini et al., 2003).
There are many evidences indicating that up regulation of reactive oxygen
species play an important role in some forms of cardiovascular disease,
including hypertension (Ademuyiwa et al., 2005; Patrick, 2006).
Thus, GSE could prevent lead induced hypertension by scavengering ROS
and/or by induction of cellular antioxidant enzymes. Furthermore, it is
likely that GSE by reduction of heart rate as shown by this study could
attenuate the lead induced hypertension. By this mean GSE could protect
against the harmful effect of ROS on cardiovascular system through its
antioxidant activity and by this mean may be have beneficial effect in
protecting the organism against oxidative stress. Whether, it is protect
the NO/cGMP, the endothelial cells or stimulate other relaxant system
or inhibits production of vasoconstrictive substances is unclear. However,
the exact mechanism by which the extract prevent lead induced hypertension
remained to be determined.
This research is supported by Research Affairs of Ahwaz
JundiShapour University of Medical Sciences (grant No. PRC-12) and is
a part of M.Sc. student thesis of Mrs. Fatemeh Zarea Mehrgerdi M.Sc. student
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