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Journal of Toxicology and Environmental Health, Part A
Current Issues
ISSN: 1528-7394 (Print) 1087-2620 (Online) Journal homepage: http://www.tandfonline.com/loi/uteh20
Pomegranate peel extract attenuates oxidative
stress by decreasing coronary angiotensin-
converting enzyme (ACE) activity in hypertensive
female rats
Roger L. dos Santos, Lais O. Dellacqua, Nathalie T. B. Delgado, Wender N.
Rouver, Priscila L. Podratz, Leandro C. F. Lima, Mariela P. C. Piccin, Silvana S.
Meyrelles, Helder Mauad, Jones B. Graceli & Margareth R. Moyses
To cite this article: Roger L. dos Santos, Lais O. Dellacqua, Nathalie T. B. Delgado, Wender
N. Rouver, Priscila L. Podratz, Leandro C. F. Lima, Mariela P. C. Piccin, Silvana S. Meyrelles,
Helder Mauad, Jones B. Graceli & Margareth R. Moyses (2016): Pomegranate peel extract
attenuates oxidative stress by decreasing coronary angiotensin-converting enzyme (ACE)
activity in hypertensive female rats, Journal of Toxicology and Environmental Health, Part A,
DOI: 10.1080/15287394.2016.1213690
To link to this article: http://dx.doi.org/10.1080/15287394.2016.1213690
Published online: 15 Aug 2016. Submit your article to this journal
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Pomegranate peel extract attenuates oxidative stress by decreasing coronary
angiotensin-converting enzyme (ACE) activity in hypertensive female rats
Roger L. dos Santos
a
, Lais O. Dellacqua
a
, Nathalie T. B. Delgado
a
, Wender N. Rouver
a
, Priscila L. Podratz
a
,
Leandro C. F. Lima
b
, Mariela P. C. Piccin
c
, Silvana S. Meyrelles
a
, Helder Mauad
a
, Jones B. Graceli
d
,
and Margareth R. Moyses
a
a
Department of Physiological Sciences, Federal University of Espirito Santo, Vitoria, Espirito Santo, Brazil;
b
Department of Physiology and
Biophysics, Federal University of Minas Gerais, Belo Horizonte, Minas Gerais, Brazil;
c
Department of Biophysics, Federal University of Rio de
Janeiro, Rio de Janeiro, Brazil;
d
Department of Morphology, Federal University of Espirito Santo, Vitoria, Espirito Santo, Brazil
ABSTRACT
Based on the antioxidant properties of pomegranate, this study was designed to investigate the
effects of pomegranate peel extract on damage associated with hypertension and aging in a
spontaneously hypertensive rat (SHR) model. The influence of pomegranate consumption was
examined on systolic blood pressure (SBP), angiotensin-converting enzyme (ACE) coronary activ-
ity, oxidative stress, and vascular morphology. Four- or 28-wk-old SHR model rats were treated for
30 d, with terminal experimental animal age being 8 and 32 wk, respectively, with either
pomegranate extract (SHR-PG) or filtered water (SHR). Data showed significant reduction in SBP
and coronary ACE activity in both age groups. The levels of superoxide anion, a measure of
oxidative stress, were significantly lower in animals in the SHR-PG group compared to SHR alone.
Coronary morphology demonstrated total increases in vascular wall areas were in the SHR group,
and pomegranate peel extract diminished this effect. Pomegranate peel extract consumption
conferred protection against hypertension in the SHR model. This finding was demonstrated by
marked reduction in coronary ACE activity, oxidative stress, and vascular remodelling. In addition,
treatment was able to reduce SBP in both groups. Evidence indicates that the use of pomegranate
peel extract may prove beneficial in alleviating coronary heart disease.
ARTICLE HISTORY
Received 27 November 2015
Accepted 30 May 2016
Systemic arterial hypertension is considered a global
public health problem with 9.4 million deaths attrib-
uted to it annually (Lima et al., 2012). As this condi-
tion is a major cardiovascular manifestation (Kearney
et al.. 2005), prevention and treatment of systemic
arterial hypertension should receive high priority.
Age is a significant risk factor in development of
cardiovascular disease; however, striking gender dif-
ferences also exist in the chronological development
of heart disease (Rosamond et al., 2007).
In human and experimental hypertension mod-
els, such as spontaneously hypertensive rats (SHR),
endothelium-dependent relaxation may be attenu-
ated, and the resulting endothelial dysfunction
contributes to increased peripheral resistance.
Endothelial dysfunction has been linked to
decreases in nitric oxide (NO) bioavailability,
reflecting impaired generation of NO and/or
enhanced inactivation of NO by free radicals
(Púzserová et al., 2010).
Free radicals are any species capable of inde-
pendent existence with at least one unpaired elec-
tron, such as superoxide anions (O
2.∙-
) (Forman
et al., 2008). The relationship between free radi-
cals and hypertension was first suggested in the
1960s (Romanowskia et al., 1960), but only in the
1990s was this association investigated exten-
sively. The administration of heparin-bound
superoxide dismutase (SOD) to SHR animals
was associated with a reduction in blood pressure
(Nakazono et al., 1991). This decrease in systolic
blood pressure (SBP) following administration of
SOD may be due to a fall in reactive oxygen
species (ROS), which may contribute to elevation
in blood pressure, either directly, related to vaso-
constrictor effects, or indirectly, by reducing the
CONTACT Prof. Roger Lyrio dos Santos, PhD rogerlyrio@hotmail.com Department of Physiological Sciences, Biomedical Center, Federal University of
Espirito Santo, Marechal Campos Avenue, 1468, 29050-755, Vitoria, ES, Brazil.
Color versions of one or more of the figures in the article can be found online at www.tandfonline.com/uteh
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A
http://dx.doi.org/10.1080/15287394.2016.1213690
© 2016 Taylor & Francis
activity of vasodilators such as NO (Reckelhoff
and Romero, 2003).
Increased sympathetic nervous system activity
(Prodel et al., 2015), upregulation of the renin–
angiotensin–aldosterone system (Probstfield and
O’Brien, 2010), and enhanced oxidative stress
(Hamilton et al., 2001) are important factors that
are modified in hypertension. These factors may
lead to changes in the vessel structure. Angiotensin
II was suggested to contribute to vascular hyper-
trophy and hypertension via stimulation of the
NADPH oxidase system to subsequently elevate
ROS levels in vascular cells (Nickenig and
Harrison, 2002).
Several epidemiological studies suggested that
regular consumption of foods and beverages that
are rich in polyphenols, such as red wine, berries,
cocoa, tea, soy, and pomegranate, is associated
with a reduction in risk of a range of pathological
conditions including hypertension, coronary heart
disease, stroke, and dementia (Ghosh and
Scheepens, 2009). Studies in rats demonstrated
that pomegranates (Mohan et al., 2010) are rich
in polyphenolic antioxidants, which include tan-
nins, anthocyanin, and flavonoids (Jurenka, 2008).
All parts of the fruit seem to possess considerable
amounts of polyphenols, but the husk appears to
contain the highest concentration of these antiox-
idants (Gil et al., 2000).
Antioxidants are well known to enhance the
biological actions of NO by protecting against
oxidative destruction mediated by ROS (Gil et al.,
2000). Pomegranate is a rich source of antioxi-
dants; however, little is known regarding the
action of pomegranate on the coronary vascular
bed. Therefore, the objective of this study was to
examine the potential of pomegranate peel extract
in protecting against damage mediated by hyper-
tension on angiotensin-converting enzyme (ACE)
activity, oxidative stress, and vascular remodelling.
Materials and Methods
Animals
Spontaneously hypertensive (SHR) Wistar female
rats (4 and 28 wk old) were randomly divided into
two groups: SHR and spontaneously hypertensive
pomegranate extract (SHR-PG). Animals were
obtained from the animal facilities at the Federal
University of Espirito Santo. Pomegranate extract
was dissolved in filtered water and administered
orally for 30 d by gavage. The control group
received filtered water. At the end of the treat-
ment, the animals were 8 and 32 wk old, respec-
tively. Rats were maintained in temperature-
controlled rooms (22°C) under a 12-h light/dark
cycle. All procedures were conducted in accor-
dance with the institutional guidelines for animal
research, and protocols were previously approved
by the Institutional Ethics Committee for Use of
Animals (CEUA 107/2011).
Noninvasive Arterial Blood Pressure Assessment
Noninvasive measurement of tail-cuff pressure as
an estimate of systolic arterial pressure was carried
out 1 d before treatment started and on the last
day (d 30) of administration. Rats were warmed in
a restraining chamber, and occluding cuffs and
pneumatic pulse transducers were placed on their
tails. A sphygmomanometer was inflated and
deflated automatically, and tail-cuff signals from
the transducer were automatically recorded using
an IITC apparatus (IITC, Inc., Woodland Hills,
CA) connected to a computer. For each blood
pressure measurement session, the mean of three
arterial blood pressure readings was recorded for
each rat.
Plant Material
The plant material of choice (Punica granatum L.)
popularly known as pomegranate, belonging to the
family Lythraceae, was collected in the city of
Vitoria, state of Espirito Santo, Brazil. Plant sam-
ples were authenticated by Dra. Valquíria Ferreira
Dutra at the Department of Biological Sciences,
Federal University of Espirito Santo, where a sam-
ple (voucher specimen number 37631) was depos-
ited in the herbarium of the VIES/UFES in botany
sector.
Preparation of Pomegranate Peel Extract
The peel of Punica granatum L. was removed and
dried in shade for 10 d before grinding. Extract
was prepared according to Lapornik et al. (2005)
2R.L.DOSSANTOSETAL.
with modification. Briefly, pomegranate was col-
lected; peel was removed, dried for 5 d, and then
ground. The ground material (85.71 g) was mixed
in 1000 ml ethanol (95°GL) in an amber bottle
until the complete extraction of peel compounds.
Subsequently, the sample was vacuum filtered,
supernatant was collected, and alcohol was evapo-
rated in a rotary evaporator at 60°C. The resultant
crude extract (hydroalcoholic extract) (68%, w/w)
was kept at 4°C in an amber bottle. Because the
hydroalcoholic extract undergoes a certain degree
of hydration, a dry weight determination was
made. The hydroalcoholic extract was diluted in
filtered water and administered for 30 d orally by
gavage at a concentration of 25 mg/100 g rat.
Determining Estrous Cycle Phase
Daily vaginal smears were taken from each female
rat as previously described (Marcondes et al.,
2002) to confirm that estrous cycles were proceed-
ing normally. The vaginal epithelial cells were
examined by microscopy for at least 7 consecutive
days before the experiment. The swabs were per-
formed between 8:00 and 10:00 a.m. to maintain
consistency. The females exhibiting normal estrous
cycles were killed at proestrus between 9:00 a.m.
and 1:00 p.m.
Isolation of Coronary Arteries
At the end of treatment, animals were anesthetized
with sodium thiopental (50 mg/kg, ip) and eutha-
nized via decapitation. The thorax cavity was
opened, and the heart was removed and placed
in a buffer solution of Tris-HCl, pH 7, with 50
mMNaCl (Carmona et al., 2006). The left anterior
descending branch and septal branch coronary
were isolated using a dissection microscope (D.F.
Vasconcelos M900, São Paulo, Brazil).
Subsequently, samples were stored at –80°C until
protein quantification (Furieri et al., 2011).
Measurement of Angiotensin-Converting Enzyme
(ACE) Coronary Activity
Angiotensin-converting enzyme (ACE) coronary
activity was determined using the fluorescence
resonance energy transfer (FRET) peptide Abz-
FRK(Dnp)P-OH (Aminotech Pesquisa and
Desenvolvimento, SP, Brazil) as a substrate
(Alves et al., 2005). Coronary samples were homo-
genized in 0.1 MTris-HCl buffer, pH 7, containing
50 mMNaCl and centrifuged at 1000 × g for 10
min. The hydrolysis rate of the Abz-FRK(Dnp)P-
OH substrate (10 μM) after incubation for 30 min
at 37°C in coronary homogenate aliquots was
assessed to obtain ACE enzymatic activity. The
assay methodology was adapted for a 96-well
plate reader. Fluorescence was measured at 320
nm excitation and 420 nm emission wavelengths
(Synergy2 BiotekR USA). Assays were performed
in triplicate, and results were averaged. Coronary
ACE activity was expressed in arbitrary fluores-
cence units (AFU/μg protein). The protein content
was determined by the Lowry et al. method (1951)
method.
Histological Analysis of Anterior Septal Coronary
At the end of treatments, each rat was anesthetized
with sodium thiopental (50 mg/kg, intraperitoneal
injection) and the heart was removed. For each
animal, the medial portion from the septal coron-
ary artery was used. Subsequently, 10 sections
every 100 µm were obtained. The morphometric
analyses of total vascular and wall areas corre-
sponded to average values obtained from 10 cross
sections. Subsequently, tissues were embedded in
optical cutting temperature (OCT) compound and
cross-sectioned on a cryostat (Jung CM1850; Leica,
Wetzlar, Germany) at a thickness of 8 μm. For
each animal, the coronary cross sections were
mounted on gelatin-coated slides and stained
with hematoxylin–eosin for morphometric analy-
sis as shown previously (Borgo et al., 2016).
Morphometry
Images of the coronary arteries were captured
using a 20× objective with a color video camera
(VKC150, Hitachi, Tokyo, Japan) connected to a
microscope (Olympus AX70, Olympus, Center
Valley, PA) and analyzed employing a National
Institutes of Health (NIH) imaging program. An
examiner blind to the experimental groups per-
formed the image analysis to prevent any bias in
the interpretation of the results.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 3
Detection of Superoxide Production
Dihydroethidium (DHE) staining was used to
determine ROS generation. Unfixed frozen coron-
ary sections (8 μm) after dehydration with a 30%
sucrose solution were incubated with 2 μmol DHE
(Molecular Probes, Sigma, D7008) in modified
Krebs solution containing 20 mmol HEPES for
30 min in a light-protected chamber at 37ºC.
Subsequently, the samples were subjected to three
washes with phosphate-buffered saline (PBS), air
dried (light-protected), and mounted with neutral
glycerine. The levels of ROS were determined
using microscopy, and coronary fluorescence was
quantified with microscope software (NIS-
Elements BR 7.0, Nikon Instruments, Inc.,
Champigny-sur-Marne, France). An enhanced
red fluorescence suggested elevated levels of super-
oxide anion. The fluorescence intensity of the cor-
onaries was quantified in 10 arbitrarily selected
coronaries, and the mean value for each islet was
calculated.
Statistical Analysis
All data are expressed as the mean ± SEM. To
identify possible outlier data, a two-sided Grubbs
test was used to identify whether at least one out-
lier was present in each dataset. When Grubbs’test
identified one outlier, an adapted ROUT method
was used to detect any outliers from that column
data and remove them according to the Q setting
at 1% (alpha = .01). For each data set, the
D’Agostino–Pearson omnibus normality test was
also performed. If data passed the normality test,
then one-way analysis of variance (ANOVA)
followed by Tukey’s post hoc test for multiple
comparisons was applied. The significance was
set at p< .05.
Results
Figure 1 shows the systolic blood pressure (SBP)
on d 30 of treatment with water or pomegranate
peel extract in the 8-wk-old animals (A) and 32-
wk-old animals (B). A significant decrease was
noted in SBP in the SHR-PG group compared
with the SHR alone after 30 d in both 8-wk-old
and 32-wk-old animals. Data in Figure 2 demon-
strate the effect of treatment with pomegranate
peel extract on coronary ACE activity. The activity
of ACE was significantly reduced in SHR-PG com-
pared with SHR in both age groups.
Figure 3 summarizes the data obtained from
coronary morphology analyses of 8-wk-old (left
panels) and 32-wk-old animals (right panels). An
increase in total vascular (1.86 ± 0.19 vs. 1.13 ± 0.1
µm
2
,Figure 3D) and wall areas (1.06 ± 0.11 vs.
0.56 ± 0.04 µm
2
,Figure 3E) was noted in SHR with
8-wk-old animals compared to age-matched con-
trols. Similarly, an elevation in total vascular (3.75
± 0.16 vs. 2.27 ± 0.16 µm
2
,Figure 3J) and wall
areas (1.82 ± 0.13 vs. 0.78 ± 0.06 µm
2
,Figure 3K)
was found in SHR at 32 wk compared to age-
matched controls. However, treatment of SHR
with pomegranate peel extract resulted in a return
to control values in total vascular area in SHR at
both 8 wk (1.86 ± 0.19 to 0.89 ± 0.07, Figure 3D)
and 32 wk (3.75 ± 0.16 to 2.25 ± 0.29 µm
2
,
Figure 3J) respectively. Further, treatment of SHR
with pomegranate peel extract also produced a
return to control levels in wall areas in SHR at 8
Figure 1. Effect of pomegranate extract on systolic blood pressure (SBP) on d 0 and on d 30 in 8-wk-old (A, n= 18) and 32-wk-old
animals (B, n=16–18). Values are expressed as means ± SEM. Asterisk indicates significant at p< .05 compared with SHR alone
(unpaired Student’st-test).
4R.L.DOSSANTOSETAL.
wk (1.06 ± 0.11 to 0.64 ± 0.03 µm
2
,Figure 3E) and
32 wk (1.82 ± 0.13 to 1.13 ± 0.09 µm
2
,Figure 3K).
An increase in wall/lumen ratios was found in
SHR at 8 wk (1.38 ± 0.10 vs. 0.89 ± 0.07 µm
2
,
Figure 3F) and 32 wk (1.28 ± 0.05 vs. 0.57 ± 0.05
µm
2
,Figure 3L) compared to age-matched con-
trols. The treatment of SHR with pomegranate
peel extract were also able to lower these values
in SHR at 8 wk (1.38 ± 0.1 to 0.97 ± 0.09 µm
2
,
Figure 3F) and 32 wk (1.28 ± 0.05 to 0.8 ± 0.13
µm
2
,Figure 3L).
The antioxidant potential effect of the pomegra-
nate peel extract treatment was observed in the cor-
onary arteries using DHE staining, as illustrated in
the microphotographs in Figure 4.ADHEoxidative
assay revealed intense fluorescence in SHR animals
but none in SHR-PG. Pomegranate peel extract
administration reduced vascular oxidative stress in
coronary arteries in the SHR at ages 8 wk (11.3 ± 0.9
AU vs. 3.66 ± 0.9 AU) and 32 wk (55.7 ± 4.9 AU vs.
25.96 ± 5.0 AU) compared with respective controls.
On average, coronary arteries from SHR-PG animals
exhibited approximately 50% less ethidium fluores-
cence than SHR.
Discussion
Our main finding was that treatment with hydroal-
coholic extracts of Punica granatum peel was able to
reduce oxidative stress and coronary ACE activity in
SHR rats. Further, the treatments lowered SBP and
prevented vascular remodeling in coronary arteries
in this hypertension model. In the present study, 30
d of treatment with pomegranate peel extract
significantly decreased SBP in 8- and 32-wk-old ani-
mals. Among the possible causes of hypertension, an
overproduction of ROS is implicated. Oxidative
stress is defined as an imbalance between ROS levels
and antioxidant defenses, which worsens with aging
and hypertension (Wind et al., 2010; Ghio et al.,
2012). Further, oxidative stress was shown to be
intimately related with endothelial dysfunction
(Hamilton et al., 2001). Wind et al. (2010)found
that ROS production increased in the aortas of
aged SHR compared with aged Wistar-matched con-
trol aortas. This augmented ROS production may
lead to elevated SBP directly by vasoconstrictor
effects or indirectly by reduced activities of vasodila-
tors (Reckelhoff and Romero, 2003). Although the
animal model used in this study was characterized by
enhanced generation of ROS, oxidative stress
appeared to increase in conditions of hypertension
and aging. Thus, greater oxidative stress was
expected in 32-wk-old versus 8-wk-old animals.
Treatment with pomegranate peel extract in both
8-wk and 32-wk SHR may have led to lower ROS
production, which may have been sufficient to lower
SBP. Further, as ROS contribute to endothelium-
dependent contraction and increase in vascular resis-
tance, antioxidant substances found in pomegranate
extracts may be associated with the observed
decrease in SBP (Kitiyakara and Wilcox, 1998).
This finding is in agreement with other studies of
advanced aging, which demonstrated a 5% decline in
SBP with a daily consumption of pomegranate juice
for 2 wk (Aviram and Dornfeld, 2001).A decrease in
coronary ACE activity was noted in the SHR-PG
group compared with SHR alone at 8 and 32 wk.
Figure 2. Influence of pomegranate extract on coronary ACE activity on d 30. Eight-week-old (A) SHR rats and SHR-PG, and 32-wk-old
(B) SHR rats and SHR-PG. Values are expressed as means ± SEM; n= 5 animals in each group. Asterisk indicates significant at p< .05
compared with SHR alone.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 5
Angiotensin-converting enzyme, a zinc (Zn) metal-
lopeptidase, possesses two large homologous
domains, the N and C domains. These domains are
often the targets of ACE inhibitors that act by bind-
ing to the Zn (Comini et al., 2007). Polyphenols have
chemical structures that favor chelation of redox-
active metals (Fraga, 2007) which may have favored
ACE inhibition observed in this study. In addition,
Mohan et al. (2010) demonstrated that consumption
of pomegranate juice for 4 wk in diabetic hyperten-
sive rats decreased serum ACE levels. In hyperten-
sion, angiotensin II is known to play a role in
vascular remodeling. This effect seems to be
mediated by a rise in production of free radicals
(Ushio-Fukai et al., 1996; Zalba et al., 2000). Thus,
coronary ACE inhibition produced by pomegranate
peel extract may have influenced the morphology of
the cells of the coronary arteries as noted in this
investigation. Althunibat et al. (2010) reported that
administration of Punica granatum peel methanol
extract improved antioxidant enzyme activities in
diabetic rats. Aviram et al. (2000) demonstrated
marked antioxidant capacity of pomegranate juice
to scavenge free radicals. Based on the antioxidant
properties of Punica granatum described in the lit-
erature (Gil et al., 2000) and its scavenging capacity,
it was postulated that treatment with pomegranate
peel extract might decrease oxidative stress in this
experimental model. Data demonstrated that coron-
ary arteries of SHR-PG exhibited a marked fall in
generation of O
2.∙-
. These results support the notion
that one of the mechanisms involved in attenuation
of damage derived from hypertension may be related
to reduced oxidative stress. Cardiovascular morpho-
physiological abnormalities have been associated
with increased oxidative stress (Lima et al., 2012).
Consequently, interventions that are able to potenti-
ate tissue antioxidant capacity, including pharmaco-
logical therapies, have been used to provide vascular
and cellular benefits (Bazargani-gilani et al., 2014;
Danesi et al., 2014). Evidence indicates that pome-
granate juice extract may also exert beneficial actions
in hypertensive individuals.
Our morphometric analyses showed that
pomegranate extract treatment prevented altera-
tions induced by hypertension and/or aging on
total vascular and coronary wall area. The
renin–angiotensin–aldosterone system has trophic
actions on the components of the arterial wall, and
angiotensin II might initiate hypertrophic pro-
cesses on vascular smooth muscles (Touyz et al.,
2003). Not surprisingly, ACE inhibition was found
to affect hypertrophied arterial walls in SHR ani-
mal models (Dedkov et al., 2006). A novel finding
Figure 3. Effect of pomegranate extract on morphometric
parameters of coronary arteries. Top panel, microphotographs
are typical cross sections of coronary arteries in SHR and SHR-
PG rats at 8 (left panel) and 32 (right panel) wk, respectively.
Bar = 100 μm. Bottom bar graphs show influence of pome-
granate peel extract on total vascular and wall area at 8 (left
panel) and 32 (right panel) wk old, respectively. Values are
means ± SEM, n=4–7 per group. Asterisk indicates significant
at p< .05, and double asterisk at p< .01 compared with
control, and
#
p< .05 compared with SHR alone.
6R.L.DOSSANTOSETAL.
of this study revealed that in SHR that received
pomegranate extract, the morphological alterations
induced by hypertension and/or aging were dimin-
ished. This finding may be attributed to a reduction
in oxidative stresses mediated by coronary ACE
inhibition and the antioxidant actions of polyphenol
compounds. These effects may be correlated with the
potent antioxidant activity of pomegranate asso-
ciated with high polyphenol content and to the spe-
cific type of polyphenols present in pomegranate,
specifically, hydrolyzable tannins, which display a
high scavenging capacity for free radicals (Aviram
and Rosenblat, 2012).
Our study corroborated the observations of Touyz
et al. (2003), which indicated that ROS might induce
morphological alterations. Indeed, ROS promoted
these alterations by (i) modifying the activity of
tyrosine kinases, metalloproteinases, and mitogen-
activated protein kinases (Baas and Berk, 1995;
Intengan and Schiffrin, 2001); (ii) acting on gene
and protein expression mechanisms by activating
transcription factors, such as nuclear factor (NF)-
κB and AP-1 (Touyz and Schiffrin, 2000); and (iii)
stimulating ion channels, such as plasma membrane
Ca
2+
and K
+
channels, leading to changes in cation
concentrations (Lounsbury et al., 2000).
Data demonstrated for the first time the influ-
ence of pomegranate peel extract administration
on the vascular remodeling process of coronary
arteries for young and elderly hypertensive female
Figure 4. Influence of pomegranate peel extract treatment on superoxide anion production in coronary arteries. The top panels
show higher fluorescence intensity (red) in SHR rats when compared to SHR-PG rats using dihydroethidium (DHE) staining. The bar
graph shows the average DHE fluorescence (AU: arbitrary units) comparing all groups (n= 6). Left panel, 8-wk-old animals and right
panel, 32-wk-old animals. Values are means ± SEM. Asterisk indicates significant at p< .01 compared with controls, and
##
p< .01
compared with SHR-PG group. Scale bar: 100 μm.
JOURNAL OF TOXICOLOGY AND ENVIRONMENTAL HEALTH, PART A 7
rats. The coronary vascular remodeling was char-
acterized by increased wall and cross-sectional
vascular areas. Thus, the coronary artery remodel-
ing in SHR might reflect an adaptive response to
elevated arterial pressures to normalize increased
wall tension (Dedkov et al., 2006; Irwin et al.,
2014).
In conclusion, this study showed that treatment
with pomegranate peel extracts was able to prevent
morphological alterations in coronary arteries
induced by hypertension and/or aging, which
likely occurred through antihypertensive actions
such as antioxidant effects and decreasing coron-
ary ACE activity. Therefore, the beneficial effects
of pomegranate peel extracts in coronary arteries
may be considered in the development of better
therapies for hypertension.
Acknowledgments
The authors thank Nazare S. Bissoli for critical review and
Polyana L. M. Dalpiaz for performing the measurement of
ACE activity.
Conflicts of Interest
All the authors disclose no financial and personal relation-
ships with other people or organizations that could inappro-
priately influence their work.
Funding
This work is supported by grants from the FAPES (54687578/
2011) and CNPq (55262312011-3).
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