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Antioxidant activities of Punica granatum (pomegranate) peel extract on brain of rats

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Abstract

Methanol extract of Punica granatum (pomegranate) peel was screened for its antioxidant activity on brain of adult male Wister albino rats. The antioxidant activity was determined by measuring reduced glutathione, catalase, superoxide dismutase, glutathione reductase, glutathione-S-transferase, and glutathione peroxidase. In addition, hydrogen peroxide (H2O2), nitric oxide (NO) and lipid peroxidation (MDA) were also measured in brain homogenate. Pomegranate peel treatment resulted in marked increase in most antioxidant parameters with reduction in oxidant H2O2, NO and MDA. On the basis of the previous results it can be concluded that pomegranate methanol peel extract is a promising natural product, which could be useful for the prevention of neurodegenerative diseases caused by oxidative stress.
Journal of Medicinal Plants Research Vol. 6(2), pp. 195-199, 16 January, 2012
Available online at http://www.academicjournals.org/JMPR
DOI: 10.5897/JMPR11.500
ISSN 1996-0875 ©2011 Academic Journals
Full Length Research Paper
Antioxidant activities of Punica granatum
(pomegranate) peel extract on brain of rats
Ahmed E. Abdel Moneim
Department of Zoology and Entomology, Faculty of Science, Helwan University, Cairo, Egypt.
E-mail: aest1977@hotmail.com. Tel: (+2) 01003499114.
Accepted 9 May, 2011
Methanol extract of Punica granatum (pomegranate) peel was screened for its antioxidant activity on
brain of adult male Wister albino rats. The antioxidant activity was determined by measuring reduced
glutathione, catalase, superoxide dismutase, glutathione reductase, glutathione-S-transferase, and
glutathione peroxidase. In addition, hydrogen peroxide (H
2
O
2
), nitric oxide (NO) and lipid peroxidation
(MDA) were also measured in brain homogenate. Pomegranate peel treatment resulted in marked
increase in most antioxidant parameters with reduction in oxidant H
2
O
2
, NO and MDA. On the basis of
the previous results it can be concluded that pomegranate methanol peel extract is a promising natural
product, which could be useful for the prevention of neurodegenerative diseases caused by oxidative
stress.
Key words: Pomegranate peel, antioxidant enzymes, brain, rats.
INTRODUCTION
Punica granatum L. (Punicaceae), commonly called
pomegranate, is a plant used in folkloric medicine for the
treatment of various diseases, such as ulcer, hepatic
damage and snakebite (Ajaikumar et al., 2005). Over the
past decade, significant progress has been made in
establishing the pharmacological mechanisms of
pomegranate and the individual constituents responsible
for them. Extracts of all parts of the fruit appear to have
therapeutic properties (Lansky and Newman, 2007), and
some studies report that the bark, roots, and leaves of
the tree have medicinal benefit as well (Jurenka, 2008).
Studies in rats and mice confirm the antioxidant
properties of a pomegranate by-product extract made
from whole fruit minus the juice, showing a 19% reduction
in oxidative stress in mouse peritoneal macrophages, a
42% decrease in cellular lipid peroxide content, and a
53% increase in reduced glutathione levels (Rosenblat et
al., 2006). In vitro assay of a fermented pomegranate
juice extract and a cold pressed seed oil extract found the
antioxidant capacity of both are superior to red wine and
similar to green tea extract (Schubert et al., 1999). A
separate study in rats with CCl
4
-induced liver damage
demonstrated pretreatment with a pomegranate peel
extract enhanced or maintained the free-radical
scavenging activity of the hepatic enzymes catalase,
super oxide dismutase, and peroxidase, and resulted in
54% reduction of lipid peroxidation values compared to
controls (Chidambara et al., 2002).
Little information has been published regarding the
antioxidant activities of pomegranate peel extract on
brain. The current study aimed to evaluate the beneficial
effect of pomegranate peel on brain antioxidant activity
that may make it one of the more important foods of the
future.
MATERIALS AND METHODS
Experimental animals
Adult male albino Wistar rats weighing 120 to 150 g were obtained
from The Holding Company for Biological Products and Vaccines
(VACSERA, Cairo, Egypt). Animals were kept in wire bottomed
cages in a room under standard condition of illumination with a 12 h
light-dark cycle at 25±1°C. They were provided with water and
balanced diet ad libitum. The experiments were approved by the
state authorities and followed Egyptian rules on animal protection.
Pomegranate peel extracts preparation
Pomegranate peels were manually separated, sun-dried and
196 J. Med. Plants Res.
ground to powder. The powder was extracted with methanol (100
ml) at 45°C for 1 h. The extract was filtered to remove the peel
particles. The extracts were concentrated under vacuum at 40°C to
get a concentrate, which was dried in a vacuum at 40 to 50°C and
stored at 3 to 4°C until used and designated as methanol extract of
pomegranate peel.
Test for tannins
The aqueous extract (1 ml) was mixed with 10 ml of distilled water
and filtered. Ferric chloride reagent (3 drops) was added to the
filtrate. A blue-black or green precipitate confirmed the presence of
gallic tannins or catechol tannins, respectively.
Reducing power
The Fe
3+
reducing power of the extract was determined according
to the method of Oyaizu (1986). The extract (2 ml) was mixed with
0.2 M phosphate buffer, pH 6.6 (2 ml) and 1% potassium
ferricyanide (2 ml). The mixture was then incubated at 50°C for 20
min. Afterwards, the mixture was stopped by adding 10%
trichloroacetic acid (2 ml) and then centrifuged at 3,000 rpm for 10
min. The upper layer of supernatant (2 ml) was mixed with distilled
water (2 ml) and 0.1% FeCl
3
solution (0.5 ml). The absorbance was
measured at 700 nm against a blank with a spectrophotometer, and
ascorbic acid was used as a standard. Higher absorbance of the
reaction mixture indicated greater reducing power. The percents of
reducing power were presented as ascorbic acid equivalents using
a calibration curve between the absorbance of the reaction and the
percent of the reducing power ability of ascorbic acid:
OD = (0.0146 × [percent]) + 0.0016, R2 = 0.9999.
Determination of total flavonoids
For the assessment of flavonoids, colorimetric method introduced
by Dewanto et al. (2002) was adapted. To determine the amount of
flavonoids by the aforementioned method, 1.50 ml of the deionized
water was added to 0.25 ml of the sample extract and then 90 ul of
5% sodium nitrite (NaNO
2
). Six min later, after addition of 180 ul of
10% AlCl
3
, mixture was allowed to stand for another 5 min before
mixing 0.6 ml of 1 M NaOH. By adding deionized water and mixing
well, final volume was made upto 3 ml. Using blank, absorbance
was measured at 510 nm. Calibration curve was prepared using
querestin acid as standard for total flavonoids which was measured
as mg querestin equivalents (QE) per gram of the sample (mg/g).
Determination of total phenolics
To analyze the total phenolic content (TPC), Kim et al. (2003)
method was followed to make the use of Folin Ciocalteu reagent.
To 0.4 ml of the extract (prepared in methanol with a concentration
of 1.0 mg/ml), 1.0 ml of (10%) Folin-Ciocalteu reagent was mixed
and solution was allowed to stand at 25°C for 5 to 8 min before
adding 0.8 ml of 7.5% sodium carbonate solution and using
deionized water, final volume was made to 10.0 ml. After two hours,
absorbance was measured at 765 nm. Calibration curve was
prepared using gallic acid as standard for TPC which was
measured as mg gallic acid equivalents (GAE) per gram of the
sample (mg/g).
Experimental protocol
To study the effect of pomegranate peel, twelve adult male Wister
albino rats were randomly divided into two groups, six rats of each.
Group I served as control (CON) and received saline (0.2 ml saline/
rat) by oral administration via epigastric tube. Group II received oral
administration of 200 mg/kg methanol extract of pomegranate peel
(Parmar and Kar, 2008) for 21 days and served as methanol extract
of pomegranate peel (MEPP) group. The animals of all groups were
cervically dislocated and blood samples were collected from retro-
orbital plexus. Blood stranded for half an hour and then centrifuged
at 500 g for 15 min at 4°C to separate serum and stored at -70°C
until analysis. Brains of rats were carefully removed, weighed and
homogenized immediately to give 50% (w/v) homogenate in ice-
cold medium containing 50 mM Tris-HCl and 300 mM sucrose
(Tsakiris et al., 2004) and centrifuged at 500 g for 10 min at 4°C.
The supernatant was used for the various biochemical
determinations.
Biochemical estimations
Lipid peroxidation
Lipid peroxidation in brain homogenate were determined according
to the method of Ohkawa et al. (1979) by using 1 ml of
trichloroacetic acid 10% and 1 ml of thiobarbituric acid 0.67%,
followed by heating in a boiling water bath for 30 min. Thiobarbituric
acid reactive substances were determined by the absorbance at
535 nm and expressed as malondialdehyde (MDA) equivalents
formed.
Nitrite/Nitrate
The assay of nitrite in brain homogenate was done according to the
method of Berkels et al. (2004). In acid medium and in the
presence of nitrite the formed nitrous acid diazotises
sulphanilamide, which is coupled with N-(1naphthyl)
ethylenediamine. The resulting azo dye has a bright reddishpurple
color which was measured at 540 nm.
Hydrogen peroxide assay
Hydrogen peroxide content in brain tissues of different groups were
determined according to the method of Fossati et al. (1980). In
briefly, In the presence of peroxidase, H
2
O
2
of brain homogenate
was reacted with 3,5-dichloro-2-hydroxybenzensulfonic acid
(DHBS) and 4-aminophenazone (AAP) to form a chromophore
determined spectrophotometrically at 510 nm.
Estimation of reduced glutathione
The reduced glutathione (GSH) of brain was determined by the
methods of Ellman (1959). The method based on the reduction of
Ellman's reagent (5,5` dithiobis, 2-nitrobenzoic acid) with GSH to
produce a yellow compound . The reduced chromogen directly
proportional to GSH concentration and its absorbance were
measured at 405 nm.
Levels of the brain anti-oxidant enzymes
Catalase activity was assayed by the method of Aebi (1984).
Catalase reacts with a known quantity of H
2
O
2
. The reaction is
stopped after exactly one minute with catalase inhibitor. In the
presence of peroxidase (HRP), the remaining H
2
O
2
reacts with 3,5-
dichloro-2-hydroxybenzene sulfonic acid (DHBS) and 4-
aminophenazone (AAP) to form a chromophore with a color
Moneim 197
Table 1. Quantitative analysis of tannins and their type in the methanol extract
of pomegranate peel.
Parameters
Amount
Gallic tannins
+++
Catechol tannins
-
+; present; -; not present.
intensity inversely proportional to the amount of catalase in the
original sample.
Brain superoxide dismutase (SOD) activity was assayed by the
method of Nishikimi et al. (1972). This assay relies on the ability of
the enzyme to inhibit the phenazine methosulphate-mediated
reduction of nitroblue tetrazolium dye.
Glutathione-S-transferase (GST) activity in brain was assayed by
the method of Habig et al. (1974). The total GST activity is
estimated by measuring the conjugation of 1-chloro-2,4-
dinitrobenzene (CDNB) with reduced glutathione. The conjugation
is accompanied by an increase in absorbance at 340 nm. The rate
of increase is directly proportional to the GST activity in the sample.
Brain glutathione peroxidase (GPx) activity was measured by the
method of Paglia and Valentine (1967). The assay is an indirect
measure of the activity of GPx. Oxidized glutathione (GSSG),
produced upon reduction of organic peroxide by GPx, is recycled to
its reduced state by the enzyme glutathione reductase. The
reaction was initiated by the addition hydrogen peroxide to the
reaction and the oxidation of NADPH to NADP
+
is accompanied by
a decrease in absorbance at 340 nm.
Glutathione reductase activity of brain was assayed by the
method of Factor et al. (1998). Glutathione reductase catalyzes the
reduction of glutathione in the presence of NADPH, which is
oxidized to NADPH
+
. The decrease in absorbance at 340 nm is
measured.
Statistical analysis
The obtained data were presented as means ± standard error.
Statistical analysis was performed using an unpaired Student’s t-
test using a statistical package program (SPSS version 17.0).
RESULTS
Tannins, reducing power, total phenolic and
flavonoids contents on pomegranate peel extract
The MEPP was given positive tests for gallic tannins
while the extract given negative result for catechol
tannins as shown in Table 1. The reducing power (RP)
and the total phenolic (TPC) and flavonoids (TFC)
contents in pomegranate peel methanol extracts are
shown in Table 2. The direct correlation between
antioxidant activity and reducing power of certain plant
extracts were reported. The presence of reductants
(antioxidants) in the extracts would result in the reduction
of iron (III) to iron (II). MEPP showed 20.8% of activity at
1 g sample/ml. The result indicated that MEPP contained
electron donors and possessed the ability to reduce iron
(III) to iron (II).
It was well-known that plant phenolics and flavonoids
are highly effective free radical scavengers and
antioxidants. P. granatum contained high amounts of
phenolic and flavonoids compounds. It was shown that
MEPP contained phenolic and flavonoids compounds at
124.34 mg GAE/g and 59.44 mg QE/g, respectively. This
result indicated that the potent antioxidant activity of
MEPP may be related to the phenolic and flavonoids
compounds in the extract.
Potential role of pomegranate peel on oxidant
molecules
In MEPP treated animals H
2
O
2
, MDA and NO as markers
for oxidative stress were determined in brain and serum
(Table 3) where H
2
O
2
reduced significantly in both serum
and brain (-12.3% and -15.6% p < 0.005), respectively,
while MDA (-10.9%) and NO (-23.6%) significantly
reduced in brain homogenates only.
Beneficial effect of pomegranate peel on antioxidant
molecules
Reduced glutathione in brain and serum changed non-
significantly (Table 4) due to MEPP treatment, while SOD
and CAT increased significantly in brain homogenates of
rats by 258.9 and 22.0%, respectively.
The activity of antioxidant enzymes (GR, GPx and
GST) were measured in brain homogenates of rats
(Table 5). MEPP administration caused significant
increase in GR and GST activity (61.8 and 11.1%,
respectively) with non-significant change in the activity of
brain GPx.
DISCUSSION
It has been reported that oxidizing biological material
leads to a rapid burst of ROS, such as superoxide (O2
),
hydrogen peroxide (H
2
O
2
) and hydroxyl (
OH) generated
primarily because of the ionizing of water molecules
(Agrawal et al., 2001), which then interact with biological
target molecules, causing lipid peroxidation and DNA
damage, and subsequently resulting in cell killing and
198 J. Med. Plants Res.
Table 2. Reducing power (RP), total phenolic content (TPC) and total flavonoids content (TFC) in
the methanol extract of pomegranate peel.
Parameters
Contents
RP (%/ g sample)
20.80 ± 1.56
a
TPC (mg GAE/g sample)
124.34 ± 5.42
a
TFC (mg QE/ g sample)
59.44 ± 3.71
a
a
Values expressed are the mean of three replications.
Table 3. Hydrogen peroxide content (H
2
O
2
) malondialdehyde (MDA) and nitrite/nitrate (NO) in brain and serum of rats treated with methanol
extract of pomegranate peel.
Parameter
groups
Brain H
2
O
2
(mM/g tissue)
Serum H
2
O
2
(mM/L)
Serum MDA
(nmol/g tissue)
Brain NO
(μmol/ g tissue)
Serum NO
(μmol/ g tissue)
Control group
0.89 ± 0.011
0.51 ± 0.004
32.37 ± 1.50
110.0 5 ± 8.54
47.33 ± 4.03
MEPP group
0.78 ± 0.009*
0.43 ± 0.003*
32.58 ± 4.68
84.11 ± 5.26*
52.67 ± 5.85
Values are means ± SE (n=6). *: significant change at p < 0.05 with respect to control group.
Table 4. Reduced glutathione (GSH), catalase (CAT) and superoxide dismutase (SOD) levels in brain and serum of rats treated
with methanol extract of pomegranate peel.
Parameter groups
Brain GSH
(mmol/g tissue)
Serum GSH
(mmol/g tissue)
Brain SOD
(U/g tissue)
Brain CAT
(U/g tissue)
Control group
18.73 ± 1.19
1.55 ± 0.34
0.56 ± 0.01
0.82 ± 0.11
MEPP group
20.40 ± 1.20
1.71 ± 0.27
2.01 ± 0.02*
1.00 ± 0.07*
Values are means ± SE (n=6). *: significant change at p < 0.05 with respect to control group.
Table 5. Glutathione reductase (GR), glutathione peroxidase (GPx) and glutathione-S-transferase (GST) on brain of rats treated with
methanol extract of pomegranate peel.
Parameter groups
Brain GR (μmol/ g tissue)
Brain GPx (U/g tissue)
Brain GST (μmol/h/ g tissue)
Control group
82.39 ± 12.90
1891.30 ± 103.79
0.36 ± 0.11
MEPP group
133.30 ± 16.96*
1945.34 ± 95.28
0.40 ± 0.14*
Values are means ± SE (n=6). *: significant change at p < 0.05 with respect to control group.
mutations (Nair et al., 2001).
Plants, vegetables, herbs and spices used in folk and
traditional medicine have been accepted currently as one
of the main sources of chemo preventive drug discovery
and development (Aruoma, 2003). It has been observed
that many plant polyphenols, such as ellagic acid,
catechins, and chlorogenic, caffeic and ferulic acids act
as potent antioxidant, antimutagenic and anticarcinogenic
agents (Ayrton et al., 1992). Ben et al. (1996) have
reported that pomegranate peel contains ellagic acid,
ellagitannins and gallic acids. The presence of these
polyphenols in the pomegranate peel may be responsible
for antioxidant and anticarcinogenic effect of peel extracts
(Gil et al., 2000). Also, in this study the methanol extract
of pomegranate peel had 210.6±7.3 mg/g total phenolics,
gallic acid equivalents. Hence, it can be suggested that
the observed antioxidant activity of pomegranate peel
methanol extract in the present study due to the presence
of these compounds. In this study, the antioxidant activity
of pomegranate peel was evaluated on brain of rats. The
present data demonstrate that MEPP reduced lipid
peroxidation and nitric oxide in both serum and brain
tissue homogenate. The ability of MEPP to reduce the
oxidant molecules, it seems likely by scavenging the
reactive oxygen radicals. Our results are in agreement
with the studies which have demonstrated that P.
granatum peel extract decreased lipid peroxidation in
hepatic, cardiac, and renal tissues (Parmar and Kar,
2008) and had a facilitatory effect on the scavenging
ability of superoxide anion and hydrogen peroxide.
Previously, Toklu et al. (2007) have shown that chronic
pomegranate peel extract supplementation alleviated
oxidative injury of the liver and improved the hepatic
structure and function in rats exposed to bile duct ligation.
Another study in rats with carbon tetrachloride-induced
liver damage demonstrated that pretreatment with
pomegranate peel extract resulted in the reduction of lipid
peroxidation, while the free-radical scavenging activity of
catalase, superoxide dismutase, and peroxidase were
significantly enhanced (Chidambara et al., 2002). In the
present study, the GSH level that was increased due to
MEPP treatment, suggesting that it may be an important
factor in protecting the tissue against oxidative injury.
Since GSH and the activities of glutathione reductase
and glutathione peroxidase, which are critical
constituents of GSH-redox cycle, provide major
protection in oxidative injury by participating in the cellular
system of defense against oxidative damage, they play a
critical role in limiting the propagation of free radical
reactions, which would otherwise result in extensive lipid
peroxidation (Toklu et al., 2009).
In conclusion, pomegranate peel is beneficial for
neuronal tissue as well as it has an antioxidant properties
and nutritive value.
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... Pomegranate and Artemiswia plants are considered having broad-spectrum activities worldwide and as an alternative natural biological source as anthelminthic medication. Pomegranate trees are cultivated in the Middle East, Mediterranean region, and China [16]. ...
... However, Pomegranate (punicagrantau) is underestimated as an agricultural waste [17]. Pomegranate (punicagrantau) known as the fruit of paradise is mentioned throughout the history and is highly appreciated in Judaism, Christianity, and Islam [16]. ...
... Pomegranate peels are a natural source that have valuable medicinal properties such as scolicidal, antihydatic and immunomodulatory effect [23], antiparasitic [18,45], antimicrobial [21], and antioxidant/anti-inflammatory effects [16]. They are also used in the treatment and prevention against cancer [20], cardiovascular disease [46], and diabetes [22]. ...
... The formation of ZnO from zinc salt is governed by the hydroxyl groups of plant extract polyphenols [27,35]. Regarding the pomegranate peel extracts, it was found that high values of phenolic and flavonoids compounds in the extract were directly correlated to the reducing power of iron (III) to iron (II) [58]. The content of phenolic and flavonoids compounds depends on the concentration of the extract, time and temperature of extraction, and the solvent used [58][59][60][61]. ...
... Regarding the pomegranate peel extracts, it was found that high values of phenolic and flavonoids compounds in the extract were directly correlated to the reducing power of iron (III) to iron (II) [58]. The content of phenolic and flavonoids compounds depends on the concentration of the extract, time and temperature of extraction, and the solvent used [58][59][60][61]. Therefore, it was essential to evaluate the total phenolic content (TPC) and total flavonoid content (TFC) of our extract. ...
... The results are presented in Table 5 and show that the TPC content was 2911.8 mg GAE/100 g and the TFC content was 765.8 mg CE/100 g. Comparing our results with those obtained in organic solvents extraction (i.e., TPC of 510 mg GAE/g, TFC of 16.4 mg quercetin/g in 60% ethanol extract [61], TPC of 124.34 mg GAE/g, and TFC of 59.44 mg quercetin/g in methanol extract [58], it is clear that the preparation of the extract in water enables very high TPC and TFC values and therefore was able to reduce the Zn precursor to ZnO-NP. Considering that the alkaline medium increases the amount of free OH − groups of cellulose, which allows higher adsorption of phytochemicals from plant extract, and consequently increases the amount of phenolic and flavonoid compounds on cotton fabric, this favors the formation of ZnO-NP on cotton. ...
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... It has a potent antioxidant potential that is essential for human health [36,74,82]. It has been previously reported that Vit E deficiency in humans and animal models leads to CNS oxidative damage and motor coordination deficits [36,62,79]. ...
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... The result proved Pomegranate as a powerful antioxidant lead for medicine development. [77] Probiotic Enhancer (Bialonska et al., 2010) reported the positive effects of Pomegranate on the gut microbial environment. The study found Pomegranate effective in enhancing the growth of probiotics for e.g Bifidobacterium spp., Lactobacillus Enterococcus species. ...
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