Nephro-protective effect of (Arachis hypogaea L.) peanut skin extracts on CCl 4 induced kidney damage in mice

Abstract

The study aimed to assessment the antioxidant activity of peanut (Arachis hypogaea) skin extracts in male albino mice with administrated intraperitoneally 3 ml/kg CCl 4 to induce nephrotoxicity. The phytochemical analysis of the peanut skin extracts were investigated, the result showed presence of flavonoids, phenols, alkaloids, glycosides and tannins in methanolic extract, while alkaloids were not detected in aqueous extract. In addition, individual phenolic composition of peanut skin was analyzed by HPLC method which showed 7 phenolic compounds were detected (chlorogenic acid, caffeic acid, epicatechin, p-coumaric acid, quercetin, luteolin and kaempferol) in methanolic extract. The nephro-protective effect of peanut skin extract was evaluated in CCl 4 induced renal toxicity. The experiment was conducted in two methods: pre-treatment groups and post-treatment groups. Mice were treated with 50 and 100 mg/kg of aqueous and methanolic peanut skin extracts for 35 days before being damaged by CCl 4 (pre-treatment group), and the other groups (post-treatment groups) which the mice were injected with CCl 4 and received 50 and 100 mg/kg of aqueous and methanolic peanut skin extracts for 35 days. Biochemical studies showed that there is decrease in the levels of serum blood urea and creatinine while increases in the levels of albumin with significant differences (p <0.01) when compared with the CCl 4 treated group. The histopathological examination of kidney obtained from mice with administrated intraperitoneally 3 ml/kg CCl 4 showed histopathological changes in the kidney represented excessive accumulation of protein material inside the proximal convoluted epithelial cells, while when treated with 100 mg/kg of peanut extract revealed look like normal structure appearance but with certain degenerative changes of the renal tubular epithelium.

Full text

Nephro-protective eect of (Arachis hypogaea L.) peanut skin extracts on CCl4 induced
kidney damage in mice
Aya K. Ibrahim1 , Ahmed H. AL-Azawi1*
1Biotechnology Department, Genetic Engineering and Biotechnology Institute for post graduate studies. University of Baghdad,
Baghdad, Iraq.
*Corresponding Author Received: 01 May 2019
E-posta: ahmedharbi_alazawi@yahoo.com Accepted: 10 October 2019
Abstract
The study aimed to assessment the antioxidant activity of peanut (Arachis hypogaea) skin extracts in male albino mice with administrated
intraperitoneally 3 ml/kg CCl4 to induce nephrotoxicity. The phytochemical analysis of the peanut skin extracts were investigated, the result
showed presence of avonoids, phenols, alkaloids, glycosides and tannins in methanolic extract, while alkaloids were not detected in aqueous
extract. In addition, individual phenolic composition of peanut skin was analyzed by HPLC method which showed 7 phenolic compounds
were detected (chlorogenic acid, caeic acid, epicatechin, p-coumaric acid, quercetin, luteolin and kaempferol) in methanolic extract. The
nephro-protective eect of peanut skin extract was evaluated in CCl4 induced renal toxicity. The experiment was conducted in two methods:
pre-treatment groups and post-treatment groups. Mice were treated with 50 and 100 mg/kg of aqueous and methanolic peanut skin extracts
for 35 days before being damaged by CCl4 (pre-treatment group), and the other groups (post-treatment groups) which the mice were injected
with CCl4 and received 50 and 100 mg/kg of aqueous and methanolic peanut skin extracts for 35 days. Biochemical studies showed that there
is decrease in the levels of serum blood urea and creatinine while increases in the levels of albumin with signicant dierences (p <0.01) when
compared with the CCl4 treated group. The histopathological examination of kidney obtained from mice with administrated intraperitoneally
3 ml/kg CCl4 showed histopathological changes in the kidney represented excessive accumulation of protein material inside the proximal
convoluted epithelial cells, while when treated with 100 mg/kg of peanut extract revealed look like normal structure appearance but with certain
degenerative changes of the renal tubular epithelium.
Keywords: Nephro-protective, CCl4, phenolic compounds, HPLC, Arachis hypogea L.
INTRODUCTION
The kidney is a vital organ in the human body and
is one of the most complicated organs in terms of both
structure and function [1]. Renal toxicity is one of the most
common kidney problems and especially occurs when the
body is subjected to chemical reagents or drugs [2]. Carbon
tetrachloride (CCl4) is one of the most potent toxins,
commonly used as a chemical inducer of experimental liver
injury. In addition, many studies showed that CCl4 can induce
kidney damage, which is widely used in scientic research to
produce experimental model that mimic the oxidative stress
in many pathophysiological situation [3]. The antioxidants
are the rst line of choice to take care of oxidative stress
, endogenous antioxidant defenses include a network of
compartmentalized antioxidant systems are necessary for
sustaining life by maintaining a delicate intracellular redox
balance and minimizing undesirable cellular damage caused
by reactive oxygen species (ROS) the same antioxidant
just due to its free radical scavenging activity may act as
disease promoter, by neutralizing the physiologically desired
ROS molecules, and as disease alleviator by removing the
excessive levels of ROS species [4].
Arachis hypogaea L. is an annual herbaceous plant [5],
belongs to the Leguminosae family [6]. Peanut seed has high
nutritional and commercial values due to the presence fatty
acids, vitamins, carbohydrates, calcium and phosphorus
[7]. Peanut skins are regarded as a low economic value by-
product of the peanut industry; Peanut skin could provide an
inexpensive source of natural antioxidants, such as catechins
and procyanidin, for use in food and dietary supplements.
Also it contains higher concentration of procyanidin trimers
and tetramers than grape seed which gives peanut skin a
comparative advantage as a source of potent antioxidants
[8]. The aim of this study is to determine the role of Arachis
hypogaea L. skin as nephro-protective against CCl4 induced
nephro-toxicity in mice.
MATERIALS AND METHODS
Chemical substances
All reagents were of the highest purity available. CCl4
were purchased from Sigma-Aldrich (Germany). Standard
phenolic compounds clorogenic acid, quercetin, luteolin,
p-coumaric acid and kaempherol were bought from company
(sigma, USA). Commercial kits of Urea, Creatinine and
Albumin were bought from company (Agappe, Swiss).
Plant collection
Raw peanuts pods were collected from the local Iraqi
markets. Pods were manually shelled and the skins were
collected from the raw peanut kernels. The skins were ground
using a grinder and stored at -20°C for further analysis.
Preparation of aqueous extract
Water extract was prepared according to [9]. Macerated
100 gram of peanut skin in 1000 ml of distilled water for
72 hours, after extraction, the mixture was vacuum ltered
through Whitman No. 1 paper and the ltrate was dried at
50°C by a rotary evaporator. The resulting extract stored in
amber glass vials at 4 °C until analyzed. The whole process
was completed under dim light to minimize light induced
degradation of phenolics, which are generally light sensitive.
Preparation of methanolic extract
Methanolic extract was prepared according to [10] by
using Soxhelt apparatus. So 50 gram of peanut skin was put
in a thimble and 350 ml of methanol was added within 40-



60 0C for 6 hours. The solution have been ltered through a
lter paper Whitman No.1 and evaporated to dryness under
vacuum at 40°C, the dried extract have been weighed and
stored in amber glass vials at 4 °C until analyzed.
Phytochemical screening of peanut skin extracts
Phytochemical test of peanut skin extracts were done
according to [10, 11, 12, 13].
High-Performance Liquid Chromatography (HPLC)
Peanut methanolic and aqueous extracts were identied by
(HPLC) according to [14], under the following conditions as
shown in table (1).
Fourier transform infrared (FTIR) assay
Fourier Transform Infrared spectroscopy is a technique
based on the vibrations of the atoms of a molecule, an
infrared spectrum is commonly obtained by passing infrared
radiation through a sample and determining what fraction
of the incident radiation is absorbed at a particular energy.
The FTIR spectrum was recorded between 4000 and 400
cm-1[15].
Experimental animals
Forty male albino mice weighing 35 ± 5 g were
obtained from Biotechnology Research Center, AL- Nahrain
University. They were kept in standard conditions, the
temperature about 22 °C, 12 hours light/dark cycle. The forty
mice were randomly divided into ten groups of four animals
each. The experiment was conducted in two methods: pre-
treatment groups and post-treatment groups.
Group 1: This group served as a negative control in
which the mice received normal feed and distilled water for
35 days
Group 2: This group was a positive control for CCl4,
which induce liver and kidney damage in mice. CCl4 was
solved in olive oil with ratio (1:3) (CCl4: olive oil) at a dose
of 3 ml/kg injected intraperitoneally (i.p.).
Group 3: This group was the pre-treatment group, in
which the mice were administered with 50 mg/kg methanolic
extract orally for 35 days and injected (i.p.) 3 ml/kg of CCl4
and olive oil mixture on the 35 day.
Group 4: This group was the pre-treatment group,
in which the mice were administered with 100 mg/kg
methanolic extract orally for 35 days and injected (i.p.) 3 ml/
kg of CCl4 and olive oil mixture on the 35 day.
Group 5: This group was the pre-treatment group, in
which the mice were administered with 50 mg/kg aqueous
extract orally for 35 days and injected (i.p.) 3 ml/kg of CCl4
and olive oil mixture on the 35 day.
Group 6: This group was the pre-treatment group, in
which the mice were administered with 100 mg/kg aqueous
extract orally for 35 days and injected (i.p.) 3 ml/kg of CCl4
and olive oil mixture on the 35 day.
Group 7: This group was the post-treatment group
in which the mice were injected (i.p.) 3 ml/kg of CCl4 and
olive oil mixture on the 1st day and treatment with 50 mg/kg
methanolic extract orally for 35 days.
Group 8: This group was the post-treatment group
in which the mice were injected (i.p.) 3 ml/kg of CCl4 and
olive oil mixture on the 1st day and treatment with 100 mg/
kg methanolic extract orally for 35 days.
Group 9: This group was the post-treatment group
in which the mice were injected (i.p.) 3 ml/kg of CCl4 and
olive oil mixture on the 1st day and treatment with 50 mg/kg
aqueous extract orally for 35 days.
Group 10: This group was the post-treatment group
in which the mice injected (i.p.) with 3 ml/kg of CCl4 and
olive oil mixture on the 1st day and treatment with 100 mg/
kg aqueous extract orally for 35 days.
Collection of blood
Blood samples were collected at the end of the
experiment; the mice were anesthetized with the injection of
200 μl (160 μl ketamine 10% + 40 μl xylazine) of anesthesia
agent. Then their abdominal areas were opened and the
blood samples were directly taken from their hearts. The
blood sample was rocked slightly and centrifuged at 3000
rpm for 5 minutes. The serum was then stored in the freezer
at -21oC until analyzed [16].
Statistical Analysis
The Statistical Analysis System (SAS) program was
used to eect of dierence factors in study parameters.
Least signicant dierence –LSD test (ANOVA) was used
to signicant compare between means in this study [17].
RESULTS AND DISCUSSION
Phytochemical screening of peanut skin extracts
Phytochemical screening means the extraction,
identication and screening of the medicinally active
substances found in plants, some of the bioactive substances
that are derived from plants are avonoids, alkaloids,
carotinoids, tannin, antioxidants, and phenolic compounds
[18]. Dierent phytochemicals have a wide range of
activities that may help in protection against various diseases
[19]. The aqueous and methanolic extracts of the peanut
skin extracts were subjected to dierent chemical tests
for the detection of dierent phyto constituents in ( Table
2 ) by using standard procedures. The results showed that
the aqueous and methanolic extracts contain (Flavonoids,
Phenols, Tannins and Glycosides) While the presence of
alkaloids in the aqueous extract was not detected.

High-performance liquid chromatography (HPLC)
Phenolic compounds of peanut skin were analyzed by
HPLC according to [14] method. In this study, 5 phenolic
compounds were detected (chlorogenic acid, quercetin,
luteolin, p-coumaric acid and kaempferol) in the methanolic
extract as shown in (Figure 1) when compared with standard
compounds as shown in (Figures 2). The results were in
agreement with the results of Mar . [20], who reported
that peanut skin contained relatively higher amounts of
phenolic compounds such as (p-hydroxybenzoic acid,
chlorogenic epicatechin, p-coumaric acid, ferulic acid,
resveratrol and quercetin). Furthermore, Al-Jubouri [21]
determined 10 phenolic compounds such as (chlorogenic
acid, caeic acid, epicatechin, p-coumaric acid, ferulic acid,
resveratrol, quercetin, daidzin, luteolin and kaempferol) in
methanolic peanut skin.
Fourier Transform Infra-Red (FTIR)
Fourier Transform Infra-Red stands for Fourier
Transform Infrared, the preferred method of infrared
spectroscopy. In infrared spectroscopy, IR radiation is
passed through a sample. Some of the infrared radiation is
absorbed by the sample and some of it is passed through
(transmitted), the resulting spectrum represents the
molecular absorption and transmission, creating a molecular
ngerprint of the sample. Like a ngerprint no two unique
molecular structures produce the same infrared spectrum,
this makes infrared spectroscopy useful for several types of
analysis [22]. Results of the FTIR Spectra of the methanolic
and aqueous extracts of Peanut skin revealed the presence
of dierent functional groups such as Phenolic– OH group
stretching, C-H stretching, Aromatic C=C and Aliphatic
C–O (Table 3). Figure (3 and 4) shows the infrared spectra
for the aqueous and methanolic peanut skin extracts.

Zavoi  ., [23] investigated the hepatoprotective
action using FTIR analysis of polyphenolic composition
of medicinal herbs viz.,  , 
,  ,  ,
  and  ) from the
wild ora of Romania. Thenmozhi  ., [24] screened
compounds in  species by using FTIR and HPLC.
Khoddami  ., [25] analyzed dierent techniques for
plant phenolic compounds. Sivakumar [26] attempted a
phytochemical study, FT-IR, GC-MS analysis of 
methanolic leaves extract.
Protective and therapeutic role of the peanut skin
extracts on kidney function enzymes
Eect of peanut skin Extracts on serum Urea level
Table (4) shows that the serum urea concentrations
were signicantly increased (p <0.01) in the CCl4 treated
group (group 2) of mice for 4 weeks (37.47 ± 1.33 mg/dl)
compared with the control group (group 1) (24.89 ± 1.05
mg/dl) indicating the induction of severe nephrotoxicity.
Treatment with methanolic extract 50 mg/kg (Group 3)
(31.08 ± 1.29 mg/dl) showed signicant decrease (p <
0.01) in concentrations of serum urea compared with the
CCl4 treated group. Furthermore the levels of serum urea
signicantly decreased (p<0.01) in methanolic treated group
100 mg/kg (Group 4) (25.37 ± 1.26 mg/dl) and it was the
best eective group when compared with the CCl4 treated
group, this indicate that the role of methanolic peanut extract
as nephroprotective. Sherkatolabbasieh. [27] mention
that the ethanolic extract of (  L.) 800
and 1600 μg/kg showed signicantly decrease (p≤0.05) in
concentrations of serum urea compared with CCl4 treated
group. Likewise when treatment with aqueous extract 50
mg/kg (Group 5) the result showed signicant decrease (p <
0.01) in concentrations of serum urea (31.61 ± 1.00 mg/dl)
when compared with CCl4 treated group, also treatment with
aqueous extract 100 mg/kg (Group 6) showed signicant
decrease (p < 0.01) in concentrations of serum urea (25.76 ±
1.28 mg/dl) compared with CCl4 treated group.
The second method was the post-treatment, the results
showed signicant decrease (p < 0.01) in concentrations
of serum urea (32.57 ± 0.93 and 25.49 ± 1.95 mg/dl)
when treatment with methanolic extract 50 and 100 mg/
kg (Group 7 and 8) respectively compared with the CCl4
treated group. Furthermore treatment with aqueous extract
50 and 100 mg/kg (Group 9 and 10) showed signicant
decrease (p < 0.01) in concentrations of serum urea (35.47
± 1.48and 30.64 ± 1.05 mg/dl) respectively when compared
with the CCl4 treated group. Ali  . [28] mention that
the avonoid chrysin, which exerts strong antioxidant and
anti-inammatory activities, was tested on adenine-induced
chronic kidney disease, chrysin, especially at 250 mg/
kg, mitigated all manifestations of adenine-induced renal
dysfunction, improved creatinine clearance, and reduced
concentrations of urea in rats.
Eect of peanut skin extracts on serum Creatinine
level
The level of serum Creatinine was signicantly increased
(p <0.01) in the CCl4 treated group (group 2) of mice (1.350
± 0.03 mg/dl) when compared to the control group (group
1) (0.803 ± 0.03 mg/dl) indicating the induction of severe
nephrotoxicity. Treatment with methanolic extract 50 mg/
kg (Group 3) showed signicant (p < 0.01) decrease in
concentrations of serum Creatinine (1.020 ± 0.02 mg/dl)
when compared with the CCl4 treated group. Furthermore
the level of Creatinine signicantly increased (p<0.01) in
methanolic treated group 100 mg/kg (Group 4) (0.873 ± 0.03
mg/dl) when compared with the CCl4 treated group. Kalantari
. [29] mention that pre-treatment with (
L.) hydroalcoholic extract at doses 500, 1000 and 2000 mg/
kg caused a signicant decrease (P < 0.001) in the level of
serum creatinine. On the other hand treatment with aqueous
extract 50 mg/kg (Group 5) showed signicant decrease (p
< 0.01) in concentrations of serum creatinine (1.006 ± 0.01
mg/dl) when compared with CCl4 treated group (1.350 ±
0.03 mg/dl). Furthermore treatment with aqueous extract 50
mg/kg (Group 5) (0.903 ± 0.012 mg/dl) showed signicant
decrease (p < 0.01) in concentrations of serum creatinine
compared with CCl4 treated group as shown in (Table 4).
Yoshioka  . [3] showed that Zn (as ZnSO4) 50 mg/kg
daily intake showed signicantly decreased in creatinine and
blood urea nitrogen levels and having protective eect from
the ability of Zn to serve as an inducer of metallothionein (a
known endogenous scavenger of free radicals) protects mice
from acute nephrotoxicity induced by CCl4.
The second method was the post-treatment, the results
showed signicant decrease (p < 0.01) in concentrations of
serum creatinine (1.076 ± 0.02 and 0.913 ± 0.04 mg/dl) when
treatment with methanolic extract 50 and 100 mg/kg (Group
7 and 8) respectively compared with the CCl4 treated group.
Furthermore treatment with aqueous extract 50 and 100 mg/
kg (Group 9 and 10) showed signicant decrease (p < 0.01)
in concentrations of serum creatinine (1.106 ± 0.01and 0.967
± 0.02 mg/dl) respectively when compared with the CCl4
treated group as shown in (Table 4). Zangeneh . [30]
suggest that ( L.) aqueous extract at doses
30, 90, and 270 mg/kg (especially 270) could signicantly
(p ≤ 0.05) reduce the raised levels of urea and creatinine as
compared to the untreated group.

Eect of Peanut Skin Extracts on Serum Albumin
Level:
Table (4) shows that the serum albumin concentrations
were signicantly decreased (p <0.01) in the CCl4 treated
group (group 2) of mice (3.51 ± 0.12 mg/dl) compared to
the control group (group 1) (4.79 ± 0.12 mg/dl) indicating
the induction of severe nephrotoxicity. Albumin is the most
abundant protein in human plasma with remarkably diverse
functions including antioxidant activity, buering properties,
binding and transport capacities for numerous substances
(free fatty acids, various ions, NO, bilirubin, peptides,
uremic toxins and drugs) [31]. Treatment with methanolic
extract 50 mg/kg (Group 3) showed signicant increase (p <
0.01) in concentrations of serum albumin (4.13± 0.19 mg/dl)
compared with the CCl4 treated group. Furthermore the level
of albumin signicantly increased (p<0.01) in methanolic
treated groups 100 mg/kg (Group 3) (4.79 ± 0.13 mg/dl) when
compared with the CCl4 treated group. Likewise the result
showed signicant increase (p < 0.01) in concentrations of
serum albumin when treatment with aqueous extract 50 and
100 mg/kg (Group 5 and 6) (4.07 ± 0.07 and 4.79 ± 0.12
mg/dl) respectively when compared with CCl4 treated group
(3.51 ± 0.12 mg/dl). Zhang. [32] mention the protective
eects of two intracellular polysaccharide (HIPS) puried
fractions (HIPS1 and HIPS2) of  having
signicant decreased (P < 0.05) in albumin levels in mice.
The second method was the post-treatment, the results
showed signicant decrease (p < 0.01) in concentrations of
serum albumin (4.05 ± 0.18 and 4.36 ± 0.12 mg/dl) when
treatment with methanolic extract 50 and 100 mg/kg (Group
7 and 8) respectively compared with the CCl4 treated group.
Furthermore treatment with aqueous extract 50 and 100 mg/
kg (Group 9 and 10) showed signicant decrease (p < 0.01)
in concentrations of serum albumin (4.03 ± 0.04 and 4.43 ±
0.14mg/dl) respectively when compared with the CCl4 treated
group. This indicates the eective role of peanut extracts
as nephrotherapeutic. Mazani. [33] administration 80
and 120 mg/kg of (  L.) methanolic
extract to rats after exposure to CCl4, signicantly increased
(p<0.001) serum albumin when compared with CCl4 control
group. The results showed there are no signicant dierences
(P<0.01) between pre- and post-treatment methods.
Histological examination of the kidneyThe light
microscopic examination by specic staining of kidney cells
in control tissues showed normal structure appearance of
glomeruli and renal tissue (proximal and distal convoluted
tubules) (Figures 5). Kidney sections obtained from the
CCl4 group (group 2) showed normal structure appearance
but with excessive accumulation of protein material inside
the proximal convoluted epithelial cells (Figure 6). Cordeiro
and Kaliwal [34] mention that kidney of the mice treated
with CCl4 at dose 2 ml/gm for 3 days showed glomeruli
were small and atrophied, loosely arranged in Bowman’s
capsule .
The pre-treatment groups, the kidney sections obtained
from the group 3 (methanolic extract 50 mg / kg + CCl4),
the results showed congestion, degenerative changes and
necrosis of renal epithelial cells with mild inammatory cells
inltration (Figure 7A), while treatment with (methanolic
extract 100 mg / kg + CCl4) in group 4 showed normal
look structure appearance but with certain degenerative
changes of the renal tubular epithelium (Figure 7B). Also

in other section showed congestion, degenerative changes
and apoptosis of renal epithelial cells when treated with
aqueous extract 50 mg / kg + CCl4 ( Group 5) as shown in
(Figure 7C). While treatment with aqueous extract 100 mg
/ kg + CCl4 (Group 6) showed look like normal structure
appearance with congestion (Figure 7D). This result agreed
with Pal  . [35] which mention that treatment with
 L. extract at dose 200 mg/kg + CCl4
showed a considerable improvement in kidney morphology
in mice.
The post-treatment groups, the kidney sections obtained
from the group 7 (CCl4 + methanolic extract 50 mg /
kg), the results showed normal looking appearance with
accumulation of protein granules inside the renal epithelial
cells of proximal convoluted tubules as shown in (Figure 8A)
. In group 8 (treated with CCl4 + methanolic extract 100 mg
/kg) the histopathological examination showed normal look
structure appearance with accumulation of proteins granules
inside the epithelial cells of renal tubules (Figure 8B). The
kidney sections of group 9 (CCl4 + aqueous extract 50 mg
/ kg) showed necrosis of renal tubules with heavy chronic
inammatory cells inltration and hyaline cast (Figure 8C).
While the histopathological examination of group 10 (CCl4
+ aqueous extract 100 mg / kg) showed normal look structure
appearance but with certain degenerative changes of the renal
tubular epithelium as shown in (Figure 8D). This explanation
was agreed with Abdulhameed . [36] which mention that
treatment with (CCl4+ aqueous extract 500 mg/kg) of ginger
() showed intact glomerules and normal
convoluted tubules structure of kidney. The ndings showed
the potential use of the methanolic and aqueous extracts of
Arachis hypogea in concentration 100 mg / kg as a novel
therapeutically useful hepato-nephroprotective (protect liver
and kidney from injuries) and hepato-nephrocurative (Cures
the injured liver and kidney) agent on CCl4 induced toxicity
in male albino mice.

CONCLUSIONS
These ndings showed that Arachis hypogaea L. pos-
sessed active compounds such as avonoids, phenols, alka-
loids, glycosides and tannins. The use of Arachis hypogaea
L. skin extracts was benecial in attenuate CCl4 induced
renal toxicity by decrease in the levels of serum blood urea
and creatinine. Arachis hypogaea L. has a novel therapeutic
potential in kidney tissues in male albino mice, against oxi-
dative damages on CCl4.
CONFLICT OF INTEREST
The authors declared that present study was performed
in absence of any conict of interest.
ACKNOWLEGEMENT
This work was supported by Biotechnology department,
Institute of Genetic Engineering and Biotechnology for
Postgraduate Studies, University of Baghdad.
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