Food &

Function
PAPER

Antioxidant and anti-inflammatory potential of

Cite this: Food Funct., 2016, 7, 3091
pomegranate rind extract to ameliorate cisplatin-
induced acute kidney injury
Ritu Karwasra,a Prerna Kalra,a Yogendra Kumar Gupta,a Deepika Saini,b Ajay Kumarb
and Surender Singh*a

Received 12th February 2016,
Accepted 23rd May 2016
DOI: 10.1039/c6fo00188b
www.rsc.org/foodfunction
Cisplatin is a chemotherapeutic agent, but the therapeutic utility is limited due to its dose dependent
nephrotoxicity. The aim of the present study was to evaluate the nephroprotective effect of pomegranate
in cisplatin-induced acute kidney injury. Wistar rats were allocated into six groups as follows: the normal
control, cisplatin-induced, pomegranate rind extract treatment (50, 100 and 200 mg kg−1) and pomegra-
nate rind extract per se group. All the experimental test drugs/vehicle were administered orally for a
period of ten days. Intraperitoneal injection of cisplatin (8 mg kg−1) was administered on day 7 to all the
groups except the normal control and pomegranate per se group. On day 10, cisplatin resulted in signifi-
cant nephrotoxicity in Wistar rats with a drastic elevation of serum creatinine and BUN, a decline in the
concentrations of GSH, MDA and superoxide dismutase (SOD), and an elevation in the TNF-α level in
renal tissues. Pathological changes in renal tissues were examined by histopathology and dysfunction in
mitochondria and proximal tubule cells was detected by transmission electron microscopy. The rate of
apoptosis and the expression of caspase-3, Il-1β and IL-6 in rat renal tissues were detected by immuno-
histochemistry. The administration of pomegranate at a dose of 200 mg per kg body weight significantly
( p < 0.001) ameliorates increased serum creatinine and BUN. In parallel to this, pomegranate also exhibits
anti-apoptotic activity through the reduction of active caspase-3 expression in kidneys. Additionally,
in-silico studies also confirmed a renoprotective effect of pomegranate. The above findings suggest that
pomegranate can be used as a dietary supplement in the treatment of cisplatin-induced kidney injury by
reducing apoptosis, oxidative stress and inflammation.

Introduction production of TNF-α and cytokines (IL-1, IL-6) in proximal

Cisplatin (cis-diaminedichloroplatinum II) is a chemothera-
peutic agent and its use in the treatment of solid tumors is
associated with many adverse effects in normal tissues such as
nephrotoxicity, ototoxicity, neurotoxicity, myelosuppression,
and vomiting. A therapeutic dose (40 mg m−2) of cisplatin,
commonly prescribed in the treatment of tumors, results in
irreversible nephrotoxicity,1 attributing to the major adverse
effect of cisplatin, which occurs mainly in 20–30% of patients.2
Several studies proposed that the proximal tubule cells which
are exposed to cisplatin activate many complex signaling path-
ways that promote cell death (caspase-3, Bcl-2, Bax, MAPK,
p53) leading to cell injury, oxidative stress, apoptosis and dys-
function of mitochondria.3 In the intervening time, cisplatin
also triggers a robust inflammatory response by inducing the
a
Department of Pharmacology, All India Institute of Medical Sciences (AIIMS),
New Delhi 110029, India. E-mail: surenderaiims@gmail.com; Tel: +011-26546460
b
Department of Pharmaceutical Chemistry, Kurukshetra University, Kurukshetra
136119, India
tubule cells that further contributes to the damage of renal
tissue.4 Thus, cisplatin in turn activates both the extrinsic
death receptor pathway of apoptosis and intrinsic mitochon-
drial pathway. In the extrinsic pathway the major death
receptors include Fas and tumor-necrosis factor-alpha recep-
tor (TNF-R1), inducing renal injury by mounting a disastrous
inflammatory response. But on the other hand, the intrinsic
or mitochondrial pathway emerged as the major apoptotic
pathway in cisplatin induced renal injury, which involves the
activation of Bcl and Bax proteins. The induction of mito-
chondrial dysfunction increases reactive oxygen species
(ROS) production. The accumulation of endogenous ROS and
oxidative stress occurs because cisplatin reacts with endogen-
ous glutathione and depletes or inactivates it.5 For many
years various renoprotective interventions were used to
curtail this side effect but the protective effects are mostly
partial, suggesting the need for alternative approaches.
Therefore, there is continuous research in exploration of
natural agents which have high therapeutic value with lower
side effects.

This journal is © The Royal Society of Chemistry 2016 Food Funct., 2016, 7, 3091–3101 | 3091
Paper
Pomegranate (Punica granatum L. Punicaceae) is one of the
oldest known edible fruit, representing a phytochemical reser-
voir with a high potential medicinal value. This fruit, grown
mainly in the mediterranean region, has been used for centuries
to treat many ailments such as parasitic and microbial infec-
tions, ulcers, diarrhea, hemorrhage and dysentery.6–8 Several
pre-clinical studies demonstrated the protective role of pomegra-
nate in mercuric chloride-induced nephrotoxicity,9 gentamicin-
induced nephrotoxicity,10 cerebral ischemia/reperfusion injury,11
carbon tetrachloride-induced nephrotoxicity,12 hexachlorobuta-
diene-induced nephrotoxicity,13 arsenic-induced hepato-
toxicity,14 and in rheumatoid arthritis.15 In addition to this,
pomegranate is endowed with antioxidant,16 wound healing17,18
and anticancer activities.19 This is why, over the past decade, the
evaluation of the nutritional and therapeutic benefits of pome-
granate (PG) have raised scientific interest with numerous publi-
cations. In point of fact, the main benefit of pomegranate has
been attributed to its unique polyphenol composition.20 Indeed,
pomegranate polyphenols have been shown to exhibit high anti-
oxidant and anti-inflammatory capacities interesting for the pre-
vention of several disorders.21 In this context, the health benefits
of pomegranate consumption in preventing cancers19 and
cardiovascular diseases22 have been widely focused. However,
only a few studies have targeted the eventual benefit of pomegra-
nate consumption towards cisplatin-induced nephrotoxicity23,24
but the mechanism is still unclear, and the rind extract is un-
explored for its nephroprotective effect to date. So, the aim of
the present study was envisaged to investigate the antioxidant
effect of standardized rind extract of pomegranate (PG) on cis-
platin induced acute kidney injury.
Materials and methods
Animals
The study was carried out on albino Wistar rats, each weighing
150–200 g. The experimental protocol was approved by Insti-
tutional Animal Ethics Committee of All India Institute of
Medical Sciences, New Delhi, India, (IAEC no. 772/IAEC/13), and
all experimental procedures were conducted in accordance to
Indian National Science Academy Guidelines for Care and Use
of Animals in Scientific research. The animals were housed in
standard laboratory conditions at a constant temperature (25 ±
2 °C) with a relative humidity of (60 ± 5%) under a 12 h light/
dark cycle. They had free access to a standard diet (Ashirwad
Industries Ltd, Chandigarh, India) and tap water ad libitum.
Test drug
The standardized hydroalcoholic rind extract of pomegranate
was procured from Natural Remedies Pvt. Ltd, Bangalore,
India (batch no. PC/PG12LOT03) and a voucher specimen (no.
SS/Pharma/014/2013) was deposited in the Inflammation lab,
Department of Pharmacology, AIIMS, New Delhi for future
reference. The standardized rind extract of pomegranate was
further quantified for the presence of punicalgin, ellagic and
gallic acid with the help of HPLC and HPTLC analysis.
3092 | Food Funct., 2016, 7, 3091–3101
Food & Function
Extraction
The coarsely powdered dried rind extract of pomegranate was
refluxed with methanol and water (1 : 1) at 65–70 °C for 1 h
and filtered; to the marc, the extraction process was repeated
2 more times and filtered, and the combined filtrate was con-
centrated and dried under vacuum, 550 mm of Hg, at a temp-
erature not more than 70 °C to obtain a brown powdered
extract of pomegranate. The yield obtained was 15% w/w.
Chemicals
The cisplatin injection was purchased from Parenteral Drugs
Limited, Madhya Pradesh (India) batch no. CISB14006. Blood
Urea Nitrogen (BUN) and serum creatinine kits were obtained
from Erba Diagnostics, Himachal Pradesh, India. ELISA kits of
TNF-α were purchased from Diclone SAS, France. The
immunohistochemistry kit was purchased from Vector labs,
USA. IL-1β, IL-6, and TNF-R1 primary antibodies were procured
from Santa Cruz, USA. All the other chemicals used were of
analytical grade.
Experimental protocol
The rats were randomly divided into 6 experimental groups
and each group consisted of 6 rats.
Group 1 (normal). Normal saline (1 ml per kg per day; p.o.)
was administered to rats for a period of 10 days.
Group 2 (cisplatin-control). Normal saline was administered
to rats for a period of 10 days and on the 7th day, a single injec-
tion of cisplatin (8 mg kg−1; i.p.) was given.
Group 3–5 ( pomegranate 50, 100, and 200 mg per kg per day
+ cisplatin). Pomegranate (50, 100, and 200 mg per kg per day;
p.o.) was administered to rats for a period of 10 days and on
the 7th day, a single injection of cisplatin (8 mg kg−1; i.p.) was
given.
Group 6 ( pomegranate per se). Pomegranate (200 mg per kg
per day; p.o.) was administered to rats for a period of 10 days.
On the 10th day, animals were anaesthetized with pentobar-
bitone sodium (60 mg kg−1; i.p.), blood was collected from the
retro-orbital plexus and then centrifuged at 1860g to separate
the serum and stored at −20 °C for estimating the BUN, serum
creatinine and TNF-α levels.3 Both the kidneys were then har-
vested; one of the kidneys was snap frozen in liquid nitrogen
and stored at −80 °C for biochemical analysis. The other
kidney was immediately kept in 10% neutral buffered forma-
lin, embedded in paraffin and used for histopathological,
immunohistochemistry (IHC) and transmission electron
microscopy (TEM).
Measurement of kidney function test
Serum creatinine and BUN levels were estimated using a diag-
nostic kit from Erba Diagnostics, Himachal Pradesh, India,
with the help of a semiauto-analyzer, Italy.
Biochemical estimation
A 10% kidney cortex homogenate was prepared in ice-chilled
phosphate buffer (0.1 M, pH 7.4) and used for the estimation
This journal is © The Royal Society of Chemistry 2016
Food & Function
Table 1 x, y and z coordinates of each receptor under study
Coordinate 4GAI 1ALU
Centre_x 7.138 2.523
Centre_y −16.485 −19.96
Centre_z −24.648 8.684
of malondialdehyde (MDA)25 and endogenous glutathione
(GSH).26 A part of the homogenate was centrifuged at 2910g
and the supernatant was used to estimate superoxide dismu-
tase (SOD)27 enzyme activities.
Determination of the serum TNF-α level
The serum TNF-α level was measured using a commercially
available ELISA kit (Diaclone, France). Initially, wells were
coated with a TNF-α capturing antibody. Subsequently,
samples and biotinylated anti-rat TNF-α antibody were added
and incubated for 3 h. Following this, a solution of Horse
Radish Peroxidase (HRP)-streptavidin complex was added to
each well and kept in incubation for 20 min. A ready-to-use
chromogen was added and the plate was kept in the dark for
10 min for color development. Absorbance was read at 450 nm
using a reference filter at 630 nm. Simultaneously, a series of
dilutions of standard TNF-α was run to obtain a standard
graph. The standard curve thus obtained was used to deter-
mine the TNF-α level and expressed as pg ml−1.
Histopathological evaluation
Kidney tissue sections were cut 6 µm thick and further
dewaxed in xylene and rehydrated in graded alcohols. Follow-
ing washing, the sections were stained with hematoxylin and
following a second wash, the sections were differentiated. The
sections were subsequently stained with eosin after washing.
Following dehydration and differentiation in alcohol, the sec-
tions were mounted and observed under a microscope (Eclipse
E200; Nikon, Tokyo, Japan).
Transmission electron microscopy (TEM) examination:
ultrastructural studies
At the end of the experiment, small pieces of renal cortex
(approximately 1–2 mm thick) were immediately fixed in 3%
glutaraldehyde for 6 h. The tissues were then washed in phos-
phate buffer (0.1 M, pH 7.4) and postfixed for 2 h in 1%
osmium tetraoxide in the same buffer at 4 °C. The specimens
were then washed in phosphate buffer, dehydrated with
acetone and then embedded in araldite CY 212 to make tissue
blocks. Ultrathin sections (70–80 nm) were cut by an Ultra-
microtome (Ultracut E, Reichert, Austria). The sections were
stained with uranyl acetate and lead acetate and examined
under a transmission electron microscope (Morgagni 268 D,
Fei Co., The Netherlands) operated at 80 kV.
Immunohistochemical analysis
IHC was done for tissue localization of caspase-3, IL-1β and
IL-6. Cryosections of kidney tissues were used for immuno-
This journal is © The Royal Society of Chemistry 2016
Paper
2ILK 2DDF 3J6C 3EY6 2IMS
18.018 24.839 32.915 9.542 0.145
45.963 28.792 57.115 −5.416 −0.472
39.206 38.260 75.095 1.111 14.813
histochemical analysis. Hydrogen peroxide (30% H2O2 in
methanol) was added to quench any endogenous peroxidase
activity in tissues. Later, sections were blocked with bovine
serum albumin for 1 h at room temperature and were incu-
bated with a primary monoclonal antibody (mAb) against
caspase-3 (1 : 200), Il-1β (1 : 200), and IL-6 (1 : 200) for 48 h. Sec-
tions were then incubated with Horse Radish Peroxidase
(HRP)-conjugated secondary antibody (1 : 2000) for 2 h. Colori-
metric immune reaction was developed with 3,3′-diamino-
benzidine (DAB). Sections were later mounted with DPX to
visualize caspase-3, Il-1β and Il-6 activity under a light micro-
scope (Nikon ECLIPSE E600, Japan).
Molecular docking study
To study the binding mode and interaction of various recep-
tors with constituents of pomegranate, docking was carried
out using AutoDock Vina 4.2.28 Ligands were drawn in Chem-
BioDraw Ultra 12.0 and converted to their 3-dimensional struc-
tures in ChemBio3D Ultra 12.0. The crystal structure of the
receptors for study, 4GAI (IL-1), 1ALU (IL-6), 2ILK(IL-10), 2DDF
(TNF-α), 3J6C(caspase-3), 3EY6(Bcl-2), and 2IMS(Bax), were
downloaded from the PDB database (http://www.rcsb.org).
Polar hydrogens were added and water of crystallization was
removed during protein preparation. Autodock Vina program
accepts the pdbqt file format for ligands as well as receptors.
Thus, both were converted into the required format for the
docking experiment. The active binding site was generated by
Autodock Vina and the dimensions for the grid for each recep-
tor are given in Table 1. Each constituent was processed 10
times for docking with each receptor to get the best confir-
mation. The binding energy and hydrogen bonds were con-
sidered for the screening of the constituents.
Statistical analysis
The data was analyzed by one way analysis of variance (ANOVA)
followed by post-hoc Tukey–Kramer multiple comparisons test
(Graphpad prism version 5.03, San Diego, CA, USA). The data
was expressed as mean ± S.E.M. A p value less than 0.05
( p < 0.05) was considered to be statistically significant.
Results
Quantification of punicalgin, ellagic and gallic acid in the
hydroalcoholic pomegranate rind extract using HPLC and
HPTLC analysis:
The total punicalgin content of pomegranate was determined
by HPLC (Fig. 1A) and was found to be 11.8% (w/w). HPTLC
Food Funct., 2016, 7, 3091–3101 | 3093
Paper
Fig. 1 HPLC chromatogram of pomegranate representing the presence
of punicalgin and a HPTLC chromatogram of pomegranate representing
gallic and ellagic acid. (A) HPLC chromatogram of the standard punical-
gin; HPLC chromatogram of the pomegranate (Punica granatum)
extract; (B) HPTLC chromatogram of the standard (ellagic and gallic
acid); HPTLC chromatogram of the pomegranate extract (Punica grana-
tum). Graph plotted between absorption unit (AU) and Rf (Retention
factor).
analysis of pomegranate was also carried out and the content
of gallic acid and ellagic acid (Fig. 1B) found in the extract was
0.00219% and 0.03743% respectively. 21.962 mg kg−1 gallic
acid and 374.398 mg kg−1 ellagic acid was present in the
pomegranate extract which was calculated with the help of the
regression equation for gallic acid (y = 4.582x + 2975, R2 =
0.993) and ellagic acid (y = 5.441x + 1035, R2 = 0.997).
3094 | Food Funct., 2016, 7, 3091–3101
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Effect of pomegranate on renal function
The cisplatin-control group exhibited a significant increase in
serum creatinine ( p < 0.001) and BUN ( p < 0.001) levels as
compared to the normal group, suggesting significant kidney
injury. On the contrary, pomegranate pretreatment reduced
the increased level of creatinine and BUN in serum with
respect to the cisplatin-control group. The significant
reduction of these parameters i.e. the serum creatinine ( p <
0.001) and BUN level ( p < 0.001) was observed at the maximum
dose (200 mg kg−1) of pomegranate (Fig. 2).
Effect of pomegranate on oxidative stress
In this study, we had assessed MDA, SOD and non-enzymatic
GSH. Cisplatin-control rats exhibited a significant increase in
tissue MDA level and a decrease in GSH and SOD enzyme
activities ( p < 0.001) as compared to the normal group.
Notably, prior treatment with pomegranate significantly sup-
pressed the renal MDA level ( p < 0.001) and attenuated the
depletion of the antioxidant defense system [GSH ( p < 0.001);
SOD ( p < 0.001)] in comparison to the cisplatin-control group
and the effect was most significant at a 200 mg kg−1 dose of
pomegranate (Fig. 3A–C).
Effect of pomegranate on the serum TNF-α level
An increase in the tissue TNF-α level would indicate increased
tissue injury as seen in the cisplatin-control group compared
to the normal group. In contrast, pomegranate treatment
resulted in a dose-dependent reduction in the serum TNF-α
level, with the most significant ( p < 0.001) suppression
observed only at a dose of 200 mg kg−1 (Fig. 3D).
Effect of pomegranate on histopathological changes in
cisplatin-induced acute kidney injury
The histopathological examination of normal rats showed
normal architecture of the cortex and medulla with no evidence
of inflammation (Fig. 4A). The kidneys of cisplatin-treated rats
showed diffuse acute tubular necrosis, denudation of epithelium
and infiltration of inflammatory cells (Fig. 4B). Pomegranate
treatment with low doses of 50 and 100 mg kg−1 showed marked
tubular damage and inflammation (Fig. 4C and D) while at the
highest dose i.e. 200 mg kg−1, tubules appeared nearly normal
and there was no inflammation (Fig. 4E). The pomegranate per
se group also showed normal architecture of the cortex and
medulla (Fig. 4F) and showed no traces of toxicity.
Effect of pomegranate on ultrastructural changes
The ultrastructure of the kidney from saline control rats was
normal in appearance showing an intact proximal tubular epi-
thelium with preserved mitochondria (Fig. 5A). In the cis-
platin-treated kidney there was extensive acute injury in the
proximal tubule and a disorganized mass of mitochondria
with disrupted cristae was seen in the tubular basement mem-
brane (Fig. 5B). Meanwhile in the cells treated with pomegra-
nate at a dose of 200 mg kg−1, there were well-defined integral
mitochondrial membranes with regular-appearing cristae
This journal is © The Royal Society of Chemistry 2016
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Fig. 2 Effect of pomegranate on kidney function tests (serum creatinine and BUN) in different experimental groups. Each bar represents the mean
± SEM of 6 animals. Statistical analysis by one-way ANOVA followed by Tukey–Kramer multiple comparisons. *p < 0.05, **p < 0.01 and ***p < 0.001
denote comparison with the cisplatin control group. #p < 0.05, ##p < 0.01 and ###p < 0.001 denote comparison with the normal group.

Fig. 3 Effect of pomegranate on the (A) MDA level, (B) GSH content, (C) SOD activity and (D) TNF-α level in different experimental groups. Each bar
represents the mean ± SEM of 6 animals. Statistical analysis by one-way ANOVA followed by Tukey–Kramer multiple comparisons. *p < 0.05, **p <
0.01 and ***p < 0.001 denote comparison with the cisplatin control group. #p < 0.05, ##p < 0.01 and ###p < 0.001 denote comparison with the
normal group.

(Fig. 5C). The pomegranate per se group also showed intact Thus, on the basis of the renal function tests, oxidative
mitochondrial membranes (Fig. 5D) and no traces of injury in stress, histopathology and microscopic changes, it is found
the proximal tubular epithelium. that 200 mg kg−1 is the most effective dose of pomegranate

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Fig. 4 Effect of pomegranate on histopathological changes in the glomerulus and proximal tubule cells (A–F) in different experimental groups. (A)
Vehicle-treated kidneys showed normal morphology with a well-preserved glomerulus and no loss of tubular epithelial cells. (B) Cisplatin-treated
kidneys showed diffuse acute proximal tubule necrosis, denudation of the epithelium and infiltration of inflammatory cells. (C and D) Kidneys from
cisplatin plus pomegranate extract at 50 & 100 mg kg−1 showed proximal tubule damage and inflammation. (E) Kidneys from cisplatin plus pomegra-
nate extract at 200 mg kg−1 where tubules appeared nearly normal and there was no inflammation. (F) Pomegranate (PG) per se group showed
normal architecture of the cortex and proximal tubule cells. Arrow (→) represents acute tubular damage. Sections were stained with H&E and photo-
graphed at ×10 magnification.

Fig. 5 Effect of pomegranate on ultrastructural changes in the renal cortex (A–D) in different experimental groups. (A: Normal control; B: cisplatin-
control group; C: PG 200 mg kg−1; D: PG per se group). M denotes mitochondria, N denotes nucleus, V denotes vacuole, AV denotes autophagic
vacuole, and L denotes lysosomes.

and hence, this dose was used for immunohistochemical
examination.
Effect of pomegranate on cisplatin induced apoptosis and
inflammation
On immunohistochemical examination, there was a significant
increase in caspase-3 levels in the cisplatin-control group.
Pomegranate pretreatment of 200 mg kg−1 significantly
lowered the elevated caspase-3 levels in cisplatin-injected rat
kidneys. In order to further measure the inflammation in renal
tissues, the expression of pro-inflammatory (Il-1β and Il-6)
cytokines were found to be elevated in the cisplatin-control
group. On the contrary, pomegranate at a dose of 200 mg kg−1
attenuates the elevated level of pro-inflammatory cytokines

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Fig. 6 Effect of pomegranate in proximal tubule cells on caspase-3, IL-1β, and IL-6 immunohistochemistry in different experimental groups. (A1–
A4) caspase-3; (B1–B4) IL-1β; (C1–C4) IL-6. Original magnification 20×.

Table 2 Results of the binding energy of protein receptors (IL-1, IL-6, IL-10, TNF-α, Bcl-2, Bax and caspase-3) with constituents of pomegranate
( punicalagin, punicallin, gallic acid, ellagic acid and punicic acid)

S. no. Compounds IL-1 4GAI IL-6 1ALU IL-10 2ILK TNF 2DDF Caspase-3 3J6C BCL 2EY6 BAX 2IMS

1. Ellagic acid −7.6 −7.2 −7.2 −7.3 −6.3 −6.9 −9.6

2. Gallic acid −5.0 −5.5 −4.9 −5.1 −4.9 −5.2 −6.6
3. Ascorbic acid −7.3 −5.2 −4.5 −5.1 −4.5 −5.7 −6.5
4. Quercitin −7.4 −6.6 −7.3 −7.0 −6.8 −6.8 −8.6
5. Punicic acid −4.2 −4.4 −5.2 −3.3 −4.0 −4.4 −4.5
6. Punicalgin −11.6 −10.6 −11.7 −12.1 −10.1 −10.8 −10.4
7. Naringin −9.0 −8.0 −9.0 −8.5 −7.6 −8.6 −8.7
8. Kaempferol −7.7 −6.4 −7.3 −7.1 −6.2 −6.7 −8.4

(Il-1β and Il-6). Thus, inflammation and apoptosis induced by in vivo data which signifies that this can be used as a sup-
cisplatin in renal proximal tubule cells was markedly sup- plement for patients treated with cisplatin chemotherapy and
pressed by pomegranate pretreatment (Fig. 6). punicalgin might be responsible for the nephroprotective
activity (Fig. 7).

Effect of pomegranate on protein–ligand interaction

The major constituents (ellagic acid, gallic acid, quercitin,
punicalgin, punicic acid, ascorbic acid, kaemferol, and narin-
gin) present in the rind part were analyzed with anti-inflamma-
tory and antiapoptotic receptors such as IL-1, IL-6, IL-10, Bcl-2,
Bax and caspase-3 and their binding energy was calculated.
Punicalgin was found to have the lowest binding energy for
IL-1 (−11.6), IL-6 (−10.6), IL-10 (−11.7), TNF-α (12.1), Bcl-2
(−10.8), Bax (−10.4) and apoptotic markers caspase-3 (−10.1),
which signifies the highest anti-inflammatory and anti-apopto-
tic activity (Table 2). Pomegranate contains the highest
amount of punicalgin (11.8% w/w), as evident by the HPLC
analysis, so it can be said that it is responsible for the nephro-
protective activity. Thus in silico data were well correlated with
Discussion
The present study demonstrated that pomegranate exhibits
antioxidant and anti-inflammatory properties and thus
reduced apoptosis in renal proximal tubule cells, which in
turn ameliorates cisplatin-induced nephrotoxicity and pre-
serves renal function.
Cisplatin is a chemotherapeutic agent commonly pre-
scribed in the treatment of various solid-organ cancers, but
unfortunately it leads to nephrotoxicity by generating reactive
oxygen species and by stimulating inflammatory and apoptotic
pathways. To combat this side effect, various renoprotective
interventions have been discovered, but the protective effects

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Fig. 7 Protein–ligand interaction of the active constituent of pomegranate (Punicalgin) on IL-1β, IL-6, IL-10, TNF-α, bcl-2, bax and caspase-3. (A)
TNF-α, (B) IL-1β, (C) IL-6, (D) caspase-3, (E) bcl-2 and (F) bax.
are partial and there is no drug available to date that can
protect the kidney from this deleterious effect of cisplatin.5 In
our present study, we found that damage to the kidney was
characterized by higher serum creatinine and BUN levels in
cisplatin treated rats compared to normal control rats, but
treatment with pomegranate in cisplatin treated rats signifi-
cantly reduces the elevated serum creatinine and BUN levels in
a dose dependent fashion. Intake of pomegranate 7 days prior
to cisplatin significantly prevented the cisplatin-induced renal
dysfunction, which is confirmed by histopathological and
ultrastructural (TEM) examination. The histologic changes in
cisplatin treated rats included tubular necrosis with interstitial
inflammatory cells, whereas the pomegranate protected group
at dose 200 mg kg−1 exhibited histologically normal tubules
with no inflammation observed.
Different mechanisms have been proposed to be involved
in the pathogenesis of cisplatin-induced nephrotoxicity,
including the production of nephrotoxic metabolites, vascular
injury, inflammation, generation of free radicals and apoptotic
pathways in which the first and foremost mechanism is the
generation of free radical species, which is an important deter-
minant of cisplatin toxicity.29,30 Normally cisplatin transports
into proximal tubule cells by organic cation transporter 2
(OCT2) and mediates its nephrotoxic effects through gene-
ration of a positively charged electrophile.31 This electrophile
in particular, accumulates in negatively charged mitochondria,
resulting in the release of reactive oxygen species (ROS) (such
as superoxides, hydroxyl and hydrogen peroxide) and inflam-
matory cytokines in proximal tubule cells contribute to the
nephrotoxic effects of cisplatin.32 The excessive ROS produced
depletes the intracellular stores of anti-oxidant enzymes such
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as superoxide dismutase, glutathione-s-transferase and gluta-
thione peroxidase, mainly GSH. Glutathione-s-transferase is
the most important endogenous anti-oxidant defense system
which maintains cell structure and integrity.33 Depletion of
GSH intensifies ROS activity triggering a cascade of lipid per-
oxidation, mitochondrial dysfunction and leads to a loss of
membrane integrity and cell death.34 On the contrary, pome-
granate supplementation decreased generation of free radicals
or ROS, suppressed the renal MDA level and increased anti-
oxidant enzyme activities (GSH, SOD) in renal tissue. These
findings substantiate the antioxidant activity of pomegranate
which is established in other nephrotoxic models such as gen-
tamicin-induced nephrotoxicity,10,35 and diabetic nephro-
pathy.36 Our observations also supported the results of Choi
et al., (2011)37 in which they have shown that pomegranate
ethanolic extract mitigated the oxidative stress in PC12 cells
and attenuated oxidative stress induced Alzheimer’s symptoms
in mice. In another study pomegranate ( juice and rind powder
extract) protected cooked chicken patties against oxidative ran-
cidity.38 Pomegranate peel extract protected against carbon
tetrachloride (CCL4) induced liver injury and attenuated oxi-
dative stress in rats by scavenging ROS, reducing MDA and
restoring activities of anti-oxidants.39 Thus, it can be hypo-
thesized that the protection afforded by pomegranate in this
study could be attributed to its anti-oxidant effect and this
finding is in harmony with the earlier reports in which it has
been reported that supplementation with antioxidant and anti-
inflammatory agents suppresses renal damage caused by
cisplatin.40,41
Another important determinant of cisplatin-induced
nephrotoxicity is inflammation which is mediated by TNF-α, a
This journal is © The Royal Society of Chemistry 2016
Food & Function
prototypical inflammatory cytokine that heralds apoptosis of
the epithelial cell, lining the tubular structure through the
death receptor pathway, persuading damage of the kidney
architecture, which is a hallmark of acute renal injury.42 Pome-
granate rind extract attenuated inflammation and injury of the
jejunum induced by E. papillata infections.43 Deng et al.,
(2001) has demonstrated an increase in the renal expression of
TNF-α and interleukin in cisplatin treated mice.44 Further-
more, it is also interesting that many of the inflammatory cyto-
kines and chemokines also get upregulated in a TNF-α
dependent fashion. Similar results have been observed in our
study with an increased TNF-α level and tubular dysfunction in
cisplatin injected rats while pomegranate pretreatment inhibi-
ted TNF-α production and maintained the structural integrity
of tubules. Pomegranate pretreatment also showed decreased
expression of pro-inflammatory cytokines (IL-1β, IL-6) as
evident from immunohistochemical analysis. Thus, it can be
stated that nephroprotection demonstrated by pomegranate is
not only due to maintenance of the redox status but also due
to its anti-inflammatory effects.
Cisplatin accumulation within the proximal tubule cells of
renal tissue also activates various downstream proteins that
mediate apoptosis and necrosis. The extensive damage caused
by cisplatin treatment affects the balance between pro- and
anti-apoptotic proteins towards the pro-apoptotic pathway and
causes activation of Bax and decreases the level of Bcl-2, an
anti-apoptotic protein. It also induces translocation of Bax
from the cytosol to mitochondria and releases cytochrome c to
the cytosol. Cytochrome c further activates caspase 3, 8 and 9
and triggers apoptosis in renal proximal tubule cells. Several
previous studies demonstrated that caspase activation is
thought to be one of the major cellular mechanisms for induc-
tion of apoptosis in renal proximal tubule cells in cisplatin-
induced acute renal injury.45 In line with these studies, we
found significant increased expressions of protein caspase-3 in
kidney tissue, as evident from immunohistochemical analysis.
Hence, pomegranate mitigated nephrotoxicity of cisplatin by
attenuating apoptosis in cisplatin injected rats. The anti-apop-
totic effect of pomegranate ( punicalgin) has been reported in a
rat model of cerebral ischemia reperfusion injury by Yaidikar
et al., (2015).46 Besides having anti-inflammatory and anti-
oxidant activity, pomegranate also exhibited anti-apoptotic
activity in cisplatin-treated kidneys. So, we can say that the
pomegranate rind extract is effective in ameliorating nephro-
toxicity not only through suppression of oxidative stress but
also by hampering the inflammatory and apoptotic pathways.
HPTLC data concluded that punicalgin is the major con-
stituent present in the pomegranate rind extract. Subsequently,
the in silico study was carried out to distinguish the active con-
stituent of the pomegranate rind extract, which may be respon-
sible for its nephroprotective role. With the aid of in silico
studies, all the active constituents of pomegranate were
docked with the receptors and the binding energy of each was
calculated. The constituent having the lowest binding energy
among all the constituents was taken and this constituent was
further analyzed for in vivo studies. The docking studies are
This journal is © The Royal Society of Chemistry 2016
Paper
conducted to screen the active constituent among various com-
pounds, thus this method is cost effective and involves the
adequate use of experimental animals. So in our present study,
we assessed the nephroprotective activity of the pomegranate
rind extract and found that it possesses significant nephro-
protective activity. Then to further analyze which constituent
in the pomegranate rind extract was responsible for the
activity, the in silico study was carried out and we found that
punicalgin has the lowest binding energy for the receptors
IL-1, IL-6, IL-10, TNF-α, Bcl-2, Bax and caspase-3 and is respon-
sible for the nephroprotective activity. Thus this compound
may be further elucidated for screening of its nephroprotective
activity.
Conclusion
It may be concluded that pomegranate treatment attenuated
cisplatin-induced functional, biochemical and morphological
changes in renal tissue and ameliorates cisplatin-induced
nephrotoxicity. Thus, the plausible mechanism underlying
pomegranate’s nephroprotective effect may be attributed to
the high content of punicalagin, the major constituent of
pomegranate. However, the favorable profile of pomegranate is
to be further elucidated to establish its role in prevention of
cisplatin-induced acute renal injury in humans.
Conflict of Interest
The authors declare that there are no conflicts of interest.
Acknowledgements
The authors are grateful to Natural Remedies, Bangalore, India
for providing the standardized extract. The authors are also
grateful to All India institute of Medical Sciences, New Delhi,
India for administrative support and Department of Pharma-
ceutical Sciences, Kurukshetra University, Kurukshetra, India
for carrying out the molecular docking study.
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