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Safety assessment of pomegranate fruit


extract: Acute and subchronic toxicity studies
ARTICLE in FOOD AND CHEMICAL TOXICOLOGY SEPTEMBER 2008
Impact Factor: 2.9 DOI: 10.1016/j.fct.2008.04.035 Source: PubMed

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Safety assessment of pomegranate fruit extract: Acute and subchronic


toxicity studies
Chintan Patel a, Paresh Dadhaniya a, Lal Hingorani b, M.G. Soni c,*
a

Pharmacology and Toxicology Department, Cadila Pharmaceuticals, 1389 Trasad Road, Ahmedabad, Gujarat, India
Pharmanza Herbals Pvt. Ltd., Khambhat, Gujarat, India
c
Soni & Associates Inc., 749, 46th Square, Vero Beach, FL 32968, USA
b

a r t i c l e

i n f o

Article history:
Received 17 December 2007
Accepted 24 April 2008

Keywords:
Pomegranate extract
Food ingredient
Safety
Toxicity
Punicalagins
.

a b s t r a c t
Pomegranate (Punica granatum L.) fruit is widely consumed as fresh fruit and juice. Because of its potential for health benets, pomegranate fruit extracts have been commonly marketed as dietary supplements in recent years. The objective of the present study was to investigate potential adverse effects,
if any, of a standardized pomegranate fruit extract in rats following subchronic administration. The
extract was standardized to 30% punicalagins, the active anomeric ellagitannins responsible for over
50% of the antioxidant potential of the juice. The oral LD50 of the extract in rats and mice was found to
be greater than 5 g/kg body weight. The intraperitoneal LD50 in rats and mice was determined as 217
and 187 mg/kg body weight, respectively. In the subchronic study, Wistar strain rats (10/sex/group) were
administered via gavage 0 (control), 60, 240 and 600 mg/kg body weight/day of the extract for 90 days.
Two additional groups received 0 and 600 mg/kg/day of the extract for 90 days, followed by a 28 day
recovery phase. Compared to the control group, administration of the extract did not result in any toxicologically signicant treatment-related changes in clinical observations, ophthalmic examinations, body
weights, body weight gains, feed consumption, clinical pathology evaluations and organ weights. The
hematology and serum chemistry parameters that showed statistical signicant changes compared to
control group were within the normal laboratory limits and were considered as biological variations
and not the toxic effect of the extract. Terminal necropsy did not reveal any treatment-related gross or
histopathology ndings. Based on the results of this study, the no observed-adverse-effect level (NOAEL)
for this standardized pomegranate fruit extract was determined as 600 mg/kg body weight/day, the highest dose tested.

1. Introduction
Pomegranate (Punica granatum L.) is one of the ancient fruit that
is widely consumed as fresh fruit and juice. Use of pomegranate
fruit dates back to Biblical times and reports of its therapeutic
qualities have echoed throughout the millennia (Longtin, 2003).
Pomegranate juice is a popular drink in the Middle East, and is also
used in Iranian and Indian cuisine. In many traditional systems of
health condition treatment, use of pomegranate for both internal
and external conditions has been documented (Seeram, 2007). In
traditional medicine pomegranate fruits have been used to treat
acidosis, dysentery, microbial infections, diarrhea, helminthiasis,

Abbreviations: ALP, alkaline phosphatase; ALT, alanine aminotransferase; AST,


aspartate aminotransferase; FDA, food and drug administration; ICH, international
conference on harmonization; NOAEL, no-observed-adverse-effect level; OECD,
organization for economic co-operation and development.
* Corresponding author. Tel.: +1 772 299 4572; fax: +1 772 299 5381.
E-mail address: sonim@bellsouth.net (M.G. Soni).

haemorrhage and respiratory pathologies (Vidal et al., 2003). In recent years several pomegranate-containing products have been
widely marketed for health benets around the world, including
the United States (Cerda et al., 2003a).
The health benets of pomegranate have been attributed to
its wide range of phytochemicals. The phytochemicals found in
pomegranate are predominantly polyphenols, including primarily
hydrolysable ellagitannins, anthocyanins and other polyphenols.
Ellagitannins found in the outer part of the fruit are largely responsible for the antioxidant activity of the pomegranate juice. It has
been demonstrated that one of the ellagitannins, punicalagins
(punicalagin anomers A and B) are responsible for over 50% of
the antioxidant activity of the pomegranate juice (Gil et al.,
2000). Since punicalagin is water soluble, commercial pomegranate juice obtained by pressing the fruit is found to contain signicant amounts of this antioxidant. Tzulker et al. (2007) reported
that the homogenates prepared from the whole fruit exhibited an
approximately 20-fold higher antioxidant activity than the level
found in the aril or seed sacs (eshy or brightly colored cover of

seed) juice. Depending on the cultivar of pomegranate and juice


processing and storage methods, punicalagin content of the juice
can vary from 1500 to 1900 mg/L (266337 mg/6 oz) (Gil et al.,
2000). For an individual weighing 60 kg and consuming one can
of 6 ounce pomegranate juice, the punicalagin intake may range
from 4.4 to 5.6 mg/kg. This suggests that the regular intake of
pomegranate juice could result in high intake of this water-soluble
hydrolysable ellagitannin. Punicalagin (CAS No.: 65995-63-3; Fig.
1), naturally abundant in pomegranate fruit and unique to pomegranate as a food source, is a proper chemical marker for the
authentication, quality control and standardization of pomegranate products (Seeram et al., 2005b).
In recent years, several investigators have attempted to unravel
the underlying mechanisms of benecial effects of pomegranate.
These investigations have focused mainly on the antioxidant, antiinammatory, and antibacterial potentials of pomegranate (Aviram
et al., 2000, 2002, 2004; Cerda et al., 2003a,b; Lansky and Newman,
2007; De Nigris et al., 2007). Pomegranate juice and fruit extracts
normalized to punicalagins and a total pomegranate tannin fraction
(TPT) from the fruit have been shown to inhibit the proliferation of
human cancer cells and modulate inammatory subcellular signaling pathways (Adams et al., 2006; Seeram et al., 2005a; Afaq et al.,
2005). The effects of pomegranate juice and extracts on molecular
and cellular mechanisms has been researched on the systemic level,
where benets for cardiovascular, prostate, dental, and metabolic
health have been shown in the clinical literature (Aviram et al.,
2000, 2002, 2004; Pantuck et al., 2006; Menezes et al., 2006; Esmaillzadeh et al., 2006). Although increasingly claimed health-benecial properties of pomegranate have been investigated to some
extent, its adverse effects, if any, have not been systematically studied, partly because it is considered as a food. The objective of the
present study was to investigate the adverse effects, if any, of a standardized pomegranate fruit extract following acute administration
to rats and mice and subchronic administration to rats. The effects
of the extract were investigated in a dose-response study.
2. Materials and methods
2.1. Acute studies
The acute toxicity study was performed in compliance with Schedule Y and
Good Laboratory Practices requirements of Govt. of India. In the acute studies, male
and female Wistar strain rats and Swiss albino mice from Cadila Pharmaceutical

Fig. 1. Molecular structure of punicalagin.

Ltd. (Dholka, India) were used. In two separate experiments designed to determine
oral LD50 of pomegranate fruit extract (POMELLA Extract) in rodents, Wistar rats
(6/sex/group; 1011 weeks of age; body weight range: males 178260 g, females
147200 g) and Swiss albino mice (6/sex/group; 1011 weeks of age; body weight
range: males 3348 g, females 3041 g) were administered a single oral (gavage)
dose of the extract at levels of 0, 50, 500 and 5000 mg/kg body weight (dosing volume 10 mL/kg). In two additional separate experiments designed to determine
intraperitoneal LD50, Wistar strain rats (6/sex/group; 1011 weeks of age; body
weight range: males 143212 g, females 125166 g) and Swiss albino mice (6/
sex/group; 1011 weeks of age; body weight range: males 3450 g, females 30
40 g) were administered pomegranate fruit extract (POMELLA Extract) dissolved
in water. In rat study, the dose levels of extract used were 0, 100, 200, 300 mg/
kg/day (dosing volume 10 mL/kg), while for mice study, the dose levels were 0,
100, 150, 200 mg/kg/day. In all these experiments, animals were observed for 14
days for any signs of morbidity or mortality. On Day 15 at completion, all the animals were euthanized and gross pathological examinations were undertaken.

2.2. Subchronic study


2.2.1. Study design
The subchronic study was performed according to a well designed protocol
based on Organization for Economic Co-operation and Development (OECD) Guidelines for Testing Chemicals, Health Effects Test Guidelines, for Repeated Dose 90Day Oral Toxicity Study in Rodents, Section 408 (Updated Guidelines, adopted
21st September 1998). The study was conducted in compliance with the OECD principles on Good Laboratory Practices.

2.2.2. Test substance


Food grade pomegranate fruit extract (POMELLA Extract) was provided by Verdure Sciences Inc. (Noblesville, IN, USA) for the present study. The test substance,
made from whole pomegranate fruit, was yellowish brown to brown in color and
standardized to contain 70% polyphenols (total), including 30% punicalagins by
HPLC (Seeram et al., 2005b). In addition to punicalagins, other chemical constituents of the extract included (not more than) 5% ellagic acid by HPLC, 0.3% gallic acid
by HPLC. The appropriate amounts of the test substance were dissolved by slowly
adding the pure water (Milli Q) and crushing the solid extract by a standardized
procedure to ensure homogeneity. The solutions containing the extract concentration of 60, 24, 6 and 0 mg/mL were prepared daily prior to administration. The extract solutions were administered to rats daily orally via gavage. The 60 mg/mL
solution contained 18 mg Punicalagins. The solutions 0, 6, 24, and 60 mg/mL prepared for administration correlated to the gavage doses of 0, 60, 240 and 600 mg
pomegranate extract/kg.

2.2.3. Animals
For the subchronic study Wistar strain rats also from Cadila Pharmaceutical Ltd.
were used. A total of 120 male and female rats 67 weeks old, were selected after
physical and behavioral examination for the subchronic study. Selected females
were nulliparous and non-pregnant. The animals were maintained according to
standard guidelines. The animals were housed in groups of ve in standard polypropylene cages with stainless steel top grill under controlled conditions in a room
ventilated with fresh air, with 1520 air changes per hour. Clean paddy husk was
used as a bedding material. The room temperature was maintained at 22 3 C with
relative humidity between 30% and 70% and a 12 h light/dark cycle. The animals
were allowed to acclimatize for a minimum of ve days before the initiation of
experiments. Amrut standard rat pellet diet (Nav Maharashtra Chackan Mills Ltd.,
India) was provided ad libitum throughout the study period. Drinking water was
provided ad libitum in polypropylene bottles with stainless steel sipper tubes.

2.2.4. Treatment
In the subchronic study, Wistar strain rats (10/sex/group) were randomly divided into six groups. At randomization, the animals were approximately 67
weeks old and their body weight was within 25% of the overall mean of each
sex (weight range for male 132220 g and females 114170 g). Rats (10/sex/group)
were treated orally (gavage) with pomegranate fruit extract at dose levels of 0
(Group I- control), 60 (Group II- low dose), 240 (Group III- mid dose), and 600
(Group IV- high dose) mg/kg body weight/day (dosing volume 10 mL/kg) for 90
days. Two additional groups of animals for recovery study received 0 (Group V)
and 600 (Group VI) mg/kg/day of the extract for 90 days, followed by no additional
treatment for 28 days. The use levels of the extract in the 90 day and recovery study
were based on a preliminary range nding study in which rats (5/sex/group) were
administered 0, 60, 240 and 600 mg/kg body weight/day for 10 consecutive days. At
the end of the preliminary study, necropsy, hematological and biochemical investigations did not reveal any adverse effects of the extract. During the course of the
subchronic study, all animals were provided ad libitum feed, until the day prior to
the scheduled euthanasia. At completion of the 90 day treatment period, all animals
in Group I to IV were euthanized. In the recovery group, after completion of the
treatment period of 90 days, animals were kept under post treatment observations
for 28 days and then euthanized.

2.2.5. Parameters investigated


2.2.5.1. Clinical observations, body weights and feed consumption. All animals were
observed twice daily for morbidity and mortality. Clinical examinations included
any abnormal physical and behavioral changes. The observations included changes
in skin, fur, eyes, mucus membrane, and autonomic activity like lacrimation, piloerection, pupil size and unusual breathing pattern. Changes in gaits, posture, response to handling, presence of clonic or tonic movements, stereotypic activities
like excessive grooming, repetitive circling, etc, were observed. The time of onset,
intensity and duration of such symptoms, if any, were recorded. Ocular examinations were conducted on all animals prior to the initiation of experiments and during day prior to euthanasia.
Individual animal body weights for treatment and recovery groups were recorded at least weekly, beginning on the day before the initiation of treatment.
Mean body weights and mean body weight changes were calculated for the corresponding intervals. Final body weights were recorded one day prior to the scheduled necropsy. The amount of feed and water consumed by each cage of animals
was recorded weekly. Feed intake was calculated as g/animal/day for the corresponding body weight intervals. The water intake was calculated as ml/animal/day.
2.2.5.2. Clinical pathology. Urine and blood samples for clinical evaluations (urinalysis, hematology, and serum chemistry) were collected from all animals in the 90 day
study and the recovery groups prior to the scheduled necropsy. Urine samples were
collected overnight from all animals a few days before termination using metabolic
cages (Nalgene, USA). Urine analysis parameters included: appearance, specic gravity, pH, total volume, protein (albumin), glucose, and microscopy of sediment. The
microscopic examinations of urine samples included RBCs, WBCs, casts, crystals,
amorphous, and epithelial cells. For hematology and clinical chemistry analysis,
blood samples were collected from all animals through cardiac puncture under carbon dioxide anesthesia. For the chemistry analysis, blood was allowed to coagulate
and serum was separated after centrifugation. Hematology parameters included:
hemoglobin concentration, red blood cell count, white blood cell count, packed cell
volume, mean corpuscular volume, mean corpuscular hemoglobin, total and differential leukocyte count, platelet count, blood clotting time, activated partial prothrombin time, and reticulocyte time. Serum chemistry parameters included:
glucose, triglycerides, cholesterol, urea, creatinine, alkaline phosphatase (ALP), alanine aminotransferase (ALT), aspartate aminotransferase (AST), gamma-glutamyl
transferase (GGT), cholinesterase, total bilirubin, blood urea nitrogen, albumin, globulin, total protein, calcium, chloride, phosphorus, potassium, and sodium.
2.2.5.3. Macroscopic and microscopic examinations. A complete necropsy was performed on all animals. Animals were euthanized under carbon dioxide inhalation
anesthesia. The necropsies included, but were not limited to, examination of the
external surface, all orices, and the cranial, thoracic, abdominal and pelvic cavities,
including viscera. Over 40 tissues and organs were collected and were placed in 10%
neutral-buffered formalin. At the scheduled necropsies the following organs were
weighed from all animals: adrenal glands, kidneys, liver, uterus + ovaries (females),
thymus, spleen, testes + epididymides (males), brain, and heart. For histopathological examinations, tissues were processed into parafn blocks, sectioned at nominal 5 lm, mounted on glass microscope slides and stained with hematoxylin and
eosin. The pathologist who performed the histological assessments of the slides
was blinded to the treatment.
2.2.6. Statistical analysis
Analyses were conducted using two-tailed Students t-test for minimum significance levels of 5%, comparing Group I to Groups II, III and IV, and Group V to VI by
sex. Values were presented as mean with the standard error of the mean (SEM) and

the number of animals (N) used to calculate the mean. Body weight, organ weight,
hematology, and clinical chemistry analysis variable were analyzed using the Students t-test, separately for each sex. Where the results of P value were <0.05 the
means were considered as statistically signicantly different.

3. Results
3.1. Acute toxicity studies
In the acute toxicity studies, oral LD50 of pomegranate extract in
Wistar rats and Swiss albino mice was greater than 5000 mg/kg
body weight. The 14-day observation period during the acute oral
toxicity study and body weight measurements did not reveal any
toxic effects in either species (data not shown). Necropsy at the
end of the study did not reveal any gross pathological abnormalities in rats and mice. These observations from oral acute toxicity
study suggest that the extract is practically non-toxic. In the acute
toxicity studies following intraperitoneal injection of pomegranate
extract, LD50 of the extract in Wistar rats was determined as
217.5 mg/kg body weight. In Swiss albino mice, the intraperitoneal
LD50 of the extract was determined as 187.5 mg/kg body weight.
Clinical observations revealed symptoms of lordosis and death occurred within 24 h of treatment (data not shown).
3.2. Subchronic study
3.2.1. Survival, clinical observations and body weights
All animals survived until the scheduled necropsy in both the
90 day study group and the recovery group. Physical and behavioral examinations did not reveal any treatment-related adverse
effects. Compared to control group, no treatment-related biologically signicant effects of pomegranate extract were noted on body
weight or body weight gain at dose levels up to 600 mg/kg/day
(Figs. 2 and 3; Table 1). Female rats treated with 60 mg/kg/day of
the extract showed a statistically signicant decrease in body
weight gain during days 4254 only. During this period the activity
of the female animals in this group was found to be normal. The
initial body weights of male and female rats in the high dose treatment group (600 mg/kg/day) were statistically signicantly higher
compared to the control group. However, at subsequent time
points no signicant changes in the body weight were noted. In
the recovery group, no signicant changes in body weight gain
were noted during the course of the study. These results suggest
that administration of pomegranate extract at levels up to
600 mg/kg/day to rats for 90 days has no adverse effects on clinical
observations and body weights. Similarly, in the recovery group
also no adverse effects of pomegranate extract were noted.

Fig. 2. Effect of pomegranate extract on body weights in male rats. Mean body weights for male rats during a 90-day oral (gavage) toxicity study and 28 day recovery study
with pomegranate extract. The values are presented as means standard error (10 rats/sex/group). *represent values signicantly different from control at p < 0.05.

Fig. 3. Effect of pomegranate extract on body weights in female rats. Mean body weights for female rats during a 90-day oral (gavage) toxicity study and 28 day recovery
study with pomegranate extract. The values are presented as means standard error (10 rats/sex/group). *represent values signicantly different from control at p < 0.05.

Table 1
Effect of pomegranate extract on body weights (g) in male and female rats
Week

0
4
8
13
17

Sex

M
F
M
F
M
F
M
F
M
F

mg/kg/day

Recovery group

60

200

600

600

174.24 5.40
138.86 3.38
293.32 4.36
207.11 4.01
324.00 6.21
229.17 3.87
316.36 3.79
243.64 3.24
NA
NA

182.45 5.55
144.78 3.13
284.78 7.21
199.66 2.88
319.86 6.82*
210.77 2.96
372.65 7.75
234.10 2.91
NA
NA

189.34 4.29
148.88 3.39
292.51 7.23
200.57 4.83
349.06 8.64
230.49 6.17
372.55 8.59
244.28 5.99
NA
NA

202.09 2.74*
153.34 3.27*
307.85 8.79
203.81 2.58
356.84 10.43
223.63 2.50
382.08 9.86
234.80 2.79
NA
NA

175.67 4.59
138.19 2.46
300.37 7.43
202.62 3.47
346.70 6.20
228.77 4.30
370.23 6.29
234.20 3.64
388.72 5.66
248.04 3.03

188.45 4.29
142.43 2.61
293.67 6.13
198.64 2.88
322.48 8.50
218.16 2.00
373.41 9.92
228.86 3.74
387.98 8.96
245.14 1.93

Values are mean SEM for 10 rats in each group; *P < 0.05. NA = not applicable.

3.2.2. Feed and water consumption


The over all feed consumption of animals receiving pomegranate extract was generally similar to that of the controls (data not
shown). Similarly, the feed consumption in the recovery group
was similar to the respective control. The results of feed consumption show that in spite of lower body weights, females consumed
similar amount of the feed compared to males. There were no
treatment-related signicant adverse effects of the extract on feed
consumption. The quantity of water consumed during the course of
study by pomegranate treated animals was comparable to the
respective controls in 90 day and recovery study groups (data
not shown). These results show that administration of the extract
at levels up to 600 mg/kg/day to rats does not affect feed and water
consumption.
3.2.3. Clinical pathology
3.2.3.1. Urinalysis. There were no treatment-related adverse effects
on urinalysis parameters in male and female rats (data not shown)
in the 90 day study groups or in the recovery group. The urine analysis parameters such as volume, specic gravity, color, pH, albumin, sugar, red blood cells, white blood cells, epithelial cells,
casts, crystals, and amorphous did not show any signicant difference from the respective control groups. Minor differences noted in
color, quantity, specic gravity, albumin and crystals between different animals were not considered as treatment-related. Compared to control group, the changes noted in these parameters in
the treatment group did not reveal any upward or downward
trends. The relationships between these changes and exposure to
the extract, as well as their toxicological signicance, are uncertain
but could be the result of normal biological variation. The individual animal data for all the urinalysis parameters was within the
historical control data of the performing laboratory. These results

suggest that administration of the extract at dose levels up to


600 mg/kg/day to rats does not affect urine parameters.
3.2.3.2. Hematology. There were no treatment-related adverse effects of pomegranate extract on hematology parameters in male
and female rats (Tables 2 and 3). However, some statistically signicant differences were noted when the control and treatment
groups were compared. In male rats, a statistically signicant decrease in mean values of packed cell volume and mean corpuscular
volume and signicant increase in corpuscular hemoglobin concentration were noted in groups treated with 200 and 600 mg/
kg/day of the extract. However, mean values of red blood corpuscle
and hemoglobin concentrations of all treatment groups were similar to control group animals. Hence, the above changes were considered as incidental and not treatment-related. A signicant
decrease in the mean values of activated partial thromboplastin
time of the group treated with 200 mg/kg/day and in mean prothrombin time in group treated with 60 mg/kg/day was noted.
However, at high dose levels, these parameters were not altered.
Hence, changes in these parameters were not considered as treatment-related.
In female rats treated with the extract, a signicant decrease in
WBC of the group receiving 60 mg/kg/day (low dose group),
packed cell volume in the group receiving 200 mg/kg/day (mid
dose group), mean corpuscular volume in groups receiving 200
(mid dose group) and 600 mg/kg/day (high dose group) was noted
(Table 3). Additionally, mean corpuscular hemoglobin concentration in animals treated with 200 and 600 mg/kg/day were signicantly increased. Similar to males, no signicant changes in RBC
and hemoglobin were noted.
In the recovery group, treatment with the extract at dose
level of 600 mg/kg/day to male rats resulted in a signicant

Table 2
Effect of pomegranate extract on hematological parameters in male rats
Parameter

Units

RBC
WBC
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
PCV
MCV
MCH
MCHC
Platelet
Hemoglobin
CT
APTT
Prothrombin time
Reticulocyte

106/cm
103/cm
%
%
%
%
%
%
fL
pg
%
103/cm
g/dL
min
s
s
%

mg/kg/day

Recovery group

60

200

600

600

8.96 0.15
13.48 1.12
18.30 1.75
81.70 1.75
0.00 0.00
0.00 0.00
0.00 0.00
55.11 0.64
61.57 0.51
17.73 0.23
28.77 0.26
909.00 32.49
15.61 0.17
1.34 0.10
22.91 1.34
20.64 0.64
0.60 0.07

9.16 0.16
12.28 0.75
14.70 1.08
85.30 1.09
0.00 0.00
0.00 0.00
0.00 0.00
55.58 0.71
60.76 0.59
17.18 0.17
28.29 0.08
945.50 33.88
15.72 0.20
1.78 0.13
19.53 1.51
18.76 0.20*
0.62 0.05

8.75 0.17
12.93 0.79
13.40 0.90
86.60 0.90
0.00 0.00
0.00 0.00
0.00 0.00
46.33 0.75*
53.01 0.35*
18.09 0.20
34.11 0.26*
979.60 40.12
15.93 0.20
1.63 0.10
17.38 1.22*
17.36 1.15
0.65 0.08

8.77 0.14
17.81 1.78
16.80 1.61
83.20 1.61
0.00 0.00
0.00 0.00
0.00 0.00
45.32 0.61*
51.72 0.34*
18.05 0.25
34.88 0.34*
954.70 24.40
15.79 0.20
1.79 0.17
22.30 1.15
18.85 0.43
0.62 0.06

8.89 0.16
13.51 0.80
11.40 0.80
88.60 0.80
0.00 0.00
0.00 0.00
0.00 0.00
52.21 0,76
58.83 0.51
16.72 0.21
28.38 0.26
1108.50 38.40
14.80 0.14
2.26 0.25
18.01 0.68
18.67 0.23
0.66 0.04

8.70 0.08
12.12 0.55
15.20 0.88*
84.80 0.88*
0.00 0.00
0.00 0.00
0.00 0.00
53.18 0.47
61.83 0.35*
16.24 0.13
26.25 0.17*
1002.10 17.27
13.96 0.10*
2.68 0.21
18.32 0.67
18.95 0.27
0.69 0.05

Values are mean SEM for 10 rats in each group. *P < 0.05; RBC = red blood cells; WBC = white blood cells; PCV = packed cell volume; MCV = mean cell volume; MCH = mean
cell hemoglobin; MCHC = mean corpuscular hemoglobin concentration; APTT = activated partial prothrombin time.

Table 3
Effect of pomegranate extract on hematological parameters in female rats
Parameter

RBC
WBC
Neutrophils
Lymphocytes
Monocytes
Eosinophils
Basophils
PCV
MCV
MCH
MCHC
Platelet
Hemoglobin
CT
APPT
Prothrombin time
Reticulocyte

Units

106/cm
103/cm
%
%
%
%
%
%
fL
pg
%
103/cm
g/dL
min
s
s
%

mg/kg/day

Recovery group

60

200

600

600

7.60 0.30
12.02 0.62
15.60 1.01
84.30 1.03
0.00 0.00
0.00 0.00
0.00 0.00
49.90 1.47
66.24 1.15
19.13 0.27
29.02 0.40
914.30 43.76
14.49 0.50
1.44 0.08
21.63 1.33
18.10 0.63
0.71 0.10

8.16 0.10
8.76 0.83*
17.20 0.92
82.80 0.92
0.00 0.00
0.00 0.00
0.00 0.00
52.35 0.61
64.16 0.50
18.37 0.25
28.60 0.26
1029.60 29.73
14.97 0.17
2.02 0.33
21.06 1.49
17.43 0.21
0.56 0.05

7.75 .09
12.99 1.14
14.50 0.91
85.50 0.91
0.00 0.00
0.00 0.00
0.00 0.00
42.16 0.45*
54.40 0.34*
18.85 0.19
34.36 0.31*
991.70 20.23
14.61 0.14
1.61 0.07
18.08 0.64
18.14 0.14
0.57 0.07

8.00 0.10
12.00 0.90
15.90 1.26
84.10 1.26
0.00 0.00
0.00 0.00
0.00 0.00
45.01 1.13
51.74 1.27*
18.14 0.63
33.78 1.14*
1040.00 50.00
14.52 0.51
1.62 0.12
26.90 2.19
22.00 1.44
0.79 0.03

7.35 0.08
9.71 0.76
13.00 1.50
87.00 1.50
0.00 0.00
0.00 0.00
0.00 0.00
46.47 0.51
63.26 0.35
19.32 0.24
30.57 0.32
990.70 22.16
14.19 0.15
2.14 0.18
18.40 0.76
17.90 0.14
0.64 0.04

7.55 0.08
11.12 0.68
13.20 0.62
86.80 0.62
0.00 0.00
0.00 0.00
0.00 0.00
47.91 0.58
63.49 0.30
17.52 0.13*
27.5 7.17*
983.30 34.95
13.20 0.13*
2.77 0.28
15.04 0.72*
18.10 0.28
0.71 0.05

Values are mean SEM for 10 rats in each group. *P < 0.05; RBC = red blood cells; WBC = white blood cells; PCV = packed cell volume; MCV = mean cell volume; MCH = mean
cell hemoglobin; MCHC = mean corpuscular hemoglobin concentration; APTT = activated partial prothrombin time.

decrease in neutrophil counts and corresponding decrease in


lymphocyte counts. Additionally a signicant decrease in mean
corpuscular hemoglobin concentration and hemoglobin and an
increase in mean corpuscular volume was noted in males receiving the pomegranate extract. The values of hemoglobin, mean
corpuscular hemoglobin concentration and corpuscular volume
in the group treated with 600 mg/kg/day were within the normal
laboratory range. In females, mean corpuscular hemoglobin,
mean corpuscular hemoglobin concentration, hemoglobin and
activated partial prothrombin time were signicantly decreased
in animals treated with 600 mg/kg/day. These variations were
within the normal laboratory range and were considered as biological variations. The above noted signicant changes following
administration of the extract in the 90 day study as well as in
the recovery group were not observed in both sexes, lacked correlating changes in other red cell parameters, were of small magnitude, and/or were not noted in a dose-related manner; hence
they were not considered test article-related. There were no
other statistically signicant differences when the respective
control and treatment groups were compared. These results

demonstrate that administration of pomegranate extract to rats


at doses up to 600 mg/kg/day had no adverse hematological
effects.
3.2.3.3. Serum chemistry. There were no treatment-related biologically signicant adverse effects of pomegranate extract on serum
chemistry parameters in male and female rats (Tables 4 and 5).
However, some statistically signicant differences were noted
when the control and treatment groups were compared. Administration of the extract at dose level of 600 mg/kg/day to male rats
resulted in a statistically signicant decrease in alanine aminotransferase (ALT) and aspartate aminotransferase (AST). As increase in the activities of ALT and AST represents liver damage,
decrease in the activities of these enzymes is not considered of
any toxicological signicance. A signicant increase in alkaline
phosphatase activity was noted in male rats treated with 60 mg/
kg/day of the extract. Although not dose-related, a signicant decrease in mean values of total bilirubin was noted in all treatment
groups. However, the values were within normal laboratory
ranges. Compared to the control group, a signicant increase in

Table 4
Effect of pomegranate extract on serum chemistry parameters in male rats
Parameter

Units

Glucose
Cholesterol
Triglycerides
Urea
Creatinine
AST
ALT
ALP
Total bilirubin
Protein
Albumin
Globulin
Sodium
Potassium
Phosphorus
Calcium
Chloride
GGT
BUN
Cholinesterase

mg/dL
mg/dL
mg/dL
mg/dL
mg/dL
IU/L
IU/L
IU/L
mg/dL
g/dL
g/dL
g/dL
mEq/L
mEq/L
mEq/L
mg/dL
mEq/L
U/L
mg/dL
U/ml

mg/kg/day

Recovery group

60

200

600

600

105.00 10.10
45.10 2.10
73.30 6.31
36.23 1.70
0.45 0.02
138.82 5.21
71.58 4.92
94.80 4.17
0.16 0.01
7.07 0.07
3.64 0.03
3.42 0.06
143.41 0.76
5.44 0.10
9.35 0.28
10.37 0.01
100.30 0.57
4.77 0.87
16.93 0.80
0.38 0.01

102.30 4.18
49.30 1.59
84.00 6.57
30.92 0.68
0.49 0.01
129.91 6.76
72.53 3.63
116.20 3.56*
0.11 0.00*
7.48 0.11*
3.80 0.03*
3.68 0.08
151.7 0.68*
6.48 0.16*
9.28 0.21
11.17 0.13*
100.70 0.52
3.56 0.29
14.45 0.32*
0.34 0.01

92.30 3.27
53.50 3.05
74.10 5.50
37.29 1.38
0.45 0.01
124.16 4.17
61.68 2.92
108.70 6.53
0.12 0.00*
7.51 0.11*
3.74 0.04
3.77 0.09*
147.00 0.42*
6.54 0.10*
9.86 0.25
10.99 0.07*
100.30 0.57
6.14 0.73
18.30 0.97
0.52 0.02*

108.70 5.53
44.90 0.81
59.70 5.47
34.21 1.46
0.42 0.01
116.13 3.23*
47.39 1.71*
98.80 4.34
0.10 0.00*
7.18 0.11
3.63 0.05
3.55 0.08
150.90 0.60*
6.39 0.18*

100.60 1.44
46.10 1.57
59.10 3.67
39.20 0.84
0.43 0.01
119.99 5.82
68.00 2.29
95.40 5.66
0.14 0.00
7.31 0.12
3.65 0.06
3.66 0.11
144.68 0.34
5.93 0.09
11.15 0.23
9.96 0.06
118.10 0.61
6.05 0.69
18.32 0.39
0.35 0.02

104.10 7.39
44.10 1.98
91.50 6.32*
33.35 0.61*
0.33 0.01*
106.08 4.43
73.76 4.13
109.90 4.33
0.10 0.01*
7.31 0.07
3.62 0.04
3.69 0.06
145.07 0.37
6.47 0.12*
13.13 0.38*
10.39 0.13*
117.70 0.44
4.01 0.84
15.58 0.29*
0.65 0.06*

9.07 0.36
10.23 0.06
99.40 0.60
3.01 0.60
15.99 0.68
0.40 0.02

Values are mean SEM for 10 rats in each group. *P < 0.05; AST = aspartate aminotransferase; ALT = alanine aminotransferase; ALP = alkaline phosphatase; GGT = gammaglutamyl transferase; BUN = blood urea nitrogen.

Table 5
Effect of pomegranate extract on serum chemistry parameters in female rats
Parameter

Glucose
Cholesterol
Triglycerides
Urea
Creatinine
AST
ALT
ALP
Total bilirubin
Protein
Albumin
Globulin
Sodium
Potassium
Phosphorus
Calcium
Chloride
GGT
BUN
Cholinesterase

Units

mg/dL
mg/dL
mg/dL
mg/dL
mg/dL
IU/L
IU/L
IU/L
mg/dL
g/dL
g/dL
g/dL
mEq/L
mEq/L
mEq/L
mg/dL
mEq/L
U/L
mg/dL
U/ml

mg/kg/day

Recovery group

60

200

600

600

77.80 2.21
49.60 2.98
56.90 3.23
34.41 1.01
0.48 0.01
136.73 6.19
54.84 3.39
66.20 6.19
0.18 0.01
7.88 0.12
4.11 0.07
3.57 0.08
144.59 0.87
5.39 0.08
8.45 0.27
10.98 0.08
102.10 0.59
4.42 0.80
16.08 0.47
0.87 0.08

99.30 3.21*
52.30 2.99
75.80 6.84
38.75 1.41*
0.58 0.01*
115.57 8.22
48.71 2.31
65.10 3.28
0.13 0.01*
8.29 0.10*
4.40 0.07*
3.89 0..05*
151.20 0.61*
6.65 0.23*
8.28 0.41
11.61 0.12*
101.40 0.85
2.23 0.37
18.71 0.77
0.99 0.06

87.20 3.82
46.10 2.29*
74.20 5.59*
39.02 1.89
0.44 0.01*
125.59 3.26
45.70 2.20
57.20 1.67
0.16 0.01
8.24 0.20
4.09 0.07
4.15 0.14*
146.20 0.44
6.48 0.12*
10.24 0.72
11.21 0.05
102.50 0.56
7.29 0.76
18.23 0.88
1.42 0.08

94.90 4.12*
42.10 3.17
54.50 3.38
39.34 0.918*
0.53 0.01*
124.49 8.33
44.66 1.93
61.00 3.06
0.13 0.008*
8.03 0.17
4.05 0.08
3.98 0.10*
150.20 0.42*
6.91 0.16*
10.79 0.70*
10.96 0.09
103.50 0.56
2.82 0.53
18.39 0.42
1.02 0.04*

98.40 3.40
56.00 2.38
76.30 3.61
43.09 1.61
0.50 0.01
112.92 3.94
54.70 2.80
57.00 2.46
0.17 0.01
8.20 0.10
4.20 0.08
4.00 0.06
144.19 0.25
6.22 0.16
10.58 0.36
10.56 0.05
120.70 0.64
7.11 0.79
20.14 0.75
1.04 0.09

115.70 7.26
58.50 2.01
70.20 5.67
36.81 1.61*
0.38 0.01*
105.23 4.90
57.17 3.67
66.00 6.54
0.16 0.01
8.01 0.12
4.11 0.07
3.93 0.12
144.68 0.45
6.36 0.15
11.67 0.32
10.84 0.07*
120.80 0.44
6.74 1.24*
17.20 0.75*
1.20 0.09

Values are mean SEM for 10 rats in each group. *P < 0.05; AST = aspartate aminotransferase; ALT = alanine aminotransferase; ALP = alkaline phosphatase; GGT = gammaglutamyl transferase; BUN = blood urea nitrogen.

total protein in groups treated with 60 and 200 mg/kg/day of the


extract were noted. Similarly a signicant increase in albumin in
rats treated with 60 mg/kg/day and globulin in rats treated with
200 mg/kg/day were noted. Slightly elevated levels of total protein
in the 60 and 200 mg/kg/day dose group, albumin in 60 mg/kg/day
dose group and globulin in 200 mg/kg/day dose group were not
considered as treatment-related. Compared to control group, a signicant increase in sodium and potassium in all treated groups and
a signicant increase in calcium in 60 and 200 mg/kg/day dose
group was noted. However, no concurrent changes in histopathology of the kidney were noted and the values were within normal
laboratory ranges for these parameters. Additionally, as compared
to control group, incidental variations such as decrease in blood
urea nitrogen and urea in 60 mg/kg/day dose group and increased

serum cholinesterase in 200 mg/kg/day dose group males was


noted.
In females, compared to control group, a signicant increase in
glucose in 60 and 600 mg/kg/day dose group and an increase in triglyceride in 200 mg/kg/day dose group were noted (Table 5). However, these variations were marginal and within normal laboratory
limits. Additionally, an increase in total bilirubin and urea was
noted in 60 and 600 mg/kg/day dose group. A signicant decrease
in mean values of creatinine was noted in 60 and 600 mg/kg/day
dose groups, while in animals treated with 200 mg/kg/day (mid
dose group) a decrease in creatinine was noted. As the changes
in these parameters were not dose-related and were not reected
by changes in other related parameters, these changes were considered as incidental. A signicant increase in total protein and

albumin in 60 mg/kg/day dose group and an increase in globulin in


all treatment groups compared to the control were noted. These
changes were not dose-related, were within normal laboratory
range and were considered as incidental. Compared to control
group, a signicant increase in sodium in 60 and 600 mg/kg/day
dose group, potassium in all treated groups, and phosphorus in
600 mg/kg/day dose group was noted. However, these changes in
electrolyte concentration were within normal laboratory range
and were not reected in concurrent histopathological changes in
kidney. A signicant increase in calcium in 60 mg/kg/day dose
group and cholinesterase in 200 mg/kg/day dose group compared
to control were noted. These changes were considered as incidental and were not related to the treatment.
In the recovery group, signicant increase in mean values of triglycerides, potassium, phosphorus, calcium and cholinesterase and
decrease in urea, creatinine, total bilirubin, and blood urea nitrogen were noted in the extract-treated male rats. All these variations were marginal and within the normal laboratory ranges. In
females, a signicant decrease in urea, blood urea nitrogen, creatinine and GGT were noted. Decreases in these values were not considered as toxicologically relevant. Additionally, calcium levels
were signicantly increased in the female treatment group. However, the values were within normal laboratory limits and were
considered as incidental. There were no other statistically signicant differences when the respective control and/or treatment
groups were compared. The results of serum chemistry analysis
from treatment and recovery groups show that administration of
pomegranate extract at doses up to 600 mg/kg/day to rats for 90
days did not cause toxicologically signicant adverse effects.
3.2.3.4. Organ weights. No treatment-related changes of biological
signicance in absolute (data not shown) and relative organ
weights were noted in male and female rats following administration of the pomegranate extract. Similarly, in the recovery group,
no treatment-related effects of pomegranate extract were noted
in male and female rats. These results suggest that feeding of the
extract had no signicant adverse effects on organ weights.
3.2.3.5. Macroscopic and microscopic examinations. There were no
treatment-related macroscopic ndings at the scheduled necropsy
following administration of pomegranate extract to rats. All macroscopic changes noted were considered to be spontaneous and/or
incidental in nature and unrelated to the treatment. There were
no treatment-related histopathological ndings. Similarly, in the
recovery group no treatment-related macroscopic or microscopic
changes were noted. All ndings observed were consistent with
normal background lesions in clinically normal rats of the age
and strain used in this study and were considered spontaneous
and/or incidental in nature and unrelated to the treatment. These
results suggest that administration of pomegranate extract at dose
levels up to 600 mg/kg/day to rats for 90 days has no adverse macroscopic or microscopic effects.

4. Discussion
In a previous study, Cerda et al. (2003a) investigated the effects
of pomegranate extract (6% punicalagin) in female Sprague-Dawley
rats following exposure to diet containing 20% of the extract for 37
days. The exposure to pomegranate extract resulted in intake of
4800 mg punicalagin/kg/day. A signicant decrease in feed consumption and body weight of the animals during the early part of
the study was noted. Except for a signicant decrease in plasma
urea and triglycerides, administration of the extract did not result
in any signicant difference in blood parameters analyzed. The
investigators suggested that the decrease in urea and triglycerides

could be due to the lower nutritional value of the punicalagin-enriched feed. Histopathological examinations of liver and kidney corroborated with the lack of adverse effects. In the present study, oral
(gavage) administration of pomegranate extract at levels up to
600 mg/kg/day for 90 days did not reveal any biologically signicant adverse effect as evaluated by several parameters including
biochemical analysis and histological examinations. The pomegranate extract in the present study was standardized to contain 30%
punicalagin. Thus the highest dose (600 mg/kg/day) of pomegranate extract used in the present study resulted in intake of 180 mg
punicalagin/kg/day, which is about 25-fold lower than that used
in the Cerda et al. (2003a) study. The ndings from the present
study following oral gavage administration of pomegranate extract
support the previous observation from feeding study that administration of pomegranate extract does not cause any adverse effects.
In the study by Cerda et al. (2003a), signicant reduction in feed
consumption and body weight appeared to be related to high levels
of the extract (20%) in the diet and the resulting punicalagin
(4800 mg/kg/day) intake from the diet. In the present study, gavage administration of the extract did not affect feed consumption
or body weight. This may be due to the relatively low (25-fold) levels of the dose and different routes of administration. Other studies
have shown that depending on the amount of polyphenol administered, body weights can be affected. A diet containing 0.4%
avonoid extract from grapefruit (Juskiewicz et al., 2002) or 2%
proanthocyanidin rich extract from grape seeds (Yamakoshi et
al., 2002) did not affect body weight, whereas, administration of
diet containing 1% polymeric grape seed tannins (Tebib et al.,
1996) or 1.9% catechin (Bravo et al., 1994) resulted in a signicant
decrease in body weight. It has been suggested that tannins
(including ellagitannins) interact with proteins and inhibit digestion of endogenous protein, which may result in weight loss (Butler
and Rogler, 1992). In the present study, gavage administration of
pomegranate extract did not affect body weight. The differential
effects on the body weight in these studies is difcult to explain
but may be related to differences in the types of polyphenols.
Lin et al. (1998) reported that subcutaneous injection of punicalagin and punicalin at doses of 25 and 12.5 mg/kg of body weight,
respectively, to rats protected against liver damage induced by carbon tetrachloride administration. The hepatoprotective effects of
punicalagin and punicalin was correlated with the decrease in both
AST and ALT levels. However, these investigators reported that larger
doses of punicalin induced liver damage. In a recent human study,
Haber et al. (2007) reported that oral ingestion of pomegranate
ellagitannin-enriched polyphenol extract in amounts up to
1420 mg/day for 28 days did not cause any adverse events or
changes in hematology, serum chemistry (including ALT, AST levels),
or urinalyses. Similarly, in the present study, oral gavage administration of pomegranate extract rich in punicalagin at doses of 600 mg/
kg body weight/day for 90 days did not cause liver toxicity or increase in serum ALT. Furthermore, biochemical parameters of liver
function such as hepatocyte integrity (AST, ALT), bile duct alterations
(ALP), and liver function (bilirubin) did not show hepatic damage following administration of punicalagin-rich pomegranate extract.
In the present study, minor changes were noted in some of the
blood parameters in some pomegranate extract-treated groups. A
statistically signicant decrease in packed cell volume and mean
corpuscular volume and signicant increase in corpuscular hemoglobin concentration were noted in male rats treated with 200 and
600 mg/kg/day of the extract. In female rats, a signicant decrease
in mean corpuscular volume in groups receiving 200 and 600 mg/
kg/day of the extract was noted. Additionally, mean corpuscular
hemoglobin concentration in animals treated with 200 and
600 mg/kg/day were signicantly increased. However, mean values of red blood corpuscle and hemoglobin concentrations of all
treatment groups were similar to control group animals. Hence,

these changes were considered as incidental and not treatment-related. As regards serum chemistry, a statistically signicant decrease in liver function test parameters such as serum ALT and
AST was noted in male rats treated with 600 mg/kg/day of the extract and in ALP in male rats treated with 60 mg/kg/day. Additionally a signicant decrease in serum total bilirubin was noted in all
treatment groups. As these parameters represent liver function, increases in their serum levels indicate liver damage. A decrease in
the activities of these liver function test enzymes is not considered
of any toxicological signicance. The decreases in serum total bilirubin values were within normal laboratory ranges. In female rats
a signicant increase in sodium in low and high dose group, potassium in all treated groups, and phosphorus in high dose group was
noted. However, these changes in electrolyte concentration were
not reected in concurrent histopathological changes in the kidney. These and other statistically signicant changes noted in
hematology and serum chemistry parameters following administration of the extract were considered as incidental and not related
to treatment, as they were either limited to one sex, lacked doseresponse, were within the normal laboratory ranges, and/or were
not supported by any other changes in related clinical parameters
or histopathological observations.
In commercially available juices, punicalagin content can range
from 1500 to 1900 mg/L (266337 mg/6 oz). The resulting exposure of punicalagin for an individual weighing 60 kg and consuming one can of 6 oz pomegranate juice will range from 4.4 to
5.6 mg/kg. This punicalagin intake is approximately 3240-fold
lower to that used in the present subchronic rat study. The highest
dose (600 mg/kg/day) of pomegranate extract (standardized to
contain 30% punicalagin) used in the present study resulted in
180 mg punicalagin/kg/day. Thus daily oral administration of punicalagin (180 mg/kg/day) in the form of pomegranate extract for 90days did not cause any adverse effects. In summary, the results of
this subchronic toxicity study suggest that oral administration of
pomegranate extract at levels up to 600 mg/kg/day does not cause
adverse effects in male and female rats. Based on the results of this
study, the no-observed effect level (NOAEL) of the extract was
found to be 600 mg/kg/day.

Conict of interest
A conicting interest exists when professional judgement concerning a primary interest (such as patients welfare or the validity
of research) may be inuenced by a secondary interest (such as
nancial gain or personal rivalry). It may arise for the authors
when they have nancial interest that may inuence their interpretation of their results or those of others. Examples of potential
conicts of interest include employment, consultancies, stock
ownership, honoraria, paid expert testimony, patent applications/
registrations, and grants or other funding.
Acknowledgement
Verdure Sciences Inc. for supplying POMELLA Pomegranate Extract for use in this study.

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