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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,
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
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.
2.2.3. 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.
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.
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.
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.
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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