Journal of Ethnopharmacology 89 (2003) 295–300

Studies on the toxicity of Punica granatum L. (Punicaceae)

whole fruit extracts
Alexis Vidal a , Adyary Fallarero a , Blanca R. Peña b , Maria E. Medina c ,
Bienvenido Gra d , Felicia Rivera a , Yamilet Gutierrez e , Pia M. Vuorela f,∗
aDepartment of Biochemistry, Faculty of Biology, University of Havana, Havana, Cuba
bDepartment of Microbiology, Faculty of Biology, University of Havana, Havana, Cuba
c Clinical Biochemistry Laboratory, Havana Pediatric Hospital, Havana, Cuba
d Pathology Laboratory, National Institute of Gastroenterology, Havana, Cuba
e Pharmacognosy Laboratory, Pharmacy and Food Sciences Institute, University of Havana, Havana, Cuba
f Department of Pharmacy, Viikki Drug Discovery Technology Center, University of Helsinki, Helsinki, Finland
Received 6 May 2003; received in revised form 1 August 2003; accepted 1 September 2003

Abstract

Current investigation focuses on the toxicity evaluation of whole fruit hydroalcoholic extract of Punica granatum L. (Punicaceae), used in
Cuban traditional medicine a.o. for the treatment of respiratory diseases. Previous findings on the anti-influenza activity of Punica granatum
extracts has given support to the ethnopharmacological application. In our study, in chick embryo model, it was found that doses of the
extract of less than 0.1 mg per embryo are not toxic. The LD50 of the extract, determined in OF-1 mice of both sexes after intraperitoneal
administration, was 731 mg/kg. Confidence limits were 565–945 mg/kg. At the doses of 0.4 and 1.2 mg/kg of extract, the repeated intranasal
administration to Wistar rats produced no toxic effects in terms of food intake, weight gain, behavioural or biochemical parameters, or results
of histopathological studies. We conclude that toxic effects of Punica granatum fruit extract occurred at higher doses than those effective in
the models where the anti-viral activity has been studied or than those doses used in Cuban folk medicine.
© 2003 Elsevier Ireland Ltd. All rights reserved.

Keywords: Punica granatum L.; Pomegranate; LD50 ; Embryotoxicity; Dose-repeated toxicity; Acute toxicity

1. Introduction plant in the treatment of respiratory disease. Punica grana-

Many extracts from roots, stems and fruits have been used
in folk medicine for the treatment of viral diseases (Vlietinck
and Vanden Bergh, 1991; Sydiskis et al., 1991; Taylor et al.,
1996). Extracts from some plant sources have in particu-
lar been shown to be effective against the influenza virus
(Serkedjieva and Manolova, 1992; Hayashi et al., 1995;
Nagai et al., 1995).
The fruits of Punica granatum L. (Punicaceae), the
pomegranate, are commonly eaten. In traditional Cuban
medicine pomegranate fruits have been used to treat acido-
sis, dysentery, microbial infections, diarrhoea, helminthia-
sis, haemorrhage and respiratory pathologies (Roig, 1974;
Jimenez et al., 1979; Seoane, 1984). Arseculeratne et al.
(1985) have also reported popular use of the pomegranate
∗ Corresponding author. Fax: +358-9-191-59138.
E-mail addresses: alvidno@yahoo.com (A. Vidal),
pia.vuorela@helsinki.fi (P.M. Vuorela).
tum would appear to have interesting anti-viral activity.
Extracts have been shown to be effective against the herpes
virus (Zhang et al., 1995) and hydroalcoholic extracts of
whole fruits have exhibited high activity against the in-
fluenza virus (Peña, 1998; Caballero et al., 2001; Peña and
Martı́nez, 2001).
However, the toxicity of Punica granatum has not been
intensively studied. Amorin (1995) observed no toxic ef-
fects in mice treated with aqueous extracts of pomegranate
similar to those used in folk medicine. Desta (1995) re-
ported that pomegranate was widely favoured in Ethiopia
as a taeniacidal drug on the basis of its relatively low tox-
icity and high potency. It is important to bear in mind that
pomegranate fruits (excluding the peel) are not toxic but
pomegranate roots and bark are (Fuentes et al., 1985). The
toxic activity of a Punica granatum bark extract was re-
lated to its alkaloid content according to Tripathi and Singh
(2000). Ferrara et al. (1989) have also reported that some
galenic preparations of pomegranate are toxic because of

0378-8741/$ – see front matter © 2003 Elsevier Ireland Ltd. All rights reserved.
doi:10.1016/j.jep.2003.09.001
296 A. Vidal et al. / Journal of Ethnopharmacology 89 (2003) 295–300
their alkaloid content. Segura et al. (1990) reported that tan-
nins from pomegranate peel were more effective than alka-
loids from pomegranate peel against Entamoeba histolytica
and Entamoeba invadens.
Accordingly, we have conducted our research on em-
bryotoxicity, acute toxicity and toxicity after repeated in-
tranasal administration of hydroalcoholic extracts of whole
Punica granatum fruits. Such extracts are traditionally used
in Cuba to treat upper respiratory disorders, for example,
those caused by the influenza virus.
2. Methodology
2.1. Plant extract
Fresh Punica granatum (pomegranate) fruits were col-
lected from wild plants in the Havana province in July
2001 and their identities confirmed by Alvarez et al. (40619
HAJB). The voucher specimen is deposited at the Havana
Botanical Garden, Havana, Cuba. The fruits (including the
peel) were macerated for 15 days in a 50% (v/v) ethanol
solution in a ratio of 1:3 (w/v). The extract was filtered
and ethanol was removed by vacuum evaporation on a
rotary evaporator at 55 ◦ C. The aqueous extract was then
lyophilized and stored at −20 ◦ C until used. The yield of
extract was 10%.
Phytochemical analysis of the lyophilized extract in-
volved determination of the following substances: reduc-
ing carbohydrates, mucilages, glycosides, phenols/tannins,
flavonoids, anthocyanidins and alkaloids. Determinations
were carried out in accordance with detailed procedures
developed by the National Medicinal Plant Programme
(1994). Flavonoids were determined according to Ogbeide
and Parvez (1991).
2.2. Embryotoxicity studies
The embryotoxicity study was performed as in Nagai et al.
(1992). Hundred microlitres of Punica granatum extracts at
concentrations of 1, 2 and 3 mg/ml or of amantadine at con-
centrations of 12.5, 25 and 50 mg/ml were administered to
chicken embryos (from CENPALAB, pathogen-free, 9–11
days of incubation) via the allantoic membrane. The em-
bryos (20–30) were randomly selected to receive each dose.
Each member of a control group received 100 ␮l of water by
the same route. All embryos were observed ovoscopically
each day for 1 week, to allow percentages surviving at the
end of the period to be calculated. The dose at which mor-
tality was similar to or lower than that in the control group
was considered non-toxic dose.
2.3. Determination of acute toxicity
OF-1 mice of both sexes (from CENPALAB), each weigh-
ing 18–22 g, were kept at room temperature and room rela-
tive humidity, exposed to the natural light–dark cycle. The
animals were randomly distributed into groups of 10 ani-
mals per dose per cage, with free access to water and pellet
feed (ALYco, Havana). Extracts of Punica granatum were
administered intraperitoneally at doses of 25, 50, 100, 200,
1000 and 2000 mg/kg of body weight. Animals were ob-
served during the first 12 h for behavioural changes and other
toxic signs, and for symptoms including time to seizure and
death. Observation continued for 15 days to confirm that the
number of animals per dose that remained alive did not al-
ter. Values for LD50 and upper and lower confidence limits
were calculated by means of the method of Litchfield and
Wilcoxon (1949) using the Microcal Origin Program.
2.4. Determination of repeated-dose toxicity
Male Wistar rats, each weighing 205–220 g (from CEN-
PALAB) were used. The animals were kept in plastic cages
and had free access to water and food (ALYco, Havana).
Four groups of 10 animals were studied for 35 days (5
weeks). Five microlitres of lyophilized extract were admin-
istered by the nasopharyngeal route through each nasal cav-
ity, using a microsyringe (Hamilton Co.). Each member of
Groups I–III received a dose of 0.4, 1.2 or 7 mg of extract
per kilogram of body weight, respectively. Reference ani-
mals (the control group) received saline solution once a day.
All of the animals were weighed and food consumption was
measured every 7 days.
At the end of the experiment the animals were decap-
itated and blood samples were taken for determination
of serum glucose, uric acid, urea, creatinine, cholesterol,
triglyceride, total protein, calcium, phosphorus, ASAT and
ALAT levels. All determinations were carried out using di-
agnostic kits (Boehringer Mannheim). A macroscopic study
was performed, during which organs were weighed. Sam-
ples of livers, lungs, kidneys, brains, hearts, stomachs, the
small and large intestines, larynxes/tracheas, spleens, the
nasal mucous membrane and fibrocartilaginous nasal tissue
were taken for histological study using the conventional
haematoxylin–eosin technique.
All the animal studies reported in this work were car-
ried out in accordance with the Cuban regulations on the
protection of animals (Código Práctico para el Uso de los
Animales de Laboratorio, Centro Nacional para la Pro-
ducción de Animales de Laboratorio, CENPALAB, 1992),
and the Declaration of Helsinki. All experimental protocols
were revised by the Animal Care and Use Committee of the
Faculty of Biology, University of Havana, and conducted
humanely.
2.5. Statistic analysis
Results are expressed as means ± standard deviations
(S.D.s). Values relating to food consumption, weight gain,
organ weights and biochemical parameters were analysed
using one-way analysis of variance, and the Duncan test
 A. Vidal et al. / Journal of Ethnopharmacology 89 (2003) 295–300 297

when variances were statistically significantly different Table 3

(∗ P < 0.05, ∗∗ P < 0.01). Food intakes (gram per animal per day) of Wistar rats (control group and
Groups I–III) treated with hydroalcoholic extracts of Punica granatum
L., after 0, 14, 28 and 35 days of treatment

3. Results Group Days of treatment

0 14 28 35
Results relating to embryotoxicity are shown in Table 1.
Control 13.3 ± 1.5a 19.7 ± 1.1b 26.4 ± 1.4c 30.0 ± 2.3d
Mortality in the control group was 5.3%, which was related I 12.5 ± 1.2a 18.9 ± 1.0b 25.5 ± 1.2c 30.5 ± 1.9d
to manipulation and other experimental factors. From results II 11.8 ± 2.0a 16.3 ± 0.9b 27.5 ± 1.5c 30.3 ± 3.1d
in Table 1, it may be assumed that doses of Punica granatum III 12.0 ± 3.0a 18.9 ± 0.8b 28.7 ± 1.6c 23.4 ± 1.5d
in a hydroalcoholic extract of less than 0.1 mg per embryo Values are expressed as means ± S.D.s. The various superscript letters
are not toxic. indicate statistically significant differences in the Duncan test, with P <
The LD50 of Punica granatum extracts administered in- 0.05.
traperitoneally to mice was 731.1 mg/kg. Confidence limits
are 565–945 mg/kg. No differences in acute toxicity in re-
lation to sex were recorded. The most evident toxic symp- As shown in Fig. 1, mean weights of animals that re-
tom in mice was piloerection. The latter mainly provides ceived repeated doses of Punica granatum extract by the
evidence for disturbance of the peripheral nervous system nasopharyngeal route did not differ statistically significant
(Wilson and Hayes, 1994). from the mean weight of animals in the control group. Mean
Results of phytochemical screening are summarized in amounts of food consumed by animals in the treated
Table 2. The extract was found to contain saponins, alkaloids groups and in the control group are shown in Table 3.
and flavonoids. Presence of these compounds could help to There is no statistically significant difference between any
account for both anti-viral activity and embryotoxicity. At groups.
present, a general relationship between structure and toxicity None of the animals exhibited any behavioural changes,
of the extract is difficult to establish. and no changes, or toxic signs or symptoms were observed. It
therefore seems likely that hydroalcoholic extracts of Punica
Table 1 granatum administered by the nasopharyngeal route are not
Embryotoxicity of Punica granatum L. extracts and amantadine (as pos- toxic at the doses studied.
itive control)
Table 4 shows that administration of Punica granatum ex-
Treatment Extract per Mortality Mortality tracts did not result in any significant changes in biochemi-
embryo (mg (D/T)a (%)
cal parameters apart from increases in serum creatinine and
per embryo)
glucose levels in animals in Group III. Statistically signifi-
Punica granatum 0.1 3/96 3.1
extract
cant differences in serum urea levels were observed between
0.2 18/95 18.9 groups but levels remained within the normal range. Ac-
0.3 58/96 61.1 cordingly, it may be concluded that extracts are not toxic, at
Amantadine 1.25 5/95 5.3 doses of 0.4 and 1.2 mg/kg, at least. Organ weights recorded
2.5 35/94 37.2 on necropsy in relation to animals in the control group and
5.0 60/95 63.2 in the groups treated with Punica granatum extracts are
Control group 0.0 5/94 5.3 summarized in Fig. 2. Liver, lung, brain, kidney and heart
(receiving water) weights in the animals treated with Punica granatum were
Each percentage mortality value represents the mean relating to three similar to those in the control animals.
experiments (involving 30 chicken embryos). Mortality was determined In histopathological studies of animals in the control
up to day 7. group and in the groups treated with Punica granatum ex-
a D/T: dead embryos per total number of embryos.
tracts no differences were noted in relation to some organs
(brain, heart, spleen, fibrous muscular tissue, nasal carti-
Table 2
Phytochemical screening of hydroalcoholic extracts of Punica granatum laginous tissue). There was slight congestion of the liver,
L. kidneys, small intestine, nasal mucous membrane and lar-
Assay Metabolite Results
ynx/trachea in 10–20% of the animals in all groups. The
mild disturbances seen were therefore not regarded as re-
Fehling Reducing carbohydrate + lated to treatment. Other abnormalities, such as superficial
Mucilages Mucilages +
Molish Glycosides +
micro-ulceration of the stomach, chronic inflammatory in-
Ferric chloride Phenols/tannins + filtration of the mucous membrane of the large intestine and
Flavonoids Flavonoids 0.63% foci of acute inflammation in the lungs, were observed in
Anthocyanidines Anthocyanidins + fewer than 10% of all treated animals. Although the animals
Dragendorff Alkaloids + in Group III had high serum creatinine levels there were no
Mayer Alkaloids −
signs of renal damage.
298 A. Vidal et al. / Journal of Ethnopharmacology 89 (2003) 295–300

360
Controls
340 Group I
Group II
320 Group III
Body weight (g)

300

280

260

240

220

200

1 2 3 4 5 6
Duration of treatments (weeks)
Fig. 1. Body weights of male Wistar rats in control group and in groups receiving hydroalcoholic extracts of Punica granatum L. by the nasopharyngeal
route for 5 weeks. Each point represents a mean relating to 10 animals.

Table 4
Biochemical parameters relating to blood of rats (control group and Groups I–III) after daily administration of 0.4, 1.2 and 7 mg/kg of hydroalcoholic
extracts of Punica granatum L. by the nasopharyngeal route for 35 days
Parameter Control group Group I Group II Group III

ASAT (U/l) 53.7 ± 3.2a,b 43.7 ± 11.7b 58.8 ± 18.2a,b 61.2 ± 14.0a
ALAT (U/l) 42 ± 7.2a 39.6 ± 11.9a 35.5 ± 6.6a 39.6 ± 5.7a
Calcium (mmol/l) 2.8 ± 0.3a 3.1 ± 0.4a 2.9 ± 0.4a 3 ± 0.3 a
Phosphorus (mmol/l) 2.4 ± 0.2a 2.2 ± 0.3a 2 ± 0.4a 2.3 ± 0.5a
Creatinine (␮mol/l) 88.3 ± 10a 88.4 ± 9a 109.8 ± 13a,b 138.2 ± 43b
Urea (mmol/l) 6.9 ± 0.7a 6.5 ± 0.9a,b 6.3 ± 0.8a,b 5.4 ± 1.0b
Uric acid (␮mol/l) 193.9 ± 11.8a 162.9 ± 66.4a 142.4 ± 23.6a 217.1 ± 171.8a
Cholesterol (mmol/l) 1.87 ± 0.42a 1.89 ± 0.30a 2.11 ± 0.43a 1.67 ± 0.46a
Triglycerides (mmol/l) 1.1 ± 0.5a 1.2 ± 0.4a 1.0 ± 0.3a 1.2 ± 0.4a
Glucose (mmol/l) 6.1 ± 1.3a 5.6 ± 0.9a 6.7 ± 0.1.2a 8.3 ± 2.5b
Total protein (g/l) 69.2 ± 9.2a 66.7 ± 5.6a 68.7 ± 6.7a 61.7 ± 7.7a
Values are means±S.D.s. The different superscript letters (for each parameter) indicate statistically significant differences in the Duncan test, with P < 0.05.

4. Discussion extract might be non-toxic. Results currently available sug-

Promising results have been recorded in recent years in
relation to searches for anti-viral activity in plant prepara-
tions (Vlietinck and Vanden Bergh, 1991; Beuscher et al.,
1994). The extract from the pomegranate (Punica granatum
L.), an ornamental plant with edible fruits, has been used
in Cuban traditional medicine to treat respiratory disease,
including that caused by the influenza virus (Roig, 1974;
Jimenez et al., 1979; Seoane, 1984).
Recently, work has been carried out in chicken embryos
in the hope of obtaining evidence that would support the tra-
ditional use of extracts of Punica granatum to treat influenza
(Peña, 1998; Peña and Martı́nez, 2001). In connection with
these studies, it became necessary to define what doses of
gest that Punica granatum extract is not embryotoxic at the
dose of 0.1 mg per embryo, which is effective as anti-viral
in the chick embryo assay (Peña, 1998). Even lower concen-
trations of the extract (0.01 mg per embryo) have also been
show to exhibit virucide activity in the chick embryo model
(Peña, 1998; Peña and Martı́nez, 2001). We therefore sug-
gest that the extract could form a basis for phytotherapeutic
preparations. Settheetham and Ishida (1995) have noted that
administration of an extract of pomegranate peel induced
apoptotic DNA fragmentation in human cell lines. This find-
ing could explain the embryotoxicity observed in our studies
at concentration over 0.2 mg of extract per embryo.
The acute toxicity of Punica granatum extracts was found
by us to be low, similar to levels reported by others. Desta
 A. Vidal et al. / Journal of Ethnopharmacology 89 (2003) 295–300
14
12
10
Organ weight
0
Fig. 2. Weights of organs from male Wistar rats in control group and groups receiving hydroalcoholic extracts of Punica granatum L. by the nasopharyngeal
route for 5 weeks. Each point represents a mean relating to 10 animals. S.D.s are also shown. There were no statistically significant differences between
any groups.
(1995) recorded a higher LD50 (2031 mg/kg) in mice than
we found. Desta used a hydroalcoholic extract of roots but an
extraction process that differed from ours and the intraperi-
toneal route of administration. Amorin (1995) also reported
a higher LD50 value (4 g/kg) in mice than we found. Amorin
administered aqueous extract of Punica granatum by mouth.
Although the fruits of Punica granatum are commonly
eaten, its roots and bark are toxic (Fuentes et al., 1985). Since
unpeeled fruits were used in our investigations, greater toxi-
city had been expected. Tripathi and Singh (2000) found no
statistically significant differences in LC50 values in snails
(Lymnaea acuminata) after using various methods of extrac-
tion and different parts of the Punica granatum plant. Tri-
pathi and Singh also suggested that the toxicity of the bark
may be related to its alkaloid content. Some parts of plants,
such as bark or fruits, can contain alkaloids, tannins or other
compounds that have toxic effects in various animal species
(Segura et al., 1990). According to Ferrara et al. (1989)
the toxic effects of some medicinal preparations of Punica
granatum may be explained by its pseudo-pelletierine con-
tent. In the studies reported here with hydroalcoholic extracts
prepared from whole fruits (including peel), alkaloids and
other compounds that may be toxic were found to be present.
Repeated consumption of Punica granatum extracts has,
under certain circumstances, been found to have serious ef-
fects in human populations. In an epidemiological study,
Ghadirian (1987) reported a direct relationship between can-
cer of the oesophagus and a foodstuff known as majum, one
component of which is pomegranate seeds. Others have re-
ported immunological disturbances following consumption
of Punica granatum fruits (Igea et al., 1991; Gaig et al.,
1992). This could be explained on the basis that some sub-
stances present cause lesions to form in the nasal cavity,
or sensitize the respiratory tract if repeatedly administered
(Blaike et al., 1997).
299
Controls
Group I
Group II
Group III
Kidneys Heart Splee
The extracts of the fruits of Punica granatum have com-
monly been used in Cuban folk medicine and the human
anti-influenza effective dose of the extract seems to be
around 0.38 mg/kg by nasopharyngeal route when repeat-
edly administered up to 5 days in the course of influenza
infection (Seoane, 1984). According to extensive literature
search, there have been no previous studies of possible toxic
effects produced by administration of repeated doses of the
extract by the nasopharyngeal route. Accordingly, there was
felt to be a need for toxicological studies of repeated doses
of Punica granatum fruit extracts.
Toxicity studies involving repeated doses administered by
the nasopharyngeal route at the dose range of 0.4–7 mg/kg,
revealed no toxic effects or behavioural alterations in rats.
Repeated administration did not alter or cause local irritation
of the nasal mucosa. This is particularly important because
of the great sensitivity of the area.
Biochemical studies revealed no disturbances following
administration of extracts except for higher creatinine val-
ues in Group III animals (administered with 7 mg/kg of the
extract) than in control-group animals. These findings could
have indicated nephrotoxicity but no kidney damage was
found in histological studies. The higher than normal glu-
cose values seen in Group III rats require more extensive
study.
5. Conclusions
The investigations described here were intended to reveal
possible toxic effects of Punica granatum extracts in view
of their anti-influenza activity. It was shown that toxic ef-
fects of Punica granatum fruit extract occurred at higher
doses than those effective in the models where its anti-viral
activity has been studied or those used in Cuban traditional
300 A. Vidal et al. / Journal of Ethnopharmacology 89 (2003) 295–300
medicine. Studies of this kind are always needed before a
phytotherapeutic agent can be generally introduced (Lapa
et al., 1999). Having regard to the high doses and lengthy
treatment times used in the dose repeated toxicity study, it
would seem that hydroalcoholic extracts of Punica grana-
tum fruit are innocuous when directly administered via the
nasal cavity.
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