Journal of Ethnopharmacology 89 (2003) 295–300

Studies on the toxicity ofPunica granatum L. (Punicaceae)whole fruit extracts

Alexis Vidala, Adyary Fallareroa, Blanca R. Peñab, Maria E. Medinac,Bienvenido Grad, Felicia Riveraa, Yamilet Gutierreze, Pia M. Vuorelaf,∗

a Department of Biochemistry, Faculty of Biology, University of Havana, Havana, Cubab Department of Microbiology, Faculty of Biology, University of Havana, Havana, Cuba

c Clinical Biochemistry Laboratory, Havana Pediatric Hospital, Havana, Cubad Pathology Laboratory, National Institute of Gastroenterology, Havana, Cuba

e Pharmacognosy Laboratory, Pharmacy and Food Sciences Institute, University of Havana, Havana, Cubaf 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 ofPunica granatum L. (Punicaceae), used inCuban traditional medicine a.o. for the treatment of respiratory diseases. Previous findings on the anti-influenza activity ofPunica granatumextracts has given support to the ethnopharmacological application. In our study, in chick embryo model, it was found that doses of theextract 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 intraperitonealadministration, 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 intranasaladministration to Wistar rats produced no toxic effects in terms of food intake, weight gain, behavioural or biochemical parameters, or resultsof histopathological studies. We conclude that toxic effects ofPunica granatum fruit extract occurred at higher doses than those effective inthe 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

Many extracts from roots, stems and fruits have been usedin folk medicine for the treatment of viral diseases (Vlietinckand 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), thepomegranate, are commonly eaten. In traditional Cubanmedicine 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).

plant in the treatment of respiratory disease.Punica grana-tum would appear to have interesting anti-viral activity.Extracts have been shown to be effective against the herpesvirus (Zhang et al., 1995) and hydroalcoholic extracts ofwhole fruits have exhibited high activity against the in-fluenza virus (Peña, 1998; Caballero et al., 2001; Peña andMartınez, 2001).

However, the toxicity ofPunica granatum has not beenintensively studied.Amorin (1995) observed no toxic ef-fects in mice treated with aqueous extracts of pomegranatesimilar to those used in folk medicine.Desta (1995)re-ported that pomegranate was widely favoured in Ethiopiaas a taeniacidal drug on the basis of its relatively low tox-icity and high potency. It is important to bear in mind thatpomegranate fruits (excluding the peel) are not toxic butpomegranate roots and bark are (Fuentes et al., 1985). Thetoxic activity of a Punica granatum bark extract was re-lated to its alkaloid content according toTripathi and Singh(2000). Ferrara et al. (1989)have also reported that somegalenic 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 againstEntamoeba histolyticaandEntamoeba invadens.

Accordingly, we have conducted our research on em-bryotoxicity, acute toxicity and toxicity after repeated in-tranasal administration of hydroalcoholic extracts of wholePunica granatum fruits. Such extracts are traditionally usedin Cuba to treat upper respiratory disorders, for example,those caused by the influenza virus.

2. Methodology

2.1. Plant extract

FreshPunica granatum (pomegranate) fruits were col-lected from wild plants in the Havana province in July2001 and their identities confirmed by Alvarez et al. (40619HAJB). The voucher specimen is deposited at the HavanaBotanical Garden, Havana, Cuba. The fruits (including thepeel) were macerated for 15 days in a 50% (v/v) ethanolsolution in a ratio of 1:3 (w/v). The extract was filteredand ethanol was removed by vacuum evaporation on arotary evaporator at 55◦C. The aqueous extract was thenlyophilized and stored at−20◦C until used. The yield ofextract 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. Determinationswere carried out in accordance with detailed proceduresdeveloped by theNational Medicinal Plant Programme(1994). Flavonoids were determined according toOgbeideand Parvez (1991).

2.2. Embryotoxicity studies

The embryotoxicity study was performed as inNagai et al.(1992). Hundred microlitres ofPunica granatum extracts atconcentrations of 1, 2 and 3 mg/ml or of amantadine at con-centrations of 12.5, 25 and 50 mg/ml were administered tochicken embryos (from CENPALAB, pathogen-free, 9–11days 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 bythe same route. All embryos were observed ovoscopicallyeach day for 1 week, to allow percentages surviving at theend of the period to be calculated. The dose at which mor-tality was similar to or lower than that in the control groupwas 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. Theanimals were randomly distributed into groups of 10 ani-mals per dose per cage, with free access to water and pelletfeed (ALYco, Havana). Extracts ofPunica granatum wereadministered 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 othertoxic signs, and for symptoms including time to seizure anddeath. Observation continued for 15 days to confirm that thenumber of animals per dose that remained alive did not al-ter. Values for LD50 and upper and lower confidence limitswere calculated by means of the method ofLitchfield andWilcoxon (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 cagesand had free access to water and food (ALYco, Havana).Four groups of 10 animals were studied for 35 days (5weeks). Five microlitres of lyophilized extract were admin-istered by the nasopharyngeal route through each nasal cav-ity, using a microsyringe (Hamilton Co.). Each member ofGroups I–III received a dose of 0.4, 1.2 or 7 mg of extractper 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 wasmeasured every 7 days.

At the end of the experiment the animals were decap-itated and blood samples were taken for determinationof serum glucose, uric acid, urea, creatinine, cholesterol,triglyceride, total protein, calcium, phosphorus, ASAT andALAT levels. All determinations were carried out using di-agnostic kits (Boehringer Mannheim). A macroscopic studywas performed, during which organs were weighed. Sam-ples of livers, lungs, kidneys, brains, hearts, stomachs, thesmall and large intestines, larynxes/tracheas, spleens, thenasal mucous membrane and fibrocartilaginous nasal tissuewere taken for histological study using the conventionalhaematoxylin–eosin technique.

All the animal studies reported in this work were car-ried out in accordance with the Cuban regulations on theprotection of animals (Código Práctico para el Uso de losAnimales de Laboratorio, Centro Nacional para la Pro-ducción de Animales de Laboratorio, CENPALAB, 1992),and the Declaration of Helsinki. All experimental protocolswere revised by the Animal Care and Use Committee of theFaculty of Biology, University of Havana, and conductedhumanely.

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 analysedusing 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(∗P < 0.05, ∗∗P < 0.01).

3. Results

Results relating to embryotoxicity are shown inTable 1.Mortality in the control group was 5.3%, which was relatedto manipulation and other experimental factors. From resultsin Table 1, it may be assumed that doses ofPunica granatumin a hydroalcoholic extract of less than 0.1 mg per embryoare not toxic.

The LD50 of Punica granatum extracts administered in-traperitoneally to mice was 731.1 mg/kg. Confidence limitsare 565–945 mg/kg. No differences in acute toxicity in re-lation to sex were recorded. The most evident toxic symp-tom in mice was piloerection. The latter mainly providesevidence for disturbance of the peripheral nervous system(Wilson and Hayes, 1994).

Results of phytochemical screening are summarized inTable 2. The extract was found to contain saponins, alkaloidsand flavonoids. Presence of these compounds could help toaccount for both anti-viral activity and embryotoxicity. Atpresent, a general relationship between structure and toxicityof the extract is difficult to establish.

Table 1Embryotoxicity of Punica granatum L. extracts and amantadine (as pos-itive control)

Treatment Extract perembryo (mgper embryo)

Mortality(D/T)a

Mortality(%)

Punica granatumextract

0.1 3/96 3.1

0.2 18/95 18.90.3 58/96 61.1

Amantadine 1.25 5/95 5.32.5 35/94 37.25.0 60/95 63.2

Control group(receiving water)

0.0 5/94 5.3

Each percentage mortality value represents the mean relating to threeexperiments (involving 30 chicken embryos). Mortality was determinedup to day 7.

a D/T: dead embryos per total number of embryos.

Table 2Phytochemical screening of hydroalcoholic extracts ofPunica granatumL.

Assay Metabolite Results

Fehling Reducing carbohydrate +Mucilages Mucilages +Molish Glycosides +Ferric chloride Phenols/tannins +Flavonoids Flavonoids 0.63%Anthocyanidines Anthocyanidins +Dragendorff Alkaloids +Mayer Alkaloids −

Table 3Food intakes (gram per animal per day) of Wistar rats (control group andGroups I–III) treated with hydroalcoholic extracts ofPunica granatumL., after 0, 14, 28 and 35 days of treatment

Group Days of treatment

0 14 28 35

Control 13.3± 1.5a 19.7 ± 1.1b 26.4 ± 1.4c 30.0 ± 2.3d

I 12.5 ± 1.2a 18.9 ± 1.0b 25.5 ± 1.2c 30.5 ± 1.9d

II 11.8 ± 2.0a 16.3 ± 0.9b 27.5 ± 1.5c 30.3 ± 3.1d

III 12.0 ± 3.0a 18.9 ± 0.8b 28.7 ± 1.6c 23.4 ± 1.5d

Values are expressed as means± S.D.s. The various superscript lettersindicate statistically significant differences in the Duncan test, withP <

0.05.

As shown inFig. 1, mean weights of animals that re-ceived repeated doses ofPunica granatum extract by thenasopharyngeal route did not differ statistically significantfrom the mean weight of animals in the control group. Meanamounts of food consumed by animals in the treatedgroups and in the control group are shown inTable 3.There is no statistically significant difference between anygroups.

None of the animals exhibited any behavioural changes,and no changes, or toxic signs or symptoms were observed. Ittherefore seems likely that hydroalcoholic extracts ofPunicagranatum administered by the nasopharyngeal route are nottoxic at the doses studied.

Table 4shows that administration ofPunica granatum ex-tracts did not result in any significant changes in biochemi-cal parameters apart from increases in serum creatinine andglucose levels in animals in Group III. Statistically signifi-cant differences in serum urea levels were observed betweengroups but levels remained within the normal range. Ac-cordingly, it may be concluded that extracts are not toxic, atdoses of 0.4 and 1.2 mg/kg, at least. Organ weights recordedon necropsy in relation to animals in the control group andin the groups treated withPunica granatum extracts aresummarized inFig. 2. Liver, lung, brain, kidney and heartweights in the animals treated withPunica granatum weresimilar to those in the control animals.

In histopathological studies of animals in the controlgroup and in the groups treated withPunica granatum ex-tracts no differences were noted in relation to some organs(brain, heart, spleen, fibrous muscular tissue, nasal carti-laginous tissue). There was slight congestion of the liver,kidneys, small intestine, nasal mucous membrane and lar-ynx/trachea in 10–20% of the animals in all groups. Themild disturbances seen were therefore not regarded as re-lated to treatment. Other abnormalities, such as superficialmicro-ulceration of the stomach, chronic inflammatory in-filtration of the mucous membrane of the large intestine andfoci of acute inflammation in the lungs, were observed infewer than 10% of all treated animals. Although the animalsin Group III had high serum creatinine levels there were nosigns of renal damage.

298 A. Vidal et al. / Journal of Ethnopharmacology 89 (2003) 295–300

1 2 3 4 5 6

200

220

240

260

280

300

320

340

360

ControlsGroup IGroup IIGroup III

Duration of treatments (weeks)

Bod

y w

eigh

t (g)

Fig. 1. Body weights of male Wistar rats in control group and in groups receiving hydroalcoholic extracts ofPunica granatum L. by the nasopharyngealroute for 5 weeks. Each point represents a mean relating to 10 animals.

Table 4Biochemical 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 hydroalcoholicextracts ofPunica 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, withP < 0.05.

4. Discussion

Promising results have been recorded in recent years inrelation 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 granatumL.), an ornamental plant with edible fruits, has been usedin 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 embryosin the hope of obtaining evidence that would support the tra-ditional use of extracts ofPunica granatum to treat influenza(Peña, 1998; Peña and Martınez, 2001). In connection withthese studies, it became necessary to define what doses of

extract might be non-toxic. Results currently available sug-gest thatPunica granatum extract is not embryotoxic at thedose of 0.1 mg per embryo, which is effective as anti-viralin the chick embryo assay (Peña, 1998). Even lower concen-trations of the extract (0.01 mg per embryo) have also beenshow to exhibit virucide activity in the chick embryo model(Peña, 1998; Peña and Martınez, 2001). We therefore sug-gest that the extract could form a basis for phytotherapeuticpreparations.Settheetham and Ishida (1995)have noted thatadministration of an extract of pomegranate peel inducedapoptotic DNA fragmentation in human cell lines. This find-ing could explain the embryotoxicity observed in our studiesat concentration over 0.2 mg of extract per embryo.

The acute toxicity ofPunica granatum extracts was foundby us to be low, similar to levels reported by others.Desta

A. Vidal et al. / Journal of Ethnopharmacology 89 (2003) 295–300 299

Kidneys Heart Splee0

2

4

6

8

10

12

14

Org

an w

eig

ht

Controls

Group I

Group II

Group III

Fig. 2. Weights of organs from male Wistar rats in control group and groups receiving hydroalcoholic extracts ofPunica granatum L. by the nasopharyngealroute for 5 weeks. Each point represents a mean relating to 10 animals. S.D.s are also shown. There were no statistically significant differences betweenany groups.

(1995) recorded a higher LD50 (2031 mg/kg) in mice thanwe found. Desta used a hydroalcoholic extract of roots but anextraction process that differed from ours and the intraperi-toneal route of administration.Amorin (1995)also reporteda higher LD50 value (4 g/kg) in mice than we found. Amorinadministered aqueous extract ofPunica granatum by mouth.

Although the fruits ofPunica granatum are commonlyeaten, its roots and bark are toxic (Fuentes et al., 1985). Sinceunpeeled fruits were used in our investigations, greater toxi-city had been expected.Tripathi and Singh (2000)found nostatistically significant differences in LC50 values in snails(Lymnaea acuminata) after using various methods of extrac-tion and different parts of thePunica granatum plant. Tri-pathi and Singh also suggested that the toxicity of the barkmay be related to its alkaloid content. Some parts of plants,such as bark or fruits, can contain alkaloids, tannins or othercompounds 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 ofPunicagranatum may be explained by its pseudo-pelletierine con-tent. In the studies reported here with hydroalcoholic extractsprepared from whole fruits (including peel), alkaloids andother compounds that may be toxic were found to be present.

Repeated consumption ofPunica 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 asmajum, onecomponent of which is pomegranate seeds. Others have re-ported immunological disturbances following consumptionof 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).

The extracts of the fruits ofPunica granatum have com-monly been used in Cuban folk medicine and the humananti-influenza effective dose of the extract seems to bearound 0.38 mg/kg by nasopharyngeal route when repeat-edly administered up to 5 days in the course of influenzainfection (Seoane, 1984). According to extensive literaturesearch, there have been no previous studies of possible toxiceffects produced by administration of repeated doses of theextract by the nasopharyngeal route. Accordingly, there wasfelt to be a need for toxicological studies of repeated dosesof Punica granatum fruit extracts.

Toxicity studies involving repeated doses administered bythe 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 irritationof the nasal mucosa. This is particularly important becauseof the great sensitivity of the area.

Biochemical studies revealed no disturbances followingadministration of extracts except for higher creatinine val-ues in Group III animals (administered with 7 mg/kg of theextract) than in control-group animals. These findings couldhave indicated nephrotoxicity but no kidney damage wasfound in histological studies. The higher than normal glu-cose values seen in Group III rats require more extensivestudy.

5. Conclusions

The investigations described here were intended to revealpossible toxic effects ofPunica granatum extracts in viewof their anti-influenza activity. It was shown that toxic ef-fects of Punica granatum fruit extract occurred at higherdoses than those effective in the models where its anti-viralactivity 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 aphytotherapeutic agent can be generally introduced (Lapaet al., 1999). Having regard to the high doses and lengthytreatment times used in the dose repeated toxicity study, itwould seem that hydroalcoholic extracts ofPunica grana-tum fruit are innocuous when directly administered via thenasal cavity.

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