Journal of Ethnopharmacology 70 (2000) 309–314

Short communication

Effect of Punica granatum Linn. (flowers) on blood glucoselevel in normal and alloxan-induced diabetic rats

M.A. Jafri a,*, M. Aslam a, Kalim Javed a, Surender Singh b

a Department of Ilmul Ad6ia, Faculty of Medicine (Unani ), Jamia Hamdard, New Delhi-110 062, Indiab Department of Pharmacology, College of Pharmacy, Pushp Vihar, New Delhi, India

Received 28 April 1999; received in revised form 30 June 1999; accepted 27 September 1999

Abstract

‘Gulnar farsi’, male abortive flowers of Punica granatum L., are used for the treatment of diabetes mellitus in Unanimedicine. Oral administration of its aqueous-ethanolic (50%, v/v) extract led to significant blood glucose loweringeffect in normal, glucose-fed hyperglycaemic and alloxan-induced diabetic rats. This effect of the extract wasmaximum at 400 mg/kg, b.w. © 2000 Elsevier Science Ireland Ltd. All rights reserved.

Keywords: Alloxan; Antihyperglycaemic; Diabetes; ‘Gulnar’; Hypoglycaemic; Punica granatum L.

www.elsevier.com/locate/jethpharm

1. Introduction

Punica granatum Linn. (Punicaceae) is a shrubor small tree and considered to be a native of Iranand Afghanistan. It is also found growing wild inthe warm valleys and outer hills of the Himalayas,and is cultivated throughout India (Satyavati etal., 1978). ‘Gulnar’ (flower of P. granatum L.) hasbeen known for a long time in Unani literature asan astringent, haemostatic, and as a remedy fordiabetes (Jurjani, 1878; Majoosi, 1889). The rootbark as well as the stem bark of the plant isastringent and also used as anthelmintic specifi-cally against tapeworms. The rind is valued as an

astringent in diarrhoea and dysentery. The juiceof the leaves and young fruits and the decoctionof the bark are used in dysentery. The powderedflower buds are useful in bronchitis. The seeds areconsidered to be stomachic and the pulp cardiacand stomachic. The green leaves are made into apaste and applied in conjunctivitis (Anonymous,1969; Satyavati et al., 1978).

The biological activities, viz. antibacterial (Cho-pra et al., 1960; Trivedi and Kazmi, 1979), anti-fungal (Janardhanan et al., 1963; Charya et al.,1979), anthelmintic (Prakash et al., 1980; Singhal,1983) and antifertility (Dhawan and Saxena, 1958;Gujral et al., 1960), of the various extracts ofdifferent parts of this plant have also been re-ported. The extracts of root of P. granatum (Car-* Corresponding author.

0378-8741/00/$ - see front matter © 2000 Elsevier Science Ireland Ltd. All rights reserved.

PII: S 0378 -8741 (99 )00170 -1

M.A. Jafri et al. / Journal of Ethnopharmacology 70 (2000) 309–314310

raz et al., 1978) and rind of this plant (Nogueiraand Pereira, 1984, 1986a,b; Zafar and Singh,1990) have been reported to exert some sugarlowering action in animals.

Since only the flowering part of the plant hasbeen recommended in Unani literature (Jurjani1878; Majoosi, 1889) as a remedy for the treat-ment of diabetes, it was, therefore, consideredworthwhile to investigate the effect of the flowersof P. granatum on blood glucose levels of glucose-fed hyperglycaemic, alloxan-induced diabetic andnormal rats and to compare it with tolbutamideas a reference standard.

2. Materials and methods

2.1. Plant material

The flowers of P. granatum were purchasedunder the Unani name ‘Gulnar farsi’ from KhariBaoli, Delhi, and its identity was confirmed by thedepartment of Botany, Faculty of Science, JamiaHamdard, New Delhi. Its voucher specimen (MA98 01) was deposited in the pharmacognosy sec-tion of the Department of Ilmul Advia, Faculty ofMedicine (Unani) Jamia Hamdard, New Delhi.

2.2. Preparation of plant extract

The flowers of P. granatum (200 g) were ex-tracted by refluxing with aqueous-ethanol (50%v/v, 1000 ml) on boiling water bath for 6 h. Theresult was filtered and residue was re-extracted inthe same manner four times. All the filtrates werecombined together and recovery of the solventunder reduced pressure yielded semisolid mass(80.20 g).

2.3. Experimental animals

Adult albino rats (Wistar strain) of either sex(120–220 g) were obtained from the AnimalHouse, Jamia Hamdard, New Delhi. Animals,described as fasted, were deprived of food for atleast 16 h but allowed free access to water.

2.4. Beha6ioural effect and toxicity

Albino mice (Swiss strain) were divided intofive groups of ten animals each. The test drugextract, suspended in vehicle (Tween 80 in dis-tilled water, 10% v/v) was administered in thedose of 200, 400, 600, 800 and 1000 mg/kg i.p. ina volume of 10 ml/kg to the animals of I, II, III,IV and V groups, respectively. Then the mice wereobserved continuously for 1 h, intermittently for 6h and at the end of 24 h for any gross behaviouralchanges and deaths.

2.5. Effect of the test drug on oral glucosetolerance

Fasted rats were divided into five groups of sixanimals each. Group I, serving as control, re-ceived only vehicle (Tween 80 in distilled water,10% v/v) orally in a volume of 10 ml/kg andgroup II received tolbutamide (500 mg/kg, p.o.)suspended in vehicle (10 ml/kg). A dose of 300,400 and 500 mg/kg of the extract of the test drugsuspended in vehicle was administered orally in avolume of 10 ml/kg to the animals of III, IV andV groups, respectively. All the animals were givenglucose (2 g/kg, p.o.) 30 min after dosing. Bloodsamples were collected from the retro-orbitalplexus just prior to and at 30 and 90 min after theglucose loading and blood glucose levels weremeasured.

2.6. Effect of the test drug on blood glucosele6els in normal fasted rats

Fasted rats were divided into five groups of sixrats each. Group I received only vehicle (Tween80 in distilled water, 10% v/v) orally in a volumeof 10 ml/kg and served as control. The extract ofthe test drug, suspended in vehicle, was adminis-tered at the doses of 300, 400 and 500 mg/kgorally in a volume of 10 ml/kg to the animals ofgroup III, IV and V, respectively. Group II re-ceived tolbutamide (500 mg/kg, p.o.) suspended invehicle (10 ml/kg). Blood samples were collectedfrom the retro-orbital plexus just prior to and at1, 2 and 3 h after dosing.

M.A. Jafri et al. / Journal of Ethnopharmacology 70 (2000) 309–314 311

2.7. Effect of the test drug on alloxan induceddiabetes in rats

Rats were made diabetic by a single intraperi-toneal injection of alloxan monohydrate 5% (w/v)in normal saline at a dose of 120 mg/kg, b.w.(Khosla et al., 1995). Then 5 days later bloodsamples were collected and glucose levels weredetermined to confirm the development of dia-betes. Of the animals receiving alloxan, �82%were found to be diabetic (fasting blood glucose259–292 mg/dl). The diabetic rats were dividedinto five groups of six animals each. Group Ireceived only vehicle (Tween 80 in distilled water,10 v/v) orally in a volume of 10 ml/kg. The testdrug extract, suspended in vehicle, was given inthe doses of 300, 400 and 500 mg/kg orally in avolume of 10 ml/kg to groups III, IV and V,respectively. Group II received tolbutamide (500mg/kg, p.o.) suspended in vehicle (10 ml/kg).Blood samples were collected just prior to and at1 and 2 h after dosing.

2.8. Estimation of blood glucose le6el

In each case 200 ml of blood sample was col-lected and estimated for glucose by o-toluidinemethod (Dubowski, 1962).

2.9. Data and statistical analysis

Data were expressed as mean9S.E.M., n=6.Statistical comparisons within the same group

were performed with Student’s t-test for pairedobservations. One-way analysis of variance(ANOVA) followed by the least significant differ-ence post-test was used to identify significantlydifferent groups. PB0.05 indicates significant dif-ferences between group means.

3. Results

3.1. Beha6ioural effect and toxicity

The extract of the test drug (P. granatum) wasfound to be safe for further biological studies asno lethality was observed at 1000 mg/kg, i.p. inmice.

3.2. Effect of the test drug on oral glucosetolerance

The effects of various doses of the extract of P.granatum on glucose tolerance are shown in Table1. By 30 min after starting the glucose tolerancetest, blood glucose concentration doubled from itsinitial level of control and then glycaemia startedto decrease gradually, without reaching its initialvalue till the end of the studies, i.e. at the 90thmin. All the doses of the test drug extract werefound to be effective in depressing the peak valueof blood sugar. Administration of tolbutamide(500 mg/kg) to glucose-fed rats induced time de-pendent hypoglycaemic effect.

Table 1Effect of oral administration of different doses of P. granatum extract and tolbutamide on oral glucose tolerance in normal ratsa

Blood glucose level (mg/dl)Sampling time(min)

P. granatum, 500P. granatum, 400P. granatum,Tolbutamide,Vehicle (10% v/v Tween 80, 10500 mg/kg 300 mg/kgml/kg) mg/kg mg/kg

0 67.3691.69 63.5091.95 59.3391.60 63.6691.72 64.0091.06110.0093.89b56.8390.86b,c133.0091.6530 99.3393.54b100.6693.85b

100.3391.65 41.3391.65b,c90 90.1693.32b 81.0094.28b 80.1692.94b

a Values are expressed as mean9S.E.M (n=6).b Statistically significant difference to the vehicle.c Statistically significant difference to the corresponding zero time value.

M.A. Jafri et al. / Journal of Ethnopharmacology 70 (2000) 309–314312

Table 2Effect of oral administration of different doses of P. granatum extract and tolbutamide on the mean blood glucose level in normalfasted ratsa

Sampling time Blood glucose level (mg/dl)(h)

Tolbutamide, P. granatum, P. granatum, 500Vehicle (10% v/v Tween 80, 10 P. granatum, 400500 mg/kg 300 mg/kgml/kg) mg/kgmg/kg

0 66.3392.4557.3391.45 63.5091.96 62.1691.66 66.8391.5460.6890.33b,c 69.3390.7166.1691.16 70.8391.491 72.5093.20

67.5090.612 46.0091.24b,c 57.5090.76b,c 53.1691.19b,c 52.0091.39b,c

3 40.1691.11b,c64.3390.84 68.0093.60 66.5093.60 69.0094.70

a Values are expressed as mean9S.E.M (n=6).b Statistically significant difference to the vehicle.c Statistically significant difference to the corresponding zero time value.

Table 3Effect of oral administration of different doses of P. granatum extract and tolbutamide on the mean blood glucose level in alloxandiabetic ratsa

Blood glucose level (mg/dl)Sampling time(h)

P. granatum, 400Tolbutamide,Vehicle (10% v/v Tween 80, 10 P. granatum, P. granatum, 500mg/kgml/kg) mg/kg500 mg/kg 300 mg/kg

267.1696.60 271.6694.07282.8394.12 280.4097.800 274.8392.82277.4096.501 255.0094.95 193.6693.96b,c 193.1692.15b,c 189.2093.42b,c

240.0095.65 205.5092.92b,c 200.0093.04b,c 198.8394.15b,c2 271.8392.15

a Values are expressed as mean9S.E.M. (n=6).b Statistically significant difference to the vehicle.c Statistically significant difference to the corresponding zero time value.

3.3. Effect of the test drug on blood glucosele6els in normal fasted rats

Table 2 shows the effects of various doses ofthe extract of P. granatum on blood glucose levelin normal fasted rats. All doses of the test drugextract showed maximum hypoglycaemic effect at2 h. Tolbutamide treatment exerted a markedtime dependent hypoglycaemic effect.

3.4. Effect of the test drug on alloxan induceddiabetes in rats

Oral administration of all the doses of the testdrug extract led to significant lowering effect inalloxan-induced diabetic rats. The fall was seen at

1 h and remained up to 2 h after administrationof the test drug extract whereas the fall in glucoselevel in the case of tolbutamide administrationwas marginal because alloxan causes diabetes bydestruction of the pancreatic cells and tolbu-tamide requires more than 30% functional pan-creas for effectiveness (Table 3).

4. Discussion

This study reports for the first time, the antihy-perglycaemic effect of the extract of P. granatum(flowers), a Unani drug frequently used by Unaniphysicians for the treatment of diabetes.

M.A. Jafri et al. / Journal of Ethnopharmacology 70 (2000) 309–314 313

A clinically used tolbutamide (a sulphonylureadrug) is known to lower the blood glucose level bystimulating b-cells to release insulin. Since alloxaninduces diabetes by destroying b-cells and impair-ing renal function, in the present study tolbu-tamide exhibited mild hypoglycaemic activity inthe alloxan diabetic rats. However, in these ratsthe extract of the flowers of P. granatum showedmarked hypoglycaemic effect. This may not bedue to potentiation of insulin release from pancre-atic cells and thus the drug may also be effectivein non insulin-dependent diabetes.

Since the blood glucose lowering effect of theextract of the flowers of P. granatum was ob-served in alloxan diabetic rats as well as in fastednormal rats, this effect could, possibly be due toincreased peripheral glucose utilization. Inhibitionof the proximal tubular reabsorption mechanismfor glucose in the kidney, if any, can also con-tribute towards blood lowering effect (Sharma etal., 1983).

The test drug extract also improved oral glu-cose tolerance in normal rats. At the presentjuncture it is not possible to pinpoint the mecha-nism of anti-hyperglycaemic action of the extractof P. granatum. However, based on an earlierreport some suggestions can be made for its possi-ble mechanism. It is reported that an infusion ofthe epicarps of P. granatum inhibited the intesti-nal absorption of glucose in rats (Nogueira andPereira, 1986b). Thus a possibility exists that re-tardation of intestinal glucose absorption mayalso be partly responsible for inhibition of hyper-glycaemia in glucose-fed rats.

The rats treated with 400 and 500 mg/kg of thetest drug extract showed almost the same bloodglucose lowering effect. The higher dose, i.e. 500mg/kg, did not further lower blood glucose levelsin normal, glucose-fed hyperglycaemic and al-loxan induced diabetic rats as compared to therats treated with 400 mg/kg. At present we mayconclude that the extract of P. granatum, in total-ity, was effective in reducing the blood glucoselevel under our experiment conditions.

Studies are underway to further elucidate themechanism of hypoglycaemic effect of the extractof the flowers of P. granatum.

Acknowledgements

The authors are thankful to the Department ofI.S.M. and H. (Ministry of Health and FamilyWelfare) for providing financial support to ourdepartment (Ilmul Advia) and a fellowship to oneof the authors (M.A.).

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