Indian J Physiol Pharmacol 2000; 44 (1): 69-74

A STUDY OF HYPOGLYCAMIC EFFECTS OF AZADIRACHTAINDICA (NEEM) IN NORMAL AND ALLOXAN DIABETIC RABBITS

P. KHOSLA*, SANGEETA BHANWRA, J. SINGH, S. SETH**AND R. K. SRIVASTAVA

Departments of Pharmacology and Biochemistry,Pt. B.D. Sharma P.G.I.M.S.,Rohtak - 124 001

( Received on May 20, 1999)

Abstract : Hypoglycaemic effect was observed with Azadirachta indicawhen given as a leaf extract and seed oil, in normal as well asdiabetic rabbits. The effect, however, was more pronounced indiabetic animals in which administration for 4 weeks after alloxaninduced diabetes, significantly reduced blood glucose levels. Hypoglycaemiceffect was comparable to that of glibenclamide. Pretreatment withA indica leaf extract or seed oil administration, started 2 weeks prior toalloxan, partially prevented the rise in blood glucose levels as compared tocontrol diabetic animals. The data suggests that A. indica could be ofbenefit in diabetes mellitus in controlling the blood sugar or may also behelpful in preventing or delaying the onset of the disease.

Key words : Azadirachta indicaalloxan diabetes

INTRODUCTION

In recent years, herbs are beingeffectively tried In a variety ofpathophysiological states. Azadirachtaindica (Neem) is one of them. It is a largeevergreen tree which freely grows all overIndia. It has been used in traditionalmedicine for its several medicinalproperties. Isolated studies have reportedanti-inflammatory, immunostimulant andhypoglycaemic effects ofA. indica leaf extract(1-3). Seed oil has been shown to haveantifertility, hypoglycaemic and antibacterialproperties (4-6).

hypoglycaemic effectblood glucose

A. indica leaf extract has been reportedto lower blood glucose in normal rats bySen et al (3). However, they have reporteda marginal increase in blood glucoselevels in restraint stress group of rats.Murty et al (7) have shown a decrease inblood glucose when leaf extract was givenintravenously in a dose of 0.15 ml/kg indogs. On the other hand, Pillai andSanthakumari (5) have shown thatdecoction of tender leaves failed to produceany significant effect on blood glucose levelsin fasting rabbits at the three dose levelsused i. e. 1, 5, 10 mllkg (1 g leaves/mldecoction), while A. indica seed oil showed

*Corresponding Author

70 Khosla et al

significant reduction in blood glucose at alower dose 2.5 mllkg but percentagereduction in blood glucose was less with ahigher dose.

The aim of the present study was toinvestigate the effects of A. indica given intwo forms i.e. leaf extract and seed oil onblood glucose in normal and alloxan diabeticrabbits given before as well as afterestablishment of diabetes to evaluate its roleas a therapeutic agent and to see itsinfluence, if any, on prevention of thedisease.

METHODS

A indica leaf extract

Method of preparation: One kg of freshlycollected, shade dried, powdered leaves ofA. indica were ground in 4 litres of distilledwater and allowed to soak overnight. Thesuspension was centrifuged at 5000 rpm for20 min and filtered through a Whatman No.1 filter paper. The supernatent fluid wasallowed to evaporate in sterile, glass petridishes under tubelight. When completely dry,the extract was collected by scraping andstored. Stock solution of aqueous extract wasprepared by dissolving 500 mg of the extractin 5 ml of distilled water (8).

A. indica seed oil: A. indica seed oil (UnjhaAyurvedic Pharmacy, Gujrat) was procuredfrom the market from an Ayurvedic Drug'sStockist and oil of the same Company wasused throughout the study period to avoidany variation.

Dosage: A. indica leaf extract was given ina dose of 500 mglkg and seed oil 5 mllkg, by

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an intragastric tube (dosages were selectedfrom the literature and after doing the pilotstudy).

Animal experiments: Albino rabbits of bothsexes, weighing 1.5-2.0 kg were used.Animals were allowed standard diet and tapwater ad libitum. The animals were dividedinto groups A and B. Group A consisted of14 normal rabbits. They were further dividedinto 2 subgroups of 7 each (Ap A2). SubgroupAJ and A2 were given A. indica leafextract and seed oil respectively, daily for 4weeks.

Blood samples were collected initially(control) and after 5 hours, 1, 2 and 4 weeksof drug administration. Group B consisted of42 diabetic rabbits. They were divided intosix subgroups of 7 each (B1-B6). These weremade diabetic by injecting alloxan as singledose of 140 mg/kg body weight, intravenously(9). Rabbits having blood glucoselevels >200 mg/100 ml after 3 days of alloxanadministration were included in the study.

Subgroup BJ - Served as control.

Subgroup B2- Was given A. indica extractdaily for 2 weeks before and4 weeks after alloxanadministration.

Subgroup B3- Was given seed oil daily for2 weeks before and 4 weeksafter alloxan administration.

Subgroup B4- In diabetic rabbits, A. indicaleaf extract was given dailyfor 4 weeks.

Subgroup B5- In diabetic rabbits, seed oilwas given daily for 4 weeks.

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Subgroup B6- In diabetic rabbits, glibenc-lamide (40 ug/kg) was givendaily, orally, for 4 weeksas standard drug forcomparison.

Blood samples were collected forestimation of blood glucose initially, after 3days of alloxan administration and then after1, 2 and 4 weeks.

Collection of blood sample: Blood sampleswere collected from the marginal ear veinof rabbits, in a test tube containing heparin20 D/ml and sodium fluoride 2 mg/ml.

Blood sugar estimation: Plasma wasseparated by centrifuging the tubes at3000 rpm. Plasma glucose was estimated byglucose oxidase/peroxidase method asdescribed by Trinder (10).

Statistical analysis: Results are expressed asmean SE and Student's 't' test was used tocheck their significance. A value of P

72 Khosla et al Indian J Physiol Pharmacol 2000; 44(1)

TABLE II: Effect of A. indica leaf extract (500 mg/kg, po) and seed oil(5 ml/kg, po) on blood glucose levels in alloxan diabetic rabbits.

Initial

Blood glucose (mg / 100 ml)

After treatmentGroups After 3 daysof alloxan

Drugs

1 week 4 weeks2 weeks

Bl Control 88A31.14 281.716.31 307.145.51 246A24.26 172.851.50

B2 LE, 2 weeks prior & 90.140.79 190.003.15* 198.003A2* 170.001.40* 119A22.22*4 weeks after ALLO

B3 SO, 2 weeks prior & 88.570.64 195.503A2* 202.001.34* 165.003.30* 112A23.32*4 weeks after ALLO

B. LE for 4 weeks 90.710.62 285.002.63 265.573.77* 188.003.15* 149.143.13*after ALLO

B5 SO for 4 weeks 91.281.17 275.500.52 255.003.03* 191.143A2* 152.572A3*after ALLO

B6 Glibenclamide for 89.571.20 270AO1.12 251.854.01 * 188.850. 71 * 145A24.05*4 weeks after ALLO

Values are mean SE; n=7;*P

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DISCUSSION

The present study was conducted to studythe hypoglycaemic effects ofA. indica in normalas well as alloxan diabetic rabbits when givenin the form of leaf extract and seed oil.

This study shows that A. indica leafextract produces a marked decrease in bloodglucose in normal as well as alloxan diabeticrabbits. Our findings are in agreement withthose reported by Murty et al in dogs (7).The hypoglycaemic effect of A. indica leafextract increased gradually and was observedto be maximum at the end of the studyperiod i.e. 4 weeks. The effect was observedboth in normal and the hyperglycaemicrabbits (Table I, II; Fig. 1, 2). However, thepercentage fall in blood glucose levels innormal rabbits was 34% while Inhyperglycaemic rabbits, it was 47% after 4weeks of extract administration.

Similar results were observed withA. indica seed oil administration. Our resultsare supported by some earlier reports whichshow the hypoglycaemic effect of A. indicaseed oil In both the normal andhyperglycaemic animals (11-14). Thepercentage fall in blood glucose levels innormal rabbits was 32% while Inhyperglycaemic rabbits, it was 44% after 4weeks of seed oil administration in our study.

It is evident from this study that bothA. indica leaf extract and seed oil havea stronger hypoglycaemic effect Inhyperglycaemic as compared to normalrabbits. The data shows that both leaf extractand seed oil could have therapeuticusefulness in disease states where bloodglucose levels are high like diabetes mellitus.

Hypoglycaemic Effect of Azadirachta Indica 73

However, A. indica oil was found to haveslightly less hypoglycaemic effect ascompared to the extract. Moreover, the oilis difficult to take orally because it isunpalatable and it has a disagreeable odour.For this reason, the oil probably is not asuitable preparation for clinical use. It seemsthat the recommended formulation for useshould be A. indica leaf extract.

In order to see any preventive effect ofA. indica on the rise of blood glucose theleaf extract and the oil were given beforeproducing experimental diabetes. It wasinteresting to note a marked attenuatingeffect, since a much less significant rise inblood glucose was observed as compared tothe control diabetic animals. Ourobservations indicate a preventive role inthe rabbits pretreated with A. indica leafextract and seed oil before the induction ofdiabetes where they have shown reductionin the severity of onset of experimentaldiabetes and improvement in the diabeticstatus when followed by leaf extract and seedoil administration. This type of effect couldbe of use in the prevention of diabetic statessince the interventions made at the earliestsign of abnormal glucose metabolism mayprevent or delay the onset of the disease.

The hypoglycaemic effect of A. indicamay be due to increased release ofinsulin from beta cells of pancreas similarto that observed after sulphonylureasadministration. Sulphonylureas, however, donot decrease blood glucose in alloxan diabeticanimals (15). In contrast, parenteraladministration of insulin is well known toproduce hypoglycaemia in normal as well asalloxan diabetic rabbits (16). In our studywith A. indica, the fall in blood glucose was

74 Khosla et al

observed in normal as well as alloxan diabeticrabbits. This would indicate that mechanismof hypoglycaemia produced by A. indica maybe similar to that of insulin. As suggestedby Sharma et al (12), the hypoglycaemicaction of A. indica may partly be due toextrapancreatic sites of action i.e. byincreased peripheral glucose utilization orby direct metabolic effect on tissuesparticularly on liver.

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In conclusion, the data in our studysuggests that A. indica may have beneficialeffects in established diabetes mellitus, andit may also delay or prevent the onset ofthe disease. A. indica thus holds the hope ofa new generation of drugs. However, thereis need for further studies on experimentalanimals and human beings with the variousactive principles to establish its usefulnessand exact mode of action.

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