COMPARATIVE STUDY OF AZADIRACHTA INDICA LEAF POWDER AND ITS DIFFERENT EXTRACTS ON GLYCEMIC, INSULINEMIC AND LIPIDEMIC STATUS OF TYPE 2 DIABETIC MODEL RATS

COMPARATIVE STUDY OF AZADIRACHTA INDICA LEAF POWDER AND ITS DIFFERENT EXTRACTS ON GLYCEMIC, INSULINEMIC AND LIPIDEMIC STATUS OF TYPE 2 DIABETIC MODEL RATS

1. A. Morshed ^1,*^2, N. Fayzunnessa ¹, M. A. Haque ^{1, 3}, A. Uddin ², A. Bhoumik ⁴, L. Ali ⁴ and B. Rokeya ⁴

Department of Biotechnology & Genetic Engineering, Faculty of Applied Science & Technology, Islamic University ¹, Kushtia, Bangladesh

Department of Pharmacy, North South University ², Bashundhara, Dhaka, Bangladesh

Department of Physiology & Cell Biology, Graduate School of Medicine, Kobe University ³, 7-5-1, Kusunoki-Cho, Chuo-Ku, Kobe, Japan

Biomedical Research Group, Bangladesh Institute of Research and Rehabilitation in Diabetes, Endocrine and Metabolic Disorders (BIRDEM) ⁴, Dhaka, Bangladesh

ABSTRACT

The present study was carried out to investigate the effect of oral feeding of A. indica leaf (powder, aqueous and 80% ethanol extract) in STZ induced type 2 diabetic rats. Diabetes was induced by single intraperitoneal injection of STZ to 48hrs old pups and experiments were carried out three months later. For chronic experiment type 2 rats were divided into 5 groups: 1) Water control, 2) Glibenclamide (5gms/kg b.w.) treated, 3) A. indica powder [mixed with rat feed (1.25 g/kg b.w.)], 4) Aqueous extract and 5) 80% ethanol extract treated. The test drugs were used at a dose of 1.25 g/kg b.w. for 28 consecutive days. Body weight was checked every week. Blood was collected by cutting the tail tip at the beginning and on the days 7, 14, 21 and by decapitation on the 28^th day. Results of the experiment showed that A. indica leaves exerted a gradual reduction in serum glucose level of type 2 diabetic rats. A significant reduction in serum glucose level was noticed on the 21 day (p = 0.001 for powder and p = 0.001 for ethanol extract) as well as on 28 day (p = 0.009 for powder, p =0.02 for aqueous extract and p = 0.001 for 80% ethanol extract). In addition to hyperglycemic effect A. indica leaf also showed hypolipidemic effect. Serum total Cholesterol and TG level decreased significantly by different extracts and powder of A. indica. Glibenclamide as expected, ameliorated diabetic condition significantly (p<0.05). Body weight of the rats tends to increase throughout the study period. No significant change was observed in serum insulin level. The obtained results suggest that A. indica leaf improves the glycemic and lipidemic status of type 2 diabetic rats. The efficacy of A.indica leaf (powder, aqueous extract and 80% ethanol extract) as hypoglycemic and anti-lipidemic agents was found ethanol extract > aqueous extract > powder.

INTRODUCTION: Hyperglycemia and hyperlipidemia are two important characters of diabetes mellitus, an endocrine disorder based disease. In modern medicine, no satisfactory effective therapy is still available to cure diabetes mellitus ¹. Though pharmaceutical drugs like sulfonylureas and biguanides are used for the treatment of diabetes but these are either too expensive or have undesirable side effects or contraindications ^{2, 3}. In recent years, there has been renewed interest in plant medicine ^{4, 5, 6} for the treatment against different diseases as herbal drugs are generally out of toxic effect ^{7, 8} reported from research work conducted on experimental model animal. Although in human, whether there is any toxic effect are not investigated. Isolated studies screened various plants having “folk medicine reputation” by biochemical test for this of antidiabetogenic effect ⁹.

Azadirachta indica (family- Meliaceae [Melioideae]), a medium-sized tree, is found throughout the South Asian region. It is one of the most versatile medicinal plants having a wide spectrum of biological activity. Every part of the tree has been used as traditional medicine for household remedy against various human ailments, from antiquity.

Traditional healers claim that the leaves of the plant possess antidiabetic properties. Scientific reports also support the hypoglycemic activity of this plant ^{10, 11, 12, 13}. Although there exists a substantial number of reports claiming the hypo/anti-hyperglycemic effect of different parts (leaves, flowers, seeds, fruits, roots and bark) of this plant in normal as well as STZ (streptozotocin) induced diabetic animals, there was no published report regarding the comparative study of A. indica leaf crude powder and different extracts (aqueous and 80% ethanol) on glucose homeostasis. So the aim of this present study is to find out the comparative study of A. indica on glycemic, insulinemic and lipidemic status of type 2 diabetic model rats.

MATERIALS AND METHODS:

Plant materials and preparation of test sample: The fresh green leaves of Azadirachta indica Linn. were collected from Kushtia, Bangladesh. The collected fresh leaves of Azadirachta indica were weighed (3.630 kg), washed and air dried hygienically. The dried leaves were homogenized to powder with the help of grinder and 1.910 kg powder was obtained. Then the powder was sieved and 1.635 kg of fine powder was obtained which was stored in the refrigerator at -20^oC.

500g each of dried leaf powder were dissolved in 4.5L of boiled water and 1.5L of 80% ethanol. These suspensions were filtered with thin and clean cloth and then filtered by filter paper. The suspensions were then dried by BUCHI Rota vapor R-114, connected with BUCHI water bath B-480 at 40^oC. Finally the semi-dried extracts were dried using a freeze-drier (HETOSICC, Heto Lab Equipment Denmark) at -55^oC temperature and finally 85.60g and 65.30g of water and ethanol extracts were obtained respectively.

Experimental Animals: The study was conducted on adult Long-Evans (both sexes) rats (weighing 150-240g) bred at the BIRDEM animal house maintained at a constant room temperature of 22±5^oC, 40-70% humidity conditions and the natural day-night cycle with an ad libitum access to food except the day of experimental procedure when animals were used after 12hrs fasting. The rats had no access to food during the whole period of blood sampling. The influence of circadian rhythms was avoided by starting all experiments at 8.30 a.m.

Induction of Type 2 Diabetes to the Rats: Diabetes stimulating Type 2 was induced by a single intra-peritoneal injection of streptozotocin (90mg/kg body weight, dissolved in 0.1 citrate buffer, pH 4.5) to 48hr old pulps of Long-Evans rats as described by Bonner-Weir et al., 1981 ¹⁴. Experiments were carried out 3 months latter to STZ injection and those rats having blood glucose level 8-12 mmol/l at fasting condition, were considered to carry out the experiments.

Experimental design: The experimental diabetic rats were divided into five groups as follows:

• Water control group: Rats of this group received deionized water at a dose of 10ml/kg bw.
• Positive control group: This group of rats received Glibenclamide at a dose of 5 mg /kg
• Test group 1(Crude powder): Azadirachta indica crude powder was given with normal food at a dose of 1.25gm/kg bw. The normal food was contained wheat (42%), wheat bran (20%), fish meal (10%), oil cake (10%), gram (6%), pulses (5%), milk (4%), soyabean oil (1%), molasses (1%) and salt (1%). The crude powder was mixed with normal food and water was added to make a cake shape and given twice a day (9am and 3pm).
• Test group 2 (Water extract): Water extract was administered orally at a dose of 1.25gm/10ml water/kg bw.
• Test group 3 (ethanol extract): Ethanol extract was administered orally at a dose of 1.25 gm/10ml water/kg bw.

Rats of all groups were kept under similar environmental conditions, and were provided with enough food and water throughout the experiment. The body weight of each rat was measured in each week.

Chronic study: The ethanol extract (1.25 gm/10 ml/kg bw), water extract (1.25gm/10ml water/kg bw), crude powder (1.25gm/kg bw), glibenclamide (5 mg/10 ml/kg bw), and water (10 ml/kg body weight) were fed to the rats for 28 consecutive days. An oral glucose tolerance test (OGTT) was performed on the 0, 7^th, 14^th, 21^st and 28^th day of the study. Blood samples were collected to measure serum glucose, total cholesterol, triglyceride and serum insulin levels.

Biochemical analysis: Serum glucose was measured by glucose-oxidase method (Sera Pak, USA). The total cholesterol and triglyceride (TG) was measured by enzymatic- colorimetric method (Randox Laboratories Ltd., UK). Serum insulin by Rat Insulin enzyme linked immunosorbent assay (ELISA) method (Crystal Chem Inc., USA). The absorbance was measured by microplate ELISA Reader (Bio-Tek EL-340, USA).

Statistical Analysis: Data from the experiments were analyzed using the Statistical Package for Social Science (SPSS) software for windows version 12 (SPSS Inc., Chicago, Illinois, USA). All the data were expressed as Mean ± SD or as Median (Range) as appropriate. Statistical analysis of the results were performed by using the student’s t-test (paired and unpaired) or ANOVA (analysis of variance) followed by Bonferroni post Hoc test or Mann Whitney (u) test. The limit of significance was set at p<0.05.

RESULTS:

Effect on the body weight (BW): There was a tendency to increase the body weight in all groups. However, only aqueous extract on 7^th day showed a significant increase (p = 0.04) in comparison with ethanol extract treated group (Table 1).

TABLE 1: CHRONIC EFFECT OF AZADIRACHTA INDICA (LEAF) ON BODY WEIGHT (GM) OF TYPE 2 DIABETIC MODEL RATS

Results are expressed as Mean ± SD; *p<0.05; WC = Water control; Gli = Glibenclamide; Powd = Crude leaf powder; Aqu_E = Aqueous extract; Eth_Ext = Ethanol extract; Between group comparison was done using one way ANOVA with post Hoc Bonferroni test

Chronic effect on glucose homeostasis: Results of fasting serum glucose (FSG) level of the studied rats at baseline  (before onset of feeding i.e., 0 day), at 7 day, 14 day, 21 and at 28 day of feeding are showed in Fig. 1. It was found that the crude powder of A. indica ameliorated diabetic condition of the model rats to a better extant than Glibenclamide [p=0.008 (7^th day); p= 0.04 (14^th day); p=0.001 (21^st day) and p=0.009 (28^th day)]. Aqueous extract of A. indica decreased serum glucose level significantly on 28^th day [FSG mmol/l (M±SD) 8.38±1.57 on 0 day vs. 6.90± 1.07 on 28^th; p=0.026]. Ethanol extract of A. indica lowered FSG significantly on 21^st and 28^th day (p=0.001).

FIG. 1: CHRONIC EFFECT OF A. INDICA (LEAF) ON FASTING SERUM GLUCOSE LEVEL OF TYPE 2 MODEL RATS

Effect on serum cholesterol: It was found that in WC group, the level of serum cholesterol increased at the end of the study period. On the contrary, Glibenclamide significantly decreased total serum cholesterol after 28 days feeding (p=0.04). In case of crude powder, there was 3% increase in comparison to the 0 day value. By the influence of aqueous extract and ethanol extract of A. indica 13% and 6% reduction were noticed respectively after 28 days study. It is noteworthy that the decrease in the total cholesterol level by aqueous extract was significant (p= 0.02) (Table 2).

TABLE 2: CHRONIC EFFECT OF AZADIRACHTA INDICA (LEAF) ON TOTAL SERUM CHOLESTEROL OF TYPE 2 DIABETIC MODEL RATS

Data are presented as Mean±SD and compared using paired‘t’ test. *p<0.05; `n’ indicates number of rats in each group

Effect on serum triglyceride (TG): It was found that all of the groups had decreased tendency regarding to serum TG level in comparison to baseline value. The reducing tendency of TG level among different groups were 17%, 18%, 60% and 24% for WC, powder, aqueous extract and ethanol extract respectively. While comparing between groups (using Mann-Whitney U test), aqueous extract showed the most significant triglyceride lowering effect (p< 0.035) (Table 3).

TABLE 3: CHRONIC EFFECT OF AZADIRACHTA INDICA (LEAF) ON SERUM TRIGLYCERIDES (TG) LEVEL OF TYPE 2 DIABETIC MODEL RATS

Results are expressed as Median (Range); p<0.05; WC = Water control, Gli = Glibenclamide, Powd= Leaf powder, Aqu_Ext = Aqueous extract, EtOH_Ext = 80% ethanol extract, between group comparison was done using Mann -Whitney U test

Effect on serum insulin level: No significant change in serum insulin level was found in case of the test samples after 28 days period (Table 4).

TABLE 4: CHRONIC EFFECT OF A. INDICA (LEAF) ON SERUM INSULIN LEVEL OF TYPE 2 DIABETIC RATS

Results are expressed as Median (Range); p<0.05; WC = Water control, Gli = Glibenclamide, Powd = Leaf powder, Aqu_Ext = Aqueous extract, EtOH_Ext = 80% ethanol extract, between group comparison was done using Mann -Whitney U test

DISCUSSION: Oral hypoglycemic agents and insulin is the mainstay of treatment of diabetes and are effective in controlling hyperglycemia, however, they have prominent side effects and fail to significantly alter the cause of diabetic complications ¹⁵. As the knowledge of heterogeneity of this disorder increases, it is needed to look for more efficacious agents with lesser side effects. Though the development of modern medicine results in the advent of modern pharmacotherapeutics (in additions to insulin, sulphonylureas and biguanides) like a-glucosidase inhibitors and thiazolidinediones, there is still a need to look for new drugs as the existing drugs do not modify the course of diabetic complications. Therefore, as the disease is progressing unabated, there is an urgent need of identifying indigenous natural resources with antidiabetic properties in order to develop them as new therapeutics.

The present study was undertaken to assess the antidiabetic effect (chronic) with underlying mechanism of action of Azadirachta indica crude leaf powder, aqueous and ethanol (80%) extract on streptozotocin induced type 2 diabetic model rats. This study also compares the antidiabetic effect of different extracts of A. indica on Type 2 diabetic model rats.

In the present study A. indica leaf powder, aqueous extract and 80% ethanol extract was fed to type2 diabetes rats for 28 consecutive days. In our study, we found that the type 2 rats from all groups gained in body weight throughout the study period. The rise was not significant except aqueous extract on 7^th day showed a significant increase (p= 0.04) in comparison with ethanol extract treated group. Our findings are in accordance with the findings of Hussain et al., ¹⁶ who also observed the gain in body weight of rats treated with aqueous extract of A. indica for 16 weeks. The probable mechanism for increased weight in Type 2 rats treated with A. indica is due to its ability to reduce hyperglycemia.

Regarding to hypoglycemic activity, it was found that continuous treatment with A. indica powder and extracts for 28 days caused a significant reduction in serum glucose level of  Type 2 diabetic rats. The results are consistent with others ^{12, 13, 17, 18, 19, 20}.

The extent of hypoglycemic effect varied considerably among different groups of extracts. Ethanol extract of A. indica lowered serum glucose level on 21^st day and 28^th day (p=0.001, while comparing within groups). Again while comparing between groups none but ethanol extract showed the most significant hypoglycemic effect on 28 day (p=0.04) in comparison to water control. Aqueous extract also reduced the serum glucose level significantly (p=0.02) but the effect was non significant while comparing between groups. Some spontaneous reduction in serum glucose level was noticed in water control group on 14^th day and 21^st day but the glucose level went back to near base line level on day 28.

The possible mechanism underlying the hypoglycemic activity of different extracts of A. indica may be potentiation of pancreatic secretion of insulin from b-cell coupled with extra pancreatic mechanisms like decreased glycogenolysis and enhanced glycogenesis by the liver; and enhanced transport of blood glucose to peripheral tissues. As it was seen from our experiment, serum insulin level did not change significantly, rather there was comparatively less serum insulin level than the base line level in A. indica treated groups.

There is another similar report claiming that A. indica does not modulate insulin effect on glycogen metabolism ²¹ rather it blocks the inhibitory effect of serotonin on insulin secretion/release in pancreas of rats mediated by glucose, eventually glucose control. In another report it was found that the increased peripheral glucose uptake was due to inhibitory action of insulin by inhibiting glycogenesis ²⁰. Hence, the obtained hypoglycemic effect by feeding A. indica for 28 days was probably, due to, increased peripheral utilization of glucose. A. indica leaves are known to have compound like flavonoids, quercetin which possess hypoglycemic activity. The presence of such constituent might be responsible for antidiabetic activity of A. indica.

Apart from the blood sugar lowering effect, beneficial changes in lipid profile have also been observed by A. indica extract and powder. Abnormalities in lipid profile are one of the most common complications in diabetes mellitus, which is found in about 40% of diabetics. Since dyslipidemia plays an important role in the pathogenesis of macro- and microvascular complications of diabetes, hence, improvement in the lipid abnormalities must play beneficial role in inhibiting the complications of diabetes.

It has been claimed that hypercholesterolemia and hypertriglyceridaemia occurred in STZ induced diabetic rats ²². Consecutive administration of A. indica powder and extracts for 28 days lowered serum triglycerides. Significant (p=0.035) reduction in triglyceride and serum cholesterol (P=0.02) level was observed with aqueous extract of A.  indica. The obtained result is supported by the findings of other investigator ²³. These findings revealed that a continuous administration of the aqueous extract of Azadirachta indica improves serum lipids secondary to the diabetic state.

Thus, it can be concluded that, Aadirachta indica (leaf) has got promising antihyperglycemic and hypolipidemic effects in respect to Type 2 diabetic model rats and the accordance of its effect of efficacy can be explained as ethanol extract > aqueous extract > powder.

ACKNOWLEDGEMENTS: We gratefully acknowledge the financial support of International Program for the Chemical Sciences (IPICS), Uppsala, Sweden; International Foundation for Sciences (IFS), Stockholm, Sweden and Diabetic Association of Bangladesh in conducting this study.

REFERENCE:

1. Ghosh S., Suryawanshi S. A.: Effect of Vinca rosea extracts in treatment of alloxan diabetes in male albino rats. Indian Journal of Experimental Biology. 2001; 39: 748-759.
2. Berger W.: Incidence of severe side effects during therapy with sulphonylureas and biguanides. Hormones Metabolic Res. 1985; 17: 111-5.
3. Rang, H. P., Dale, M. M. and Rittar, J. M.: The endocrine system Pharmacology. In: Pharmacology. Longman Group Ltd., UK. 1991; 504-508.
4. Dubey, G.P., Dixit, S.P., Alok, S.: Alloxan-induced diabetes in rabbits and effect of herbal formulation D-400. Indian Journal of Pharmacology. 1994; 26: 225-226.
5. Prince, P.S., Menon, V.P., Pari, L.: Hypoglycemic activity of Syzigium cuminii seeds: effect on lipid peroxidation in alloxan diabetes rats. Journal of Ethnopharmacology. 1998; 61: 1-7.
6. Ladeji, O., Omekarah, I., Solomon, M.; Hypoglycemic properties of aqueous bark extract of Ceiba pentandra in streptozotocin-induced diabetic rats. Journal of Ethnopharmacology. 2003; 84: 139-142.
7. Geetha, B. S., Biju, C. M., Augusti, K. T.: Hypoglycemic effect of leucodelphidin derivative isolated from Ficus bengalensis (Linn.). Indian Journal of Pharmacology. 1994; 38: 220-222.
8. Rao, B. K., Sudarshan, P. R., Rajasekhar, M. D., Nagaraju, N., Rao, C. A.: Antidiabetic activity of Terminalia pallida fruit in alloxan-induced diabetic rats. Journal of Ethnopharmacology. 2003; 85: 169-172.
9. Vats, V., Grover, J. K., Rathi, S. S.: Evaluation of antihyperglycemic effect of Trigonella foenum-graecum Occium sanctum Linn. and Pterocarpus marsupium Linn., in normal and alloxanised diabetic rats. Journal of Ethnopharmacology. 2002; 79: 95-100.
10. Murty, K. S., Rao, D. N., Rao, D. K. and Murty, L. B. G.: Indian J. 1978; 10: 247–250.
11. Khosla, P., Bhanwra, S., Singh, J., Seth, S. and Srivastava, R. K.: Indian J. Physiol. Pharmacol. 2000; 44: 69–74.
12. Chattopadhyay, R.R.: Possible mechanism of antihyperglycemic effect of Azadirachta indica leaf extracts part 1V. Pharmacal Col. Letrs. 1996; 27(3): 431-434.
13. Chattopadhyay, R.R.: A comparative evaluation of some blood sugar lowering agents of plant origin. Ethnopharmacology. 1999; 67: 367-372.
14. Bonner-Weir, S.; Trent, D.F.; Honey, R.N. and Weir, G.C.: Responses of neonatal rat islets on streptozotocin limited beta cell regeneration and hyperglycemia. Diabetes: 1981; 30: 64-69
15. Rang, H. P., Dale, M. M. and Rittar, J. M.: The endocrine system Pharmacology. In: Pharmacology. Longman Group Ltd., UK. 1991; 504-508.
16. Halim Eshrat M. Ali Hussain.: Reversal of diabetic retinopathy in STZ induced diabetic rats using traditional Indian plant, Azadirachta indica (L.). Indian Journal of Clinical Biochemistry. 2002; 17 (2): 115-123.
17. Chattopadhyay, R.R., Chattopadhyay, R.N., Nandy, A.K., Poddar, G. and Maitra, S.K.: The effect of fresh leaves of Azadirachta indica on glucose uptake and glycogen content in the isolated rat hemi diaphragm. Bull. Calcutta Sch. Trop. Med., 1987b; 35: 8-12.
18. Gholap, S. and Kar, A.: Hypoglycemic effects of some plant extracts are possibly mediated through inhibition in corticosteroid concentration. Pharmazie. 2004; 59: 876-878.
19. Kar, A., Choudhary, B.K. and Bandyopadhyay, N.G.: Comparative evaluation of hypoglycaemic activity of some Indian medicinal plants in alloxan diabetic rats. Ethnopharmacology. 2003; 84:105-108.
20. Sonia, B. and Srinivasan, B.P.: Investigations in to the Anti-diabetic activity of Azadirachta indica. Indian Journal of Pharmacology. 1999; 31: 138-141
21. Chatopadhyay, R.R., Medda, C., Das, S. and Basu, T.K.: Hypoglycemic and antihyperglycemic effect of Gymnema sylvestre leaf extract in rats. Fitoterapia. 1993; 64: 450-454.
22. Sharma, S.R., Dwivedi, S.K., Varshney, V.P. and Swarup, D.: Antihyperglycemic and insulin release effect of marmelos leaves in streptozotocin diabetic rats. Phytotherapy Research. 1996; 10: 426-428.
23. Chattopadhyay R. R., Bandyopadhyay M.: Possible mechanism of hepatoprotective activity of Azadirachta indica leaf extract against paracetamol-induced hepatic damage in rats: Part III. Indian Journal of Pharmacology. 2005; 37(3): 184-185.

type 2 diabetic rat