Antidiabetic activity of alcoholic extract of neem (Azadirachta indica) root bark.
Neem plant was tested for different activities. Isolation of active gradient from neem was started by Siddiqui in 1942. After that more than 135 compounds were isolated from different parts of neem.  Different types of extracts from different part of plant were used for different activities e.g. aqueous extract leaf-immuno-stimulant activity, ethanolic extract of the flowers- hypolipidemic activity, methanolic leaf extract--antipyrectic activity, chloroform extract of stem bark--anti-inflammatory activity, acetone leaf extract-CNS depressant activity, Hexane extract of neem seed- antifertility effect. [2-7] But antidiabetic activity of neem root bark was not evaluated. Considering these things in our study we evaluated whether 70% of alcoholic neem root bark extract has antidiabetic activity?
MATERIALS AND METHODS
Plant Material: Neem roots were collected from neem tree in Navodaya Medical College campus. The root bark was shade dried in department of pharmacology. Shade dried root bark was powdered. Alcoholic extract of bark was obtained by continuous extraction in percolator using 70% ethyl alcohol. Fresh solution was prepared by dissolving extracts in distilled water before each experiment.
Animals: Wistar albino rats of either sex weighing 200-250 grams were used in present study. The rats were provided standard laboratory feed and tap water. They were exposed to an alternate light and dark cycle of 12 hours and had free access to food and water. The experimental protocol was approved by institutional animal ethics committee. The whole project was carried out as per guidelines of the Committee for the Purpose of Control and Supervision of Experiments on Animals (CPCSEA).
Reagents and Drugs: Alloxan s.d. fine -chem Ltd. Boisar, Tab. Glibenclamide:-5 mg in each tablet (Daonil- Aventis)
Instruments: Blood glucose levels were measured with the help of Glucometer [Prestige smart system (Andheri, Mumbai)]. Percolator was purchased from Maharashtra Emporium, Wardha, Maharashtra.
Methods for Evaluating Antidiabetic Activity
1. Glucose Tolerance Test : Animals were overnight fasted. Fasting blood sugar levels were detected by taking blood samples. Then the test drugs were administered orally 60 minutes prior to administration of glucose to the 5 groups of animals each containing 6 animals.
Group I (Control): Received 2 ml of distilled water
Group II (Standard): Received Glibenclamide (0.5 mg/kg) 
Group III: Received NRE (200 mg/kg) single dose
Group IV: Received NRE (400 mg/kg) single dose
Group V: Received NRE (800mg/kg) single dose
After 60 minutes glucose (in the dose of 1.25 grams/kg) was administered orally to each rat. Blood samples were drawn every half hourly up to 4 hours and blood sugar levels were detected by glucometer. The blood was taken by chopping tail of rat. [10,11]
2. Alloxan Induced Diabetes: Overnight fasted animals were given 100 mg/kg alloxan monohydrate.  After 48 hours blood sugar levels were estimated by glucometer. Then the animals were divided into 5 groups. In each group 6 rats were kept. All rats received drugs orally for 15 days. In pilot study NRE in single daily dose reduced blood sugar levels but they were not significant. So we gave NRE twice daily.
Group I (Control): Received 2 ml of distilled water daily.
Group II (Standard): Received Glibenclamide 0.5 mg/kg daily 
Group III: Received NRE 200 mg/kg twice daily
Group IV: Received NRE 400 mg/kg twice daily
Group V: Received NRE 800 mg/kg twice daily
The blood was taken by chopping tail of rat.  Blood sugar level was measured on 48 hours, 5, 10 and 15 days by using glucometer.
Statistical Analysis: Analysis was done with one way ANOVA followed by Dunnet test.
For glucose tolerance test Glibenclamide was given in the dose of 0.5 mg/kg showed significant (p< 0.01) reduction in blood sugar levels. It reduced blood sugar level to basal line after 4 hours as comparison to control. The NRE was given in the dose of 200 and 400 mg/kg showed reduction in blood sugar level, but it was not statistically significant. The NRE was given in the dose of 800 mg/kg showed significant (p < 0.05) reduction in blood sugar level. It reduced blood sugar level by 54% after 4 hours as comparison to control. But in comparison to glibenclamide it was not showing significant result (Table 1, Fig 1)
In alloxan induced diabetes Glibenclamide was given in the dose of 0.5 mg/kg showed significant (p < 0.01) reduction in blood sugar levels. It reduced the blood sugar level to basal line on 10th day. NRE reduced blood sugar levels with dose 200 and 400 mg/kg but it was not significant. In dose 800 mg/kg it showed significant (p < 0.05) reduction in blood sugar levels as comparison to control. Maximum reduction of blood sugar level was 76%. This was seen on 15th day with same dose. (Table.2, Fig.2)
[FIGURE 1 OMITTED]
[FIGURE 2 OMITTED]
Nimbidin is a mixture of tetranortriterpenes and is the major active principle of the seed oil of Azadirachta indica A. Juss (Meliaceae). In study carried out by Pillai et al 1981 in oral glucose tolerance test nimbidin was studied in rabbits. In this study nimbidin (200 mg/kg) significantly delayed rise in blood glucose level after oral glucose administration as compared to control. Nimbidin brought down blood sugar level to base line in 4th hour.  In our study alcoholic root bark extract delayed the rise in blood sugar level. It shows that NRE has antihyperglycemic activity.
For anti-diabetic activity evaluation of neem many studies were done. Khosla et al. (2000) showed that when aqueous extract of neem leaf (500 mg/kg orally) when administered for 4 weeks after alloxan induced diabetes in rabbits, it significantly (P < 0.001) reduced blood glucose levels.  In the study carried out by Kar et al 95% alcoholic extract of neem leaf in the dose of 250 mg/kg twice daily orally for one week reduced blood sugar level by 55% and urine sugar by 100% (p < 0.05) in alloxan induced diabetes in rats.  In our study with NRE given twice daily for 15 days reduced blood sugar levels significantly. This effect was less significant and less potent than glibenclamide. Neem root bark contains terpanoids like nimbin and nimbidin).  Nimbidin is having anti-diabetic activity and it may be responsible for the activities seen in our study. As NRE is delaying onset of diabetes produced by alloxan it may be used as prophylactic agent in diabetes. As it is reducing levels of sugar in alloxan induced diabetes it can be used as adjuvant.
70% alcoholic neem root extract has antidiabetic activity. Further studies are required to know which active ingredients are responsible for this action.
Cite this article as: Patil PR, Patil SP, Mane A, Verma S. Antidiabetic activity of alcoholic extract of Neem (Azadirachta Indica) root bark. Natl J Physiol Pharm Pharmacol 2013; 3:142-146.
Source of Support: Nil
Conflict of interest: None declared
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Prabhakar Patil (1), Sudha Patil (2), Abhay Mane (3), Sushilkumar Verma (4)
(1) Department of Pharmacology, Navodaya Medical College, Raichur, Karnataka, India
(2) Department of Anatomy, Navodaya Medical College, Raichur, Karnataka, India
(3) Department of Community Medicine, Navodaya Medical College, Raichur, Karnataka, India
(4) Department of Pharmacology, MGIMS, Sewagram, Maharashtra, India
Table-1: Oral Glucose Tolerance Test Using Azadirachta Indica (Neem) Root Bark Extract in Albino Rats Blood Sugar Level (mg/dl) (Mean [+ or -] SEM) A.D.A. Drugs Doses B.D.A. 0.5 hr mg/kg Distilled Water 2 ml 80 [+ or -] 2.1 260 [+ or -] 1.3 Glibenclamide 0.5 80 [+ or -] 2 190 [+ or -] 1.8 ** NRE200 200 78 [+ or -] 2.6 210 [+ or -] 1.6 NRE 400 400 77 [+ or -] 3 198 [+ or -] 1.2 NRE800 800 83 [+ or -] 2.1 180 [+ or -] 2.3 * Blood Sugar Level (mg/dl) (Mean [+ or -] SEM) A.D.A. Drugs 1 hr 1.5 hr Distilled Water 250 [+ or -] 6.8 246 [+ or -] 9.8 Glibenclamide 123 [+ or -] 2.3 ** 114 [+ or -] 1.6 ** NRE200 220 [+ or -] 9.1 210 [+ or -] 9 NRE 400 195 [+ or -] 7.3 198 [+ or -] 7.8 NRE800 185 [+ or -] 4.6 * 178 [+ or -] 6.2 * Blood Sugar Level (mg/dl) (Mean [+ or -] SEM) A.D.A. Drugs 2 hr 2.5 hr Distilled Water 240 [+ or -] 9.8 228 [+ or -] 9.2 Glibenclamide 99 [+ or -] 2.7 ** 90 [+ or -] 1.2 ** NRE200 204 [+ or -] 8.5 199 [+ or -] 7.6 NRE 400 188 [+ or -] 7.8 184 [+ or -] 7.1 NRE800 167 [+ or -] 5 * 155 [+ or -] 6.3 * Blood Sugar Level (mg/dl) (Mean [+ or -] SEM) A.D.A. Drugs 3 hr 3.5 hr Distilled Water 220 [+ or -] 14 212 [+ or -] 13 Glibenclamide 82 [+ or -] 1.5 ** 82 [+ or -] 2 ** NRE200 195 [+ or -] 7.3 190 [+ or -] 8.5 NRE 400 181 [+ or -] 7.7 178 [+ or -] 7.9 NRE800 130 [+ or -] 5.9 130 [+ or -] 6.4 * Blood Sugar Level (mg/dl) (Mean [+ or -] SEM) A.D.A. Drugs 4 hr Distilled Water 196 [+ or -] 13 Glibenclamide 80 [+ or -] 2** NRE200 187 [+ or -] 8.2 NRE 400 173 [+ or -] 7.5 NRE800 128 [+ or -] 5.7 * * p < 0.05; ** p < 0.01; *** p < 0.001; B.D.A.--Before drug administration; A.D.A.--After drug administration; n--Number of animals Table-2: Anti-Diabetic Effect of Azadirachta Indica (Neem) Root Bark in Albino Rats (Alloxan Induced Diabetes) Group Drug and Doses B.D.A. I Distilled Water 2ml 86 [+ or -] 1.1 orally II Glibenclamide 0.5 86 [+ or -] 1.6 mg/kg orally III NRE 87 [+ or -] 1.6 (200 mg/kg orally) IV NRE 86 [+ or -] 3.1 (400 mg/kg orally) V NRE 87 [+ or -] 1.8 800 mg/kg orally) Blood Sugar Level (mg/dl) (Mean [+ or -] S.E.M) A.D.A Group Drug and Doses 48 hrs 5 days I Distilled Water 2ml 360 [+ or -] 15 355 [+ or -] 12 orally II Glibenclamide 0.5 120 [+ or -] 14 ** 87 [+ or -] 1.5 ** mg/kg orally III NRE 87 [+ or -] 7.6 270 [+ or -] 7.6 (200 mg/kg orally) IV NRE 86 [+ or -] 10 250 [+ or -] 10 (400 mg/kg orally) V NRE 86 [+ or -] 12* 178 [+ or -] 11 * 800 mg/kg orally) Blood Sugar Level (mg/dl) (Mean [+ or -] S.E.M) A.D.A Group Drug and Doses 10 days 15 days I Distilled Water 2ml 356 [+ or -] 13 360 [+ or -] 14 orally II Glibenclamide 0.5 86 [+ or -] 1 ** 86 [+ or -] 1.1 ** mg/kg orally III NRE 250 [+ or -] 7.6 232 [+ or -] 8.3 (200 mg/kg orally) IV NRE 236 [+ or -] 10 220 [+ or -] 7.8 (400 mg/kg orally) V NRE 165 [+ or -] 12 * 152 [+ or -] 18 * 800 mg/kg orally) * p < 0.05; ** p < 0.01; *** p < 0.001; B.D.A.--Before drug administration; A.D.A.--After drug administration; n--Number of animals
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|Title Annotation:||RESEARCH ARTICLE|
|Author:||Patil, Prabhakar; Patil, Sudha; Mane, Abhay; Verma, Sushilkumar|
|Publication:||National Journal of Physiology, Pharmacy and Pharmacology|
|Date:||Jul 1, 2013|
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