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Antidiabetic activity of alcoholic extract of neem (Azadirachta indica) root bark.

INTODUCTION

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. [1] 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 [8]: 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) [9]

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. [12] 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 [9]

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. [11] 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.

RESULTS

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]

DISCUSSION

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. [8] 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. [13] 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. [12] 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). [14] 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.

CONCLUSION

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

REFERENCES

[1.] Biswas K, Chattopadhayay I, Banerjee RK, Bandyopadhyay U. Biological activities and medicinal properties of neem (Azadirachta indica) Current science 2002; 11:1336-1345.

[2.] Sen P, Mediratta PK, Ray A. Effect of Azadirachta indica A Juss on some biochemical, immunologicla and visceral parameters in normal and stressed rats. Indian Journal of experimental biology 1992; 30:1170-75.

[3.] Purohit A, Daradka HM. Hypolipidemic effects of neem flower (Azadirachta indica A Juss) in rabbits Geobios 1999; 26:146-48.

[4.] Okpanyi SN, Ezeukwv GC. Anti-inflammatory antipyretic activities of Azadirachta indica Planta med 1981; 41:34-39.

[5.] 5. Tidjani MA, Dupont C, Wapierre J. Perspectives on ethno-phytotherapy of "Yoruba" medicinal herbs and preparations. J. Planta Med.Phytother. 1989; 23:259-266.

[6.] Singh PP, Junnarkar AY, Thomas GP, Tripathi RM, Verma RK. Antidepressant activity of Azadirachta indica ibid 1980; 61:164-68.

[7.] Mukharjee S, Garg S, Talwar GP. Early post implantation contraceptive effects of purified fractions of neem (Azadirachta indica) seeds, given orally in rats: possible mechanism involved. Journal of Ethnopharmacology 1999; 67:287-96.

[8.] Pillai NR, Santhakumari G. Hypoglycemic activity of Melia azadirachta Linn.(Neem) Indian Journal of Medical Research 1981; 74:931-33.

[9.] Ghosh R, Sharatchandra K, Rita S, Thokchom IS. Hypoglycemic activity of Ficus hispida (bark) in normal and diabetic albino rats. Indian J Pharmacol 2004; 36:222-5.

[10.] Ghosh MN, Fundamentals of Experimental Pharmacology, 3rd edi. Kolkotta: Hilton and company. 2005. P. 192.

[11.] Farris EJ, Griffith JQ. The Rat in Laboratory Investigations 1st ed. New York: Lippincott. 1967. P. 406-407.

[12.] Kar A, Choudhary BK, Bandopadhyay NG. Comparative evaluation of hypoglycemic activity of some Indian medicinal plants in alloxan diabetic rats. Journal of Ethopharmacology 2003; 84:105-108.

[13.] Khosla P, Bhanwra S, Singh J, Seth S, Srivastva RK. A study of hypoglycemic effect of Azairachta indica in normal and alloxan diabetic rabbits. Indian J Pharmacol 2000; 4(1):69-74.

[14.] Daniel M. Medicinal Plants: chemistry and properties. Enfield, NH, USA: Science publishers. 2006. P. 113.

Received: 27.12.2012

Accepted: 11.02.2013

DOI: 10.5455/njppp.2013.3.134

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

Correspondence to:

Prabhakar Patil

(drprpatil2006@gmail.com)
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
Words:2161
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