STUDY OF ANTI-DIABETIC PROPERTIES IN THE BARK OF TERMINALIA ARJUNA USING ALLOXAN INDUCED MICE MODEL

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1 International Journal of Advanced Research in Engineering and Technology (IJARET) Volume 9, Issue 2, March - April 201, pp. 10 1, Article ID: IJARET_09_02_002 Available online at ISSN Print: and ISSN Online: IAEME Publication STUDY OF ANTI-DIABETIC PROPERTIES IN THE BARK OF TERMINALIA ARJUNA USING ALLOXAN INDUCED MICE MODEL J. Aparajita Department of Biotechnology, T.M Bhagalpur University, Bihar, India K. K Sinha Department of P.G Botany, T.M Bhagalpur University, Bihar, India D. Kumar Department of Veterinary Surgery, Jabalpur Veterinary University, Jabalpur, M.P, India. ABSTRACT The medicinal plant T.arjuna is a large evergreen tree with buttressed trunk. It is an important cardiotonic plant described in the ayurveda. Recently the antioxidant activities of the chloroform extract of T.arjuna in diabetic rats have been reported.bark of Terminalia arjuna was shade dried and its ethanolic extract was prepared. Dosage was further defined 250, 500 and 600 mg/kg body weight of mice for Herbal extract and 10 mg/kg body weight for. The three parameters that measured were blood glucose, blood cholesterol, and blood triglycerides via kit method of Erba Diagnostics. Cholesterol was measured by modified Roeschlau s Method, Triglycerides was measured by method of Wako and the modifications by Gowan and Fosssati. The whole work was divided between the period of 0 days and readings were taken at the intervals of seven days. After diabetes was induced in mice, immediately herbal and allopathic was started and after of 7 days it is found that there is positive effect of though mice are still diabetic when compared to control. There was progressive reduction in the level of glucose as the moved step forward. Lastly when is carried out for one month, diabetic status is cured and all mice have reached to normal level of blood cholesterol, glucose and triglyceride level. Key words: T.arjuna, Ethanolic extract,. Cite this Article: J. Aparajita, K. K Sinha and D. Kumar, Study of Anti-Diabetic Properties in the Bark of Terminalia Arjuna Using Alloxan Induced Mice Model. International Journal of Advanced Research in Engineering and Technology, 9(2), 201, pp editor@iaeme.com

2 J. Aparajita, K. K Sinha and D. Kumar 1. INTRODUCTION Diabetes mellitus or Diabetes is an endocrine disorder, characterized by a loss of glucose homeostasis resulting from defects in insulin secretion, or insulin action or both resulting in impaired metabolism of glucose and other energy yield fuels such as lipid and protein (WHO, 195). pancreatic β cells can compensate for the insulin resistance by increasing insulin secretion; however, extensive exposure of pancreatic β cells to high glucose levels causes β cell dysfunction that is associated with impaired insulin secretion and bio-synthesis.` According to World Health Organization projections, the diabetes population is likely to increase to 00 million or more by the year 2025.It has emphasized the need of evaluation of the potential of plants as effective therapeutic agents, especially in areas where we lack safe modern drugs (WHO, 1994). A multitude of herbs, spices and other plant materials have been described for the of diabetes throughout the world (Kesari et al., 2006). Few of the plants used for the of diabetes recommend that this area warrant further attention (Sepici et al., 2004). There are 1200 plant species worldwide that are used in the of Diabetes mellitus and a substantial number of plants have shown effective hypoglycemic activity after laboratory testing (Eddouks et al., 2005; Sivaraj, et al., 2009). The medicinal plants provide a useful service of oral hypoglycemic compounds for the development of new pharmaceutical products as well as a dietary supplement to existing therapies (Bailey and Day, 199). India has about 45,000 plant species and many of them have medicinal properties (Sivaraj et al., 2009). 2. REVIEW OF LITERATURE Bajpai et al. (2011) identified promising sources of anti-oxidants of some food and medicinal plants. They studied the total phenolic contents and anti-oxidant activity of these plants and found that the leaves, bark and fruits of T.arjuna, T.bellorica, T.chebula and T.mulleri were found to have high phenolic contents ( mg/g) and high anti-oxidant activity ( %). Fruits of T.bellorica and T.chebula were rich source of gallic acid, whereas bark of T.arjuna was rich source of ellagic acid. Dwivedi (2011) reported that T.arjuna bark possesses glycosides, large quantities of flavonoids, tannins and minerals. He also reported that flavonoids exerts anti-oxidant, antiinflammatory, and lipid lowering effect while glycosides are cardiotonic, thus making T.arjuna unique amongst currently used medicinal plants. Manna et al. (2011) reported that a triterpenoid saponin, arjunolic acid (AA) has been isolated from the bark of T.arjuna. It possesses free radical scavenging activity, and could enhance the cellular anti-oxidant capability against NaAsO(2)-induced cyto-toxicity. They further reported induced cardiac oxidative stress and cardio-protective action of the active phyto- constituents of the T.arjuna against the oxidative result. Hemlatha et al. (2010) reported that the scientific basis for the use of arjunolic acid in ayurvedic medicine has been clearly proved by its versatile effects i.e. prevention of myocardial necrosis, platelet aggregation, anticoagulant property, free radical scavenging property, anti-oxidant property, metal ch-elating property, antimicrobial activity etc. More studies have to be undertaken to unravel the molecular mechanisms by which arjunolic acid exerts preventive role against various cellular stress condition. Manna et al. (2009) suggested that increasing evidences in both experimental and clinical studies suggest that oxidative stress is involved in the pathogenesis of diabetic tissue damage editor@iaeme.com

3 Study of Anti-Diabetic Properties in the Bark of Terminalia Arjuna Using Alloxan Induced Mice Model Pancreatic β-cell death is the cause of decreased insulin production in diabetes. Streptozotocin is widely used to induce experimental diabetes due to its ability to selectively target and destroy insulin producing pancreatic β-cells via the formation of both reactive oxygen species(ros) and reactive nitrogen species(rns).treatment of animals with Arjunolic acid (T.arjuna) at a dose of 20 mg/kg body weight orally both prior and post to the Streptozotocin administration effectively reduced these adverse effects by inhabiting the excessive ROS and RNS formation as well as by down regulating the pathways leading to apoptotic cell death. Thus, the study suggests that arjunolic acid plays some beneficial roles against Streptozotocin -induced diabetes. Parveen et al. (2011) reported that T.arjuna is a potent anti-diabetic and beneficial in the control of diabetes related abnormalities in serum lipid profile renal markers and oxidative damage in liver and pancreas of streptozotocin induced rat model. They also reported that the antioxidant activity of T.arjuna bark extract is contributed due to rich concentration of its active constituents include tannins, triterpenoid, saponins (arjunic acid, arjunolic acid, arjungenin, arjunglycosides), flavonoids (arjunone, arjunolone, luteolin), gallic acid, ellagic acid, oligomeric proanthocynidins phytosterols, calcium, magnesium, zinc, and copper. It is thought that the flavonoids and oligomeric proanthocynidins provide free radicals scavenging activity and enhance the antioxidant status in body. Antibiotic activity of T.arjuna may be due to the stimulation of β cells of the pancreatic islets.. MATERIALS AND METHODS The permision for using animal model for the experiment was taken from Instituitional Animal Ethical Commitee, College of Veterinary Science and Animal Husbandry, Nanaji Deshmukh Veterinary Science University. Jabalpur(M.P) (11/IAEC/Sci/2011) in the year Clinical Examination of Anti-Diabetic Properties of T.Arjuna, and an Allopathic Medicine Animal models are indispensably used for the clinical evaluation of the effect of medicinal plants. In the present study mice model has been used for the study of the effect of the antidiabetic principles of herbal plant Terminalia arjuna and the anti-diabetic allopathic medicine. About 24 mice were purchased with prior permission of ethical society of Jabalpur, Madhya Pradesh. These mice were divided into four s having 6 mice in each. The s were normal mice without alloxan and without, T.arjuna bark ; Jamun leaves, and. All the mice were freely fed for ten days and then they were induced with diabetes via alloxan. Diabetes is induced within 4 hours. After induction of diabetes, was started. Three parameters were considered i.e. blood glucose, cholesterol, and triglycerides and their readings were taken before diabetic induction, after diabetic induction and at regular intervals of 7, 15, 25 and 0 days after herbal and allopathic..2. Preparation of the Plant Extract Bark of Terminalia arjuna were shade dried for 10 to 15 days. Further these plant tissues were grounded to powdered form and about kg of the powdered form were soaked in ethanol whose volume was kept double the volume of the powder. It was left for 2 to days. After this the soaked powder was squeezed with the help of muslin cloth properly and the extract were dried on petri-plates for several days till all the moisture was evaporated. Then these dried form were scratched and stored in dark coloured bottles and their bread or flour pellets 12 editor@iaeme.com

4 J. Aparajita, K. K Sinha and D. Kumar were made and fed to the alloxan-induced diabetic mice on per day basis for two times after their proper feeding... Dosage Fixed For Herbal Extract and Allopathic Medicine Three different doses were defined i.e. 250, 500 and 600 mg/kg body weight of mice for herbal medicine (T.arjuna) and 10 mg/kg body weight for allopathic medicine ()..4. Measurement of Blood Glucose, Cholesterol and Triglycerides The three parameters that measured were blood glucose, blood cholesterol, and blood triglycerides via kit method of Erba Diagnostics. Cholesterol was measured by modified Roeschlau s Method, Triglycerides was measured by method of Wako and the modifications by Gowan et al (19) and Fosssati et al (190). Standard error and mean analysis of mice model S E = Where S is the sample standard deviation. is the size (number of observations) of the sample 4. RESULTS The whole work was divided between the period of 0 days and readings were taken at the intervals of seven days. Thus whole readings were divided into three s and readings were taken at the interval of seven days. First reading was taken when mice were not injected with either alloxan or herbal extract and was considered as normal parameter reading. In this reading normal cholesterol level was found to be less than 200mg/dl, normal triglyceride level was found to be less than 150mg/dl, and normal glucose level was found to be in between the range mg/dl. Second reading was taken after alloxan was injected and mice were made diabetic. In case of diabetes the blood sugar was 200mg/dl, blood cholesterol was in between mg/dl range, and blood triglyceride was in between mg/dl. Now third reading was taken after seven days interval. These readings indicated that there was positive effect of though mice were still diabetic. Fourth reading was taken after fifteen days interval and positive results were seen. The readings indicated that blood cholesterol, blood sugar, and blood triglycerides were reaching to the normal level due to the effect of herbal extracts and allopathic medicine on mice. Fifth reading was taken at twenty five days interval which indicates a very fast recovery from diabetic status due to of herbal extract and. We can see that arjuna extract are more effective than allopathic medicine or closely effective when compared to. Sixth reading was taken at the interval of thirty days and it showed that when was carried out for one month diabetic status was cured and all mice had reached to normal level of blood cholesterol, glucose and triglyceride. The results have been interpreted in terms of, Mean ± S.E, analysis of variance (ANOVA), and Correlation Matrix of mice model for blood glucose, blood cholesterol, and blood triglyceride. It has been divided into six readings Standard Error and Mean Analysis of Mice Model Initially normal blood glucose, blood cholesterol and blood triglyceride level in mice were measured.(table 1).The normal cholesterol level was less than 200mg/dl ( 200mg/dl), normal triglyceride level less than 150mg/dl ( 150mg/dl) and normal glucose level was in between 1 editor@iaeme.com

5 Study of Anti-Diabetic Properties in the Bark of Terminalia Arjuna Using Alloxan Induced Mice Model the range mg/dl. In case of diabetic mice the blood sugar is 200mg/dl, blood cholesterol is between mg/dl range, and blood triglyceride is between mg/dl. Further mice were induced to diabetes after alloxan (4hrs after injecting alloxan intravenously). (Table 2). In this case except 4 th i.e. normal control mice, all other s were intravenously injected with alloxan who had developed diabetes after 4hrs which were confirmed with the levels of blood sugar, blood cholesterol, and blood triglyceride in the treated mice. After diabetes was induced in mice, immediately herbal and allopathic was started and after of 7 days there was slight decrease in the levels of blood glucose, blood cholesterol and blood triglyceride level (Table ).These readings indicated that there is positive effect of though mice were still diabetic. After of 15 days, positive results were seen and readings indicated that blood cholesterol, blood sugar, and blood triglycerides were reaching to the normal level due to the effect of herbal extracts and allopathic medicine on mice (Table 4). After of 25 days very fast recovery from diabetic status could be seen due to of herbal extract and. We can see that arjuna extract were more effective than allopathic medicine or closely effective when compared to (Table 5). Lastly when is carried out for one month diabetic status is cured and all mice have reached to normal level of blood cholesterol, glucose and triglyceride level (Table 6) Standard Error and Mean Analysis of Mice Model Table-1 Before Treatment of Alloxan GROUP GLUCOSE Untreated arjuna extract Untreated mice Group Untreated arjuna extract Untreated mice 7.± ± ± ± ± ± ± ± ±7.06 Table 2 After Alloxan Treatment (Diabetic Mice) GLUCOSE 20± ± ± ± ± ± ± ± ± editor@iaeme.com

6 GROUP arjuna extract mice GROUP J. Aparajita, K. K Sinha and D. Kumar Table 1 ST Reading after Herbal and Allopathic Treatment (7 Days) DOSAGE mg/kg Body weight GLUCOSE (mg/dl) ±S.E ±2 215±2 157± ± ± ± ± ±5 175± ± ± ± ± ± ±7.06 Table 4 2 ND Reading After Herbal And Allopathic Treatment (15 Days) DOSAGE mg/kg Body weight GLUCOSE (mg/dl)±s.e (mg/dl) ±S.E (mg/dl) ±S.E arjuna extract mice ± ± ± ± 217.5± ± ± ± ± ± ± ± ± ± ±7.06 GROUP Table 5 rd Reading After Herbal and Allopathic Treatment (25 Days) DOSAGE mg/kg Body weight GLUCOSE (mg/dl) ±S.E (mg/dl)±s.e (mg/dl) ±S.E arjuna extract mice ± ± ± ±14 175±15 15± ± ± ± ± ± ± ± ± ± editor@iaeme.com

7 Study of Anti-Diabetic Properties in the Bark of Terminalia Arjuna Using Alloxan Induced Mice Model GROUP arjuna extract mice Table 6 4 th Reading after Herbal and Allopathic Treatment (0 Days) DOSAGE mg/kg Body weight GLUCOSE (mg/dl) ±S.E (mg/dl) ±S.E (mg/dl)±s.e ± ±4.5 0± ±4 ± ± ±5 79.5± ± ± ±4.02.± ± ± ± Analysis of Variance of Mice Model for Blood Glucose, Blood Cholesterol and Blood Triglyceride Above results are significant at. Table 7 Before Alloxan Treatment freedom (df) Table After Alloxan Treatment (Diabetic Mice) freedom (df) Above results are insignificant or very less significant at Table 9 After Diabetes Induction Herbal and Allopathic Treatment Of 7 Days freedom (df) Above results is comparatively more significant at editor@iaeme.com

8 J. Aparajita, K. K Sinha and D. Kumar Table 10 After Diabetes Induction Herbal And Allopathic Treatment Of 15 Days freedom (df) Above table is comparatively more significant at Table 11 After Diabetes Induction Herbal and Allopathic Treatment of 25 Days freedom (df) Above results are very much significant at Table 12 After Diabetes Induction Herbal and Allopathic Treatment of 0 Days freedom (df) Above results are highly significant at CONCLUSIONS As far as standard error and mean calculation is concerned the normal cholesterol level is less than 200mg/dl (<200mg/dl), normal triglyceride level is less than 150mg/dl (150mg/dl) and normal glucose level is between the range mg/dl as indicated. In case of diabetic patients the blood sugar is (>200mg/dl), blood cholesterol is between mg/dl range, and blood triglyceride is between mg/dl. After induction of diabetes via alloxan (4hrs after injecting alloxan intravenously). In this table except 4 th i.e. control, all mice of other s were intravenously injected with alloxan and after 4hrs were discovered with diabetes. In case of diabetes the blood sugar is (>200mg/dl), blood cholesterol is between mg/dl range, and blood triglyceride is between mg/dl and results confirms the above range which indicates diabetes. After diabetes was induced in mice, immediately herbal and allopathic was started and after of 7 days it is found that there is positive effect of though mice are still diabetic when compared to control. After of 15 days, positive results are seen and readings indicate that blood cholesterol, blood sugar, and blood triglycerides are reaching to the normal level due to the effect of herbal extracts and allopathic medicine on mice. Further we find a very fast recovery from diabetic status due to of herbal extract and for 25 days. We can see that arjuna extract are more effective than allopathic medicine or closely 17 editor@iaeme.com

9 Study of Anti-Diabetic Properties in the Bark of Terminalia Arjuna Using Alloxan Induced Mice Model effective when compared to. Lastly when is carried out for one month diabetic status is cured and all mice have reached to normal level of blood cholesterol, glucose and triglyceride level. ACKNOWLEDGEMENT Gratuitous praise and profound thanks are the least that I can offer in return for the untiring help,the able guidance, the constant encouragement extended to me by my reverend supervisor and mentor late Prof (Dr) Arun Kumar Singh, University Deptt Botany and my present supervisor Prof (Dr) K.K Sinha and my Co-Supervisor Mrs Dr(Prof), U.K Chauhan, Head of the deptt of Biotechnology, APS University, Rewa, Madhya Pradesh and Prof(Dr)V.B Lal Co-Ordinator (P.G Biotechnology), T.M Bhagalpur University. The constant guidance and scholarly supervision by my mentors made it possible for me to carry out the entire research work. REFERENCES [1] WHO Guideline: sugars intake in adults and children. Geneva: World Health Organization; [2] Policy brief: Global nutrition targets 2025: Childhood overweight. Geneva: World Health Organization; [] Johnsson, IW, Haglund B, Ahlsson F, Gustafsson J. A high birth weight is associated with increased risk of type 2 diabetes and obesity. Pediatric Obesity. 2015; 10(2):77. [4] Wendland EM, Torloni MR, Falavigna M, Trujillo J, Dode MA, Campos MA, et al. Gestational diabetes and pregnancy outcomes a systematic review of the World Health Organization (WHO) and the International Association of Diabetes in Pregnancy Study (IADPSG) diagnostic criteria. BMC Pregnancy Childbirth. 2012; 12 :(1)2. [5] Seuring T, Archangelidi O, Suhrcke M. The economic costs of type 2 diabetes: A global systematic review. PharmacoEconomics. 2015; (): [6] Transforming our world: the 200 Agenda for Sustainable Development (A/RES/70/1). New York: United Nations General Assembly; [7] Imamura F, O Connor L, Ye Z, Mursu J, Hayashino Y, Bhupathiraju SN, Forouhi NG. Consumption of sugar-sweetened beverages, artificially sweetened beverages, and fruit juice and incidence of type 2 diabetes: systematic review, meta-analysis, and estimation of population attributable fraction. British Medical Journal. 2015; 51:h576. [] Luo J, Rossouw J, Tong E, Giovino GA, Lee CC, Chen C, et al. Smoking and diabetes: does the increased risk ever go away? American Journal of Epidemiology. 201; 17 :(6) [9] Wong E, Backholer K, Gearon E, Harding J, Freak-Poli R, Stevenson C, et al. Diabetes and risk of physical disability in adults: a systematic review and meta-analysis. Lancet Diabetes Endocrinology. 201;1:(2) [10] Seuring T, Archangelidi O, Suhrcke M. The economic costs of type 2 diabetes: A global systematic review. PharmacoEconomics. 2015; (): [11] IDF Diabetes Atlas, 6th ed. Brussels, International Diabetes Federation; 201. [12] Bloom DE, Cafiero ET, Jané-Llopis E, Abrahams-Gessel S, Bloom LR, Fathima S, et al. The global economic burden of noncommunicable diseases (Working Paper Series). Geneva: Harvard School of Public Health and World Economic Forum; [1] Transforming our world: the 200 Agenda for Sustainable Development (A/RES/70/1). New York: United Nations General Assembly; editor@iaeme.com