Assessment of Anti-Hyperglycemic and Anti-Oxidant Activities of Tinospora Cordifolia & Juglans Regia Composite Extract in STZ Induced Diabetes in Wistar Rats

The present study explored the assessment of the antidiabetic potential of Tinospora cordifolia & Juglans regia composite extract in STZ induced diabetes in wistar rats. As streptozotocin -associated infiltrations of increase glucose level has been reported to be responsible for diabetes. We evaluated the glucose lowering potential of Tinospora cordifolia & Juglans regia on the basis of its anti-diabetic property. Rats were administered streptozotocin (55 mg/kg i.p., once) with nicotinamide (120mg/kg) to induce experimental toxicity. The development of diabetes was assessed biochemically as well as histologically 72 hours after induction of diabetes. Body weight and blood glucose levels were determined in (0, 7th, 14th, 21st, 28th) days. Serum lipid profile and enzyme estimated, (kidney, liver, pancreas) tissue was measured at the end of the experimental period. Treatment with composite extracts TCAE high dose (350 gm/kg b.w.) & JRAE high dose (800 mg/kg b.w.) and TCHE high dose (350 gm/kg b.w.) & JRHE high dose (800 mg/kg b.w.) were noted to be more effective against the streptozotocin- induced toxicity as compared to Glibenclamide (5 mg/kg b.w.). it may be concluded that streptozotocin-induced glucose may be accountable for the induction of diabetes toxicity in rats. Interestingly, improvement in body weight, glucose level, lipid profiles, biochemical parameters and histopathological changes in kidney, liver and pancreas was observed following herbal treatment in STZ induced diabetic rats. Furthermore, composite extract of TCAE (350mg/kg b.w.) & JRAE (800mg/kg b.w.) was found to be efficacious than the composite extract of TCHE (350mg/kg b.w.) & JRHE (800mg/kg b.w.).

The present study explored the assessment of the antidiabetic potential of Tinospora cordifolia & Juglans regia composite extract in STZ induced diabetes in wistar rats. As streptozotocin -associated in iltrations of increase glucose level has been reported to be responsible for diabetes. We evaluated the glucose lowering potential of Tinospora cordifolia & Juglans regia on the basis of its anti-diabetic property. Rats were administered streptozotocin (55 mg/kg i.p., once) with nicotinamide (120mg/kg) to induce experimental toxicity. The development of diabetes was assessed biochemically as well as histologically 72 hours after induction of diabetes. Body weight and blood glucose levels were determined in (0, 7th, 14th, 21st, 28th) days. Serum lipid pro ile and enzyme estimated, (kidney, liver, pancreas) tissue was measured at the end of the experimental period. Treatment with composite extracts TCAE high dose (350 gm/kg b.w.) & JRAE high dose (800 mg/kg b.w.) and TCHE high dose (350 gm/kg b.w.) & JRHE high dose (800 mg/kg b.w.) were noted to be more effective against the streptozotocin-induced toxicity as compared to Glibenclamide (5 mg/kg b.w.). it may be concluded that streptozotocin-induced glucose may be accountable for the induction of diabetes toxicity in rats. Interestingly, improvement in body weight, glucose level, lipid pro iles, biochemical parameters and histopathological changes in kidney, liver and pancreas was observed following herbal treatment in STZ induced diabetic rats. Furthermore, composite extract of TCAE (350mg/kg b.w.) & JRAE (800mg/kg b.w.) was found to be ef icacious than the composite extract of TCHE (350mg/kg b.w.) & JRHE (800mg/kg b.w.).

INTRODUCTION
Diabetes mellitus (DM) is a global public health problem with an escalating incidence and prevalence, particularly in developing and newly industrialized countries. DM is a disease which decreases the body capacity to make or use insulin. Insulin is a hormone conveyed in the pancreas (beta-cells) that facilitates shipping (glucose) from the dissemination framework into the cells so they can isolate it and use it for body fuel. People cannot survive without insulin (Riaz, 2009). We currently watch high rates of DM-associated removals, cerebrovascular contamination, coronary heart-related problems, and kidney disease in masses that became now not as of late appreciate for those trying out medicinal issues (Stratton et al., 2000). Diabetes includes more treatment in different ways, like "broken red coral, oil of roses, unre ined quinces and slop, dates, sweet almonds and new blossoms of outwardly hindered weeds (Lakhtakia, 2018). Glibenclamide is an II -generation sulfonylurea that decrease blood glucose level by increasing insulin secretion from pancreatic beta cells. Glibenclamideinduced hypoglycemia more likely in the elderly in patients with irregular eating habits, and in renal impairment (Naidoo et al., 2014). Tinospora cordifolia is very important herb in ayurvedic system of medicine. Tinospora cordifolia are used in various somatic, psychosomatic and lifestyle disorder of human being since times immune Systemic and proper use of Giloy can cure various life-threatening disorder like diabetes, arthritis, fever, malignancy etc (Biswas et al., 2015). Juglans regia is important herb in ayurvedic system of medicine. These are used in various diseases like diabetes, in lammation, cancer etc. On the basis of ability of Tinospora cordifolia & Juglans regia to decrease the glucose level. The present study has been designed to investigate the anti-diabetic potential of a composite extract of Tinospora cordifolia and Juglans regia against Glibenclamide-induced diabetic toxicity. Dried Tinospora cordifolia Stem & Juglans regia kernel were powdered mechanically through mesh sieve. The drug powder of Tinospora cordifolia & Juglans regia was extracted with alcohol (Ethanol 90%) and Hydroalcoholic (Ethanol 50% and 50% water) solution using the Soxhlet method (Mishra et al., 2014).

Experimental groups
All the experimental animal was divided into 13 groups with each group consisting of 6 animals as follows: Group 1-Control: This group was used for studying the baseline values of the parameters studied. Group 2-Positive control: This group consisted of streptozotocin (55mg/kg/b.w.) + Nicotinamide (120mg/kg/b.w.) induced diabetic rats. Group 3-Diabetic rats treated with (5 mg/kg b.w.) Glibenclamide. Group 4-Diabetic rats treated with (350 mg/kg b.w.) high dose of alcoholic extract of TC.

Drug treatment
The dosing of all the groups were given from 0 to 28th once daily orally. Parameter i.e. Body Weight, Glucose have been evaluated on day 0, 7th, 14th, 21th, 28th, Animals were senseless with diethyl ether and blood was compiled by retro orbital puncture. The serum evaluated for serum Blood Glucose on day 0, 7th, 14th, 21th, 28th and HDL, LDL, VLDL and were assed 28th day AST, ALT, ALP, ALB, Bilirubin.

Body weights and blood glucose levels
Body weight and glucose level of rats were measured by using a weighing balance (Ekambaram et al., 2010) and Blood sample was collected from retro-orbital of rat and measured glucose level by Dr. Morepen glucometer was bought from Morepen Laboratories constrain (Chougale et al., 2009).

High density lipoprotein (HDL)
Measuring of High-density Lipoprotein (HDL) Was done by Phosphotungstic acid Method with the procedure from Germany kit. The HDL level was measured by using the formula given below

Total Cholesterol
Estimating of TC was done by CHOD/PAP method with the procedure from Germany kit.

Albumin (ALB) level measurement
Measured of Albumin (ALB) was done by Bromocresol green (BCG) Dye Method with the procedure from Germany kit.

AST
Measured of Serum glutamic-oxaloacetic transaminase (SGOT/AST) was done by international federation of clinical chemistry (IFCC) method with the procedure from Germany kit. The SGOT level was measured with the formula

ALT
Measured of Serum glutamic pyruvic transaminase (SGPT/ALT) was done by international federation of clinical chemistry (IFCC) method with the procedure from Germany kit (Diagnostic System International) cat no.10 135 021. The ALT level was measured with the formula

Statistical analysis
All values were expressed as mean ± SEM. One-way ANOVA, followed by Tukey's multiple comparison test, was applied for statistical analysis of the data obtained from different groups. For statistically signi icance, p-value ≤0.05 was considered.

Effect of Alcoholic and Hydro-alcoholic extracts on Body weight in wistar rats.
The body weight of rats was signi icantly (P < 0.05) reduced as compared to control and standard groups. Oral administration of TCAE at 350mg/kg/b.w. and 100mg/kg/b.w. signi icantly (P < 0.05) improved as comparision with positive control. TCHE at 350mg/kg/b.w. signi icantly (P < 0.05) improved body weight at 14th, 21st and 28thday time point while the improvement was seen to be on 14th and 28th days respectively at 100 mg/kg b.w. which was approximately equal to standard diabetic group. JRAE at 800mg/kg b.w. and 200mg/kg b.w. signi icantly (P < 0.05) improved body weight on 14th, 21st and 28th days, respectively. JRHE at 800mg/kg/b.w. signi icantly (P < 0.05) improved which was almost better to standard diabetic group. Combination dose of alcoholic TCAE (H.D) + JRAE (H.D) signi icantly (P < 0.005) increased body weight which was better as compared to standard diabetic group. Combination dose of hydroalcoholic TCHE (H.D) + JRHE (H.D) signi icantly (P < 0.05) increased the body weight observed on 7th,14th, 21st and 28th days respectively. Body weight changes among different groups are shown in Table 1 and Figure 1.

Effect of Alcoholic and Hydro-alcoholic extracts on glucose levels.
Glucose level of STZ induced positive control group rats (after 28 days) was signi icantly (P < 0.05) elevated as comparison with control and standard group. Oral administration of TCAE at 100 mg/kg/b.w. signi icantly (P < 0.05) decreased glucose level as comparison with positive control and at 350 mg/kg/b.w. glucose level reduction was observed on 14th, 21st and 28th days respectively. TCHE at 350mg/kg/b.w. signi icantly (P < 0.05) evaluated which was almost equal as compared to standard diabetic group. TCHE at 100 mg/kg/b.w. signi icantly (P<0.05) decreased glucose level as compared with positive control. JRAE at 800mg/kg/b.w signi icantly (P<0.05) reduced glucose level as compared with positive control and at 200 mg/kg/b.w. signi icantly (P<0.02) reduced glucose level when compared with positive control group. JRHE at 200 mg/kg/b.w. signi icantly (P<0.02) decrease glucose level observed on 14th and 28th day only and at 800mg/kg/b.w. significantly (P<0.05) reduced glucose level which was better as compared to standard diabetic group. Combination dose of alcoholic TCAE (H.D) + JRAE (H.D) signi icantly (P < 0.05) decreased glucose level which was better as compared to standard diabetic group. Combination dose of Hydroalcoholic TCHE

Biochemical pro ile of alcoholic and hydroalcoholic extracts
Effect of alcoholic and hydro-alcoholic extracts on lipid pro ile and enzymatic parameters are mentioned below.  Table 3 and Figure 3.  Table 4 and Figure 4.

Liver
Histopathological pro ile of liver necrosis and ibrotic changes with conspicuous evidence of fatty deposits in the diabetic liver when comparison with normal control group presenting the central vein with radiating cords of hepatocytes. Glibenclamide group shows normal portal tract (PT   Histological changes in islets of Langerhans in pancreas after treatment with streptozotocin. Control bunch demonstrating ordinary engineering of the pancreas. The exocrine segment types of pancreas irmly pressed by acinar cells and masterminded into little lobules. The islets showed up softly recoloured than the encompassing acinar cells. Positive benchmark group uncovered neurotic changes of both exocrine and endocrine parts. The acinar cells were swollen and little vacuoles were seen in practically all acinar cells. Islet β-cells are primarily lost in STZ-rewarded rodents. Glibenclamide indicating bending of the overall design. Most exocrine acini uncovered acinar harm spoke to by cytoplasmic vacuolation and cell decay. Wider interlobular and intralobular duct were observed. Treatment TCAE 800 mg/kg demonstrated an ordinary structure of islets of Langerhans. Atrophic difference in the acinar cells was less serious and the fringe among exocrine and endocrine parts turned out to be progressively unmistakable.  Figure 7

DISCUSSION
Diabetes mellitus is a metabolic disorder that in luences individuals of all age gatherings and from varying backgrounds. The executives of diabetes with no symptoms is as yet a test in the clinical ield, as by and by accessible medications for diabetes have at least one antagonistic impact. Since the current medications for the treatment of diabetes mellitus do not satisfy our need totally, the quest new medications continue (Shobha, 2015). Other than commendable improvement in the ield of allopathic drugs, the herbal drug additionally has their radiant importance as they have no side effect not at all like allopathic medications. Therefore, an ef icient approach has been done to ind out the utility of Tinospora cordifolia and Juglans regia in composite against diabetes. Review of literature Tinospora cordifolia revealed that this plant various pharmacological activities like -diabetes, microbial, obesity, in lammatory, pain-relieving, depressant, oxidant, cancer etc. (Tripathi et al., 2011). and Juglans regia revealed that this plant possesses pharmacological activities like diarrhea, stomachache, arthritis, asthma, eczema, skin disorders, and various endocrine diseases such as diabetes mellitus, thyroid dysfunctions, anorexia, cancer and infectious diseases etc. The Present study was title to evaluated the antidiabetic activity of alcoholic and hydroalcoholic extracts of stem of Tinospora cordifolia and kernel of Juglans regia in STZ induced diabetes in wistar rats for 28 days. Its Lipid pro ile, enzymatic activity was investigated on 28 days. And histopathological study was examined in the kidney, liver, pancreas.
Data analysis of this present study revealed that the following groups such as all treated groups improved body weight but body weight increased the most in the combination group, normalized the blood glucose level and restored the lipid pro ile as well as liver biochemical parameters towards the normal range. Our inding revealed that all treated group had a positive effect in lowering the blood glucose and body weight on 28th day as compared to the initial observation on 0th day. A previous study had shown that the methanolic extract of stem Tinospora cordifolia 250mg/kg.b.w. Decreased the blood glucose level and activities of glucose-6phosphatase, fructose-1,6disphosphatase (Rajalakshmi et al., 2009). Previous study showed that Tinospora cordifolia has signi icant (P < 0.05) antidiabetic activity in diabetic animals and has viability of 40% to 80% compared to insulin. Tinospora cordifolia administration in diabetic animals' recovery of pancreatic β cells but showed increased hepatic glycogen synthase and decreased glycogen phosphorylase activity (Ekambaram et al., 2010). Alcoholic extract of Juglans regia has protective effect on streptozotocin-nicotinamide induced diabetic rats. Streptozotocin-nicotinamide injection can give rise diabetes mellitus, and ruin the β-cells in the islets of Langerhans. The increase of blood glucose could damage β cells and lead to reduction in consumption of glucose by muscle tissues. Alcoholic extract of Juglans regia (400mg/kg/b.w.) decreased the blood glucose level and regenerate of β cells (Puranik et al., 2010).
In our study alteration in lipid pro ile such as HDL, LDL, VLDL, triglycerides and total Cholesterol was observed on 28th days in diabetic rats when compared to treatment and standard group. Elevated cholesterol, LDL, VLDL and HDL were normalized in diabetic rats after the treatment of some treated groups did not produce signi icant effect on lipid pro ile. The previous study of (Kannadhasan and Venkataraman, 2012) had shown that the ethanolic extract of Tinospora cordifolia (1000 mg/kg) reduced the total cholesterol, triglyceride level near to the normal and increased in HDL and more signi icantly decrease of LDL compared with diabetes control (Kannadhasan and Venkataraman, 2012). Ethanolic extract of Tinospora cordifolia (200mg/kg/b.w.) The levels of total cholesterol, triglycerides LDL-C and VLDL-C were increased in diabetic rats whereas the level of HDL-C were reduced in diabetic rats when compared to the control normal rats. Administration of Ethanolic extract of Tinospora cardifolia to alloxon induced diabetic rats restored all these changes to near normal levels by reduction of the level of total cholesterol, triglycerides, LDLC and VLDLC of diabetic rats and signi icant increase in the level of HDL-C (Ravikiran et al., 2015).

Extracts of stem Tinospora cordifolia and kernel
Juglans regia not only decrease the level of triglyceride and total cholesterol but also the restore the function of the hepatic enzyme by inhibition of oxidative stress. Study by (Bostani et al., 2014) had shown that the ethanolic extract of leaf Juglans regia (200mg/kg) decreased the serum AST and ALT level compared to diabetes control group. Elevation of renal enzymes such as AST, ALT, ALP, Albumin, and Bilirubin was also evaluated in diabetic rats pointed to trained liver function due to hepatic damage (Khedekar, 2016). Treatment with herbal formulation at the dose of 200 and 400 mg/kg/day along with alcohol showed reduced levels of SGPT, SGOT, albumin, creatinine and increased the levels of total protein as compared with alcohol treated group (Sreshta et al., 2018). 28 days treatment with TCAE and TCHE at (350mg/kg/b.w.) and (100 mg/kg/b.w.), JRAE and JRHE at (800mg/kg/b.w.) and (200mg/kg/b.w.) and composite of TCAE (HD) + JRAE (HD) and TCHE (HD) + JRHE (HD) restored al the biochemical parameters toward the normal levels.
Histopathology, kidney section of STZ induced diabetes mice showed damage epithelium cells, proteinuria and necrosis, while the extract of Tinospora cordifolia (50, 100, 200 mg/kg/b.w) normal tubules with congested glomerulus, and showed congested glomerulus and tubules with vacuolated epithelial cells (Gupta and Sharma, 2011). Which was similar to the extracts of Tinosora cordifolia, Juglans regia and their combination. Liver section of STZ induced diabetic rats showed necrosis and ibrotic changes with conspicuous evidence of fatty deposits, which got restored by extract of Tinospora cordifolia, Juglans regia and their composite treatment. The extract of Tinospora cordifolia produced protective effects in the kidney against lead toxicity (Sharma and Pandey, 2010). The extract of Tinospora cordifolia (100mg/kg) changed in the centrilobular region and vacuolar congestion and graded 1-2 by hepatoprotective system (Gurav et al., 2017). Pancreas section of STZ induced diabetes rats showed damaged pancreatic islets cells and the extract of Tinospora cordifolia comparatively less degeneration of Islets of Langerhans and degranulation (Sreshta et al., 2018) which was also similar to our inding.

CONCLUSIONS
Taken together, the present study for the irst time reported the effect of Tinospora cordifolia and Juglans regia (Alcoholic and Hydroalcoholic) and their composite extract in STZ induced diabetes rats. Interestingly, improvement in body weight, glucose level, lipid pro iles, biochemical parameters and histopathological changes in kidney, liver and pancreas was observed following herbal treatment in STZ induced diabetic rats. Furthermore, composite extract of TCAE (350mg/kg b.w.) and JRAE (800mg/kg b.w.) was found to be more ef icacious than the composite extract of TCHE (350mg/kg b.w.) and JRHE (800mg/kg b.w.).