الفهرس 
AcknowledgementOnly 14 pages are availabe for public view
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Abstract
There is a sharp increase in the incidence of T2DM and obesity as a result of aging, dietary habits and decreased physical activities. The majority of diabetic patients consume sweeteners with low calories to decrease their calorie intake. Most artificial sweeteners, such as cyclamates and saccharine, are a source of high calorie sugars and are potential carcinogens. The increased incidence of diabetes and obesity and the growing concern over the safety of some chemical sweeteners such as aspartame, cyclamate, saccharin, sucralose, etc., have stimulated the use of natural non-calorie sweeteners. In addition, the currently available hypoglycemic agents for management of diabetes mellitus are ineffective and have certain drawbacks. The United Kingdom Prospective Diabetes Study (UKPDS) showed that monotherapy with oral agents often fails to maintain glycemic control over time, and many patients have to switch to treatment with combinations of oral agents or insulin therapy. Therefore, there is a demand for new natural-based medicinal compounds with diversed actions. Stevia rebaudiana Bertoni is a medicinal plant with multiple potential benefits. Nevertheless, the need still exists for more thorough investigation of the pharmacological activity of stevia and, particularly; its possible use and interaction with the other known marketed anti-diabetic agents. Thus, the present study was undertaken to study the anti-diabetic effect of S. rebaudiana alone and in combination with other anti-diabetic drugs in rats to prove a new strategy for treatment of patients with diabetes mellitus or to use it in combination with small doses of oral anti-diabetic drugs in patient with overt diabetes mellitus. Thus, this thesis is devoted to evaluate the potential effectiveness of stevia extract alone and in adjunct with anti-diabetic agents in treating streptozotocin-induced diabetes in albino rats. In the first set of experiments we evaluated the acute single administered effect of stevia on the blood glucose levels (BGL) in euglycemic rats. The rats were divided into 5 groups, 8 rats in each. Animals of group-I received saline, group- II were treated with stevia extractorally (150 mg/kg), group- III were treated with stevia extract orally (300 mg/kg), group- IV were treated with stevia extract orally (450 mg/kg), group- V were treated with stevia extract orally (600 mg/kg). In the second set of experiments, we evaluated the acute single administered effect of different doses of stevia extract on the BGLs in diabetic rats. The diabetes was induced in the rats by a single intraperitonial injection (I.P) of 230 mg/kg of nicotinamide (NA) followed by 55 mg/kg of streptozotocin (STZ), which was freshly prepared in citrate buffer, after an overnight fasting. The rats with FBG higher than 200 mg/dL after 72 h of STZ injection were considered diabetic and were selected for the study. Diabetic rats were divided into 5 groups of 8 animals in each group. The animal groups were divided as the following: group I received saline (control); group II: treated with stevia extract orally (150mg/kg); group III: treated with stevia extract orally (300mg/kg); group IV: treated with stevia extract orally (450mg/kg); group V: with stevia extract orally (600mg/kg). In the Third set of experiments, we evaluated the acute combined effect of administration of stevia and other anti-diabetic drugs on BGL of diabetic rats. The diabetic rats were divided into 5groups, 8 rats each. group I received saline (Control-diabetic); group II recieved saline with 0.5 % tween80 (Control); group III treated with stevia extract (300mg/kg) plus repaglinide (0.3mg/kg); group IV treated with stevia extract (300mg/kg) plus glimipiride (1mg/kg) and group V treated with stevia extract (300mg/kg) plus metformin (250mg/kg). Control animals were treated likewise with pure vehicles.
In the fourth set of experiments, we evaluated of the chronic administered effect of stevia and/or other anti-diabetic drugs on BGL and certain pharmacological parameters in diabetic rats. The rats were divided into 10 groups of 8 animals in each group. group I received saline (Control- non diabetic); group II received saline (Control- diabetic); group III received with 0.5 % tween 80 (Control-diabetic); group IV diabetic treated with stevia extract (300mg/kg); group V diabetic treated with repaglinide (0.3mg/kg); group VI diabetic treated with stevia extract (300mg/kg) plus repaglinide (0.3mg/kg); group VII diabetic treated with glimipiride (1mg/kg); group VIII diabetic treated with stevia extract (300mg/kg) plus glimipiride (1mg/kg); group IX diabetic treated with metformin (250mg/kg); group X diabetic treated with stevia extract (300mg/kg) plus metformin (250mg/kg). The diabetic rats were treated with the stevia extract and /or anti-diabetic agents for 3 weeks. Control animals were treated likewise with pure vehicles for the same duration. At the end of experimental duration, the overnight fasted rats were sacrificed by decapitation. Blood, liver and kidney tissues were obtained from each animal for biochemical measurements. A part of each blood sample (5ml from each animal) was used for estimation of glucose level in the blood. The other part of each blood samples was centrifuged for 10 min using Eppendorff centrifuge (Hettich EBA 12, ZENTRIFUGEN, Tuttlingen, Germany). After centrifugation, the serum was collected for estimation of the activities of aspartate aminotransferase (AST/GOT), and alanine aminotransferase (ALT/GPT) (i.e liver function tests); the levels of creatinine and urea (i.e kidney function tests); insulin level; adiponectin level; tumor necrosis factor-alpha (TNF-α) as well as total cholesterol level, triglycerides and high density lipoprotein levels (i.e lipid profile). A part of each liver and one kidney from each animal were kept in 10% formalin for histopathological and immunohistochemical studies and the other part of the each liver and the other kidney were rinsed in ice-cold saline, dissected, cleaned from fat and other tissues and blotted carefully. Part of each liver and kidney tissues were cut into small pieces and a suitable weight from liver and kidney tissues were homogenized in 10% w/v phosphate buffer (pH 7.4) or saline by using a motor-driven Teflon pestle (Glas-Col, USA).The homogenate was centrifuged for 10 min at 10,000 rpm and the supernatant was used for estimation of malondialdehyde (MDA). Acute treatment of euglycemic rats with different doses of stevia extract (150, 300, 450 and 600) mg/kg orally produced a dose dependant decrease in the blood glucose levels.Similarly, acute treatment of diabetic rats with different doses of stevia extract (150, 300, 450 and 600) mg/kg orally produced a dose dependant decrease in the blood glucose levels of diabetic rats more evident after 4h and 6h intervals. Acute combined treatment of diabetic rats with a single dose of stevia extract 300 mg/kg and repaglinide 0.3 mg/kg orally produced a significant decrease in the blood glucose levels which appear early after 1h and persist up to 8h. Acute combined treatment of diabetic rats were with a single dose of stevia extract 300 mg/kg and glimipiride 1 mg/kg orally produced a significant decrease in blood glucose levels continue up to 8h. Diabetic rats treated with a single dose of stevia extract 300 mg/kg and metformin 250 mg/kg orally showed prominent decrease in blood glucose levels at time intervals (1h, 2h, 4h, 6h and 8h.). Unlike the other used anti-diabetic drugs, the peak effect of metformin appeared delayed after 4h. However the peak effect of repaglinide and glimipiride appeared after 1h and 2h respectively. chronic treatment of diabetic rats with 300 mg/kg/day stevia extract orally for 3 weeks produced a significant decrease (p< 0.001) on the blood glucose levels in comparison to initial diabetic values. Diabetic rats treated with 250 mg/kg/day metformin orally for 3 weeks showed insignificant decrease (p> 0.05) in blood glucose levels all over the duration of the therapy. While diabetic rats treated with combination of stevia extract 300 mg/kg/day and metformin 250 mg/kg/day orally for the same duration showed significant decrease (p< 0.001) in blood glucose levels. Concurrent administration of stevia extract 300 mg/kg/day and glimipride 1 mg/kg/day orally for 3 weeks showed significant decrease (p< 0.001) on blood glucose level. The combination therapy of stevia extract and glimipride caused an earlier decrease (170.5±9.5) on blood glucose levels at the day15 versus to diabetic rats treated with glimipride alone (283.8±13). Similarly, diabetic rats treated with combination of stevia extract 300 mg/kg/day and repaglinide 0.3 mg/kg/day orally for 3 weeks showed significant decrease (p< 0.001) on blood glucose levels. The addition of stevia extract to repaglinide caused more decrease in blood glucose levels (126.3±2.4) than diabetic rats treated with repaglinide alone (176.3±21. 4) especially at the day 21. Therefore the combination therapy of stevia extract and anti-diabetic drugs showed more decrease in the blood glucose levels versus to anti-diabetic drugs alone. Diabetic animals treated with 300 mg/kg/day stevia extract orally for 3 weeks showed lower mean of renal injuries than those treated with metformin, glimipiride or repaglinide alone. Combination therapy of stevia & metformin or stevia & repaglinide or stevia & glimipiride significantly reduced the renal injuries than those treated with stevia alone. Improvement of the hepatic injuries was noted in stevia treated group with insignificant difference in comparison to those treated by drugs alone. It is of note that, combination of stevia & metformin or stevia & repaglinide or stevia & glimipiride significantly reduced the liver injuries than those treated with stevia or the drug alone. Daily treatment of diabetic rats with 300 mg/kg/day stevia extract orally for three weeks produced a significant decrease in the serum AST and ALT activities as well as the serum urea and creatinine levels. Also, daily combined treatment of diabetic rats with stevia & metformin or stevia & repaglinide or stevia & glimipiride significantly reduced AST and ALT activities as well as the serum urea and creatinine levels. Important effect of stevia extract was detected in increasing the serum insulin level of diabetic rats. Moreover, the combination of stevia extract and insulin releaser anti-diabetic drugs like glimepiride and repaglinide showed additive increase in the insulin level Besides, combination of stevia & metformin showed significant increase in the serum insulin level of diabetic rats versus diabetic rats treated with metformin alone. The impact of administration of stevia extract and/or anti-diabetic drugs on the lipid profile showed a significant improvement. While combinations of stevia extract and anti-diabetic drugs showed remarkably higher improvements regarding decreasing the levels of TC and TGs and increasing the levels of HDL than that of extract or drug alone. The results indicated that aqueous extract of stevia has a significant effect on increasing the serum level of adiponectin. Similarly, the effect on serum adiponectin level was more prominent on combination groups. In addition, notable modulation of oxidative stress marker (MDA) by stevia extract at a dose 300mg/kg/day was observed in the renal and hepatic tissues of diabetic rats after treatment for 3 weeks compared to control group. Similar effects were noted in combination of stevia and anti-diabetic drugs which showed significant reduction in both renal and hepatic MDA levels. A prominent anti-inflammatory activity in stevia extract a dose 300mg/kg/day and combination therapy of stevia and anti-diabetic drugs was noted in the rat’s serum by significant reduction in the level TNF-α after 3 weeks of treatment. In this study, the immunohistochemical analysis of the kidney tissues showed that, administration of 300 mg/kg/day stevia extract orally diabetic rats for three weeks significantly decreased the intensity of eNOS immunostaining compared to diabetic rats. Concomitant administration of 300 mg/kg/day stevia extract with metformin or repaglinide or glimipiride orally to diabetic rats for three weeks significantly decreased the protein expressions of eNOS in the kidney tissues. Thus, stevia extract could protect the microstructure of the kidney from injury due to oxidative stress in STZ-induced diabetic rats. Results of this work lead to the following conclusions The finding of the present study throw some light on a new effective and safe anti-diabetic combination based on natural zero calorie sweetener (stevia rebaudiana bertoni) with the commonly used anti-diabetic drugs in the treatment of type 2 diabetes mellitus. This natural plant will offer a natural, inexpensive and safe anti-diabetic agent instead of less effective, expensive agents, which also accompanied by adverse effects. The results of the present study demonstrated that stevia extract alleviated the STZ- induced diabetic changes as evidenced by: Decrease in blood glucose level (BGL) Decrease in the biomarkers of STZ-induced hepatotoxicity (AST and ALT activities) and nephrotoxicity (urea and creatinine).Improvement in lipid profile (decrease in TC and TGs as well as increase in HDL-C).Improvement of glycometabolic parameters which evidenced by increase in serum insulin and adiponectin level. Ameliorating oxidative stress marker (decrease in renal and hepatic MDA) Anti-inflammatory activity and enhancement of insulin sensitivity through reduction of the inflammatory cytokine TNFα Increase the renal protection through decrease the expression of eNOS protein.It is recommended that combination of aqueous extract of stevia at a concentration 300 mg/kg plus 250 mg/kg metformin or repaglinide 0.3 mg/kg or glimipiride 1 mg/kg may provide an effective therapeutic anti-diabetic combination for the treatment of diabetes and its associated complications.