Egyptian Journal of Basic and Clinical PharmacologyVol. 10 (2020), Article ID 101455, 15 pagesdoi:10.32527/2020/101455 www.kenzpub.com
Research Article
The Potential Efficacy of Stevia Extract, Glimepirideand Their Combination in Treating Diabetic Rats:A Novel Strategy in Therapy of Type 2 DiabetesMellitus
AbdelAzim Assi1, Doaa H. Abd Elhamid1, Mahran S AbdelRahman1, Esraa E. Ashry1,Soad AI Bayoumi2, and AsmaaM. Ahmed3
1Department of Pharmacology, Faculty of Medicine, Assiut University, Assiut, Egypt2Department of Pharmacognosy, Faculty of Pharmacy, Assiut University, Assiut, Egypt3Department of Pathology, Faculty of Medicine, Assiut University, Assiut, Egypt
Abstract. Background: The currently therapy of type 2 diabetes is unsatisfactory. Nowadays, there is a great interest in usingthe medicinal plants for treatment of diabetes. Therefore, we studied the efficacy of stevia extract alone and in combination withcommonly used sulfonylureas ”glimepiride” in a trial to introduce a new effective therapeutic regimen for type 2 diabetes mellitus.Methods: Nicotinamide (230 mg/kg, IP) followed by streptozotocin (65 mg/kg, IP) were injected for induction of type 2 diabetesin male rats. The diabetic groups were treated for 21 days as the following, the first with stevia extract (300 mg/kg), the secondwith glimepiride (1 mg/kg), and the third with a combination of glimepiride and stevia extract. Many parameters were measuredto evaluate the alleviation of the toxic effect of streptozotocin by stevia and/or glimepiride. Immunohistochemical expression ofendothelial nitric oxide (eNOS) was assessed in renal tissues. Results: The stevia extract reduced the blood glucose, triglycerides,cholesterol, ALT, AST, urea, creatinine, tumour necrosis factor (TNFα) levels and malondialdehyde concentration compared tocontrol diabetic group. Stevia treatment improved insulin and adiponectin levels. Stevia reduced eNOS expression in renal tissuescompared to the diabetic rats. All these changesweremore significant with combined treatment. Conclusion: Stevia and glimepiridemay be a new putative therapeutic regimen for management of type 2 diabetes and its complications.
Keywords: Type 2 diabetes mellitus, stevia, glimepiride, streptozotocin and nitric oxide synthase.
1. Introduction
Diabetes mellitus (DM) is a global problem, character-ized by high levels of glucose in the blood. The WorldHealth Organization demonstrates that diabetes affects 366million people all over the world and new predictionsestimate that 552 million people will be diabetic on2030 [1]. With high rates in the incidence of obesity,
the prevalence of T2DM is also increased. Thus, iden-tification of new therapeutic antidiabetic regimens thatare beneficial for patients with T2DM is of great need[2].
The current therapies for T2DM are unsatisfactory. Forregulation of the blood glucose levels as near to normalvalues as possible, diet control, exercise, and hypoglycemicagents are needed. Most of diabetic patients often complain
2 Egyptian Journal of Basic and Clinical Pharmacology
of secondary failure after prolonged treatment with sulfony-lureas (SUs) [3].
Sulfonylureas, although commonly used as a second linetherapy, often cause hypoglycemia and weight gain [4].Besides, having many adverse effects, none of the oral antidi-abetic agents successfully maintain euglycemic state andcontrol the micro- and macro-vascular complications. Thus,United Kingdom Prospective Diabetes Study (UKPDS)demonstrated that single therapy of oral antidiabetic drugsusually shows a failure in the regulation of euglycemic statethrough a long time, and most of diabetics need to changethis monotherapy to eighter combinations of oral antidiabeticdrugs or insulin therapy [3].
Moreover type 2 Diabetes show complex pathophysi-ological changes, so that the ideal antidiabetic combina-tion strategy: provides multiple mechanisms of action thatmaintain the function of pancreatic beta cell, improve theproblem of insulin resistance, be well tolerated with littlerisk of hypoglycemia or weight gain, as well as providecardiovascular and renal protective benefits [4, 5]. Therefore,the need for newer and safer agents for treatment of T2DMis impelling. Nowadays, there is a huge interest in the useof herbal agents for the management of T2DM. Thus, manyclinical studies are done to estimate the antihyperglycemicaction of medicinal plant. Among these herbal plants “Ste-via rebaudiana Bertoni” which was extensively used as atraditional medicine to control hypertension, hyperglycemia,and hyperlipidemia. Stevia is famous due to its sweetnessproperty and its usefulness action in blood glucose control[6]. Glycosides of stevia like stevioside and rebaudiosideA have been established as natural zero-calorie/low-caloriesweeteners [7]. Awing to the sweetening power and potentialtherapeutic benefits of its leaves; Stevia has attracted botheconomic interest and scientific research.
Studies reported that the stevia exhibits significant phar-macological activities such as: antihypertensive, antihyper-glycemic, anticancer, antiobesity, anti-diarrheal and enzymeinhibitory activities [7, 8]. Treatment with stevia extract isleading to enough antihyperglycemic action, with a little riskfor hypoglycemia, reduced body weight, and other beneficialhealth effects like antioxidant, hepatic and renal protection[9, 10]. However, thorough investigation of the pharmaco-logical interaction between the stevia and commonly usedantidiabetic agents (glimepiride) are needed.
Therefore, this study was designed to provide a newexperimental strategy for the effectiveness of stevia extractin combination with glimepiride (tradename AmarylTM)compared to glimepiride alone in treating T2DM.
2. Materials andMethods
2.1. Chemicals. Streptozotocin powder was purchasedfrom Sigma-Aldrich Chemical Company (Germany)and Glimepiride powder (AmarylTM) was given frompharmaceutical companies (EIPICO) company, Egypt as agift.
2.2. Plant materials. Leaves of Stevia rebaudiana Bertoni,family Asteraceae were obtained from farm in Assiut, Egyptduring the flowering stage (April to July 2016). Stevia leaveswere cleaned with water to eliminate the dust particles. Then,the leaves of stevia were dried.
2.3. Extraction of the plant. The dried powdered leaves (5kg)of stevia rebaudiana Bertoni was macerated in 70% ethanol(10 L x 3) for extraction. Followed by concentration of thealcoholic extract and the solvent free residue was (835 g)(16.7% w/w). Then, 500 mL of distilled H2O was added toa Part of the alcoholic extract (425 g) and repeated solventfractionations with dichloromethane was done till completeexhaustion using Rotary flash evaporator, HeidolphWB2000(Germany). Concentration of the dichloromethane fractionand aqueous fractionwere done thus, the solvent free residueswere (87 g) and (336 g) respectively.
2.4. Experimental animals. Forty eight adult male ratsweighing 180-200 g obtained from the animal home ofFaculty of Medicine, Assiut University and were lived instainless-steel cages (five rats in each cage). Rats were givenwater and food ad libitum. The animals were housed withideal conditions of temperature (22 ± 2°C) and humidity(55 ± 5%) with 12-light/12-dark cycle. Animal handlingand rights were maintained on accordance with the EthicalCommittee guidelines of the Faculty of Medicine, AssuitUniversity.
2.5. Induction of diabetes. For induction of T2DM, 230mg/kg nicotinamide and 65 mg/kg of streptozotocin, whichwas freshly prepared in citrate buffer (0.1 M, pH 4.5) wereinjected I.P in male rats [11]. The STZ-injected rats wereinfused glucose (20%) in the first 24 h to avoid the initial STZ-induced deaths due to hypoglycemia. Occurrence of T2DMwas insured by measuring the blood glucose level, 72 h fromI.P administration of steptozotocin. STZ-injected rats withblood glucose levelsmore than 200mg/dLwere experimentalmodels of T2DM and included in the experiment.
2.6. Experimental design. Forty eight wistar rats were classi-fied into following groups: Group I: received saline (Control-non diabetic); Group II: received saline (control diabetic);Group III: received 0.05% tween 80 (control diabetic); GroupIV: diabetic rats treated with 300 mg/kg aquatic extractof stevia; Group V: diabetic rats treated with 1 mg/kgglimepiride; Group VI: diabetic rats treated with 300 mg/kgaquatic extract of stevia combined with 1 mg/kg glimepiride.
Stevia extract was prepared as solution (dissolve in saline),while glimepiride was dissolved in 0.5 % tween 80. Theaqueous extract of stevia and glimepiride were given orallyby stomach tube every day for 21 days.Control rats weretreated likewise with pure vehicles. Doses of glimepiride andstevia were determined in accordance with our preliminarytests and from literature review [12, 13].
Egyptian Journal of Basic and Clinical Pharmacology 3
2.7. Biochemical analysis. At the end of the experiment andbefore scarifying the experimental animals, blood sampleswere collected from the orbital sinus for determination ofglucose, insulin, adiponectin, TNFα levels, lipid profile,liver and kidney function test. Blood glucose was measuredby glucometer (Accu-Chek, Germany). Total cholesterol,triglycerides, high density lipoprotein (HDL), liver enzymesand renal function test were measured colorimetrically bySpectophotomer. The serum insulin, TNF-α and adiponectinwere estimated by using an enzyme-linked immunosorbentassay (ELISA) kits purchased from (Calbiotech., USA, SinoBiotech Co., Ltd and Elbascience, USA) respectively. Thelevel of renal and hepatic MDA was evaluated by a kitobtained from Biodiagnostic, Egypt. The hepatic and renaltissue from each rat, which were used for histopathologi-cal and immunohistochemical analysis, were kept in 10%formalin. The kidney and part of hepatic tissues weredissected to pieces for homogenization. The renal and hepatichomogenate were putted in the centrifuge for determinationof malondialdehyde (MDA) level from the supernatant.
2.8. Histopathological examination. After scarification ofthe animals, slices from the hepatic and renal tissues werefixed in buffered formalin (10%) and then processed for pho-tomicroscopic assessment [14]. The histopathologic changeswere scored according to the percentage of tissue affectioninto: 0 = (without pathologic changes), mild affection = 1%-30%, moderate affection = 31%-70% and severe affection(>70%).
2.9. Immunohistochemical analysis. Deparaffinization andrehydration of the sections (four µm thick) were done.After that, blocking of the endogenous peroxides activityby H2O2 (3%) was performed. Then, the sections wereimmersed in 10 mmol/l citrate buffer at pH 6.0 with furtherexposure to heating in a microwave at 80◦C for fifteenminutes to retrieve the antigens. The sections were incubatedwith the primary antibody (Rabbit Polyclonal Endothelialnitric oxide synthase (eNOS) dilution 1:100, ElabscienceBiotechnology Inc, USA) at room temperature overnight.The secondary staining kit (Thermo Scientific Corporation;Fremont, CA, USA) was used based on manufacturer’sinstructions. Immunoreactivity score of eNOS was evaluatedin the blood vessels, glomeruli and tubules. The stainingintensity was scored as: 0: negative, 1: weak, 2: moderateand 3: strong. The percentage of positive cells was scored as:0 = negative, 1 = (<10%), 2= (10% - 50%) and 3(> 50%).Then, both scores were added to provide a single score (0-6).
2.10. Statistical analysis. The results are represented as(means ± S.E.M.). The results analyzed by the GraphPad Prism (Graph Pad; San Diego CA, USA) program.The analysis of difference between groups was done usingthe (ANOVA) test and post hoc test, Bonferroni test. The
significance level was taken at P < 0.05, P < 0.01 and P <0.001.
3. Results
3.1. The Effect of different treatments on blood glucose levels(BGL). Stevia extract at a dose of (300 mg/kg) produced asignificant change in BGL in the form of decreasing the meanblood glucose levels upon daily administration for diabeticrats compared to control rats as represented in (Table 1).These findings were in accordance with previous results [12].It’s obvious from the (Table 1) that the stevia extract hasa significant ant-hyperglycemic action in diabetic rats. Ourfinding demonstrated a significant reduction in blood glucoselevel in glimepiride treated rats. The combined group ofstevia extract (300 mg/kg) and glimepiride (1 mg/kg) showeda more reduction in the BGL compared to glimepiride alone.It’s evident from the Table 1, that the combination therapyof stevia extract and glimepride caused an earlier decrease(170.5 ± 9.5) on BGL of diabetic rats at the day15 versus todiabetic rats treated with glimepride alone (283.8 ± 13).
3.2. Effect of different treatments on serum insulin level. Asshown in Figure 1, treatment of the rats with STZ showeda significant change in the serum insulin (p < 0.001) inthe form of decreasing the insulin level in comparison tonon diabetic rats. While diabetic rats treated with a 300mg/kg/day of stevia extract orally for three weeks showed asignificant increase (p < 0.001) in the serum insulin levelin comparison to control rats. Administration of insulinreleasing drug like glimepiride (1 mg/kg/day) to diabeticrats for the same duration showed a significant increase (p< 0.001) in the insulin level in comparison to control ratsfor (Figure 1). Combined administration of glimepiride andstevia to diabetic rats for the same period showed a moreelevation in the insulin level as compared to diabetic ratstreated with glimepiride alone.
3.3. Effect of different treatments on the serum adiponectinlevel. As shown in Figure 2, administration of 300mg/kg/day of stevia extract to diabetic rats for three weeksshowed a significant elevation in the adiponectin level.Combined administration of glimepiride 1 mg/kg/day andstevia extract 300 mg/kg/day to diabetic rats for the sameduration caused a significant elevation (p < 0.05) in theadiponectin level as compared to diabetic rats administeredglimepiride alone.
3.4. Effect of different treatment on lipid profile level
3.4.1. Influence on cholesterol level. Treatment of diabeticrats with stevia extract (300 mg/kg/day) caused a significantdecrease in the serum cholesterol (P< 0.05, Figure 3) in com-parison to untreated rats. Combined administration of stevia
4 Egyptian Journal of Basic and Clinical Pharmacology
Table 1: Influence of stevia and/or glimepiride on blood glucose levels of diabetic rats.
Regimens Blood glucose levels (mg/dl) at time intervalsGroups Treatment Dose and duration (mg/kg) Day 0 Day 5 Day 10 Day 15 Day 21
1 Control(non-diabetic)
Saline Saline Equal volumes single I.PEqual volumes orally for
** Significant difference at P < 0.01 vs. initial diabetic value
*** Significant difference at P < 0.001 vs. initial diabetic value.
N.B: There is no difference between different control vehicles.
0
10
20
30
40
50
Control-diabetic
Stevia
Glimipiride
***
control-non diabetic
Glimipiride+Stevia
***
***
++
In s
u lin
con
c. (µ
I U / m
l)
Values represent mean ± S.E.M.
- ++ Significant difference at P< 0.01 vs. non diabetic values
- *** Significant difference at P< 0.001 vs. control values.
Figure 1: Influence of daily administration of stevia and/or glimepiride for 21 days to diabetic rats on the insulin level.
extract (300 mg/kg/day) plus glimepiride (1 mg/kg/day) todiabetic rats showed a significant decrease in the cholesterollevel (P < 0.001, Figure 3) in comparison to diabetic ratsadministered glimepiride alone.
3.4.2. Effect on serum triglycerides. Treatments of dia-betic rats with stevia extract (300 mg/kg/day) orally forthree weeks caused insignificant decrease in serum (TG).Diabetic rats treated with glimepiride (1 mg/kg/day) forthe same period showed a significant reduction (P <
0.01, Figure 4) in serum (TG). Concurrent administra-tion of stevia extract (300 mg/kg/day) plus glimepiride (1mg/kg/day) to diabetic rats for a similar period produceda significant decrease in the (TG) level as compared tountreated diabetic rats (p < 0.001, Figure 4). The steviaenhanced the influence of glimepiride on the level oftriglycerides.
3.4.3. Effect on serum high density lipoprotein. Diabeticrats treated with stevia extract 300 mg/kg/day for three
Egyptian Journal of Basic and Clinical Pharmacology 5
0
2
4
6
8Control-non diabetic
Control diabetic
Stevia
Glimipiride
Glimipiride + Stevia
*** ***
+++
#
***Ad
ipon
ectin
(ng/
ml)
Values represent mean ± S.E.M.
· +++ Significant difference at P< 0.001 vs. non diabetic value.
· *** Significant difference at P< 0.001 vs. diabetic value.
· #Significant difference at P< 0.05 vs. glimepiride value.
Figure 2: Influence of daily administration of stevia and/or glimepiride for 21 days to diabetic rats on the adiponectin level.
0
10
20
30
40
50
60
70
80Control-non diabetic
Control diabetic
Stevia
Glimipiride
Glimipiride+Stevia
+++
* * #***
T C
(m g
/d l)
Values represent mean ± S.E.M.
- +++ Significant difference at P< 0.001 vs. non diabetic value.
- *Significant difference at P< 0.05 vs. diabetic value.
- ***Significant difference at P< 0.001 vs. diabetic value.
- #Significant difference at P< 0.05 vs. glimepiride value .
Figure 3: Influence of daily administration of stevia and/or glimepiride for 21 days to diabetic rats on the total cholesterol level.
weeks showed a significant elevation (p < 0.001, Fig-ure 5) in the HDL level as compared to control dia-betic rats. However, combined administration of steviaextract 300 mg/kg/day and glimepiride 1 mg/kg/day todiabetic rats orally caused a significant increase (p <0.05, Figure 5) in the HDL level as compared to ratstreated with glimepiride alone. Stevia extract increasethe effect of glimepiride on serum high density lipopro-tein.
3.5. Effect of different treatment of liver function test.Table 2, shows that treatment of the rats with STZ causedelevation of the liver function parameters. Administrationof 300 mg/kg/day stevia extractfordiabetic rats expresseda significant reduction in hepatic parameters (ALT andAST). Co-administration of stevia extract and glimepiride todiabetic rats caused more improvement in liver function.
3.6. Effect on kidney function test. As shown in Table 3, ratstreated with STZ exhibited elevation of the kidney functionparameters. Treatment of diabetic rats with stevia extractexpressed a significant decrease in renal indicators (ureaand creatinine). Combined treatment administration of stevia
extract and glimepiride to diabetic rats withcaused moreimprovement in renal function.
3.7. Effect of different treatments on level of tumour necrosisfactor-α (TNF-α). AS shown from Figure 6, treatment ofthe rats with STZ caused a significant elevation (p < 0.001)in the serum level of TNF-α. Combined administration ofglimepiride 1 mg/kg/day and stevia extract 300 mg/kg/dayto diabetic rats showed a significant reduction (p < 0.001) inthe tumor necrosis factor level as compared to control rats.Stevia extract increase the inhibitory effect of glimepiride onTNF-α, which support the anti-inflammatory effect of stevia.
3.8. Effect of different treatment on renal and hepaticlevel of Malondialdehyde (MDA). Diabetic rats administered300 mg/kg/day stevia extract for three weeks showed asignificant reduction (p < 0.001, Figure 7) in the hepaticMDA level. Combined administration of stevia extract 300mg/kg/day and glimepiride 1 mg/kg/day to diabetic rats forthe same duration expressed a significant reduction (p <0.001) in hepatic MDA level as compared to rats treated withglimepiride alone.
6 Egyptian Journal of Basic and Clinical Pharmacology
Table 2: Influence of stevia and/or glimepiride on liver function test of diabetic rats.
Regimens Liver function test
Groups Treatment Dose and duration (mg/kg) ALT(U/I) AST(U/I) Total proteinconc. (mg/dl)
1 Control(non-diabetic)
Saline Saline Equal volumes single I.PEqual volumes orally for 3weeks
26.7 ± 2.8 18.7 ± 2.3 7.7 ± 0.2
2 Diabetic control STZ + NAVehicle(Saline
/Tween)
Equal volumes single I.PEqual volumes orally for 3weeks
62.3 ± 4.2 51 ± 5.3 9.4 ± 0.4
3 Stevia extract STZ + NA Steviaextract
STZ 55 mg/kg single I.P + NA 230 mg/kg300 mg/kg stevia orally for 3weeks
32.6 ± 4.6*** 31.7 ± 3.7** 8.6 ± 0.5
4 Glimepiride STZ + NAGlimepiride
STZ 55 mg/kg single I.P + NA 230 mg/kg1 mg/kg glimepiride orally for 3weeks
28.5 ± 2.4*** 23.5 ± 0.9*** 7.8 ± 0.1**
5 Glimepiride +Stevia extract
STZ + NAGlimepiride +Stevia extract
-STZ 55 mg/kg single I.P + NA 230 mg/kg(1 mg/kg glimepiride + 300 mg/kg stevia)
orally for 3weeks
22.3 ± 1.7*** 18.1 ± 1.2*** 7.2 ± 0.2***
Values represent mean ± S.E.M.
*Significant difference at P < 0.05 vs. diabetic value.
** Significant difference at P < 0.01 vs. diabetic value.
*** Significant difference at P < 0.001 vs. diabetic value.
N.B: There is no difference between different control vehicles.
Table 3: Effects of stevia and/or glimepiride on kidney function test of diabetic rats.
Regimens Kidney function test (mg/dl)
Groups Treatment Dose and duration (mg/kg) Urea Creatinine
1 Normal control(non-diabetic)
Saline Saline Equal volumes single I.PEqual volumes orally for 3weeks
39.1 ± 1.8 0.8 ± 0.04
2 Diabetic control STZ + NAVehicle(Saline
/Tween)
Equal volumes single I.PEqual volumes orally for 3weeks
60.6 ± 4.3 1.3 ± 0.07
3 Stevia extract STZ + NA Steviaextract
STZ 55 mg/kg single I.P + NA 230 mg/kg300 mg/kg stevia orally for3weeks
48.4 ± 2.7* 0.87 ± 0.04***
4 Glimepiride STZ + NAGlimepiride
STZ 55 mg/kg single I.P + NA 230 mg/kg1 mg/kg glimepiride orally for 3weeks
43 ± 1.7*** 0.8 ± 0.02***
5 Glimepiride +Stevia extract
STZ + NAGlimepiride +Stevia extract
-STZ 55 mg/kg single I.P + NA 230 mg/kg(1 mg/kg glimepiride + 300 mg/kg stevia) orally for 3weeks
40.5 ± 2.6*** 0.72 ± 0.02***
Values represent mean ± S.E.M.
*Significant difference at P < 0.05 vs. diabetic value.
** Significant difference at P < 0.01 vs. diabetic value.
*** Significant difference at P < 0.001 vs. diabetic value.
N.B: There is no difference between different control vehicles.
Egyptian Journal of Basic and Clinical Pharmacology 7
0
50
100
150Control-non diabetic
Control diabetic
Stevia
Glimipiride
Glimipiride+Stevia
+++
******
T G
s (m
g /d
l)
Values represent mean ± S.E.M.
- +++ Significant difference at P< 0.001 vs. non diabetic value.
- * Significant difference at P< 0.05 vs. diabetic value.
- ** Significant difference at P< 0.01 vs. diabetic value.
- *** Significant difference at P< 0.001 vs. diabetic value.
- #Significant difference at P< 0.05 vs. glimepiride value.
Figure 4: Influence of daily administration of stevia and/or glimepiride for 21 days to diabetic rats on the level of triglycerides.
0
20
40
60Control-non diabetic
Control diabetic
Stevia
Glimipiride
Glimipiride+Stevia
*** **+++
#***
H D
L ( m
g / d
l)
Values represent mean ± S.E.M.
- +++ Significant difference at P< 0.001 vs. non diabetic value
- ** Significant difference at P<0.01 vs. diabetic value.
- *** Significant difference at P< 0.001 vs. diabetic value.
- #Significant difference at P< 0.05 vs. glimepiride value.
Figure 5: Influence of daily administration of stevia and/or glimepiride for 21 days to diabetic rats on the high density lipoprotein level.
Similarly, diabetic rats treated with stevia extract pro-duced a significant decrease (p < 0.001, Figure 8) in renalMDA content. Combined daily administration of glimepiride1mg/kg/day plus stevia extract 300mg/kg/day to diabetic ratsorally for the same period expressed a significant reduction(p < 0.001) in renal MDA content. The addition of steviaextract to glimepiride significantly (p< 0.05) decreased renalMDA content in comparison to diabetic rats treated withglimepiride alone (Figure 8).
3.9. Histopathological results
3.9.1. Histologic evaluation of the hepatic tissue. Sectionsof liver tissue from normal control group showed normalliver architecture without any pathological changes (Figure9A.). Liver tissues from the diabetic control rats revealedsignificant changes, which appeared as excessive hydropicdegeneration (2.66 ± 0.21), lobular inflammation (1.33± 0.33) (Figure 9B.), vascular congestion (1.50 ± 0.22)(Figure 9C.), portal inflammation (1.16 ± 0.30) (Figure
9D.), necrosis (0.66 ± 0.21), sinusoidal dilatation (1.66± 0.21) and congestion (Figure 9E.). The mean score ofhepatic injuries (1.49 ± 0.27) was significantly elevated indiabetic rats in comparison to normal group (p = 0.002).Significant decrease of the liver injuries was appeared inthe group ofstevia (Figure 9F&G). The mean score ofliver injuries (0.38 ± 0.09) was significantly less thandiabetic control rats (p = 0.005). Treatment with glimepiridesignificantly improved all the hepatic injuries except formild hydropic degeneration (0.66 ± 0.42) and vascularcongestion (0.16 ± 0.16) (Figure 9H). The mean score ofliver injuries (0.13± 0.10) was significantly less than diabeticcontrol rats (p = 0.004). Co-administration of glimepirideand stevia extract produced significant improvement of allthe hepatic injuries except for mild hydropic degenera-tion (0.16 ± 0.16) (Figure 9I). The mean score of liverinjuries (0.02 ± 0.02) was significantly lower than diabeticcontrol rats (p = 0.003). However, insignificant differencewas detected as compared withglimepiride treatment (p =0.461).
8 Egyptian Journal of Basic and Clinical Pharmacology
0
20
40
60
80
100Control non diabetic
Control
Stevia
Glimipiride
*** *** Glimipiride+Stevia
***
+++
T N F
a (n g
/ g w
. w t)
Values represent mean ± S.E.M.
- +++ Significant difference at P< 0.001 vs. non diabetic value.
- *** Significant difference at P< 0.001 vs. diabetic value.
Figure 6: Influence of daily administration of stevia and/or glimepiride for 21 days to diabetic rats on the tumour necrosis factor level.
0
50
100
150
200Control non diabetic
Control diabetic
Stevia
Glimipiride
Glimipiride+Stevia
+++
****** ###
***
Hepa
tic M
D A
conte
nt
(n m
o l /
g w.w
t.)
Values represent mean ± S.E.M.
- +++ Significant difference at P< 0.001 vs. non diabetic value.
- *** Significant difference at P< 0.001 vs. diabetic value.
- ### Significant difference at P< 0.001 vs. glimepiride value.
Figure 7: Influence of daily administration of stevia and/or glimepiride for 21 days to diabetic rats on the hepatic MDA level.
3.9.2. Histologic evaluation of the renal tissue. The kidneysections from the normal control group showed normalappearance of the glomeruli and tubules without any patho-logical changes (Figure 10A.). In the diabetic control group,the renal tissues showed signs of earlier diabetic changesas hydropic tubular degeneration (2.66 ± 0.21) (Figure10B.), vascular congestion (2.33 ± 0.21) and mild dilationof glomeruli with glomerular capillaries congestion (1 ±0.0) (Figure 10C).The mean score of the renal injuries(1.99 ± 0.50) was significantly more than normal group(p = 0.037). Improvement of all renal injuries with onlymild glomerular enlargement, tubular degeneration and mildvascular congestion was seen in the stevia treated group(Figure 10D).The mean score of renal injuries (0.44 ±0.05) was significantly decreasedin the stevia group thanthat of diabetic control rats (p = 0.046). Regarding therenal injuries in the glimepiride treated group, reductionof the tubular degeneration (1.33 ± 0.21) and vascu-lar congestion (0.33 ± 0.21) (Figure 10E) together withabsence of any glomerular injury was observed (Figure10). Although the mean score of renal injuries in theglimepiride treated group (0.55 ± 0.40) was lower thanuntreated diabetic group, the difference was statisticallyinsignificant (p = 0.127). Co-administration of glimepirideand stevia extract produced significant improvement of allthe renal injuries except for mild vascular congestion (0.33± 0.21) (Figure 10F). The mean score of renal injuries
(0.11 ± 0.11) was significantly lower than untreated diabeticrats (p = 0.046). However, insignificant difference wasdetected in comparison to the glimepiride treated group (p= 0.346).
3.10. Immunohistochemical results
3.10.1. Evaluation of eNOS immunohistochemical expressionin the renal tissues of the normal control group. In thenormal control group, moderate immunoreactivity of eNOSwas seen in the cytoplasm of the endothelial cells in most ofthe glomeruli, arterioles, interlobular arteries and peritubularcapillaries (Figure 11A). While eNOS expression was absentin the proximal and distal convoluted tubules.
3.10.2. Evaluation of eNOS immunohistochemical expressionin the renal tissues of the diabetic control rats. The dia-betic group demonstrated positive eNOS immunostaining inendothelial cells in most of glomeruli, arterioles, interlob-ular arteries and peritubular capillaries. Also, strong eNOSprotein expression was detected in most tubules occupyingthe whole cytoplasm in the proximal and distal convolutedtubules and most of the collecting tubules (Figure 11B).The expression of eNOS was significantly higher in diabeticgroup (5.33± 0.21) as compared with the normal group (3.33± 0.21) (p = 0.002).
Egyptian Journal of Basic and Clinical Pharmacology 9
0
50
100
150Control non diabetic
Control diabetic
Stevia
Glimipiride
Glimipiride+Stevia
+++
*** *** #
***
Rena
l M D
A co
ntent
(n m
o l/ g
w.w
t.)
Values represent mean ± S.E.M.
- +++ Significant difference at P< 0.001 vs. non diabetic value.
- *** Significant difference at P< 0.001 vs. diabetic value.
- # Significant difference at P< 0.05 vs. glimepiride value.
Figure 8: Influence of daily administration of stevia and/or glimepiride for 21 days to diabetic rats on the renal MDA level.
Figure 9: Hematoxylin and eosin stained liver section. A; normal control group showing preserved liver architecture without any pathologicalchanges (x200). Liver sections from diabetic control group showing: severe hydropic degeneration and lobular inflammation (B, x400),vascular congestion (C, x400), portal inflammation (D, x400) & sinusoidal dilatation and congestion (E, x400). Liver sections from steviatreated group showing: improvement of all the hepatic injuries with only mild hydropic degeneration, sinusoidal dilatation and focal mildlobular inflammation (F, x400) andMild vascular congestion (G, x400). Liver sections from the glimepride treated group showing attenuationof the hepatic injuries with mild hydropic degeneration, mild vascular congestion (H, x400). Liver sections from the glimepride and steviatreated group showing marked improvement of all the hepatic injuries with only mild focal hydropic degeneration (I, x400).
3.10.3. Evaluation of eNOS immunohistochemical expressionin renal tissues of the stevia group. Significant decrease ofthe eNOS protein expression was noted in the Stevia treatedgroup (2.3 ± 0.21) (Figure 11C). The mean of eNOS proteinexpression was significantly less than diabetic rats (p =0.003).
3.10.4. Evaluation of eNOS immunohistochemical expres-sion in renal tissues of glimepiride group. Treatments withglimepride result in decreased expression of eNOSin theendothelial cells and in the tubules (4.8 ± 0.16) (Figure11D). Although a lower mean of eNOS protein expressionwas detected in glimepride group in comparison with thediabetic group, the difference was statistically insignificant(p = 0.092).
3.10.5. Evaluation of eNOS immunohistochemical expressionin the renal tissues of the combined stevia and glimepride
group. Significant decrease of the eNOS protein expressionwas noted in group treated by combination of glimepride andstevia extract (3.6 ± 0.42) (Figure 11E). The mean of eNOSprotein expression was significantly less than glimepridetreated group (p = 0.045) and diabetic control rats (p =0.014).
4. Discussion
Type 2 Diabetes represents a worldwide challenge for healthcare. The World Health Organization (WHO) revealed thatthe diabetic prevalence in 2014 increased up to 8.5% inthe adult peoples, while most of them were obese. Diabetesmellitus was the 7th cause of mortalities by ranking of theWorld Health Organization in 2016 and is projected to moveup by 2030 [15, 16].
10 Egyptian Journal of Basic and Clinical Pharmacology
Figure 10: Hematoxylin and eosin stained renal sections of the normal control group showing normal glomeruli and tubules (A, x200). Kidneysections from diabetic control group showing: severe hydropic degeneration in tubules (B, x400), vascular congestion and enlargementinthe glomeruli with marked enlarged congested capillary tuft of glomeruli(C, x400). Kidney sections from steviatreated group showingimprovement of all the renal injuries with mild glomerular enlargement, mild tubular degeneration and mild vascular congestion (D, x400).Kidney sections from glimepride treated group showing mild improvement of the renal injuries with moderate hydropic degeneration of thetubules and vascular congestion (E, x400). Kidney sections from glimepride and stevia treated group showing marked improvement of allthe renal injuries with only mild vascular congestion (F, x400).
Figure 11: Immunohistochemistry of eNOS: kidney sections from the normal control group showing week to moderate expression inendothelial cells of the glomeruli and peritubular capillaries (A, x400). Sections from the diabetic group show strong expression in endothelialcells of the glomeruli, peritubular capillaries and the proximal convoluted tubules (B, x400). Sections from stevia treated group showingweek expression in the glomeruli, peritubular capillaries and the proximal convoluted tubules(C, x400). Glimepiride treated group showingmoderated expression in the glomeruli, peritubular capillaries and the proximal convoluted tubules (D, x400). Glimepiride and stevia treatedgroup showing week expression in the glomeruli, peritubular capillaries and the proximal convoluted tubules (E, x400).
The increased prevalence of T2DM and obesity is dueto aging, dietary habits, increased consumption of sug-ars and decreased physical activities [17]. The majorityof diabetic peoples take sweeteners with low calories inorder to reduce the caloric entry. However, many of arti-ficial sweeteners like aspartame, cyclamates and saccha-rine, considered high calorie sugars and are potentiallycarcinogenics. The high prevalence of diabetes and obesity
and need for safety of some artificial sweeteners, haveenhanced the search for natural little calorie sweetener[18].
Among the new effective antidiabetic agents the herbStevia rebaudiana Bertoni have been used in management ofD.M in South America [19]. Stevioside and its related gly-cosides have been tested as natural zero-calorie sweeteners[7].
Egyptian Journal of Basic and Clinical Pharmacology 11
The currently marketed ant-diabetic agents are less effec-tive and having many drawbacks. Besides, these side effects,none of the available ant-diabetic drugs successfully estab-lishing euglycemic state and treating long term micro- andmacro-vascular complications. United Kingdom ProspectiveDiabetes Study (UKPDS) revealed that the treatment ofdiabetes with single oral hypoglycemic agent mostly failedto establish euglycemic state for long time, and most ofdiabetics have to use combined antidiabetic agents [3]. Steviais a medicinal herb with multi potential health benefits [20].
Sulfonylureas as glimepiride lower the high blood glucoselevels by inducing insulin release via the closing the ATP-sensitive potassium channels on beta cells. However, sulfony-lureas have no influence on insulin resistance (IR), the maincause and complication in type 2 diabetes mellitus. There-fore, many patients with type 2 have to use sulfonylureas inadjunct with other agents that enhance insulin sensitivity and/or diabetic control [21].
In a consistence with other studies, our findings revealedthat rats with type 2 diabetes treated with stevia extract (300mg/kg/day) orally for 21 days showed a good control of bloodglucose levels. Similary, combined treatment of diabeticrats with stevia extract (300 mg/kg/day) and glimepiride(1 mg/kg/day) for a similar period caused a more decreasein the BGLs in a comparison to diabetic rats treated withglimepiride alone. This finding is collaborated with theprevious results that approved potential benefits of steviaplant on hyperglycemia on diabetic animals [22–24].
The results also showed that diabetic rats administeredstevia extract expressed a significant elevation in insulinrelease in a similar level like insulin releaser drugs likeglimepiride. Moreover the diabetic rats administered com-bination of glimepiride and stevia extract expressed a moreenhancement in the insulin release reflecting on decreasingthe blood glucose levels. These findings demonstrated thatstevia extract having a good efficacy on controlling thehyperglycemia in diabetic rats especially when combinedwith other hypoglycemic agents, like glimepiride.
Previous data revealed that the adiponectin is a mainfactor in the development obesity, IR, MS, and T2DM.It cause insulin-sensitization and anti-inflammatory action,and sometimes decreases body weight [25, 26]. Decreasedadiponectin level is mostly linked to obesity, MS and T2DM.Thus, it has an important role as a therapeutic agent inmanagement of MS and T2DM.
Several studies have demonstrated that glimepirideincrease adiponectin gene expression in adipocytes [27] andother data have also revealed that glimepiride may causeenhancement in insulin sensitivity which linked to increasedserum adiponectinemia [28]. However, it is still unknownwhether the treatment with glimepiride in T2D could improvethe serum adiponectin level for better glycemic controlor not. Our findings demonstrate that stevia extract hasa significant action on enhancing the serum adiponectin.Also treatment of diabetic rats with glimepiride significantly
increases the adiponectin level. Moreover, the diabetic ratsadministered combination of stevia and glimepiride showedadditive effects on elevating the adiponectin level. Thus thecombined administration of stevia and glimepiride may bepotentially a therapeutic target for management of obesityand type 2 diabetes.
The United Kingdom Prospective Diabetes Study(UKPDS) stated that, the prognosis of diabetics withT2DM depends on management of elevated blood glucoselevel, and other associated problems, like hypertension andhyperlipidemia [29]. Thus, the pharmacologic treatment forT2DM should be directed to control BGL and improve thelipid profile.
Our diabetic rat model showed the main features ofdiabetic hyperlipidemia: an elevated triglyceride and totalcholesterol levels and a decrease in the HDL-cholesterol.These features is mostly due to elevation in the rate of lipol-ysis from insulin-resistant adipocytes [30] which, decreasesthe glucose uptake in the muscles, enhances triglycerideformation, exaggerates glucose synthesis in the liver, andleading to failure of β-cell [31]. Thus, improving the serumlipid profile could help in suppressing the progress ofmetabolic syndrome and T2DM.
It was obvious from our findings that diabetic rats withtreated stevia extract showed a significant reduction in theTC and TG levels and increased HDL level. Glimepiridetreatment significantly improved the levels of triglyceride,cholesterol, and HDL-cholesterol and its effect was enhancedwhen it combined with stevia extract. These results arecollaborated with previous result [32–34], that stevia extractdecreased total cholesterol level and elevated the HDL-cholesterol level. Also, data on humans revealed that admin-istration of stevia extract elevated the level of HDL anddecreased the levels of cholesterol, triglycerides [35]. Ahmadet al. 2018 [36] revealed that stevia extract may be consideredas a natural anti-hyperlipidemic agent in management ofdyslipidemia and its related conditions. Therefore, it appearsthat stevia extract and glimepiride could give a potential ther-apeutic effects in diseases linked to impairment of glucosetolerance, hyperlipidemia and increased insulin resistance.
The pro-inflammatory cytokine tumour necrosis factorTNF-α has been involved as a key element in the developmentand progression of obesity, insulin resistance and T2DM,doing its effect via the process of inflammation and immunity[37]. Our findings showed that the stevia extract significantlyreduced the level of TNF-α in diabetic group. Concurrentadministration of glimepiride plus stevia extract to diabeticrats reverses the increase in TNF-a level. The influence ofglimepiride on TNF- α was more enhanced with its combi-nation with stevia resulting in a significant reduction in itslevel in comparison to diabetic rats administered glimepiridealone. This action is consistent with the data obtained frominsulin-resistant mice model showed that stevioside was alsoable to down regulate expression of TNF-α [38] and micemodel with cisplatin (CP)-induced kidney injury treated withStevia ethanolic extract (SE) or stevioside. Both SE and
12 Egyptian Journal of Basic and Clinical Pharmacology
stevioside inhibited CP nephrotoxicity by reducing the levelof TNF- α [39].
The combined treatment of glimepiride and stevia extractremarkably improved insulin resistance, explained by a sig-nificant reduction in TNF- α and lipid profile. Moreover, themodification of adipocytokine concentration (i.e., elevatedserum adiponectin) may also being the cause of improvingthe insulin resistance
Diabetic nephropathy (DN), considered a major end-termmicro-vascular complication and it is the major cause ofmorbidities and early mortalities in diabetics. Since thatthe hyperglycemia is the main cause in the pathogenesisof DN [40]. Management of DN needs controlling ofmany factors like: blood glucose level, hypertension andkidney function. DN was also identified by testing certainbiochemical indices in the blood. STZ induced-renal damageis confirmed by both diagnostic biochemical parameters andhistopathological changes [10]. Our results showed that thelevels of renal biomarkers like urea and creatinine wereelevated in the diabetic rats with impairment in the renalstructure and this collaborated with previous results [41].The levels of renal biomarkers were significantly reducedin stevia and combined stevia & glimepiride groups. More-over, the histopathological analysis of the kidney showedimprovement in renal tissue with lower mean of injuries wasseen in stevia and combined stevia & glimepiride groupsas compared to control diabetic rats. These results are inagreement of previous observed data that concerning theeffect of stevia has a in amelioration of renal injury inthe STZ-injected rats in addition to its antihyperglcemicaction [10, 42]. Our findings support the potential roleof stevia in combination with other hypoglycemic agentfor treatment of T2DM and other diabetes-related renalconditions.
Diabetic nephropathy represents a complex metabolicprocess characterized with patho-physiological events thatboth stimulate and depress intra-renal nitric oxide (NO)production. The net effect on renal NO level relies onthe stage of the disease. Most of literature has built upabnormalities and the role of intra-renal nitric oxide release inthe development of diabetic nephropathy. These differencescould be explained by the different methods used forevaluation of NO, the NOS isoforms studied and the stageof the disease [43].
The studies demonstrate that early diabetic nephropathy isassociatedwith increased constitutive NOproduction derivedmainly from eNOS and possibly iNOS and this effect maybe related to intra glomerular hemodynamic changes notedin early diabetic renal disease [44]. On the other hand, laterstages of diabetic nephropathy characterized by hypertension,progressive renal insufficiency is associated with decreasedNO production (especially iNOS derived) resulting in a NOdeficient state [45].
Our immunohistochemical results are in line with thesedata with regard to strong eNOS protein expression in
the renal tissue of STZ- injected rats. While, signifi-cant decrease of eNOS protein expression was noted instevia treated group. Importantly, the mean of eNOSprotein expression was significantly lower in glimepiride& stevia treated group compared to glimepiride treatedgroup.
Both T2DM and non-alcoholic fatty liver disease(NAFLD) are global problems [46]. The prevalence ofNAFLD was very high among diabetic patients reachingupto 70% between these patients [47]. Thus, T2DM is usedas estimator for the development of NAFLD. The mainproblems in diabetes that leading to hepatogenous diabetesare insulin sensitivity and β- cell failure [48].
Our findings demonstrated that stevia extract reducedthe serum level of hepatic enzymes in diabetic rats. Thereis a significant reduction of the level hepatic biomarkersin stevia and combined stevia& glimepiride groups, whichwas represented in the form of improvement in the hep-atic function and structure. The combination of stevia &glimepiride significantly reduced the mean score of hepaticinjuries than those treated with glimepiride alone. Theseresults are in accordance with [10] and [49] who revealed thehepatoprotective effect of stevia on diabetes induced hepaticchanges in diabetic rats. Glimepiride and stevia extract couldimprove both metabolic and hepatic dysfunction as a classeffect. The alleviation of hepatic dysfunction was mediatedpartly through the control of hyperglycemia, reduction ofelevated hepatic enzymes, antioxidant effect and partly viaimproving in insulin resistance.
Oxidative stress is also claimed to have an important rolein pathogenesis of T2DM. It is reported that the permanentelevation of blood glucose levels occurring in most of dia-betics causes glucose autoxidation and glycation of proteinsend products [50, 51], which leading to depletion of theantioxidant defense system and thus enhancing free radicalproduction. MDA has a main role in pathogenesis of manychronic diseases like diabetes [24]. Thus, attention has beendirected toward natural antioxidants that able to inhibit freeradical production [52], especially, the plant-basedmedicineswhich currently investigated in the prevention and treatmentof diabetes.
Our findings demonstrated that diabetic rats treated withextract from the plant leaves of stevia rebaudiana signif-icantly decreased the hepatic and renal content of MDA.Combined administration of glimepiride plus stevia extractto diabetic rats showed a marked reduction of renal andhepatic MDA in comparison to diabetic rats treated withglimepiride alone. In consistence with the results of thecurrent work, many experimental studies revealed that steviaextract has a potent antioxidant property, as the plant containslarge amounts of flavonoids and total phenols [32, 49].Taken together with the antidiabetic action and antioxidantproperties of stevia leaves, the extract of stevia could providea new therapeutic antidiabetic agent in prevention andmanagement of T2DM.
Egyptian Journal of Basic and Clinical Pharmacology 13
5. Conclusion
The combined use of glimepiride and stevia extract consid-ered as a novel approach to glycemic control and is accompa-nied by multiple demonstrable metabolic and renal benefitsbeyond its glucose-lowering effect. The co-administration ofstevia and glimepiride represents a new effective therapeuticstrategy for better controlling diabetes and its complications.Our study results provide further support to the recent use ofstevia rebaudiana bertoni as antidiabetic agent either alone orcombined with the commonly used oral hypoglycemic agentslike glimepiride.
Funding Resource
The present study is a part of project grant from Science andtechnology development fund (STDF) Grant No, 12667.
Acknowledgement
The present study was financially supported by Science andtechnology development fund (STDF) Grant No, 12667.
(STZ): Streptozotocin(TC): Total cholesterol(TGs): Triglycerides(TNFα): Tumour necrosis factor-α(Type 2D.M): Type 2 diabetes mellitus(UKPDS): United Kingdom Prospective Diabetes Study(WHO): World Health Organization
Competing Interests
The authors declare no competing interests.
References
[1] R. Saravanan, K. Vengatash babu, and V. Ramachandran,“Effect of Rebaudioside A, a diterpenoid on glucose homeosta-sis in STZ-induced diabetic rats,” Journal of Physiology andBiochemistry, vol. 68, no. 3, pp. 421–431, 2012.
[2] C. Hu and W. Jia, “Diabetes in China: Epidemiology andGenetic Risk Factors and Their Clinical Utility in PersonalizedMedication,” Diabetes, vol. 67, no. 1, pp. 3–11, 2017.
[3] J. Chen, P. B. Jeppesen, I. Nordentoft, and K. Hermansen,“Stevioside counteracts the glyburide-induced desensitizationof the pancreatic beta-cell function in mice: studies in vitro,”Metabolism, vol. 55, no. 12, pp. 1674–1680, 2006.
[4] M. J. Davies, D. A. D’Alessio, J. Fradkin et al., “Correctionto: Management of hyperglycaemia in type 2 diabetes, 2018.A consensus report by the American Diabetes Association(ADA) and the European Association for the Study of Dia-betes (EASD) (Diabetologia, (2018), 61, 12, (2461-2498),10.1007/s00125-018-4729-5),” Diabetologia, 2019.
[5] A. J. Garber, M. J. Abrahamson, J. I. Barzilay et al., “Con-sensus Statement by the American Association of ClinicalEndocrinologists and American College of Endocrinology onthe Comprehensive Type 2 Diabetes Management Algorithm –2018 Executive Summary,” Endocrine Practice, vol. 24, no. 1,pp. 91–120, 2018.
[6] J.-E. Park and Y.-S. Cha, “Stevia rebaudiana Bertoni extractsupplementation improves lipid and carnitine profiles inC57BL/6J mice fed a high-fat diet,” Journal of the Science ofFood and Agriculture, vol. 90, no. 7, pp. 1099–1105, 2010.
[7] P. B. Jeppesen, S. Gregersen, K. K. Alstrup, and K. Hermansen,“Stevioside induces antihyperglycaemic, insulinotropic andglucagonostatic effects in vivo: Studies in the diabetic Goto-Kakizaki (GK) rats,” Phytomedicine, vol. 9, no. 1, pp. 9–14,2002.
[8] V. Chatsudthipong and C. Muanprasat, “Stevioside and relatedcompounds: therapeutic benefits beyond sweetness,” Pharma-cology & Therapeutics, vol. 121, no. 1, pp. 41–54, 2009.
[9] P. B. Jeppesen, S. Gregersen, C. R. Poulsen, and K. Hermansen,“Stevioside acts directly on pancreatic β cells to secreteinsulin: actions independent of cyclic adenosine monophos-phate and adenosine triphosphate-sensitive K+-channel activ-ity,” Metabolism, vol. 49, no. 2, pp. 208–214, 2000.
[10] N. Shivanna, M. Naika, F. Khanum, and V. K. Kaul, “Antiox-idant, anti-diabetic and renal protective properties of Steviarebaudiana,” Journal of Diabetes and its Complications, vol.27, no. 2, pp. 103–113, 2013.
14 Egyptian Journal of Basic and Clinical Pharmacology
[11] S. I. Rabbani, K. Devi, and S. Khanam, “Inhibitory effect ofGlimepiride on nicotinamide-streptozotocin induced nucleardamages and sperm abnormality in diabeticWistar rats,” IndianJournal of Experimental Biology (IJEB), vol. 47, no. 10, pp.804–810, 2009.
[12] B. Mehta, D. Jain, H. Misra, M. Soni, N. Silawat, and D.Mehta, “Antidiabetic activity of medium-polar extract from theleaves of Stevia rebaudiana Bert. (Bertoni) on alloxan-induceddiabetic rats,” Journal of Pharmacy and Bioallied Sciences, vol.3, no. 2, p. 242, 2011.
[13] A. K. Sharma and B. Srinivasan, “Triple verses glimepirideplus metformin therapy on cardiovascular risk biomarkers anddiabetic cardiomyopathy in insulin resistance type 2 diabetesmellitus rats,” European Journal of Pharmaceutical Sciences,vol. 38, no. 5, pp. 433–444, 2009.
[14] S. Asyura, H. Hamzah, R. Shaari, S. Sithambaram, and N.Mustapha, “Blood Profiles and Histopathological Changes ofLiver and Kidney Tissues from Male Sprague Dawley RatsTreated with Ethanol Extracts of Clinacanthus nutans Leaf,” JClin Toxicol, vol. 329, pp. 2161–0495, 2016.
[15] WH. Organization, World health statistics 2016: monitoringhealth for the SDGs sustainable development goals, WorldHealth Organization, 2016.
[16] WH. Organization, World health statistics 2018: monitoringhealth for the SDGs, sustainable development goals, Worldhealth statistics 2018, monitoring health for the SDGs, 2018.
[17] P. Z. Zimmet, D. J. McCarty, andM. P. De Courten, “The globalepidemiology of non-insulin-dependent diabetes mellitus andthe metabolic syndrome,” Journal of Diabetes and its Compli-cations, vol. 11, no. 2, pp. 60–68, 1997.
[18] G. Brahmachari, L. C. Mandal, R. Roy, S. Mondal, and A. K.Brahmachari, “Stevioside and Related Compounds - Moleculesof Pharmaceutical Promise: A Critical Overview,” Archiv derPharmazie, vol. 344, no. 1, pp. 5–19, 2011.
[19] D. D. Soejarto, C. M. Compadre, P. J. Medon, S. K. Kamath,and A. D. Kinghorn, “Potential sweetening agents of plant ori-gin. II. field search for sweet-tastingStevia species,” EconomicBotany, vol. 37, no. 1, pp. 71–79, 1983.
[20] A. Carrera-Lanestosa, Y. Moguel-Ordóñez, and M. Segura-Campos, “Stevia rebaudiana Bertoni: A Natural Alternativefor Treating Diseases Associated with Metabolic Syndrome,”Journal of Medicinal Food, vol. 20, no. 10, pp. 933–943, 2017.
[21] H. M. El-sheikh, S. M. El-Haggar, and T. A. Elbedewy,“Comparative study to evaluate the effect of L-carnitine plusglimepiride versus glimepiride alone on insulin resistance intype 2 diabetic patients,” Diabetes & Metabolic Syndrome:Clinical Research& Reviews, vol. 13, no. 1, pp. 167–173, 2019.
[22] R. S. Kujur, V. Singh, M. Ram et al., “Antidiabetic activity andphytochemical screening of crude extract of Stevia rebaudianain alloxan-induced diabetic rats,” Pharmacognosy Research,vol. 2, no. 4, pp. 258–263, 2010.
[23] S. Akbarzadeh, F. Eskandari, H. Tangestani et al., “The effect ofStevia rebaudiana on serum omentin and visfatin level in STZ-induced diabetic rats,” Journal of Dietary Supplements, vol. 12,no. 1, pp. 11–22, 2015.
[24] R. Assaei, P. Mokarram, S. Dastghaib et al., “Hypoglycemiceffect of aquatic extract of Stevia in pancreas of diabeticrats: PPARγ-dependent regulation or antioxidant potential,”Avicenna Journal of Medical Biotechnology, vol. 8, no. 2, pp.65–74, 2016.
[25] K. Ghoshal, “Adiponectin: Probe of the molecular paradigmassociating diabetes and obesity,” World Journal of Diabetes,vol. 6, no. 1, p. 151, 2015.
[26] “Adiponectin, a Therapeutic Target for Obesity, Diabetes, andEndothelial Dysfunction,” International Journal of MolecularSciences, vol. 18, no. 6, p. 1321, 2017.
[27] Y. Kanda, M. Matsuda, K. Tawaramoto et al., “Effects ofsulfonylurea drugs on adiponectin production from 3T3-L1adipocytes: Implication of different mechanism from pioglita-zone,” Diabetes Research and Clinical Practice, vol. 81, no. 1,pp. 13–18, 2008.
[28] T. Tsunekawa, T. Hayashi, Y. Suzuki et al., “Plasmaadiponectin plays an important role in improving insulinresistance with glimepiride in elderly type 2 diabetic subjects,”Diabetes Care, vol. 26, no. 2, pp. 285–289, 2003.
[29] I. M. Stratton, A. I. Adler, H. A. W. Neil et al., “Associationof glycaemia with macrovascular and microvascular complica-tions of type 2 diabetes (UKPDS 35): prospective observationalstudy,” British Medical Journal, vol. 321, no. 7258, pp. 405–412, 2000.
[30] A. D. Mooradian, “Dyslipidemia in type 2 diabetes mellitus,”Nature Clinical Practice Endocrinology & Metabolism, vol. 5,no. 3, pp. 150–159, 2009.
[31] B. Mlinar, J. Marc, A. Janež, and M. Pfeifer, “Molecularmechanisms of insulin resistance and associated diseases,”Clinica Chimica Acta, vol. 375, no. 1-2, pp. 20–35, 2007.
[32] S. Singh and V. Garg, “Antidyslipidymic and AntioxidativePotential of Methanolic Root Extract of Stevia Rebaudiana(Bertoni) on Alloxan Induced Diabetic,” J Pharm Pharm Sci,vol. 3, no. 7, p. 72, 1859.
[33] “Evaluación de la expresión del gen GLP-1 (péptido 1 homol-ogo al glucagón) en ratas inducidas a Diabetes Mellitustipo 2 tratadas con extracto metanólico de Schkuhria pinnata(Canchalagua),” Revista ECIPeru, pp. 98–103.
[34] A. Aghajanyan, Z. Movsisyan, and A. Trchounian, “Antihy-perglycemic and Antihyperlipidemic Activity of HydroponicStevia rebaudiana Aqueous Extract in Hyperglycemia Inducedby Immobilization Stress in Rabbits,” BioMed Research Inter-national, vol. 2017, Article ID 9251358, 6 pages, 2017.
[35] N. Sharma, R. Mogra, and B. Upadhyay, “Effect of SteviaExtract Intervention on Lipid Profile,” Studies on Ethno-Medicine, vol. 3, no. 2, pp. 137–140, 2017.
[36] U. Ahmad, R. S. Ahmad, M. S. Arshad, Z. Mushtaq, S.M. Hussain, and A. Hameed, “Antihyperlipidemic efficacy ofaqueous extract of Stevia rebaudiana Bertoni in albino rats,”Lipids in Health and Disease, vol. 17, no. 1, 2018.
[37] D. E. Moller, “Potential role of TNF-α in the pathogenesis ofinsulin resistance and type 2 diabetes,” Trends in Endocrinology& Metabolism, vol. 11, no. 6, pp. 212–217, 2000.
[38] Z.Wang, L. Xue, C. Guo et al., “Stevioside ameliorates high-fatdiet-induced insulin resistance and adipose tissue inflammationby downregulating the NF-κB pathway,” Biochemical andBiophysical Research Communications, vol. 417, no. 4, pp.1280–1285, 2012.
[39] I. Potočnjak, D. Broznić, M. Kindl, M. Kropek, S. Vladimir-Knežević, and R. Domitrović, “Stevia and stevioside protectagainst cisplatin nephrotoxicity through inhibition of ERK1/2,STAT3, and NF-κB activation,” Food and Chemical Toxicol-ogy, vol. 107, pp. 215–225, 2017.
[40] X. Tong, H. Ma, S. W. Amadi, L. Ma, and G. Wu, “Reno-protection of G004, a novel anti-diabetic sulfonylurea in db/db
Egyptian Journal of Basic and Clinical Pharmacology 15
mice,” Naunyn-Schmiedeberg’s Archives of Pharmacology,vol. 388, no. 8, pp. 831–841, 2015.
[41] M. Raish, A. Ahmad, B. L. Jan et al., “Momordica charantiapolysaccharides mitigate the progression of STZ induced dia-betic nephropathy in rats,” International Journal of BiologicalMacromolecules, vol. 91, pp. 394–399, 2016.
[42] C. Ozbayer, H. Kurt, S. Kalender et al., “Effects of Steviarebaudiana (Bertoni) Extract andN-Nitro-L-Arginine on RenalFunction and Ultrastructure of Kidney Cells in ExperimentalType 2 Diabetes,” Journal of Medicinal Food, vol. 14, no. 10,pp. 1215–1222, 2011.
[43] S. Sharma, “Nitric oxide and the kidney,” Indian J Nephrol, vol.14, no. 3, pp. 77–84, 2004.
[44] H. Sugimoto, K. Shikata, M. Matsuda et al., “Increasedexpression of endothelial cell nitric oxide synthase (ecNOS)in afferent and glomerular endothelial cells is involved inglomerular hyperfiltration of diabetic nephropathy,”Diabetolo-gia, vol. 41, no. 12, pp. 1426–1434, 1998.
[45] H. Trachtman, S. Futterweit, E. Pine, J. Mann, and E. Valder-rama, “Chronic diabetic nephropathy: role of inducible nitricoxide synthase,” Pediatric Nephrology, vol. 17, no. 1, pp. 20–29, 2002.
[46] ZM. Younossi, AB. Koenig, D. Abdelatif, Y. Fazel, L. Henry,and M. Wymer, “Global epidemiology of nonalcoholic fattyliver diseasemeta-analytic assessment of prevalence, incidence,and outcomes,” Hepatology, vol. 64, no. 1, pp. 73–84, 2016.
[47] R. Kwok, K. C. Choi, and G. L.-H. Wong, “Screening diabeticpatients for non-alcoholic fatty liver disease with controlledattenuation parameter and liver stiffness measurements: aprospective cohort study,” Gut, 2015.
[48] E. Orsi, V. Grancini, S. Menini, A. Aghemo, and G. Pugliese,“Hepatogenous diabetes: Is it time to separate it from type 2diabetes?” Liver International, vol. 37, no. 7, pp. 950–962,2017.
[49] L. S, S. Chaudhary, and R. R.S, “Hydroalcoholic extractof Stevia rebaudiana bert. leaves and stevioside amelio-rates lipopolysaccharide induced acute liver injury in rats,”Biomedicine & Pharmacotherapy, vol. 95, pp. 1040–1050,2017.
[50] S. P. Wolff and R. T. Dean, “Glucose autoxidation and proteinmodification: the potential role of ‘autoxidative glycosylation’in diabetes,” Biochemical Journal, vol. 245, no. 1, pp. 243–250,1987.
[51] J. L. Rains and S. K. Jain, “Oxidative stress, insulin signaling,and diabetes,” Free Radical Biology &Medicine, vol. 50, no. 5,pp. 567–575, 2011.
[52] S. Ghanta, A. Banerjee, A. Poddar, and S. Chattopadhyay,“Oxidative DNA Damage Preventive Activity and AntioxidantPotential of Stevia rebaudiana (Bertoni) Bertoni, a NaturalSweetener,” Journal of Agricultural and Food Chemistry, vol.55, no. 26, pp. 10962–10967, 2007.
