Anti-obesity and Anti-diabetic Effect of Ursolic Acid against Streptozotocin/High Fat Induced Obese in Diabetic Rats

order that leads to severe complications, such as higher reactive oxygen species （ ROS ） , destruction of antioxidant enzymes, hyperglycemia, insulin signalling and cellular damage changes caused by ROS 6, 7 ） . All of this alteration is directed to secondary complications associated with diabetes, such as nephropathy, retinopathy, nephropathy, cardiovascular morbidity and neuropathy 4 ） . Investigational scientific research findings Abstract: Obesity is occurring due to continue taken high fat diet; this is the fast-growing problem reaching epidemic proportion globally. Ursolic acid altered the abnormal glucose metabolism in diabetic rats. In this experimental protocol, we examine ursolic acid (UA) anti-obesity effect against streptozotocin (STZ) and high-fat diet-induced obesity in rats. Orally administered the ursolic acid (2.5, 5 and 10 mg/kg) dose to the hyperglycemic rats for 8 weeks and estimated the blood glucose level at different time intervals. Biochemical, hepatic, lipid, renal and antioxidant parameters were estimated. Traf-4, Mapk-8, Traf-6 and genes such as Ins-1, ngn-3 and Pdx-1 mRNA expression were estimated using qRT-PCR to scrutinize the molecular mechanism in MAPK downstream JNK cascade and insulin pathway signalling pathways. Ursolic acid significantly ( p <0.001) down-regulated the blood glucose level at dose dependent manner. Its also reduced the plasma insulin level, non-essential fatty acid and increased the level of adiponectin as compared to obese control group rats. Ursolic acid treated group rats reduced the level of total cholesterol and triglycerides. Ursolic-acid-treated rats have been shown to decrease oxidative stress in pancreatic tissue by restoring the free radical effect of scavenging, suppress the Traf-6, Mapk-8 and Traf-4 mRNA expression, enhance the expression of Pdx-1, Ins-1 and Ngn-3 and ensure the regeneration of pancreas β cells and therefore pancreas insulin. The current result suggested the anti-obese effect of ursolic acid against high fat diet (HFD) induced obese rats via alteration of insulin and JNK signaling pathway.

shown a powerful association between hyperglycemia-induced oxidative stress and diabetes, since excessive quantities of production of ROS occur via glucose oxidation 8 . Excessive ROS also attacks the pancreatic β cells as a result of cellular damage due to the free and weak potential for radical scavenging. Oxidative stress also affects numerous signals, start the production of proinflammatory cytokines, the development of advanced glycation end products AGEs and cell death 9, 10 .
Various antidiabetic/Oral antihyperglycemic agents are marketed available to control the glycemic but numerous side effects are caused by these agents such as hepatic disorders, abdominal pain and kidney injury 11 . According to clinical research, it is well recognized that products derived from the traditional/medicinal plant have confirmed its beneficial therapeutic activity without having any side effects. Because of their broad spectrum of valuable therapeutic properties, polyphenolics richer plants gained considerable attention. Polyphenols have been reported to produce glucose-like insulin activity, reduce ROS production and increase the free radical scavenging mechanism 12,13 . These bioaction-based compounds protect against oxidising stress, stimulate the pathway of insulin signalling and regulate transcription factors, hormones, inflammatory tracts and peptides to manage diabetes-related hyperglycemia and complications 14 16 . The aim of the current experimental study was to scrutinize the anti-obesity effect of ursolic acid against Streptozotocin/High Fat induced Obese in Diabetic rats via alteration of insulin and c-Jun Nterminal kinases JNKs signaling pathway.

Chemicals
Ursolic acid 90 and streptozotocin STZ was purchased from Sigma Aldrich, U.S.A. Rest of the chemical and regents used in the current protocol was analytical grade.

Animals
In the current experimental studies, 7 weeks old Wistar 150-200 g, male rats received from the Institutional animal house and kept under standard experimental conditions 22 3 , relative humidity 60 5, 12/12 h dark/light cycle in under polyethylene cage. The rats received water ad libitum. The entire animal experimental study was approved by the Committee on Institutional Animal Ethics.

Preparation of STZ
For induction the diabetes mellitus, first prepared the solution of STZ by dissolved in freshly prepared citrate buffer pH 4.5 and stored in cold conditions until use. Table 1 showed the high fat diet food composition used in the current experimental study.

Induction of diabetes
After 4 weeks, intraperitoneal STZ injection 40 mg/kg was applied to the rats for diabetes induction. After 7 days, the blood sample was taken by punching all group rat retro-orbital plexuses for blood glucose level estimation. The fasting blood glucose FBG level of the rats was over 16.7 mmol/L.

Preclinical study
The diabetic rats are divided randomly into the following groups and contain 12 animals in each group. Group I: normal rats received only saline, Group I: normal control NC , Group II: diabetic rats DC alone, Group III: DC UA 2.5 mg/kg, body weight , Group IV: DC UA 5 mg/kg, body weight , Group V: DC UA 10 mg/kg, body weight and Group VI: DC GM 10 mg/kg, body weight , respectively. The aforementioned treatment was given to group rats for 12 weeks. The group rat blood glucose levels have been estimated at a regular interval. At the end of the experiment, all group rats were placed in single metabolic cages and the urine was collected for 24 hr. Regular interval was recorded for water intake, food and body weight.
Rats fasted over the night and ketamine 20 mg/kg / xylazine 12.5 mg/kg was used for anesthetize the rats to collected the blood samples via puncturing the retro orbital plexus. The collected blood was centrifuged at 10000 rpm for 15 min for separating the serum. The rats were scarified with excess anesthesia and renal and hepatic tissues were collected. Skeletal muscle was excised and maintained at 80 until necessary.

Serum parameters
The blood glucose level was estimated using the Accu-Chek glucometer Roche Diagnostic, Germany . Total cholesterol and triglycerides OSR60118 and non-esterified fatty acids MBS748204 were estimated using kits Beckman Coulter, Inc. Brea, California, USA and MyBio-Source, Inc. San Diego, USA . The estimated levels of adiponectin KHP0041 and serum insulin KAQ1251 have been rats insulin and mouse/rata ELISA kits Thermo fisher scientific, USA .

Antioxidant parameters
Antioxidant parameters viz., glutathione peroxidase GPx ab102530 , malonaldehyde MDA ab243066 and super oxide dismutase SOD ab141496 were scrutinized via using the standard kits Abcam. USA following the manufacture protocol.

Renal parameters
Blood urea nitrogen BUN MBS168363 , urine protein and serum creatinine MBS2504918 were estimated using the ELISA kits following the manufacture instruction My-BioSource, Inc. San Diego, USA .

Serum leptin, PYY and serum amylin
Serum amylin NBP2-76735 , peptide YY and serum leptin ab100773 were estimated using the ELISA kits following the manufacture protocol Novus Biologicals, USA and RayBio ® , Norcross, GA, USA .

In ammatory parameters
Inflammatory parameters include Transforming growth factor beta 1 TGF-β1 14-8348-62 and Nuclear factor kappa B NF-κB 14-6731-81 were scrutinized via using the standard kits eBioscience, Inc., San Diego. USA following the manufacture protocol.

RNA preparation and RT-PCR quanti cation
Isogen based on guanidine isothiocyanate has been used for isolation of total RNA from the muscle tissue via following the manufacture protocol Nippon Gene, Tokyo, Japan . The isolated RNA has then been treated with RNase-free Dnase at 37 for 10 min before using the RNeasy Mini kits Qiagen . The final absorbance was estimated at the wavelengths of 260 and 280 nm and agarose gel electrophoresis was carried out for qualitative RNA analysis. To synthesise cDNA, total RNA 4 mg was used. In the presence of oligo, dNTP 10 mmol/L or primers at 65 during 5 min and incubated into 20-μL with TrisHCl 50 mmol/L inhaling DTT 0.1 mol/L superScript III reverse transcriptase 50 U , at 25 for 5 min for 60 min for 50 and 15 min for 70 , Total RNA was denatured. The cDNA liquots have been used by Applied Biosystems for the subsequent quantitative PCR using the manufacturing instructions. The target sequences were estimated using cDNA-specific primers Table 2 .

Statically analysis
The data is described as mean SEM in the current experimental analysis. The statistical study involved repeated steps, with one method followed by ANOVA via the Dennett test. A substantial difference between the averages was found to be p 0.05. Mineral Mix S10022M 0 g (0 kcal) 35 g (0 kcal)

Effect of UA on food consumption
Normal rats showed the uniform food consumption throughout the experimental protocol. DC group rats increased the food consumption as compared to treated and normal rats. UA and GM treated rats showed the less consumption of food as compared to DC rats Table 3 .

Effect of UA on body weight
DC rats demonstrated the increased body weight as compared to initial weight and other group rats. UA treatment significantly p 0.001 reduced the body weight as compared to DC group rats. A similar momentum was observed in the GM group rats Table 4 .

Assessment of UA on biochemical parameters FBG,
TC and TG in diabetic rats During treatment with ursolic acid, the levels of fasting blood glucose were analyzed after the rats subjected to fast for 12 h at 0, 4 and 8 weeks Fig. 1 . The study shows that at week 0 levels of fat/streptozotocin-induced diabetic rats were significantly improved the FBG level compared to the normal group at weeks 0. In addition to standard drug and ursolic acid after 4 and 8 weeks, FBG levels were significantly reduced as compared to group II dose-related, as illustrated in Fig. 1. The levels of total cholesterol TC and triglyceride TG in the serum of the ursolic acid-treated rats were drastically lesser, whereas this content was higher in HFD/STZ induced diabetes group rats Fig. 2 . In Table 2 Sequences of primers used for quantitative RT-PCR.

Gene
Forward primer Reverse primer   addition, the increased level of triacylglycerol, non-esterified fatty acids and reduced level of plasma insulin and adiponectin was observed in HFD/STZ rats. UA treatment showed a significant reduced the level of triacylglycerol, non-esterified fatty acids and increased the level of insulin and adiponectin Fig. 3 .    Figure 4 illustrated the assessment of ursolic acid on renal function. The level of serum renal function such as blood urea nitrogen BUN , creatinine SCr and urinary protein excretion for 24 h was obviously bigger in group II as relative to the normal control rats. Although, after treatment of the HFD induced/STD induced diabetic rats with glibenclamide and ursolic acid at different dose levels, these renal function indices were decreased. Additionally, compared to group II, the level of BUN at a lower dose level ursolic acid treated animals was decreased with no significance Fig. 4 . 3.5 Assessment of UA on serum amylin, leptin and peptide YY levels in diabetic rats Figure 5 reveals the impact of ursolic acid treatment fol-lowing 8 weeks on the activities of leptin, Peptide YY and amylin in the serum of the diabetic rats. Supplementation of the ursolic acid to diabetic rats demonstrated a significant reduction in serum leptin compared to group I. Oral supplementation with UA significantly reversed the activity of serum amylin and peptide YY in diabetic rats in dose dependent manner compared to the HFD induced/STD induced diabetic rats Fig. 5 . 3.6 Assessment of UA on oxidative parameters in diabetic rats Oxidative parameters have been assessed with the identification of GSH-Px and SOD activities and MDA levels in all group rats. The diabetic rats group induced by HFD/STZ revealed a significant decrease in activities of GSH-Px and SOD, while MDA levels were increased in comparison with

Assessment of UA on the protein expressions of NF-κB and TGF-β1
The results of the expression of TGF-β1 and NF-κB were significantly up-regulated in group II compared to the normal rats, which showed no significant boost in those proteins expressions. Comparative to the HFD/STZ induced diabetic rats, ursolic acid-treated groups displayed dose-dependent standardized levels of NF-κB and TGF-β1 Fig. 7 .
3.8 Assessment of UA on Traf-4, MAPK-8 and Traf-6 expression The expression of Traf-6 and Traf-4 were higher in retort to a high production of ROS in the diabetic rats, which differed considerably from the normal control rats. Oral supplementation in low doses of ursolic acid led to a significant decrease in ROS level, which shows reduced Traf-6 and Traf-4 in diabetic rats induced by HFD/STZ compared to Group II. ROS-dependent activation of Traf-6 and Traf-4, as illustrated in Fig. 8, shows an enhancement of the MAPK-8 gene in HFD/STZ induced obese rats.
3.9 Assessment of UA on gene expression ngn-3, Pdx-1 and Ins-1 in diabetic rats The RT-PCR analysis determined the impact of UA on the insulin signaling pathway via genes expression ngn-3, Pdx-1 and Ins-1 to classify the underlying mechanism accepted by UA in the β cells regeneration and enhanced production of insulin. In the diabetic rats, levels of gene expression such as Ins-1, ngn-3 and Pdx-1 in tissues of pancreas were significantly decreased compared to normal control rats. The findings suggest that UA significantly enhanced the protein expression of ngn-3, Pdx-1 and Ins-1 and improved pancreatic β cells regeneration as displayed in Fig. 9.

Discussion
The study finded that the protective action of UA on obese diabetes mellitus induced by a high fat diet and streptozotocin has been substantially verified and prevent-  ed by alleviating oxidative stress and improving renal function by inhibiting the expression pathways NF-κB/TGF-β1. Obesity was induced in a 4-week high-fat model and streptozotocin injection that only acts on β-islet cells and results in insulin resistance, increased blood glucose levels and a reduced insulin level. The high-fat diet and streptozotocin treatments of rats were characterized by the hyperglycemia and insulin resistance of animals that imitate the symptom of diabetes in humans. Diabetes is associated with multiple complication in humans and most common being the cardiac vascular dysfunction 17 . Glibenclamide is a member of the sulfonylurea class of drugs and has been used in the clinic as an oral hypoglycaemic agent. Recent findings concerning the benefits of glibenclamide GBC as a neuroprotective drug have initiated a number of new prospective studies 18 . Recently it has been established that it prevents the stroke in human patients 20 . A single drug with multiple pharmacological action prompt us to use glibenclamide 24 as a positive control. Similar studies have been already carried out earlier to support the claim 22 .
Because the HFD promotes the formation of a positive energy balance, which leads to an increase in visceral fat deposition, it has been linked to obesity, particularly abdominal obesity 26 . Furthermore, previous study observed that HFD feeding is convoyed through molecular adaptations that favor deposition of fat in the muscle rather than oxidation. In this experimental study, the rats were feded with HFD as compared to normal rats and they showed a massive increase in perirenal visceral adipose tissue mass, showing that extra energy contributed to adiposity buildup 28,30,32 . HFD received rats demonstrated the enhanced  body weight. Rats received the HFD showed the consumption of more kilocalories roughly 27 5 and exhibited the increased body weight. Rats fed a palatable dietary fat in addition to a conventional chow diet consume roughly 10 more calories per day than rats fed only the chow diet, becoming obese and exhibiting a number of obesityrelated problems over time 26,34,36 . Our result exhibited significant reduction in the whole-body weight and food intake after the ursolic acid treatment.
In this study, the levels of SCr and BUN as well as 24-hr urinary protein excretion in serum were scrutinized to recognize an alteration in the diabetic rats renal function. The profile of renal function was significantly enhanced in group II compared with the normal control rats, whereas dose dependently UA alleviated all these changes. The data have revealed that UA could be a compound capable of having valuable effects on the tissue of kidney. In patients having diabetes mellitus type 2, generally hyperglycemia was associated with dyslipidemia. The higher level of the TC and TG in serum was found in the early phase of DM.
Leptin is an adipokine, which functions to reduce appetite under normal physiological conditions, control the balance of energy and improve the consumption of glucose and insulin sensitivity 19 . Conversely, due to a decrease in leptin receptors in peripherally situated organ and the increased leptin concentration, obesity and diabetes following leptin resistance are present in the diseased condition 21 . High serum leptin levels were reported in diabetic rats that predict a greater likelihood of developing diabetes. Due to adipocyte-insulin-axis deregulation in pancreatic β-cells under hyperleptinemia which leads to hyperinsulinemia 23 . By lowering the leptin in serum after the supplementation of UA significantly reserved the HFD induced obesity diabetics in a dose dependent manner.
PYY is an intestinal hormone produced in the ileum and colon, which responds by slowing down the rate of gastric motility to alleviate appetite and weight loss 25 . High levels of PYY in rats diabetes , which may be interrelated with impassive signaling cascade of leptin as established by leptin resistant, according to finding of data. Enhanced levels of amylin are related to JNK cascade activation, oxidative stress production and ultimately pancreatic β cell apoptosis 25 . Thus, level of amylin could be regarded as a vital target for β cell regeneration and diabetes treatment. This factual judgment in the literature reported that higher content of amylin are linked to the of β cells apoptosis 27 . The figure suggests that UA significantly restored the levels of serum PYY and amylin in the diabetic rats.
Chronic hyperglycemia produces a significant alteration in the markers of oxidative stress 4 . Patients with diabetes demonstrate significantly greater oxidative stress as compare to the healthy people. In the meantime, podocyte apoptosis induced by stress from endoplasmic reticulum played a serious role in DN development 16 .
Oxidative stress developed during the condition of hyperglycemia degrades the body s antioxidant defense mechanism and as a result increases the level of MDA and decrease antioxidant enzyme activity 4, 7 . MDA is known to be an indicator of lipid peroxidation, as it is produced by membrane lipid oxidation. Data illustrated the usage of UA in dose dependent manner significantly reduced the MDA level and boost the activities of GPx and SOD. Superoxide radicals mainly generate ROS and oxidative stress from various sources, and it is important to eliminate SOD to perform the key functions of cells. Glutathione, a major antioxidant, excessively occurs in all cells. It quenches lipid peroxides and GSH-Px hydrogen peroxide by providing an electron with water and oxygen 4, 7 . This outcome validated the improvement by UA in the re-storage of the enzymatic antioxidant parameters similar to normal groups and prevent the organelles of cell from the impact of oxidative stress.
TGF-β1 is a cytokine type with a variety of regulatory impacts on immune reactions that also interfere with ECM production. ROS induced obese diabetes mediates the transcription factor that finally stimulates the profibrotic gene TGF-β1 activation, that promote fibrosis 29,31 . Documentation also shows the low doses therapy of TGF-β1 in untimely DN ameliorates renal hypertrophy and oxidative stress. Activated NF-κB is believed to activate the profibrotic gene TGF-β1 expression that develops ECM accumulation of ECM through a mechanism where activated NF-κB shifted to the nucleus, unite with the DNA sequence of TGF-β1 and activate the TGF-β1 transcription, finally leading to promote the formation of renal fibrosis 33 . From the figure, it is clear levels of NF-κB and TGF-β1 protein enhanced in group II and in a similar manner UA at different dose levels prevented the protein levels NF-κB and TGF-β1 in HFD/STZ induced obese diabetes in rats.
A mitogen-activated protein kinase MAPK downstream route caused by JNK oxidative stress has proven to play a critical role in the development of diabetes. Numerous stress stimuli for example environmental stresses and inflammatory cytokines and in response to that series of phosphorylation on threonin and tyrosine residues act on MKK4 and MKK7 and activate the JNK proteins 35 . Oxidative stress triggers TNF receptor-associated factors 4 and 6 TRAF-4 and TRAF-6 for ASK1 activation and following stimulation of MAPK downstream JNK signaling in various tissues, involving islet β-cells of pancreas 11 . In case of diabetes, various tissues show the stimulation of the JNK pathway and insulin resistance is the resultant of the activation. Although, JNK pathway inhibition in various research studies has also led to a decline in insulin resistance and overall diabetes 11 .
The intention to scrutinizing the TRAF-4, MapK-8 and TRAF-6 expression in normal control and HFD induced diabetic rats to determine how polyphenolics compound UA improve the diabetic conditions with underlying mechanisms 37 . Genes expression levels TRAF-4, MapK-8 and TRAF-6 were significantly increased in diabetic rats, according to the results indicated 11 . Alternatively, treatment with UA significantly inhibited JNK pathway activation as produced by high glucose diet. Consequently, we observed that UA restored the raised level of TRAF-6 and TRAF-4 resultant into the MAPK-8 expression inhibition.
UA and GM treated rats showed the less consumption of food as compared to DC rats Table 3 . Illustrated in section of results. Emphasizing the diet consumption is directly linked to diabetes manifestations. This is supported by study by Srour et al. in this large observational prospective study, a higher proportion of UPF in the diet was associated with a higher risk of T2D 38 . Further the findings suggest that UA significantly enhanced the protein expression of ngn-3, Pdx-1 and Ins-1 and improved pancreatic β cells regeneration as displayed in Fig. 9, responsible for the optimum secretion of insulin by the pancreas.
No the effects are not simila to other polyphenol and flavonoids, as the concept of Structure activity relationship in drug action will fail in the latest development of scientific information.
In continuation with above stated parameter, we have also demonstrated that a mitogen-activated protein kinase MAPK downstream route caused by JNK oxidative stress has proven to play a critical role in the development of diabetes.

Conclusion
As reported in the literature that UA is a polyphenolic compound and over biochemical parameters and protein expressions indicates that the UA dose-dependent alleviates the HFD/STZ induced diabetes in rats are encouraging. More validated proof is required before recommended the usage of UA in the obese type diabetes mellitus. Ursolic acid exhibited the anti-obesity effect via  Reduction of blood glucose level and increased the plasma insulin  Reduced the urine parameters  Reduced the lipid parameters  Reduced the inflammatory parameters such as NF-κB and TGF-β1  Finally reduced the MAPK downstream signaling pathway Kindly Provide the Acknowledgement