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European Journal of Pharmacolo

Antioxidant and pancreas-protective effect of aucubin on rats withstreptozotocin-induced diabetes

Lei Jin a, Hong-Yu Xue a, Li-Ji Jin a,b, Shu-Ying Li c, Yong-Ping Xu a,b,c,⁎

a Department of Bioscience and Biotechnology, Dalian University of Technology, Dalian, Liaoning, PR Chinab State Key Laboratory of Fine Chemicals, Dalian University of Technology, Dalian, Liaoning, PR China

c Dalian SEM Bioengineer and Biotech Ltd., Dalian, Liaoning, PR China

Received 2 July 2007; received in revised form 27 November 2007; accepted 11 December 2007Available online 23 December 2007

Abstract

Oxidative stress has been suggested as a contributory factor in development and complication of diabetes. The aim of the present study was todetermine the protective effect of aucubin on lipid peroxidation and activities of antioxidant defense systems and to conduct immunohistochemicalevaluation of pancreas in streptozotocin-induced diabetic rats. Lipid peroxidation was determined by assessing the concentration ofmalondialdehyde and activities of antioxidant enzymes — catalase, glutathione peroxidase and superoxide dismutase in liver and kidneys of ratswere determined. Changes of blood glucose and immunohistochemical evaluation on pancreas were also investigated as part of the pathology ofdiabetes. In our study, aucubin treatment lowered blood glucose. Diabetic rats exhibited an increase in the level of lipid peroxidation and decreasein activities of antioxidant enzymes in liver and kidneys as compared to control rats. Administration of aucubin to diabetic rats for 15 dayssignificantly reversed damage associated with diabetes. In addition, diabetic rats showed an obvious decrease in insulin immunoreactivity and thenumber of β cells in pancreas, but the pancreas of aucubin-treated rats were improved and the number of immunoreactive β cells weresignificantly increased. These results indicated that aucubin may have value as a safe preventive or therapeutic agent against diabetes mellitus.© 2007 Elsevier B.V. All rights reserved.

Keywords: Diabetes mellitus; Blood glucose; Lipid peroxidation; Antioxidant enzymes; Pancreas

1. Introduction

Diabetesmellitus is a serious, complex chronic conditionwhichis a major source of ill health all over the world. This metabolicdisorder affects approximately 4% of the population worldwideand is expected to increase by 5.4% in 2025 (Kim et al., 2006).Diabetes mellitus is characterized by hyperglycemia and isassociated with disturbances in carbohydrate, protein and fatmetabolism which occurs secondary to an absolute (type І) orrelative (type ІІ) lack of insulin (Alberti and Zimmet, 1998).

Oxidative stress occurs when the balance between oxidant andantioxidant systems shifts in favour of the former leading to thegeneration of free oxygen radicals. Reactive oxygen species are

⁎ Corresponding author. Department of Bioscience and Biotechnology, DalianUniversity of Technology, No. 2 Linggong Road, Ganjingzi District, Dalian116024, PR China. Tel.: +86 411 8470 6345; fax: +86 411 8470 6359.

E-mail address: rayking007@gmail.com (Y.-P. Xu).

0014-2999/$ - see front matter © 2007 Elsevier B.V. All rights reserved.doi:10.1016/j.ejphar.2007.12.011

involved in the pathogenesis of many diseases including hypoxia,hypercholesterolemia, atherosclerosis, hypertension, ischemiareperfusion injury and heart failure (Taniyama and Griendling,2003; Wilcox and Gutterman, 2005). It has been shown thatpatients with diabetesmellitus have increased oxidative stress andimpaired antioxidant defense systems, which appear to contributeto the initiation and progression of diabetes-associated complica-tions (Maritim et al., 2003). There is convincing experimental andclinical evidence that the generation of reactive oxygen species isincreased in both types of diabetes. Hyperglycemia is the primarysymptom of diabetes and is blamed for the complications ofdiabetes because elevated glucose concentration directly injurescells and induces lipid peroxidation (Davi et al., 2005). Normally,the level of oxidative stress is modulated by antioxidant defensesystems (Saxena et al., 1993). Diabetes-linked alterations inantioxidant defense system enzymes such as catalase, glutathioneperoxidase, superoxide dismutase have been demonstrated(Maritim et al., 2003). Diabetes mellitus also results in severe

163L. Jin et al. / European Journal of Pharmacology 582 (2008) 162–167

metabolic imbalances and non-physiologic changes in manytissues especially the pancreas which is the organ that secretesinsulin (Baynes and Thorpe, 1999). Increases in oxidative stressmarkers in pancreatic islets in experimental diabetic rats havebeen reported (Ihara et al., 1999). Taken together, these studieshint that oxidative stress plays a role in diabetes mellitus.

Streptozotocin, a monofunctional nitrosourea derivative, is oneof the most commonly used substances to induce diabetes inexperimental animals (Szkudelski, 2001). Evidences suggest thatthe diabetogenic capacity of streptozotocin may depend on itsability to damageβ-cell and induce oxidative stress (Ohkuwa et al.,1995). Aucubin (1,4a,5,7a-tetra-5-hydroxy-7-(hydroxymethyl)cyclopenta(c)pyran-1-yl-β-D-glucopyranoside) is a common iri-doid glucoside isolated from Plantago asiatica, Eucommiaulmoides, the leaves of Aucuba japonica (Pailer and Haschke-Hofmeister, 1969; Bianco et al., 1974; Bernini et al., 1984) andmore recently from butterfly larva (Suomi et al., 2001). Aucubinhas been known to have diverse biological activities such asinhibiting the synthesis of RNA and proteins in the liver of mice,protecting against liver damage induced by carbon tetrachloride orα-amanitin in mice and rats, and antimicrobial activity (Changet al., 1983; Davini et al., 1986; Chang, 1998). Aucubin wasselected for this investigation because of its in vitro antioxidativecapacity (Li et al., 2004).

To our knowledge, this is the first biochemical investigationon the effect of aucubin on the antioxidant status of experimentaldiabetic rats. We report on the effect of aucubin on elements ofdiabetes mellitus including blood glucose, oxidative parametersand immunohistochemical evaluation in rats with streptozoto-cin-induced diabetes.

2. Materials and methods

2.1. Chemicals and reagents

Streptozotocin was obtained from Sigma-Aldrich Inc. (St.Louis, Mo). Analytical grade aucubin (productN99% purity) waspurchased from Genay EXTRASYNTHÊSE Lyons, France.Mouse monoclonal insulin antibody (INS05) and biotinylatedgoat anti-mouse immunoglobulin were purchased from LabVision Corp., Fremont, CA. The activities of catalase, glutathioneperoxidase, superoxide dismutase as well as the concentrations ofthe malondialdehyde and the protein in the supernatant were alldetermined by commercially available kits (Nanjing JianchengBioengineering Institute, Jiangsu, China). Blood glucose SpanDiagnostic kit and Jinque test strips was obtained from ShanghaiMicroSence Inc., Shanghai, China.

2.2. Animals

Male Wistar rats (200 g to 230 g) were procured from theExperimental Animal Center, Dalian Medical University, China.The rats were randomly divided into four groups. Group I: controlanimals (n=8). Group II: control animals given aucubin alone(n=8). Group III: streptozotocin-diabetic animals (n=12). GroupIV: streptozotocin-diabetic animals given aucubin (n=12). Ratswere maintained in an air-conditioned room (25±1 °C) with a

12 h light:12 h dark cycle (07:00 h on 19:00 h off). Standard ratsfeed andwater were provided ad libitum. The rats were allowed toacclimatize to the laboratory environment for 7 days before thestart of the experiment. Throughout the study, rats were assessedweekly for body weight.

All experimental procedures were conducted in conformitywith institutional guidelines for the care and use of laboratoryanimals in China (Permit: SCXK 2002-0002), and theinternational guidelines on the ethical use of animals (NIHpublications No. 80-23).

2.3. Induction of diabetes in rats

Diabetes was induced in Groups III and IV rats followingovernight fasting by an intraperitoneal injection of streptozo-tocin in a single dose of 60 mg/kg. Streptozotocin was dissolvedin a freshly prepared 0.01 M citrate buffer (pH 4.5) whilecontrol rats (Groups I and II) were injected with buffer alone.Streptozotocin-injected animals (Groups ІІІ and IV) weregiven 5% glucose (2 ml/kg body weight) at 24 h followingstreptozotocin injection to prevent initial drug-induced hypo-glycemic mortality. Blood was drawn from the tail plexus ofconscious rats using a heparinized inoculator 72 h later.Concentration of blood glucose was tested using the SpanDiagnostic kit with Jinque test strips. Blood glucose wasmeasured at 3, 10, 20, 30, 35, 40 and 45 days to assess the bloodglucose-lowered effect of aucubin. Rats with bloodglucoseN11.1 mmol/L were considered diabetic.

2.4. Aucubin treatment of rats

Intraperitoneal injections of aucubin (5 mg/kg body weight)to animals in Groups II and IV respectively after the rats weremade diabetic for 30 days, the rats in Groups I and III wereintraperitoneally injected with normal physiological salinealone. Aucubin was administered for 15 days starting withintraperitoneal injections twice daily for the first 5 days,followed by single injections daily for the last 10 days.

At the end of the 45-day experiment, the number of rats stillalive in each treatment wasGroup І, 8/8;Group ІІ, 8/8; Group III,7/12 and Group IV, 9/12. For statistical analysis, 7 surviving ratsfrom each group were randomly selected. These 28 rats selectedwere deprived of food overnight, sacrificed by decapitation, andthen used in series of studies.

2.5. Lipid peroxidation and antioxidant enzymes activities

Following the termination of the rats, liver and kidneys wereimmediately removed and rinsed in ice-cold saline. 10% (M/V)tissue homogenate was prepared with PBS buffer, pH 7.4. Aftercentrifugation at 2000 rpm for 10 min, the supernatant wascollected for immediate measurement of lipid peroxidation andantioxidant enzymes activities.

The activities of catalase, glutathione peroxidase, superoxidedismutase as well as the concentrations of the malondialdehydeand the protein in the supernatant were determined by com-mercially available kits as the manufacturer's instructions. Lipid

Table 1Effect of aucubin on body weight gains of rats

Group І(control)

Group ІІ(control+aucubin)

Group ІІІ(diabetic)

Group ІV(diabetic+aucubin)

Initial bodyweight (g)

216.57±6.80 212.14±6.84c 220.00±8.48c 214.28±8.63c

Final bodyweight (g)

311.86±20.04 322.14±25.35c 181.85±17.28a 211.71±18.93b

Body weight was expressed as mean±S.D. (n=7). Groups ІІ and ІІІ werecompared with Group І, Group ІV was compared with Group ІІІ.aPb0.001; bPb0.05; cPN0.05.

Table 2Effect of aucubin on lipid peroxidation and antioxidant enzymes in liver

164 L. Jin et al. / European Journal of Pharmacology 582 (2008) 162–167

peroxidation was assessed by measuring the concentration ofmalondialdehyde. The level of malondialdehyde was expressedas nmol malondialdehyde per milligram protein. The activitiesof antioxidant enzymes were expressed as units per milligramprotein.

2.6. Immunohistochemical evaluation on pancreas

The pancreaswas removed immediately from the animals aftersacrificing and rinsed in ice-cold saline. The tissue samples werefixed in Paraformalclehyde, dehydrated in a graded series ofethanol, and embedded in paraffin wax before sectioning. Sec-tions were dewaxed and rehydrated. After the step of washing inphosphate-buffered saline, sections were immersed in a solutionof 3% H2O2 for 10 min. The sections were then pre-incubatedwith nonimmune serum for 15 min and subsequently replacedwith the mouse anti-insulin antibody (1:200, INS05, Lab Vision,CA) for incubation at 4 °C for 16 h. Biotinylated goat anti-mouseimmunoglobulin was used as a secondary antibody. They werelabelled with streptavidin peroxidase following incubation withthe secondary antibody at 37 °C for 30min. The localization of theantigen was indicated by a brown color obtained with 3-amino-9-ethyl-carbazole (AEC) as chromogenic substrate for peroxidaseactivity. Slides were counterstained with hematoxylin for mic-roscopic observation.

The specificity of the immunohistochemical staining waschecked by omission of the primary antibody, or by using aninappropriate antibody (anti-gastrin). All these controls gavenegative results. Control pancreas sections with (+) signals wereused as a positive control.

Fig. 1. Changes in blood glucose levels of different experimental groups. GroupI: control animals. Group II: control animals given aucubin. Group III:streptozotocin-diabetic animals. Group IV: streptozotocin-diabetic animalsgiven aucubin.

2.7. Image analysis for insulin immunoreactivity

In order to evaluate insulin immunoreactivity, the intensity ofthe corresponding signals from the tissue sections was measured.More than 10 islets in each rats group were randomly selected andtransferred to a pathology image analyzing system (VNT, Beijing,China). Staining signals of the islets selected on the capturedimage were converted to gray density which can be automaticallycalculated as a staining intensity per unit area (mm2). The insulinimmunoreactivity was calibrated from 0 to 10.

2.8. Statistical analysis

All data were analyzed using the SPSS statistical software(version 13.0, SPSS, Chicago, IL), with Pb0.05 values con-sidered as statistically significant. Results were expressed asmean±S.D. Comparisons between the groups were assessed bypaired-samples t test.

3. Results

3.1. Body weight gains of rats

As shown in Table 1, initial body weights in all groups weresimilar. At the end of the study, rats in both control groups I andII had grown to much greater than their starting weights.However, the streptozotocin-diabetic rats (Group III) had lostbody weight even when compared with their initial weight suchthat their final body weight was significantly less than controlgroups I and II. While the body weight of diabetic rats givenaucubin (Group IV) were significantly less than those in thecontrol groups I and II, it was still significantly greater than thestreptozotocin-treated rats in Group III.

3.2. Changes in blood glucose level

Fig. 1 shows the changes in blood glucose levels of differentexperimental groups over the experimental period. Thestreptozotocin-diabetic rats (Group III and IV) showed a

Group І(control)

Group ІІ(control+aucubin)

Group ІІІ(diabetic)

Group ІV(diabetic+aucubin)

Malondialdehyde(nmol/mg protein)

5.07±0.37 4.41±0.20c 7.06±0.95a 5.77±0.36b

Catalase(U/mg protein)

19.80±1.64 21.67±0.90c 13.48±1.92b 16.23±1.44c

Glutathioneperoxidase(U/mg protein)

13.24±1.56 15.02±1.04c 8.68±0.63b 10.81±1.68c

Superoxidedismutase(U/mg protein)

9.14±0.67 11.61±1.35b 7.42±0.76a 10.24±0.94a

Data were expressed as mean±S.D. (n=7). Groups ІІ and ІІІ were comparedwith Group І, Group ІV was compared with Group ІІІ.aPb0.001; bPb0.01; cPb0.05.

Table 3Effect of aucubin on lipid peroxidation and antioxidant enzymes in kidney

Group І(control)

Group ІІ(control+aucubin)

Group ІІІ(diabetic)

Group ІV(diabetic+aucubin)

Malondialdehyde(nmol/mg protein)

2.83±0.57 1.86±0.23b 4.92±0.81a 2.99±0.29a

Catalase(U/mg protein)

15.09±1.16 17.52±1.23c 10.47±1.43a 13.28±0.59a

Glutathioneperoxidase(U/mg protein)

7.46±0.90 8.76±0.83c 5.05±0.64a 6.01±0.48c

Superoxidedismutase(U/mg protein)

14.58±0.94 16.49±1.31c 9.24±0.55a 11.56±1.39b

Data were expressed as mean±S.D. (n=7). Groups ІІ and ІІІ were comparedwith Group І, Group ІV was compared with Group ІІІ.aPb0.001; bPb0.01; cPb0.05; dPN0.05.

Fig. 3. Comparative evaluation of the expression of insulin immunoreactivity inthe pancreatic islets. Intensity of the immunoreaction in each group wasmeasured by the image analyzing system and recorded with the range from 0 to10. Mean±S.D. Levels of significance were determined by paired-samples ttest. ⁎Pb0.01 vs control rats. ⁎⁎Pb0.01 vs diabetic rats.

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significant increase in the level of blood glucose followingstreptozotocin treatment. Aucubin administration to diabeticrats (Group IV) significantly lowered the observed bloodglucose level. There were no differences between control rats(Groups І and ІІ).

3.3. Effect on lipid peroxidation and antioxidant enzymes

To study the effect of aucubin on oxidative stress, the con-centration of malondialdehyde and activities of catalase,glutathione peroxidase, superoxide dismutase in liver and kidneywere measured. Tables 2 and 3 depict the levels of lipidperoxidation and activities of antioxidant enzymes in liver and

Fig. 2. Immunohistochemical evaluation on pancrea. (400×): (A) Control ratshowing normal structure and β cells (→); (B) Rats administered with aucubinalone showing normal structure and β cells (→); (C) streptozotocin-diabetic ratsshowing the decrease in insulin immunoreactivity and the number of immunor-eactive β cells (→); (D) Diabetic rats treated with aucubin showing increase ofinsulin immunoreactivity and the number of immunoreactive β cells (→).

kidneys of experimental rats. In control rats (Groups І and ІІ),significant differences in antioxidant activity between control andaucubin-treated rats demonstrates the obvious antioxidant abilityof aucubin. Diabetic rats (Group III) showed marked elevation inlipid peroxidation and a decrease in activities of antioxidantenzymes, whereas, aucubin-treated diabetic rats (Group IV)showed a decrease in the level of lipid peroxidation and elevationof the activities of antioxidant enzymes significantly compared todiabetic rats in Group III.

3.4. Immunohistochemical evaluation on pancreas

Fig. 2(A–D) demonstrates the immunohistochemical resultson panceras in experimental rats. In control rats (Groups І andІІ), the islets showed the normal structure with a large centralcore formed by insulin-secreting β cells. The control groupgiven aucubin alone was no different from the intact controlgroup considering the insulin immunoreactivity and the numberof β cells (Fig. 2A and B).

In the pancreatic islets of the diabetic group ІІІ, a significantdecrease in insulin immunoreactivity and the number ofimmunoreactive β cells was observed by comparison with thecontrol groups (Fig. 2C). On the other hand, in the pancreaticislets of the diabetic rats given aucubin in group IV, ansignificant increase in insulin immunoreactivity and the numberof immunoreactive β cells was observed as compared withuntreated diabetic rats.(Fig. 2D).

3.5. Image analysis for insulin immunoreactivity

Fig. 3 shows the result of image analysis for insulinimmunoreactivity of theβ cells by immunohistochemical intensity.It represented a remarkable decrease in the diabetic rats in groupІІІ when compared to control rats (Groups І and ІІ). Afteradministration of aucubin, the insulin immunoreactivity in groupIV was relatively more numerous than that of diabetic rats.However, it was still weaker than that of control rats.

4. Discussion

Diabetes mellitus, the most common endocrine disease, isnot a single disease but a group of disorders of varying etiologyand pathogenesis. The management of diabetes is considered aglobal problem and a cure has yet to be discovered. Modern

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drugs, including insulin and other hyperglycemia agents suchas biguanides, sulphonylureas etc. control the blood glucoselevel only when they are regularly administered, but thesetreatments are tedious and have several disadvantages(Upadhyay et al., 1996). However, medicinal herbs may offera similar degree of efficacy without so many troublesome sideeffects. It has been reported that chemicals with antioxidanteffects may help to regenerate β-cells and protect pancreaticislets against cytotoxic effects of streptozotocin (Alvarez et al.,2004). Several studies have shown increased lipid peroxidationin the diabetic state (Sekeroglu et al., 2000; Ugochukwu et al.,2004). Lipid peroxide-mediated tissue damage have beenobserved in the development of all types of diabetes mellitus(Feillet-Coudray et al., 1999). Diabetes mellitus is associatedwith generation of reactive oxygen species leading to oxidativedamage particularly in liver and kidney (Mohamed et al.,1999). In our study, there were significant increases in lipidperoxidation in liver and kidney of the diabetic group, whichwas in agreement with others (Venkateswaran and Pari, 2002;Latha and Pari, 2003). Moreover, following intraperitonealinjection of aucubin, the concentration of malondialdehyde inaucubin-treated rats was significantly decreased both in controland diabetic groups, which indicates a powerful antiperox-idative effect of aucubin.

Increased lipid peroxidation under diabetic conditions can bedue to increased oxidative stress in the cell as a result ofdepletion of antioxidant protective systems. Antioxidant pro-tective systems against reactive oxygen species and the break-down products of peroxidized lipids and oxidized protein andDNA are provided by many enzyme systems such as catalase,glutathione peroxidase and superoxide dismutase. Oxidativestress in diabetes is coupled to a decrease in the antioxidantstatus, which can increase the deleterious effects of free radicals(Picton et al., 2001). Catalase and superoxide dismutase are thetwo major scavenging enzymes that remove radicals in vivo. Adecrease in the activity of these antioxidants can lead to anexcess availability of superoxide anion (O2

•−) and hydrogenperoxide (H2O2), which in turn generate hydroxyl radicals(•OH), resulting in initiation and propagation of lipid peroxida-tion. Superoxide dismutase can catalyze dismutation of O2

•− intoH2O2, which is then deactivated to H2O by catalase or glutathioneperoxidase (Aebi, 1984; Kumuhekar and Katyane, 1992).Normally, superoxide dismutase works in parallel with selenium-dependent glutathione peroxidase, which plays an important role inthe reduction of hydrogen peroxide in the presence of reducedglutathione (GSH) forming oxidized glutathione (GSSG), and thus,it protects cell proteins and cell membranes against oxidative stress.Glutathione peroxidase has a key role in enzymatic defense systemsand reduces organic peroxides (H2O2, lipid or organic peroxides)into their corresponding alcohols. A significant decrease inglutathione peroxidase activity could suggest inactivation by reac-tive oxygen species, which are increased in diabetic rats. Thedecrease may also be due to the decreased availability of itssubstrate, GSH, which has been shown to be depleted duringdiabetes (Jain, 1998; Ugochukwu et al., 2004). In this study,activities of antioxidative enzymes were seriously depressed in thestreptozotocin-diabetic rats compared to control groups. Decreased

activities of catalase, glutathione peroxidase and superoxidedismutase may be due to inactivation caused by radicals. Indiabetes, non-enzymatic glycation due to persistent hyperglycemiamay also inactivate the antioxidant enzymes (Saxena et al., 1993).However, administration of aucubin could reverse progress of thedisease. Compared with control groups, the increased activities ofcatalase, glutathione peroxidase and superoxide dismutase inaucubin-treated rats suggest that aucubin has free radical scaven-ging activity, which may exert a beneficial effect againstpathological alterations caused by reactive oxygen species.Aucubin increases the activities of antioxidant enzymes instreptozotocin-treated rats implying that aucubin reactivates theantioxidant defense system, thereby increasing anti-diabeticactivity.

As the most predominant characteristic of diabetes mellitus,hyperglycemia is in itself, very dangerous for diabetic patients. Theelevated blood glucose levels in diabetes are thought to lead to celldeath through oxidative stress induction that occurs as a commonsequel of diabetes-induced modification of sugar moieties onproteins and lipids (Donnini et al., 1996). Hyperglycemia increasesoxidative stress through the overproduction of reactive oxygenspecies, which results in an imbalance between free radicals andthe antioxidant defense systems of the cells. The administration ofaucubin decreases blood glucose concentration in diabetic ratsdemonstrating the blood glucose-controlling ability of aucubin andits role as an essential trigger for the liver and kidney to revert totheir normal antioxidative abilities.

The protective effect of aucubin is probably due to itsantioxidant nature as it counteracts free radicals. While itsprotective effect is also evident from the immunohistochemicalevaluation on pancreas. The present study confirms that pancreaticβ-cells are destroyed by streptozotocin (Bolkent et al., 2005;Murugan and Pari, 2006). In the streptozotocin-diabetic rats, asignificant decrease in insulin immunoreactivity and the numberof immunoreactiveβ cells was observed as distinguished from thecontrol rats in Groups І and ІІ. However, after the treatment ofaucubin, the insulin immunoreactivity was improved and anincrease in the number of immunoreactiveβ cells was observed incomparison to the diabetic group. According to the immunohis-tochemical results obtained, aucubin may have the ability toenhance insulin sensitivity or regenerate the β cells of insulindependent diabetic rats. The protective effect of aucubin could bedue to its direct influence on the endocrine pancreatic function indiabetic animals like drug THC (Pari and Murugan, 2005).Aucubin acts on the stimulation of glucose intake into cells andthus a lowering of the blood glucose level is encountered. Thus,aucubin treatment in rats may maintain the blood glucosehomeostasis, which in turn prevents the autoxidation of glucoseby insulin secreted from the pancreatic β-cells.

In conclusion, the present investigation shows that aucubinpossesses several healthful properties including control ofhyperglycemia, antioxidant effects and pancreatic β-cell protec-tion that taken together contribute to its protective effect instreptozotocin-diabetic animals. Thus, aucubin may be impli-cated as a preventive agent against diabetes mellitus. However,more work is warranted to elucidate its myriad mechanisms ofaction.

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Acknowledgement

The financial support was provided by National NaturalScience Foundation of China (No. 30371053) and NationalOutstanding Youth Foundation of China (No. 30125034). Theauthors are also grateful to Professor Jun-jun Zhao in DalianMedical University, China for guide on Histopathologicalexamination and Professor Nicole Buckley in Canada SpaceAdministration (CSA), Canada for revision on manuscript.

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