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Phytoecdysteroids from Ajuga iva act as potential antidiabetic agent against alloxan-induced diabetic male albino rats
Biomedicine & Pharmacotherapy 96 (2017) 480–488
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Original article
Phytoecdysteroids from Ajuga iva act as potential antidiabetic agent against alloxan-induced diabetic male albino rats Jin-Jun Wang, Hao Jin, Shao-Ling Zheng, Peng Xia, Yong Cai, Xiao-Jie Ni
MARK
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Department of Transplantation, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Phytoecdysteroid Diabetes Alloxan Pancreas Rats
The present study investigated the protective effect of phytoecdysteroids extracted from the Ajuga iva plant on body weight changes, blood glucose, insulin total protein, blood urea nitrogen (BUN), creatinine, triglycerides (TG), cholesterol, lipid peroxidation, antioxidant enzymes, pancreatic histopathology and hexokinase-I expression in the alloxan-induced diabetic rats. Experimental diabetes was induced following 15 day intraperitoneal administration of alloxan. The rats were divided into four groups. Group I served as a sham group, and group II served as the diabetic control. Group III served as a treatment for phytoecdysteroids (10 mg/kg), and group IV served as a treatment for phytoecdysteroids (20 mg/kg). Phytoecdysteroids restored body weight loss to its antihyperglycemic effect. Blood glucose was reduced 19.2 and 52.9% in group III and IV respectively. Blood insulin (54.9 and 105.88%) and total protein (25 and 72.2%) was increased in group III and IV respectively. BUN, creatinine, TG, cholesterol and lipid peroxidation was significantly reduced following treatment. Catalase, superoxide dismutase (SOD), and glutathione peroxidase activity were significantly increased following treatment. Islet β-cells are lost in alloxan-induced diabetic rats. Regeneration of islets and reduced atrophy of acinar cells were noted. The number of insulin-secreting cells was tremendously reduced in alloxan-induced diabetic rats. Insulin-secreting cells were increased 48 and 61% in group III and IV respectively. Hexokinase-I mRNA (28.3 & 93.5%) and protein (27.9 and 55.3%) expression were significantly increased following treatment. Taking all these data together, it is suggested that the phytoecdysteroid could be a potential therapeutic agent against experimental diabetes.
1. Introduction Diabetes mellitus is recognized as a clinical syndrome that is characterized by hyperglycemia due to deficiency of insulin. A striking feature of diabetes is the shift in the form of fuel usage from carbohydrates to fats [1]. Diabetes is recognized as a common disease that leads to chronic conditions like neuropathy, nephropathy, vascular diseases associated with heart, kidney, brain, peripheral blood vessels and retinopathy [2]. In diabetes, the body either produces little insulin or ceased to produce insulin, or became progressively resistant to insulin action [3]. Diabetes mellitus is a metabolic disorder that affects millions of people worldwide. Diabetes present in sixteen million peoples in the United States and more than 165 million peoples worldwide [4]. Increased oxidative stress leads to the excessive production of free radicals, which plays an important role in the development of diabetes and its complications [5]. Accelerated generation of free radicals leads to the increased tissue damage, metabolic stress, and cell death [6].
Hyperglycemia can substantially increase the production of reactive oxygen species (ROS) and lead to several complications, including nephropathy [7,8]. Diet can notably contribute for the treatment of several diseases, including diabetes and immune system-associated disorders [9–11]. Also, Fomes fomentarius exhibits as a potential therapeutic agent against streptozotocin-induced diabetic rats [12]. Although, insulin is a wellknown remedy for the prevention and management of diabetes, searching an alternative from the plant and synthetic sources when insulin is not readily available has been attempted [12]. Furthermore, several herbs can be used as an alternative to insulin for the treatment of diabetes when insulin is not available [13]. For instance, antidiabetic and anticholesterolemic potential of phytoecdysteroids extracted from Ajuga iva has been reported [14–16]. Therefore, the present study was aimed to evaluate the protective effect of phytoecdysteroids in alloxaninduced diabetic rats.
Abbreviations: SOD, superoxide dismutase; ROS, reactive oxygen species; TG, triglycerides; PEG, polyethylene glycol; BUN, blood urea nitrogen; TBST, tris-buffered saline with Tween20; qPCR, real-time quantitative PCR; HRP, horseradish peroxidase; PVDF, polyvinylidene difluoride; SEM, sandard error of the mean ⁎ Corresponding author at: Department of Transplantation, The First Affiliated Hospital of Wenzhou Medical University, 2 Fuxue Lane, Wenzhou, Zhejiang 325000, PR China. E-mail address: [email protected] (X.-J. Ni). http://dx.doi.org/10.1016/j.biopha.2017.10.029 Received 2 September 2017; Received in revised form 3 October 2017; Accepted 6 October 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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(125I) insulin, for the limited binding sites on a specific antibody. At the end of incubation, the antibody bound and free insulin was separated by the second antibody-polyethylene glycol (PEG) aided separation method [20]. The insulin concentration in the blood was then quantitated by measuring the radioactivity associated with the antibody bound fraction of the samples.
2. Materials and methods 2.1. Animals A total of 24 healthy male albino Wistar strain rats (6 weeks old) were obtained from the animal house in Shangai (Shanghai Laboratory Animals Center, Shanghai, China). The rats weighed 160–200 g and were kept in polypropylene cages at a temperature of 25 ± 0.5° C with a relative humidity of 60 ± 5% and a light/dark cycle of 12 h. Rats were allowed free access to food and water. Ethical approval was obtained from the Ethics Committee of Wenzhou Medical University (approval no. 201308807).
2.8. Determination of serum total proteins The amount of total protein in the blood was determined according to Lowry et al. [21]. Briefly, 0.l ml of a blood sample, 0.5 m1 of l0% TCA was added, and contents were mixed and centrifuged. To the precipitate, 0.l ml of 0.l N NaOH was added. From this solution, 0.l ml was taken and 5 ml alkaline copper reagent was added mixed, and the contents were allowed to stand at room temperature for l0 min. Then 0.5 ml of Folin's-Phenol (1N) reagent was added to the reaction tube and mixed well. The blue color developed in each of the reaction tubes was measured at 680 nm employing a spectrophotometer. Values are expressed in mg/dl.
2.2. Preparation of phytoecdysteroids An aqueous solution of Ajuga iva was prepared according to the method described by Sharma et al. [17]. Briefly, Ajuga iva was supplied by Xinran Biotechnology Co., Ltd (Shangai, PR China). The 100 g of Ajuga iva leaves were air-dried and extracted (distilled water) via refluxing for 36 h at 90° C. Subsequently, the aqueous extract was evaporated using a vacuum and then converted into a powder at 90° C for 12 h.
2.9. Determination of blood urea nitrogen (BUN) Quantitative and competitive enzyme immunoassay technique used in the BUN assay [22]. This method utilizing a monoclonal antibody and a conjugate to BUN. Horseradish peroxidase (HRP) conjugate was incubated with sample for 1 h in a pre-coated assay plate. Wells were repeatedly washed at the end of incubation and incubated with a substrate for HRP enzyme. Blue colored complex was formed as a product of the enzyme-substrate reaction. The blue color turned into yellow following the addition stop solution, and intensity was measured at 450 nm.
2.3. Experimental diabetes induction Experimental diabetes was induced by the use of alloxan. Briefly, a freshly prepared solution of alloxan (60 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in 0.1 M citrate buffer (pH 4.5) was injected intraperitoneally. A booster (secondary) dose of alloxan was administered at 7th day following the primary dose administration. Following 15 days of alloxan administration, rats were exhibiting blood glucose more than 250 mg/dl were selected for the experiment [18].
2.10. Determination of blood creatinine 2.4. Experimental groups The blood levels of creatinine were determined Muthuviveganandavel et al. [23]. Creatinine was measured with a rateblanked and compensated picric acid colorimetric assay. The finally liberated hydrogen peroxide is used to form a chromogen by oxidase. The color intensity is directly proportional to creatinine level in the sample. Values were expressed in U/L.
The rats were divided into 4 groups, which contained 6 rats each. Group I served as a sham group and treated with normal saline once daily for 15 consecutive days. Group II served as the diabetic control and treated with normal saline once daily for 15 consecutive days. Group III served as a treatment for phytoecdysteroids (10 mg/kg) once daily for 15 consecutive days. Group IV served as a treatment for phytoecdysteroids (20 mg/kg) once daily for 15 consecutive days.
2.11. Determination of blood triglycerides and cholesterol Triglycerides (TG) and cholesterol were determined according to Muthuviveganandavel et al. [23]. Briefly, mix 1000 μl of working reagent with 10 μl of the sample and incubated at 37 °C for 5 min. The absorbance was measured at 546 nm within 60 min. Cholesterol was estimated by mixing 0.1 ml of serum with 4.9 m1 of ferric chloride and was taken in a screw-capped centrifuge tube and allowed to stand for 15 min. The sample was centrifuged at 3000 × g for l0 min. 1.5 ml of concentrated sulphuric acid was added to 2.5 m1 of the clear supernatant and was incubated for 20 min at room temperature. The intensity of the developed color was read against a blank at 560 nm. Values are expressed in mmol/L.
2.5. Determination of body weight The body weight of each animal was observed before and after the treatment. 2.6. Determination of blood glucose Blood glucose levels were determined in the control and extracttreated rats as described by Muthuraman et al. [19]. Briefly, 0.1 ml of a blood sample, 3.8 ml of isotonic sodium sulfate-copper sulfate solution and 0.l ml of 10% sodium tungstate solution was added to precipitate the proteins. The samples were centrifuged at 1500 rpm for 10 min to obtain a protein-free solution. Alkaline tartarate (1 ml) was then added to l ml of the clear supernatant and placed in a boiling water bath for l0 min. This was then cooled, and 3 ml phosphomolybdic acid and 3 ml water was added and mixed thoroughly. The solution was allowed to stand for 5 min for color development. The developed color was read against a blank at 630 nm. Values are expressed in mg/dl.
2.12. Determination of lipid peroxidation Lipid peroxidation was estimated as the content of malondialdehyde (MDA) [24]. Briefly, 0.4 ml of blood samples, 0.05 ml of butylated hydroxytoluene, 1.6 ml of phosphate buffer (pH 7.4), and 1 ml of 30% trichloroacetic acid (TCA) were incubated together for 2 h at −20 °C. The mixture was then centrifuged (500g) for 15 min. Then, 1 ml of the supernatant was added to 0.075 ml of 0.1 M EDTA, and 0.25 ml of 1% TBA contained in a tube for 20 min in a boiling water bath. The absorbance was measured at 532 and 600 nm in a spectrophotometer. Values are expressed as nmol/mg of protein.
2.7. Determination of blood insulin The radioimmunoassay method employed was based on the competition of unlabeled insulin present in the blood, with radio-iodinated 481
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1 mM PMSF, 15 μg/ml leupeptin, and 15 μg/ml aprotinin. The protein which is present in the lysate was run on SDS-PAGE. Polyvinylidene difluoride (PVDF) membrane was used for transferring in the SDSPAGE. Tris-buffered saline with Tween-20 (TBST) was used for the nonspecific blocking proteins. The membrane probed for 12 h with an antibody against hexokinase-I. Membranes were washed twice with TBST and incubated with HRP-conjugated goat anti-rabbit IgG (St. Louis, MO 63178 USA) for 60 min. The protein level of hexokinase-I was determined using enhanced chemiluminescence method [26].
2.13. Determination of antioxidant enzymes The concentration of antioxidant enzymes, including catalase, glutathione peroxidase and superoxide dismutase (SOD), was determined according to Muthuraman et al. [24] and values were expressed as μmol/mg of protein. 2.14. Histopathology For the histopathological studies, the rats were anesthetized with intramuscular injection of ketamine hydrochloride (100 mg/kg) + Xylazine (11 mg/kg) (Sigma-Aldrich; Merck KGaA), and pancreas tissue was removed and placed in 10% formaldehyde for fixation for 3 h at room temperature and embedded in paraffin. Tissues section (5-μmthick) were prepared and stained with Hematoxylin and Eosin (H & E) for 5 min at room temperature. Stained tissue sections were qualitatively analyzed as described previously [23] and images were captured using a light microscope at a magnification of ×40. Image analysis was performed with the R package CRImage (http://www.lepem.ufc.br/ jaa/colorout_1.1-0.tar.gz) and analyzed by a pathologist.
2.19. Statistical analysis Values are expressed as the mean ± standard error of the mean (SEM) experiments performed three times. The statistical significance of differences between the control and treatment was evaluated using ANOVA and followed by Student’s t-test (SPSS v. 17; SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant difference. 3. Results
2.15. Immunohistochemical analysis
3.1. Effect of phytoecdysteroids on body weight
The pancreas was surgically removed from the rat animals following decapitation and rinsed in ice-cold normal saline. Paraformaldehyde was used for fixation of pancreas tissue and dehydrated with ethanol (graded series). Then, tissues were embedded in paraffin wax. Tissues were dewaxed and rehydrated before sectioning. Sections were made and incubated with mouse anti-insulin antibody (1:300, Abcam, USA) for overnight at 4 °C. After repeated washing with PBS, sections were incubated with HRP-conjugated secondary antibody at 37 °C for 60 min. Sections were counterstained with hematoxylin [19]. Insulinpositive cells were counted in visible sections at a magnification of ×40. The percentage of insulin-secreting cells were calculated via the number of insulin-immunoreactive cells was divided by the total number of cells. A total of 20 random islets were analyzed from a total of 10 slides in control and treated group.
The administration of phytoecdysteroids to alloxan induced diabetic rats reversed their body weight loss. The potential of phytoecdysteroids to restore body weight loss appears to be due to its antihyperglycemic effect (Fig. 1). 3.2. Effect of phytoecdysteroids on blood glucose Blood glucose levels were determined in control and treated rats (Fig. 2). The blood glucose reached a diabetic level in alloxan-induced diabetic male albino rats (group II) compared with group I (P < 0.05). However, phytoecdysteroid administration significantly reduced the blood glucose by19.2 and 52.9% in group III and IV respectively, compared with group II (P < 0.05). 3.3. Effect of phytoecdysteroids on blood insulin
2.16. RNA isolation and cDNA synthesis Blood insulin level was determined in control and treated rats (Fig. 3). The blood insulin reached a diabetic level in alloxan-induced
Liver tissue was removed surgically from experimental rats. Tissues were homogenized and lysed in the Trizol reagent. Total RNA was isolated from the control and treated samples based on the manufacturer protocol. DNase I was used to removing the residual DNA from samples. M-MLV reverse transcriptase with the anchored oligo d(T)12–18 primer was used to convert the 1 μg of RNA to cDNA and used for the amplification [25]. 2.17. Real-time quantitative PCR (qPCR) Hexokinase-I mRNA expression was measured using qPCR. The specific hexokinase-I primer was used for the amplification of mRNA. Forward (5′-GACCAAGTCAAAAAGATTGA-3′) and reverse (5′TCTTCTCGTGGTTCACCTGC-3′) were used in this study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as PCR internal control. The reaction was carried out in 15 μl using SYBR Green Master Mix (Bio-Rad). Relative ratios were calculated based on the 2−△△ method [26]. The qPCR reaction was monitored with the use of CT Mini Opticon Real-Time PCR System (Bio-Rad). 2.18. Western blot analysis Liver tissue was homogenized and centrifuged at 12000g for 30 min at 4 °C. The total protein content was estimated in the supernatant. Cell homogenate was washed with PBS, and lysed with 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP- 40, 50 mM NaF, 2 mM EDTA (pH 8.0),
Fig. 1. Phytoecdysteroids restore body weight loss appears to be due to its antihyperglycemic effect. Body weight changes were noted before and after the treatment and values are expressed as g. Data shown are the mean of six different sets of experiments (mean ± SEM).
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Fig. 4. Phytoecdysteroids reduced total serum protein concentration in the alloxan-induced diabetic rat. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as mg/dl. Data shown are the mean of six different sets of experiments (mean ± SEM).
Fig. 2. Phytoecdysteroids reduced blood glucose levels in alloxan-induced diabetic rats. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as mg/dl. Data shown are the mean of six different sets of experiments (mean ± SEM).
induced diabetic rats. Serum total protein was increased by 25 and 72.2% in group III and IV respectively, compared with group II (P < 0.05). 3.5. Effect of phytoecdysteroids on BUN BUN was determined in control and treated rats (Fig. 5). BUN was increased in alloxan-induced diabetic male albino rats compared with group I (P < 0.05). Phytoecdysteroid administration significantly reduced BUN in alloxan-induced diabetic rats. BUN was increased by 33.6 and 58.4% in group III and IV respectively, compared with group II (P < 0.05).
Fig. 3. Phytoecdysteroids increased blood insulin concentration in the alloxan-induced diabetic rat. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as microunit/ml. Data shown are the mean of six different sets of experiments (mean ± SEM).
diabetic male albino rats (group II) compared with group I (P < 0.05). However, phytoecdysteroid administration significantly increased blood insulin by 54.9 and 105.88% in groups III and IV respectively, compared with group II (P < 0.05). 3.4. Effect of phytoecdysteroids on serum total protein Blood protein levels were determined in control and treated rats (Fig. 4). Serum total protein was reduced in alloxan-induced diabetic male albino rats compared with group I (P < 0.05). Phytoecdysteroid administration significantly increased serum total protein in alloxan-
Fig. 5. Phytoecdysteroids reduced blood urea nitrogen in the alloxan-induced diabetic rat. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as mg/dl. Data shown are the mean of six different sets of experiments (mean ± SEM).
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Fig. 7. Phytoecdysteroids reduced serum TG and cholesterol levels in alloxan-induced diabetic rats. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Values are expressed as mmol/L. Data shown are the mean of six different sets of experiments (mean ± SEM).
Fig. 6. Phytoecdysteroids reduced blood creatinine in the alloxan-induced diabetic rat. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as U/L. Data shown are the mean of six different sets of experiments (mean ± SEM).
3.6. Effect of phytoecdysteroids on creatinine Blood creatinine was determined in control and treated rats (Fig. 6). Blood creatinine was increased in alloxan-induced diabetic male albino rats compared with group I (P < 0.05). Phytoecdysteroid administration significantly reduced blood creatinine in alloxan-induced diabetic rats. Creatinine content was reduced by 27.2 and 42% in group III and IV respectively, compared with group II (P < 0.05). 3.7. Effect of phytoecdysteroids on serum TG and cholesterol The TG and cholesterol levels were determined in control and treated rats (Fig. 7). TG and cholesterol levels were tremendously increased in alloxan-induced diabetic rats compared with group I (P < 0.05). However, phytoecdysteroid administration significantly reduced TG and cholesterol levels, with the TG level being significantly reduced by 33.3% and 58.6% in groups III and IV respectively, compared with group II (P < 0.05). Also, the cholesterol level was significantly reduced by 31.6% and 52% in groups III and IV respectively, compared with group II (P < 0.05). 3.8. Effect of phytoecdysteroid on lipid peroxidation Fig. 8. Phytoecdysteroids reduced lipid peroxidation in alloxan-induced diabetic rats. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Values are expressed as nmol/mg. Data shown are the mean of six different sets of experiments (mean ± SEM).
Lipid peroxidation was determined in control and treated rats (Fig. 8). MDA was tremendously increased in alloxan-induced diabetic rats compared with group I (P < 0.05). Phytoecdysteroid administration attenuated the MDA content, with a reduction of 31.6 and 62.6% in group III and IV respectively, compared with group II (P < 0.05).
and IV respectively, compared with group II (P < 0.05). Glutathione peroxidase level was increased by 47.2 and 103% in group III and IV respectively, compared with group II (P < 0.05).
3.9. Effect of phytoecdysteroid on antioxidant enzymes
3.10. Effect of phytoecdysteroid on pancreatic tissue morphology
Catalase, SOD, and glutathione peroxidase levels were determined in control and treated rats (Fig. 9). The levels of these antioxidant enzymes were drastically reduced in alloxan-induced diabetic rats (group II vs. group I; P < 0.05). The administration of phytoecdysteroids attenuated this reduction. Catalase level was increased by 56.9% and 100% in group III and IV respectively, compared with group II (P < 0.05). SOD level was increased by 34.8 and 102% in group III
Histopathological analysis was performed on specimens from the control and treated rats (Fig. 10). Microscopic investigation of pancreas sections of sham control rats indicated the normal appearance of islets of Langerhans (Fig. 10a). The cellular architecture of the diabetic pancreas tissue samples was markedly altered compared with group I. 484
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3.11. Effect of phytoecdysteroid on insulin immunoreactivity Insulin immunoreactivity was significantly reduced in the control group compared to sham (Fig. 11a, b). However, phytoecdysteroid administration significantly increased insulin immunoreactivity (Fig. 8c, d). The number of insulin-secreting cells was tremendously reduced in alloxan-induced diabetic rats. However, phytoecdysteroid administration increased insulin-secreting cells 48 and 61% in group III and IV respectively, compared with group II (Fig. 11B, P < 0.05). 3.12. Effect of phytoecdysteroid hexokinase-I mRNA expression Hexokinase-I mRNA expression was significantly reduced in diabetic rats (group II) compared to the sham control (group II). However, phytoecdysteroid treatment significantly upregulates hexokinase-I mRNA expression (Fig. 12). Hexokinase-I mRNA was increased 28.3 and 93.5% in groups III and IV respectively, compared with group II (Fig. 12B, P < 0.05). 3.13. Effect of phytoecdysteroid hexokinase-I protein expression Hexokinase-I protein expression was drastically reduced in diabetic rats (group II) compared to the sham control (group I). However, phytoecdysteroid treatment significantly upregulates hexokinase-I protein expression (Fig. 13). Hexokinase-I protein was increased 27.9 and 55.3% in groups III and IV respectively, compared with group II (Fig. 13B, P < 0.05).
Fig. 9. Phytoecdysteroids increased SOD, catalase and glutathione peroxidase activity in alloxan-induced diabetic rats. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Values are expressed as μmol/mg. Data shown are the mean of six different sets of experiments (mean ± SEM).
Small vacuoles were found, and acinar cells were swollen. Flattened epithelium with interlobular ducts was observed. Islet β-cells are lost in alloxan-induced diabetic rats (Fig. 10b). However, phytoecdysteroid administration notably attenuated the atrophic changes of acinar cells. Regeneration of islets was observed in phytoecdysteroid treated diabetic rats (Fig. 10c, d).
4. Discussion Insulin-dependent diabetes is a metabolic disorder in which the pancreas fails to produce sufficient amounts of insulin to maintain circulating blood glucose [19] Increased blood glucose has been associated with the increased production of ROS, which leads to the damage Fig. 10. Phytoecdysteroids attenuate the alteration of pancreatic tissue morphology in alloxan-induced diabetic rats. Normal appearance of islets of Langerhans (a). Vacuoles, swollen acinar cells and loss of Islet of β-cells in alloxan-induced diabetic rats (b). Attenuated atrophic changes in acinar cells and regeneration of islets were observed (c, d). Image analysis was performed with the R package CRImage. H & E, ×40. Representative images from six different sets of experiments.
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Fig. 12. Phytoecdysteroid upregulates hexokinase-I mRNA expression. Representative agarose gel electrophoretic bands of hexokinase-I (A). Densitometry analysis of agarose gel bands (B). Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Data shown are the mean of six different sets of experiments (mean ± SEM).
Fig. 11. an Immunohistochemical examination of pancreas tissue (A). Normal insulin immunoreactivity (a), reduced insulin immunoreactivity (b) and increased insulin immunoreactivity (c, d). Quantitation of insulin immunoreactivity in the pancreatic cells (B). Insulin-positive cells were counted in visible sections at a magnification of ×40. The percentage of insulin-secreting cells were calculated via the number of insulin-immunoreactive cells was divided by the total number of cells. A total of 20 random islets were analyzed from a total of 10 slides in control and treated group. Values are expressed as%. Data shown are the mean of six different sets of experiments (mean ± SEM).
of cellular lipids, proteins and nucleic acids [27]. Furthermore, reduced levels of antioxidant enzymes have been reported in diabetes [27]. The activation of protein kinase-c and several transcription factors, and advanced glycation end products, have been associated with glucosemediated toxicity [28]. In the present study, blood glucose and insulin levels were significantly reduced in alloxan-induced diabetic rats following the administration of phytoecdysteroids. This effect may be due to stimulation of remaining pancreatic β-cells to produce insulin. This theory was supported by the histopathological results from pancreatic tissue specimens, which revealed the regeneration of cells. El-Hilaly et al., [29] have reported that the reduced level of TG and cholesterol in streptozotocin-induced diabetic rats following the treatment of Ajuga iva. Our results agree with above finding of El-Hilaly et al. [29]. Bhor et al., [30] have reported that the hyperglycemia increases lipid peroxidation, which further leads to chronic tissue damage. Reduced lipid peroxidation following treatment with phytoecdysteroids matched with results of Taleb-Senouci et al., [31], who have reported that the reduced lipid peroxidation following 4 week treatment of Ajuga iva. Phytoecdysteroids have been known to reduce blood glucose levels through the direct stimulation of pancreatic β-cells [32,33]. A previous study reported that the administration of phytoecdysteroids in the form of phytoestrogen inhibits glucogenic and lipogenic enzymes [34]. In the present study, the administration of phytoecdysteroids extracted from
Fig. 13. Phytoecdysteroid upregulates hexokinase-I protein expression. Representative western blot bands of hexokinase-I (A). Densitometry analysis of Western blot bands (B). Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Data shown are the mean of six different sets of experiments (mean ± SEM).
the Ajuga iva plant significantly increased levels of antioxidant enzymes including catalase, glutathione peroxidase, and SOD, indicating that phytoecdysteroids reduce ROS levels and thus oxidative stress and glucose oxidation. These results are in agreement with those of a previous study [34]. The degradation of catalase, SOD and glutathione peroxidase and their associated co-factors are known to be essential for the reduced secretion of pancreatic insulin [35]. Furthermore, the degradation of 486
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these enzymes leads to increased lipid peroxidation in renal and other tissues [35]. Hyperglycemia has been associated with increased levels of TG, urea, creatinine, and cholesterol [36]. Houghton and Raman [37] have reported that the presence of several flavonoids, tannins, terpenes, and steroids in Ajuga iva. Flavonoids are well known for their antioxidant and hypocholesterolemic activity [38]. Furthermore, flavonoids are very good free radical scavenger which leads to reduced lipid peroxidation [39]. The results of the present study also indicated that phytoecdysteroid administration reduces renal toxicity through the reduction of BUN, lipid peroxidation, and creatinine, TG and cholesterol levels. This is in agreement with the results of previous studies [40,41]. Histopathological examination revealed that the phytoecdysteroids improved the histological architecture of the islets of Langerhans of diabetic rats following treatment. Histopathological study of diabetic control rats showed the almost complete destruction of beta cells due to alloxan. Experimental diabetes can be induced partially due to an insufficient dose of alloxan [42]. Therefore, it is believed that the phytoecdysteroid exhibits antihyperglycemic effect through secretion of insulin from the intact beta-cells of the islets. Furthermore, it is supported by regeneration of pancreatic islets histological analysis. Hexokinase is key glycolytic, and insulin-dependent enzyme plays a vital role in carbohydrate utilization [43,44]. We have determined the expression level of hexokinase-I in liver lysates by qPCR and Western blot analysis. Hexokinase-I expression was reduced in alloxan-induced diabetic rats. However, phytoecdysteroid treatment significantly upregulates hexokinase-I expression, which indicates the increased expression of hexokinase-I reflects the increased cellular glucose utilization [45]. Phytoecdysteroid administration showed the therapeutic effect on diabetes through immunomodulation [46]. In conclusion, the results of the present study indicate that phytoecdysteroid administration attenuates the metabolic changes caused by diabetes. Therefore, phytoecdysteroids from Ajuga iva could serve as a potential therapeutic agent for the treatment of diabetes in the future. Further studies are required to validate the results of the present study.
[9]
[10]
[11] [12]
[13]
[14] [15]
[16] [17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
Conflict of interest [25]
Authors declare that they no conflict of interest. [26]
Acknowledgements
[27]
The present study was supported by the Science and Technology Department of Wenzhou (grant no: Y20130199), the Zhejiang Provincial Natural Science Foundation of China (grant no: LQ13H100003) and the National Natural Science Foundation of China (grant no: 81501382).
[28] [29]
References
[30]
[1] B. Andallu, Control of hyperglycemia and retardation of cataract by mulbeny (Morus indica. L) leaves in streptozotocin-diabetic rats, Indian J. Exp. Biol. 40 (2000) 791–795. [2] H. King, R.E. Aubert, W.H. Herman, Diabetes and Risk factors for coronary heart disease, Diabetes Care 21 (1998) 1414–1431. [3] A. Annapurna, D. Mahalakshmi, The antidiabetic activity of a polyherbal preparation (Tincture Punchapuma) in normal and diabetic rats, Indian J. Exp. Biol. 39 (2001) 500–501. [4] K. Maiese, Triple play: promoting neurovascular longevity with nicotinamide, WNT, and erythropoietin in diabetes mellitus, Biomed. Pharmacother. 62 (2008) 218–232. [5] S. Wild, G. Roglic, A. Green, R. Sicree, H. King, Global prevalence of diabetes: estimates for the year 2000 and projections for 2030, Diabetes Care 27 (2004) 1047–1053. [6] J.W. Baynes, Role of oxidative stress in the development of complications in diabetes, Diabetes 40 (1991) 405–412. [7] A. Eidi, M. Eidi, M. Sokhteh, Effect of fenugreek Trigonella foenum-graecum L; seeds on serum parameters in normal and streptozotocin-induced diabetic rats, Nutr. Res. 27 (2007) 728–733. [8] E.O. Farombi, O.O. Ige, Hypolipidemic and antioxidant effects of ethanolic extract
[31]
[32] [33]
[34]
[35]
[36]
[37]
487
from dried calyx of Hibiscus sabdariffa in alloxan-induced diabetic rats, Fund Clin. Pharmacol. 21 (2007) 601–609. A.I. Galan, E. Palacios, F. Ruiz, A. Dıez, M. Arji, M. Almar, C. Moreno, J.I. Calvo, M.E. Munoz, M.A. Delgado, R. Jimenez, Exercise, oxidative stress, and risk of cardiovascular disease in the elderly. Protective role of antioxidant functional foods, BioFactors 26 (2006) 167–183. J. Juskiewicz, Z. Zdunczyk, A. Jurgonski, L. Brzuzan, I. Godycka-Klos, E. ZarySikorska, Extract of green tea leaves partially attenuates streptozotocin-induced changes in antioxidant status and gastrointestinal functioning in rats, Nutr. Res. 28 (2008) 343–349. H.S. Parmar, A. Kar, The antidiabetic potential of Citrus sinensis and Punica granatum peel extracts in alloxan-treated male mice, BioFactors 31 (2007) 17–24. J.S. Lee, Effects of Fomes fomentarius supplementation on antioxidant enzyme activities, blood glucose, and lipid profile in streptozotocin-induced diabetic rats, Nutr. Res. 25 (2005) 187–195. A. Pandey, P. Tripathi, R. Pandey, R. Srivastava, S. Goswami, Alternative therapies useful in the management of diabetes: a systematic review, J. Pharm. Bioallied Sci. 3 (2011) 504–512. M. Wessner, B. Champion, J.P. Girault, M. Kaouadji, B. Saidi, R. Lafont, Ecdysteroids from Ajuga iva, Phytochemistry 31 (1992) 3785–3788. A. Chenni, D.A. Yahia, F.O.J. Boukortt, J. Prost, M.A. Lacaille-Dubois, M. Bouchenak, Effect of aqueous extract of Ajuga iva supplementation on plasma lipid profile and tissue antioxidant status in rats fed a high-cholesterol diet, J. Ethnopharmacol. 109 (2007) 207–213. V.N. Syrov, Comparative experimental investigations of the anabolic activity of ecdysteroids and steranabols, Pharm. Chem. J. 34 (2000) 193–197. A. Sharma, M.K. Sharma, M. Kumar, Protective effect of mentha piperita against arsenic-induced toxicity in liver of swiss albino mice, Basic Clin. Pharmacol. Toxicol. 100 (2007) 249–257. P. Muthuraman, K. Srikumar, A comparative study on the effect of homobrassinolide and gibberellic acid on lipid peroxidation and antioxidant status in normal and diabetic rats, J. Enzyme Inhib. Med. Chem. 24 (2009) 1122–1127. P. Muthuraman, R. Senthilkumar, K. Srikumar, Alterations in beta-islets of Langerhans in alloxan-induced diabetic rats by marine Spirulina platensis, J. Enzyme Inhib. Med. Chem. 24 (2009) 1253–1256. M. Serrano-Rios, F. Ramos, J.L. Radrogez-Minor, I. Vivanco, Studies in prediabetic insulin response to oral glucose infusion in genetic prediabetes, Diabetologica 6 (1970) 392–398. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1955) 265–275. R.A. Kohn, M.M. Dinneen, E. Russek-Cohen, Using blood urea nitrogen to predict nitrogen excretion and efficiency of nitrogen utilization in cattle, sheep, goats, horses, pigs, and rats, J. Anim. Sci. 83 (2005) 879–889. V. Muthuviveganandavel, P. Muthuraman, S. Muthu, K. Srikumar, A study on low dose cypermethrin is induced histopathology, lipid peroxidation and marker enzyme changes in male rats, Pestic. Biochem. Physiol. 9 (2008) 12–16. P. Muthuraman, K. Ramkumar, D.H. Kim, Analysis of the dose-dependent effect of zinc oxide nanoparticles on the oxidative stress and antioxidant enzyme activity in adipocytes, Appl. Biochem. Biotechnol. 174 (2014) 2851–2863. J. Kishalay, K.B. Tushar, G. Debidas, Antidiabetic effects of Eugenia jambolana in the streptozotocin-induced diabetic male albino rat, Biomark. Genomic Med. 7 (2015) 116–124. P. Muthuraman, K. Ramkumar, D.H. Kim, Analysis of the dose-dependent effect of zinc oxide nanoparticles on the oxidative stress and antioxidant enzyme activity in adipocytes, Appl. Biochem. Biotechnol. 174 (2014) 2851–2863. P. Newsholme, E.P. Haber, S.M. Hirabara, E.L.O. Rebelato, J. Rebelato, D. Morgan, H.C. Oliveira-Emilio, A.R. Carpinelli, R. Curi, Diabetes-associated cell stress and dysfunction: the role of mitochondrial and non-mitochondrial ROS production and activity, J. Physiol. 583 (2007) 9–24. P. Pacher, J.S. Beckman, L. Liaudet, Nitric oxide and peroxynitrite in health and disease, Physiol. Rev. 87 (9) (2007) 315–424. J. El-Hilaly, A. Tahraoui, Z.H. Israili, B. Lyoussi, Hypolipidemic effects of acute and sub-chronic administration of an aqueous extract of Ajuga iva L. whole plant in normal and diabetic rats, J. Ethnopharmacol. 105 (2006) 441–444. V.M. Bhor, N. Raghuram, S. Sivakami, Oxidative damage and altered antioxidant enzymes activities in the intestine of streptozotocin-induced diabetic rats, Int. J. Biochem. Cell Biol. 36 (2004) 89–97. D. Taleb-Senouci, H. Ghomari, D. Krouf, S. Bouderbala, J. Prost, M.A. LacailleDubois, M. Bouchenak, Antioxidant effect of Ajuga iva aqueous extract in streptozotocin-induced diabetic rats, Phytomedicine 16 (2009) 623–631. M.S. Islam, H. Choi, Green tea, anti-diabetic or diabetogenic: a dose-response study, BioFactors. 29 (9) (2007) 45–53. A. Wiederkehr, C.B. Wollheim, Impact of mitochondrial calcium on the coupling of metabolism to insulin secretion in the pancreatic β-cell, Cell Calcium 44 (2008) 64–76. J. Vina, C. Borras, M.C. Gomez-Cabrera, W. Orr, Role of reactive oxygen species and (phyto)oestrogens in the modulation of adaptive response to stress, Free Radic. Biol. Med. 40 (2006) 111–119. M. Ohnishi, T. Matuo, T. Tsuno, A. Hosoda, E. Nomura, H. Taniguchi, H. Sasaki, H. Morishita, Antioxidant activity and hypoglycemic effect of ferulic acid in STZinduced diabetic mice and KK-Â{y} mice, BioFactors 21 (2004) 315–319. K. Ravi, B. Ramachandran, S. Subramanian, Protective effect of eugenia jambolana seed kernel on tissue antioxidants in streptozotocin-induced diabetic rats, Biol. Pharm. Bull. 27 (2004) 1212–1217. P.J. Houghton, A. Raman, Laboratory Handbook for the Fractionation of Natural Extracts, first ed., ITPs, London, 1998.
Biomedicine & Pharmacotherapy 96 (2017) 480–488
J.-J. Wang et al.
[42] D.G. Lyman, E.G.D. Murray, The pathology of the pancreas in experimental diabetes mellitus, Am. J. Med. Sci. 210 (9) (1945) 381–396. [43] L.D. Griffin, B.D. Gelb, V. Adams, E.R.B. McCabe, Developmental expression of hexokinase I in the rat, Biochim. Biophys. Acta 1129 (1992) 309–317. [44] L. Pari, G. Saravanan, Antidiabetic effect of cogent db, an herbal drug in alloxaninduced diabetes mellitus, Comp. Biochem. Physiol. 131 (2002) 19–25. [45] C. Postic, A. Leturque, R.L. Printz, P. Maulard, M. Loizeau, D.K. Granner, J. Girard, Development and regulation of glucose transporter and hexokinase expression in the rat, J. Am. Physiol. Soc. 266 (9) (1994) 548–559. [46] D.S. Trenin, V.V. Volodin, 20-Hydroxyecdysone as a human lymphocyte and neutrophil modulator: in vitro evaluation, Arch. Insect Biochem. Physiol. 41 (1999) 156–161.
[38] H. Wagner, M.A. Lacaille-Dubois, Recent pharmaco-logical results on bioflavonoids, in: S. Antus, M. Gabor, K. Vetschera (Eds.), Flavonoids and Bioflavonoids, Akademiai Kiado, Budapest, 1995, pp. 53–57. [39] C. Soto, R. Recoba, H. Barron, C. Alvarez, L. Favari, Sylimarin increases antioxidant enzymes in alloxan-induced diabetes in rat pancreas, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 136 (2003) 205–212. [40] K. Hamden, S. Carreau, S. Lajmi, D. Aloulou, D. kchaou, A. Elfeki, Protective effect of 17β-estradiol on hyperglycemia, stress oxidant, liver dysfunction and histological changes induced by alloxan in male rat pancreas and liver, Steroids 94 (2008) 495–501. [41] S. Lortz, M. Tiedge, T. Nachtwey, A.E. Karlsen, J. Nerup, S. Lenzen, Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes, Diabetes 49 (2000) 1123–1130.
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Biomedicine & Pharmacotherapy journal homepage: www.elsevier.com/locate/biopha
Original article
Phytoecdysteroids from Ajuga iva act as potential antidiabetic agent against alloxan-induced diabetic male albino rats Jin-Jun Wang, Hao Jin, Shao-Ling Zheng, Peng Xia, Yong Cai, Xiao-Jie Ni
MARK
⁎
Department of Transplantation, The First Affiliated Hospital of Wenzhou Medical University, Wenzhou, Zhejiang 325000, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords: Phytoecdysteroid Diabetes Alloxan Pancreas Rats
The present study investigated the protective effect of phytoecdysteroids extracted from the Ajuga iva plant on body weight changes, blood glucose, insulin total protein, blood urea nitrogen (BUN), creatinine, triglycerides (TG), cholesterol, lipid peroxidation, antioxidant enzymes, pancreatic histopathology and hexokinase-I expression in the alloxan-induced diabetic rats. Experimental diabetes was induced following 15 day intraperitoneal administration of alloxan. The rats were divided into four groups. Group I served as a sham group, and group II served as the diabetic control. Group III served as a treatment for phytoecdysteroids (10 mg/kg), and group IV served as a treatment for phytoecdysteroids (20 mg/kg). Phytoecdysteroids restored body weight loss to its antihyperglycemic effect. Blood glucose was reduced 19.2 and 52.9% in group III and IV respectively. Blood insulin (54.9 and 105.88%) and total protein (25 and 72.2%) was increased in group III and IV respectively. BUN, creatinine, TG, cholesterol and lipid peroxidation was significantly reduced following treatment. Catalase, superoxide dismutase (SOD), and glutathione peroxidase activity were significantly increased following treatment. Islet β-cells are lost in alloxan-induced diabetic rats. Regeneration of islets and reduced atrophy of acinar cells were noted. The number of insulin-secreting cells was tremendously reduced in alloxan-induced diabetic rats. Insulin-secreting cells were increased 48 and 61% in group III and IV respectively. Hexokinase-I mRNA (28.3 & 93.5%) and protein (27.9 and 55.3%) expression were significantly increased following treatment. Taking all these data together, it is suggested that the phytoecdysteroid could be a potential therapeutic agent against experimental diabetes.
1. Introduction Diabetes mellitus is recognized as a clinical syndrome that is characterized by hyperglycemia due to deficiency of insulin. A striking feature of diabetes is the shift in the form of fuel usage from carbohydrates to fats [1]. Diabetes is recognized as a common disease that leads to chronic conditions like neuropathy, nephropathy, vascular diseases associated with heart, kidney, brain, peripheral blood vessels and retinopathy [2]. In diabetes, the body either produces little insulin or ceased to produce insulin, or became progressively resistant to insulin action [3]. Diabetes mellitus is a metabolic disorder that affects millions of people worldwide. Diabetes present in sixteen million peoples in the United States and more than 165 million peoples worldwide [4]. Increased oxidative stress leads to the excessive production of free radicals, which plays an important role in the development of diabetes and its complications [5]. Accelerated generation of free radicals leads to the increased tissue damage, metabolic stress, and cell death [6].
Hyperglycemia can substantially increase the production of reactive oxygen species (ROS) and lead to several complications, including nephropathy [7,8]. Diet can notably contribute for the treatment of several diseases, including diabetes and immune system-associated disorders [9–11]. Also, Fomes fomentarius exhibits as a potential therapeutic agent against streptozotocin-induced diabetic rats [12]. Although, insulin is a wellknown remedy for the prevention and management of diabetes, searching an alternative from the plant and synthetic sources when insulin is not readily available has been attempted [12]. Furthermore, several herbs can be used as an alternative to insulin for the treatment of diabetes when insulin is not available [13]. For instance, antidiabetic and anticholesterolemic potential of phytoecdysteroids extracted from Ajuga iva has been reported [14–16]. Therefore, the present study was aimed to evaluate the protective effect of phytoecdysteroids in alloxaninduced diabetic rats.
Abbreviations: SOD, superoxide dismutase; ROS, reactive oxygen species; TG, triglycerides; PEG, polyethylene glycol; BUN, blood urea nitrogen; TBST, tris-buffered saline with Tween20; qPCR, real-time quantitative PCR; HRP, horseradish peroxidase; PVDF, polyvinylidene difluoride; SEM, sandard error of the mean ⁎ Corresponding author at: Department of Transplantation, The First Affiliated Hospital of Wenzhou Medical University, 2 Fuxue Lane, Wenzhou, Zhejiang 325000, PR China. E-mail address: [email protected] (X.-J. Ni). http://dx.doi.org/10.1016/j.biopha.2017.10.029 Received 2 September 2017; Received in revised form 3 October 2017; Accepted 6 October 2017 0753-3322/ © 2017 Elsevier Masson SAS. All rights reserved.
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(125I) insulin, for the limited binding sites on a specific antibody. At the end of incubation, the antibody bound and free insulin was separated by the second antibody-polyethylene glycol (PEG) aided separation method [20]. The insulin concentration in the blood was then quantitated by measuring the radioactivity associated with the antibody bound fraction of the samples.
2. Materials and methods 2.1. Animals A total of 24 healthy male albino Wistar strain rats (6 weeks old) were obtained from the animal house in Shangai (Shanghai Laboratory Animals Center, Shanghai, China). The rats weighed 160–200 g and were kept in polypropylene cages at a temperature of 25 ± 0.5° C with a relative humidity of 60 ± 5% and a light/dark cycle of 12 h. Rats were allowed free access to food and water. Ethical approval was obtained from the Ethics Committee of Wenzhou Medical University (approval no. 201308807).
2.8. Determination of serum total proteins The amount of total protein in the blood was determined according to Lowry et al. [21]. Briefly, 0.l ml of a blood sample, 0.5 m1 of l0% TCA was added, and contents were mixed and centrifuged. To the precipitate, 0.l ml of 0.l N NaOH was added. From this solution, 0.l ml was taken and 5 ml alkaline copper reagent was added mixed, and the contents were allowed to stand at room temperature for l0 min. Then 0.5 ml of Folin's-Phenol (1N) reagent was added to the reaction tube and mixed well. The blue color developed in each of the reaction tubes was measured at 680 nm employing a spectrophotometer. Values are expressed in mg/dl.
2.2. Preparation of phytoecdysteroids An aqueous solution of Ajuga iva was prepared according to the method described by Sharma et al. [17]. Briefly, Ajuga iva was supplied by Xinran Biotechnology Co., Ltd (Shangai, PR China). The 100 g of Ajuga iva leaves were air-dried and extracted (distilled water) via refluxing for 36 h at 90° C. Subsequently, the aqueous extract was evaporated using a vacuum and then converted into a powder at 90° C for 12 h.
2.9. Determination of blood urea nitrogen (BUN) Quantitative and competitive enzyme immunoassay technique used in the BUN assay [22]. This method utilizing a monoclonal antibody and a conjugate to BUN. Horseradish peroxidase (HRP) conjugate was incubated with sample for 1 h in a pre-coated assay plate. Wells were repeatedly washed at the end of incubation and incubated with a substrate for HRP enzyme. Blue colored complex was formed as a product of the enzyme-substrate reaction. The blue color turned into yellow following the addition stop solution, and intensity was measured at 450 nm.
2.3. Experimental diabetes induction Experimental diabetes was induced by the use of alloxan. Briefly, a freshly prepared solution of alloxan (60 mg/kg; Sigma-Aldrich; Merck KGaA, Darmstadt, Germany) in 0.1 M citrate buffer (pH 4.5) was injected intraperitoneally. A booster (secondary) dose of alloxan was administered at 7th day following the primary dose administration. Following 15 days of alloxan administration, rats were exhibiting blood glucose more than 250 mg/dl were selected for the experiment [18].
2.10. Determination of blood creatinine 2.4. Experimental groups The blood levels of creatinine were determined Muthuviveganandavel et al. [23]. Creatinine was measured with a rateblanked and compensated picric acid colorimetric assay. The finally liberated hydrogen peroxide is used to form a chromogen by oxidase. The color intensity is directly proportional to creatinine level in the sample. Values were expressed in U/L.
The rats were divided into 4 groups, which contained 6 rats each. Group I served as a sham group and treated with normal saline once daily for 15 consecutive days. Group II served as the diabetic control and treated with normal saline once daily for 15 consecutive days. Group III served as a treatment for phytoecdysteroids (10 mg/kg) once daily for 15 consecutive days. Group IV served as a treatment for phytoecdysteroids (20 mg/kg) once daily for 15 consecutive days.
2.11. Determination of blood triglycerides and cholesterol Triglycerides (TG) and cholesterol were determined according to Muthuviveganandavel et al. [23]. Briefly, mix 1000 μl of working reagent with 10 μl of the sample and incubated at 37 °C for 5 min. The absorbance was measured at 546 nm within 60 min. Cholesterol was estimated by mixing 0.1 ml of serum with 4.9 m1 of ferric chloride and was taken in a screw-capped centrifuge tube and allowed to stand for 15 min. The sample was centrifuged at 3000 × g for l0 min. 1.5 ml of concentrated sulphuric acid was added to 2.5 m1 of the clear supernatant and was incubated for 20 min at room temperature. The intensity of the developed color was read against a blank at 560 nm. Values are expressed in mmol/L.
2.5. Determination of body weight The body weight of each animal was observed before and after the treatment. 2.6. Determination of blood glucose Blood glucose levels were determined in the control and extracttreated rats as described by Muthuraman et al. [19]. Briefly, 0.1 ml of a blood sample, 3.8 ml of isotonic sodium sulfate-copper sulfate solution and 0.l ml of 10% sodium tungstate solution was added to precipitate the proteins. The samples were centrifuged at 1500 rpm for 10 min to obtain a protein-free solution. Alkaline tartarate (1 ml) was then added to l ml of the clear supernatant and placed in a boiling water bath for l0 min. This was then cooled, and 3 ml phosphomolybdic acid and 3 ml water was added and mixed thoroughly. The solution was allowed to stand for 5 min for color development. The developed color was read against a blank at 630 nm. Values are expressed in mg/dl.
2.12. Determination of lipid peroxidation Lipid peroxidation was estimated as the content of malondialdehyde (MDA) [24]. Briefly, 0.4 ml of blood samples, 0.05 ml of butylated hydroxytoluene, 1.6 ml of phosphate buffer (pH 7.4), and 1 ml of 30% trichloroacetic acid (TCA) were incubated together for 2 h at −20 °C. The mixture was then centrifuged (500g) for 15 min. Then, 1 ml of the supernatant was added to 0.075 ml of 0.1 M EDTA, and 0.25 ml of 1% TBA contained in a tube for 20 min in a boiling water bath. The absorbance was measured at 532 and 600 nm in a spectrophotometer. Values are expressed as nmol/mg of protein.
2.7. Determination of blood insulin The radioimmunoassay method employed was based on the competition of unlabeled insulin present in the blood, with radio-iodinated 481
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1 mM PMSF, 15 μg/ml leupeptin, and 15 μg/ml aprotinin. The protein which is present in the lysate was run on SDS-PAGE. Polyvinylidene difluoride (PVDF) membrane was used for transferring in the SDSPAGE. Tris-buffered saline with Tween-20 (TBST) was used for the nonspecific blocking proteins. The membrane probed for 12 h with an antibody against hexokinase-I. Membranes were washed twice with TBST and incubated with HRP-conjugated goat anti-rabbit IgG (St. Louis, MO 63178 USA) for 60 min. The protein level of hexokinase-I was determined using enhanced chemiluminescence method [26].
2.13. Determination of antioxidant enzymes The concentration of antioxidant enzymes, including catalase, glutathione peroxidase and superoxide dismutase (SOD), was determined according to Muthuraman et al. [24] and values were expressed as μmol/mg of protein. 2.14. Histopathology For the histopathological studies, the rats were anesthetized with intramuscular injection of ketamine hydrochloride (100 mg/kg) + Xylazine (11 mg/kg) (Sigma-Aldrich; Merck KGaA), and pancreas tissue was removed and placed in 10% formaldehyde for fixation for 3 h at room temperature and embedded in paraffin. Tissues section (5-μmthick) were prepared and stained with Hematoxylin and Eosin (H & E) for 5 min at room temperature. Stained tissue sections were qualitatively analyzed as described previously [23] and images were captured using a light microscope at a magnification of ×40. Image analysis was performed with the R package CRImage (http://www.lepem.ufc.br/ jaa/colorout_1.1-0.tar.gz) and analyzed by a pathologist.
2.19. Statistical analysis Values are expressed as the mean ± standard error of the mean (SEM) experiments performed three times. The statistical significance of differences between the control and treatment was evaluated using ANOVA and followed by Student’s t-test (SPSS v. 17; SPSS, Inc., Chicago, IL, USA). P < 0.05 was considered to indicate a statistically significant difference. 3. Results
2.15. Immunohistochemical analysis
3.1. Effect of phytoecdysteroids on body weight
The pancreas was surgically removed from the rat animals following decapitation and rinsed in ice-cold normal saline. Paraformaldehyde was used for fixation of pancreas tissue and dehydrated with ethanol (graded series). Then, tissues were embedded in paraffin wax. Tissues were dewaxed and rehydrated before sectioning. Sections were made and incubated with mouse anti-insulin antibody (1:300, Abcam, USA) for overnight at 4 °C. After repeated washing with PBS, sections were incubated with HRP-conjugated secondary antibody at 37 °C for 60 min. Sections were counterstained with hematoxylin [19]. Insulinpositive cells were counted in visible sections at a magnification of ×40. The percentage of insulin-secreting cells were calculated via the number of insulin-immunoreactive cells was divided by the total number of cells. A total of 20 random islets were analyzed from a total of 10 slides in control and treated group.
The administration of phytoecdysteroids to alloxan induced diabetic rats reversed their body weight loss. The potential of phytoecdysteroids to restore body weight loss appears to be due to its antihyperglycemic effect (Fig. 1). 3.2. Effect of phytoecdysteroids on blood glucose Blood glucose levels were determined in control and treated rats (Fig. 2). The blood glucose reached a diabetic level in alloxan-induced diabetic male albino rats (group II) compared with group I (P < 0.05). However, phytoecdysteroid administration significantly reduced the blood glucose by19.2 and 52.9% in group III and IV respectively, compared with group II (P < 0.05). 3.3. Effect of phytoecdysteroids on blood insulin
2.16. RNA isolation and cDNA synthesis Blood insulin level was determined in control and treated rats (Fig. 3). The blood insulin reached a diabetic level in alloxan-induced
Liver tissue was removed surgically from experimental rats. Tissues were homogenized and lysed in the Trizol reagent. Total RNA was isolated from the control and treated samples based on the manufacturer protocol. DNase I was used to removing the residual DNA from samples. M-MLV reverse transcriptase with the anchored oligo d(T)12–18 primer was used to convert the 1 μg of RNA to cDNA and used for the amplification [25]. 2.17. Real-time quantitative PCR (qPCR) Hexokinase-I mRNA expression was measured using qPCR. The specific hexokinase-I primer was used for the amplification of mRNA. Forward (5′-GACCAAGTCAAAAAGATTGA-3′) and reverse (5′TCTTCTCGTGGTTCACCTGC-3′) were used in this study. Glyceraldehyde 3-phosphate dehydrogenase (GAPDH) was used as PCR internal control. The reaction was carried out in 15 μl using SYBR Green Master Mix (Bio-Rad). Relative ratios were calculated based on the 2−△△ method [26]. The qPCR reaction was monitored with the use of CT Mini Opticon Real-Time PCR System (Bio-Rad). 2.18. Western blot analysis Liver tissue was homogenized and centrifuged at 12000g for 30 min at 4 °C. The total protein content was estimated in the supernatant. Cell homogenate was washed with PBS, and lysed with 10 mM Tris-HCl (pH 7.5), 100 mM NaCl, 1% NP- 40, 50 mM NaF, 2 mM EDTA (pH 8.0),
Fig. 1. Phytoecdysteroids restore body weight loss appears to be due to its antihyperglycemic effect. Body weight changes were noted before and after the treatment and values are expressed as g. Data shown are the mean of six different sets of experiments (mean ± SEM).
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Fig. 4. Phytoecdysteroids reduced total serum protein concentration in the alloxan-induced diabetic rat. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as mg/dl. Data shown are the mean of six different sets of experiments (mean ± SEM).
Fig. 2. Phytoecdysteroids reduced blood glucose levels in alloxan-induced diabetic rats. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as mg/dl. Data shown are the mean of six different sets of experiments (mean ± SEM).
induced diabetic rats. Serum total protein was increased by 25 and 72.2% in group III and IV respectively, compared with group II (P < 0.05). 3.5. Effect of phytoecdysteroids on BUN BUN was determined in control and treated rats (Fig. 5). BUN was increased in alloxan-induced diabetic male albino rats compared with group I (P < 0.05). Phytoecdysteroid administration significantly reduced BUN in alloxan-induced diabetic rats. BUN was increased by 33.6 and 58.4% in group III and IV respectively, compared with group II (P < 0.05).
Fig. 3. Phytoecdysteroids increased blood insulin concentration in the alloxan-induced diabetic rat. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as microunit/ml. Data shown are the mean of six different sets of experiments (mean ± SEM).
diabetic male albino rats (group II) compared with group I (P < 0.05). However, phytoecdysteroid administration significantly increased blood insulin by 54.9 and 105.88% in groups III and IV respectively, compared with group II (P < 0.05). 3.4. Effect of phytoecdysteroids on serum total protein Blood protein levels were determined in control and treated rats (Fig. 4). Serum total protein was reduced in alloxan-induced diabetic male albino rats compared with group I (P < 0.05). Phytoecdysteroid administration significantly increased serum total protein in alloxan-
Fig. 5. Phytoecdysteroids reduced blood urea nitrogen in the alloxan-induced diabetic rat. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as mg/dl. Data shown are the mean of six different sets of experiments (mean ± SEM).
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Fig. 7. Phytoecdysteroids reduced serum TG and cholesterol levels in alloxan-induced diabetic rats. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Values are expressed as mmol/L. Data shown are the mean of six different sets of experiments (mean ± SEM).
Fig. 6. Phytoecdysteroids reduced blood creatinine in the alloxan-induced diabetic rat. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.01). Values are expressed as U/L. Data shown are the mean of six different sets of experiments (mean ± SEM).
3.6. Effect of phytoecdysteroids on creatinine Blood creatinine was determined in control and treated rats (Fig. 6). Blood creatinine was increased in alloxan-induced diabetic male albino rats compared with group I (P < 0.05). Phytoecdysteroid administration significantly reduced blood creatinine in alloxan-induced diabetic rats. Creatinine content was reduced by 27.2 and 42% in group III and IV respectively, compared with group II (P < 0.05). 3.7. Effect of phytoecdysteroids on serum TG and cholesterol The TG and cholesterol levels were determined in control and treated rats (Fig. 7). TG and cholesterol levels were tremendously increased in alloxan-induced diabetic rats compared with group I (P < 0.05). However, phytoecdysteroid administration significantly reduced TG and cholesterol levels, with the TG level being significantly reduced by 33.3% and 58.6% in groups III and IV respectively, compared with group II (P < 0.05). Also, the cholesterol level was significantly reduced by 31.6% and 52% in groups III and IV respectively, compared with group II (P < 0.05). 3.8. Effect of phytoecdysteroid on lipid peroxidation Fig. 8. Phytoecdysteroids reduced lipid peroxidation in alloxan-induced diabetic rats. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Values are expressed as nmol/mg. Data shown are the mean of six different sets of experiments (mean ± SEM).
Lipid peroxidation was determined in control and treated rats (Fig. 8). MDA was tremendously increased in alloxan-induced diabetic rats compared with group I (P < 0.05). Phytoecdysteroid administration attenuated the MDA content, with a reduction of 31.6 and 62.6% in group III and IV respectively, compared with group II (P < 0.05).
and IV respectively, compared with group II (P < 0.05). Glutathione peroxidase level was increased by 47.2 and 103% in group III and IV respectively, compared with group II (P < 0.05).
3.9. Effect of phytoecdysteroid on antioxidant enzymes
3.10. Effect of phytoecdysteroid on pancreatic tissue morphology
Catalase, SOD, and glutathione peroxidase levels were determined in control and treated rats (Fig. 9). The levels of these antioxidant enzymes were drastically reduced in alloxan-induced diabetic rats (group II vs. group I; P < 0.05). The administration of phytoecdysteroids attenuated this reduction. Catalase level was increased by 56.9% and 100% in group III and IV respectively, compared with group II (P < 0.05). SOD level was increased by 34.8 and 102% in group III
Histopathological analysis was performed on specimens from the control and treated rats (Fig. 10). Microscopic investigation of pancreas sections of sham control rats indicated the normal appearance of islets of Langerhans (Fig. 10a). The cellular architecture of the diabetic pancreas tissue samples was markedly altered compared with group I. 484
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3.11. Effect of phytoecdysteroid on insulin immunoreactivity Insulin immunoreactivity was significantly reduced in the control group compared to sham (Fig. 11a, b). However, phytoecdysteroid administration significantly increased insulin immunoreactivity (Fig. 8c, d). The number of insulin-secreting cells was tremendously reduced in alloxan-induced diabetic rats. However, phytoecdysteroid administration increased insulin-secreting cells 48 and 61% in group III and IV respectively, compared with group II (Fig. 11B, P < 0.05). 3.12. Effect of phytoecdysteroid hexokinase-I mRNA expression Hexokinase-I mRNA expression was significantly reduced in diabetic rats (group II) compared to the sham control (group II). However, phytoecdysteroid treatment significantly upregulates hexokinase-I mRNA expression (Fig. 12). Hexokinase-I mRNA was increased 28.3 and 93.5% in groups III and IV respectively, compared with group II (Fig. 12B, P < 0.05). 3.13. Effect of phytoecdysteroid hexokinase-I protein expression Hexokinase-I protein expression was drastically reduced in diabetic rats (group II) compared to the sham control (group I). However, phytoecdysteroid treatment significantly upregulates hexokinase-I protein expression (Fig. 13). Hexokinase-I protein was increased 27.9 and 55.3% in groups III and IV respectively, compared with group II (Fig. 13B, P < 0.05).
Fig. 9. Phytoecdysteroids increased SOD, catalase and glutathione peroxidase activity in alloxan-induced diabetic rats. Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Values are expressed as μmol/mg. Data shown are the mean of six different sets of experiments (mean ± SEM).
Small vacuoles were found, and acinar cells were swollen. Flattened epithelium with interlobular ducts was observed. Islet β-cells are lost in alloxan-induced diabetic rats (Fig. 10b). However, phytoecdysteroid administration notably attenuated the atrophic changes of acinar cells. Regeneration of islets was observed in phytoecdysteroid treated diabetic rats (Fig. 10c, d).
4. Discussion Insulin-dependent diabetes is a metabolic disorder in which the pancreas fails to produce sufficient amounts of insulin to maintain circulating blood glucose [19] Increased blood glucose has been associated with the increased production of ROS, which leads to the damage Fig. 10. Phytoecdysteroids attenuate the alteration of pancreatic tissue morphology in alloxan-induced diabetic rats. Normal appearance of islets of Langerhans (a). Vacuoles, swollen acinar cells and loss of Islet of β-cells in alloxan-induced diabetic rats (b). Attenuated atrophic changes in acinar cells and regeneration of islets were observed (c, d). Image analysis was performed with the R package CRImage. H & E, ×40. Representative images from six different sets of experiments.
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Fig. 12. Phytoecdysteroid upregulates hexokinase-I mRNA expression. Representative agarose gel electrophoretic bands of hexokinase-I (A). Densitometry analysis of agarose gel bands (B). Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Data shown are the mean of six different sets of experiments (mean ± SEM).
Fig. 11. an Immunohistochemical examination of pancreas tissue (A). Normal insulin immunoreactivity (a), reduced insulin immunoreactivity (b) and increased insulin immunoreactivity (c, d). Quantitation of insulin immunoreactivity in the pancreatic cells (B). Insulin-positive cells were counted in visible sections at a magnification of ×40. The percentage of insulin-secreting cells were calculated via the number of insulin-immunoreactive cells was divided by the total number of cells. A total of 20 random islets were analyzed from a total of 10 slides in control and treated group. Values are expressed as%. Data shown are the mean of six different sets of experiments (mean ± SEM).
of cellular lipids, proteins and nucleic acids [27]. Furthermore, reduced levels of antioxidant enzymes have been reported in diabetes [27]. The activation of protein kinase-c and several transcription factors, and advanced glycation end products, have been associated with glucosemediated toxicity [28]. In the present study, blood glucose and insulin levels were significantly reduced in alloxan-induced diabetic rats following the administration of phytoecdysteroids. This effect may be due to stimulation of remaining pancreatic β-cells to produce insulin. This theory was supported by the histopathological results from pancreatic tissue specimens, which revealed the regeneration of cells. El-Hilaly et al., [29] have reported that the reduced level of TG and cholesterol in streptozotocin-induced diabetic rats following the treatment of Ajuga iva. Our results agree with above finding of El-Hilaly et al. [29]. Bhor et al., [30] have reported that the hyperglycemia increases lipid peroxidation, which further leads to chronic tissue damage. Reduced lipid peroxidation following treatment with phytoecdysteroids matched with results of Taleb-Senouci et al., [31], who have reported that the reduced lipid peroxidation following 4 week treatment of Ajuga iva. Phytoecdysteroids have been known to reduce blood glucose levels through the direct stimulation of pancreatic β-cells [32,33]. A previous study reported that the administration of phytoecdysteroids in the form of phytoestrogen inhibits glucogenic and lipogenic enzymes [34]. In the present study, the administration of phytoecdysteroids extracted from
Fig. 13. Phytoecdysteroid upregulates hexokinase-I protein expression. Representative western blot bands of hexokinase-I (A). Densitometry analysis of Western blot bands (B). Group II was compared with group I (*P < 0.05) and group III and IV were compared with group II (#P < 0.05). Data shown are the mean of six different sets of experiments (mean ± SEM).
the Ajuga iva plant significantly increased levels of antioxidant enzymes including catalase, glutathione peroxidase, and SOD, indicating that phytoecdysteroids reduce ROS levels and thus oxidative stress and glucose oxidation. These results are in agreement with those of a previous study [34]. The degradation of catalase, SOD and glutathione peroxidase and their associated co-factors are known to be essential for the reduced secretion of pancreatic insulin [35]. Furthermore, the degradation of 486
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these enzymes leads to increased lipid peroxidation in renal and other tissues [35]. Hyperglycemia has been associated with increased levels of TG, urea, creatinine, and cholesterol [36]. Houghton and Raman [37] have reported that the presence of several flavonoids, tannins, terpenes, and steroids in Ajuga iva. Flavonoids are well known for their antioxidant and hypocholesterolemic activity [38]. Furthermore, flavonoids are very good free radical scavenger which leads to reduced lipid peroxidation [39]. The results of the present study also indicated that phytoecdysteroid administration reduces renal toxicity through the reduction of BUN, lipid peroxidation, and creatinine, TG and cholesterol levels. This is in agreement with the results of previous studies [40,41]. Histopathological examination revealed that the phytoecdysteroids improved the histological architecture of the islets of Langerhans of diabetic rats following treatment. Histopathological study of diabetic control rats showed the almost complete destruction of beta cells due to alloxan. Experimental diabetes can be induced partially due to an insufficient dose of alloxan [42]. Therefore, it is believed that the phytoecdysteroid exhibits antihyperglycemic effect through secretion of insulin from the intact beta-cells of the islets. Furthermore, it is supported by regeneration of pancreatic islets histological analysis. Hexokinase is key glycolytic, and insulin-dependent enzyme plays a vital role in carbohydrate utilization [43,44]. We have determined the expression level of hexokinase-I in liver lysates by qPCR and Western blot analysis. Hexokinase-I expression was reduced in alloxan-induced diabetic rats. However, phytoecdysteroid treatment significantly upregulates hexokinase-I expression, which indicates the increased expression of hexokinase-I reflects the increased cellular glucose utilization [45]. Phytoecdysteroid administration showed the therapeutic effect on diabetes through immunomodulation [46]. In conclusion, the results of the present study indicate that phytoecdysteroid administration attenuates the metabolic changes caused by diabetes. Therefore, phytoecdysteroids from Ajuga iva could serve as a potential therapeutic agent for the treatment of diabetes in the future. Further studies are required to validate the results of the present study.
[9]
[10]
[11] [12]
[13]
[14] [15]
[16] [17]
[18]
[19]
[20]
[21] [22]
[23]
[24]
Conflict of interest [25]
Authors declare that they no conflict of interest. [26]
Acknowledgements
[27]
The present study was supported by the Science and Technology Department of Wenzhou (grant no: Y20130199), the Zhejiang Provincial Natural Science Foundation of China (grant no: LQ13H100003) and the National Natural Science Foundation of China (grant no: 81501382).
[28] [29]
References
[30]
[1] B. Andallu, Control of hyperglycemia and retardation of cataract by mulbeny (Morus indica. L) leaves in streptozotocin-diabetic rats, Indian J. Exp. Biol. 40 (2000) 791–795. [2] H. King, R.E. Aubert, W.H. Herman, Diabetes and Risk factors for coronary heart disease, Diabetes Care 21 (1998) 1414–1431. [3] A. Annapurna, D. Mahalakshmi, The antidiabetic activity of a polyherbal preparation (Tincture Punchapuma) in normal and diabetic rats, Indian J. Exp. Biol. 39 (2001) 500–501. [4] K. Maiese, Triple play: promoting neurovascular longevity with nicotinamide, WNT, and erythropoietin in diabetes mellitus, Biomed. Pharmacother. 62 (2008) 218–232. [5] S. Wild, G. Roglic, A. Green, R. Sicree, H. King, Global prevalence of diabetes: estimates for the year 2000 and projections for 2030, Diabetes Care 27 (2004) 1047–1053. [6] J.W. Baynes, Role of oxidative stress in the development of complications in diabetes, Diabetes 40 (1991) 405–412. [7] A. Eidi, M. Eidi, M. Sokhteh, Effect of fenugreek Trigonella foenum-graecum L; seeds on serum parameters in normal and streptozotocin-induced diabetic rats, Nutr. Res. 27 (2007) 728–733. [8] E.O. Farombi, O.O. Ige, Hypolipidemic and antioxidant effects of ethanolic extract
[31]
[32] [33]
[34]
[35]
[36]
[37]
487
from dried calyx of Hibiscus sabdariffa in alloxan-induced diabetic rats, Fund Clin. Pharmacol. 21 (2007) 601–609. A.I. Galan, E. Palacios, F. Ruiz, A. Dıez, M. Arji, M. Almar, C. Moreno, J.I. Calvo, M.E. Munoz, M.A. Delgado, R. Jimenez, Exercise, oxidative stress, and risk of cardiovascular disease in the elderly. Protective role of antioxidant functional foods, BioFactors 26 (2006) 167–183. J. Juskiewicz, Z. Zdunczyk, A. Jurgonski, L. Brzuzan, I. Godycka-Klos, E. ZarySikorska, Extract of green tea leaves partially attenuates streptozotocin-induced changes in antioxidant status and gastrointestinal functioning in rats, Nutr. Res. 28 (2008) 343–349. H.S. Parmar, A. Kar, The antidiabetic potential of Citrus sinensis and Punica granatum peel extracts in alloxan-treated male mice, BioFactors 31 (2007) 17–24. J.S. Lee, Effects of Fomes fomentarius supplementation on antioxidant enzyme activities, blood glucose, and lipid profile in streptozotocin-induced diabetic rats, Nutr. Res. 25 (2005) 187–195. A. Pandey, P. Tripathi, R. Pandey, R. Srivastava, S. Goswami, Alternative therapies useful in the management of diabetes: a systematic review, J. Pharm. Bioallied Sci. 3 (2011) 504–512. M. Wessner, B. Champion, J.P. Girault, M. Kaouadji, B. Saidi, R. Lafont, Ecdysteroids from Ajuga iva, Phytochemistry 31 (1992) 3785–3788. A. Chenni, D.A. Yahia, F.O.J. Boukortt, J. Prost, M.A. Lacaille-Dubois, M. Bouchenak, Effect of aqueous extract of Ajuga iva supplementation on plasma lipid profile and tissue antioxidant status in rats fed a high-cholesterol diet, J. Ethnopharmacol. 109 (2007) 207–213. V.N. Syrov, Comparative experimental investigations of the anabolic activity of ecdysteroids and steranabols, Pharm. Chem. J. 34 (2000) 193–197. A. Sharma, M.K. Sharma, M. Kumar, Protective effect of mentha piperita against arsenic-induced toxicity in liver of swiss albino mice, Basic Clin. Pharmacol. Toxicol. 100 (2007) 249–257. P. Muthuraman, K. Srikumar, A comparative study on the effect of homobrassinolide and gibberellic acid on lipid peroxidation and antioxidant status in normal and diabetic rats, J. Enzyme Inhib. Med. Chem. 24 (2009) 1122–1127. P. Muthuraman, R. Senthilkumar, K. Srikumar, Alterations in beta-islets of Langerhans in alloxan-induced diabetic rats by marine Spirulina platensis, J. Enzyme Inhib. Med. Chem. 24 (2009) 1253–1256. M. Serrano-Rios, F. Ramos, J.L. Radrogez-Minor, I. Vivanco, Studies in prediabetic insulin response to oral glucose infusion in genetic prediabetes, Diabetologica 6 (1970) 392–398. O.H. Lowry, N.J. Rosebrough, A.L. Farr, R.J. Randall, Protein measurement with Folin phenol reagent, J. Biol. Chem. 193 (1955) 265–275. R.A. Kohn, M.M. Dinneen, E. Russek-Cohen, Using blood urea nitrogen to predict nitrogen excretion and efficiency of nitrogen utilization in cattle, sheep, goats, horses, pigs, and rats, J. Anim. Sci. 83 (2005) 879–889. V. Muthuviveganandavel, P. Muthuraman, S. Muthu, K. Srikumar, A study on low dose cypermethrin is induced histopathology, lipid peroxidation and marker enzyme changes in male rats, Pestic. Biochem. Physiol. 9 (2008) 12–16. P. Muthuraman, K. Ramkumar, D.H. Kim, Analysis of the dose-dependent effect of zinc oxide nanoparticles on the oxidative stress and antioxidant enzyme activity in adipocytes, Appl. Biochem. Biotechnol. 174 (2014) 2851–2863. J. Kishalay, K.B. Tushar, G. Debidas, Antidiabetic effects of Eugenia jambolana in the streptozotocin-induced diabetic male albino rat, Biomark. Genomic Med. 7 (2015) 116–124. P. Muthuraman, K. Ramkumar, D.H. Kim, Analysis of the dose-dependent effect of zinc oxide nanoparticles on the oxidative stress and antioxidant enzyme activity in adipocytes, Appl. Biochem. Biotechnol. 174 (2014) 2851–2863. P. Newsholme, E.P. Haber, S.M. Hirabara, E.L.O. Rebelato, J. Rebelato, D. Morgan, H.C. Oliveira-Emilio, A.R. Carpinelli, R. Curi, Diabetes-associated cell stress and dysfunction: the role of mitochondrial and non-mitochondrial ROS production and activity, J. Physiol. 583 (2007) 9–24. P. Pacher, J.S. Beckman, L. Liaudet, Nitric oxide and peroxynitrite in health and disease, Physiol. Rev. 87 (9) (2007) 315–424. J. El-Hilaly, A. Tahraoui, Z.H. Israili, B. Lyoussi, Hypolipidemic effects of acute and sub-chronic administration of an aqueous extract of Ajuga iva L. whole plant in normal and diabetic rats, J. Ethnopharmacol. 105 (2006) 441–444. V.M. Bhor, N. Raghuram, S. Sivakami, Oxidative damage and altered antioxidant enzymes activities in the intestine of streptozotocin-induced diabetic rats, Int. J. Biochem. Cell Biol. 36 (2004) 89–97. D. Taleb-Senouci, H. Ghomari, D. Krouf, S. Bouderbala, J. Prost, M.A. LacailleDubois, M. Bouchenak, Antioxidant effect of Ajuga iva aqueous extract in streptozotocin-induced diabetic rats, Phytomedicine 16 (2009) 623–631. M.S. Islam, H. Choi, Green tea, anti-diabetic or diabetogenic: a dose-response study, BioFactors. 29 (9) (2007) 45–53. A. Wiederkehr, C.B. Wollheim, Impact of mitochondrial calcium on the coupling of metabolism to insulin secretion in the pancreatic β-cell, Cell Calcium 44 (2008) 64–76. J. Vina, C. Borras, M.C. Gomez-Cabrera, W. Orr, Role of reactive oxygen species and (phyto)oestrogens in the modulation of adaptive response to stress, Free Radic. Biol. Med. 40 (2006) 111–119. M. Ohnishi, T. Matuo, T. Tsuno, A. Hosoda, E. Nomura, H. Taniguchi, H. Sasaki, H. Morishita, Antioxidant activity and hypoglycemic effect of ferulic acid in STZinduced diabetic mice and KK-Â{y} mice, BioFactors 21 (2004) 315–319. K. Ravi, B. Ramachandran, S. Subramanian, Protective effect of eugenia jambolana seed kernel on tissue antioxidants in streptozotocin-induced diabetic rats, Biol. Pharm. Bull. 27 (2004) 1212–1217. P.J. Houghton, A. Raman, Laboratory Handbook for the Fractionation of Natural Extracts, first ed., ITPs, London, 1998.
Biomedicine & Pharmacotherapy 96 (2017) 480–488
J.-J. Wang et al.
[42] D.G. Lyman, E.G.D. Murray, The pathology of the pancreas in experimental diabetes mellitus, Am. J. Med. Sci. 210 (9) (1945) 381–396. [43] L.D. Griffin, B.D. Gelb, V. Adams, E.R.B. McCabe, Developmental expression of hexokinase I in the rat, Biochim. Biophys. Acta 1129 (1992) 309–317. [44] L. Pari, G. Saravanan, Antidiabetic effect of cogent db, an herbal drug in alloxaninduced diabetes mellitus, Comp. Biochem. Physiol. 131 (2002) 19–25. [45] C. Postic, A. Leturque, R.L. Printz, P. Maulard, M. Loizeau, D.K. Granner, J. Girard, Development and regulation of glucose transporter and hexokinase expression in the rat, J. Am. Physiol. Soc. 266 (9) (1994) 548–559. [46] D.S. Trenin, V.V. Volodin, 20-Hydroxyecdysone as a human lymphocyte and neutrophil modulator: in vitro evaluation, Arch. Insect Biochem. Physiol. 41 (1999) 156–161.
[38] H. Wagner, M.A. Lacaille-Dubois, Recent pharmaco-logical results on bioflavonoids, in: S. Antus, M. Gabor, K. Vetschera (Eds.), Flavonoids and Bioflavonoids, Akademiai Kiado, Budapest, 1995, pp. 53–57. [39] C. Soto, R. Recoba, H. Barron, C. Alvarez, L. Favari, Sylimarin increases antioxidant enzymes in alloxan-induced diabetes in rat pancreas, Comp. Biochem. Physiol. C Toxicol. Pharmacol. 136 (2003) 205–212. [40] K. Hamden, S. Carreau, S. Lajmi, D. Aloulou, D. kchaou, A. Elfeki, Protective effect of 17β-estradiol on hyperglycemia, stress oxidant, liver dysfunction and histological changes induced by alloxan in male rat pancreas and liver, Steroids 94 (2008) 495–501. [41] S. Lortz, M. Tiedge, T. Nachtwey, A.E. Karlsen, J. Nerup, S. Lenzen, Protection of insulin-producing RINm5F cells against cytokine-mediated toxicity through overexpression of antioxidant enzymes, Diabetes 49 (2000) 1123–1130.
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