|Year : 2015 | Volume
| Issue : 4 | Page : 151-158
Ginger extract attenuates preliminary steps of streptozotocin-mediated oxidative stress in diabetic rats
Mostafa I Waly, Nejib Guizani, Sithara Suresh, Mohammad Shafiur Rahman
Department of Food Science and Nutrition, College of Agricultural and Marine Sciences, Sultan Qaboos University, Muscat, Oman
|Date of Web Publication||19-Oct-2015|
Department of Food Science and Nutrition, Sultan Qaboos University, PO Box - 34, Al-Koud - 123, Muscat
Source of Support: None, Conflict of Interest: None
| Abstract|| |
Objective: Although the role of streptozotocin (STZ) in the pathogenesis of diabetes in rats has been well investigated, as evidenced by several citations, to our knowledge no study has been carried out yet to examine the preliminary steps of STZ-mediated oxidative stress in pancreatic rat tissues. This study aimed to evaluate the hypoglycemic and potential antioxidant properties of ginger extract (GE) in diabetic rats. Materials and Methods: Forty-eight male Sprague-Dawley rats weighting 250-300 g were allocated to groups as follows: Nondiabetic control group (n = 12) that received chow diet; nondiabetic control group that received chow diet plus oral feeding of GE (n = 12); diabetic group (n = 12) that received chow diet; and diabetic group (n = 12) that received chow diet plus oral feeding of GE. The drug STZ was used as a diabetogenic agent in a single intraperitoneal injection dose of 60 g/kg body weight, and the blood glucose level for each rat was measured twice a week. After 12 weeks, all animals were overnight fasted and sacrificed; serum was collected for biochemical measurements of glucose, insulin, and oxidative stress indices [advanced oxidation protein products (AOPP), protein carbonyls, and nitrates plus nitrites]. The pancreas tissues were dissected and homogenized for antioxidant measurements [glutathione (GSH) and total antioxidant capacity (TAC)]. Results: Diabetic rats treated with GE showed a significant protective effect against STZ-induced hyperglycemia and oxidative stress as compared with the control group. Conclusion: Our results suggested that GE possesses potential benefits in controlling type 2 diabetes mellitus (T2DM) and that it may also prevent pancreas damage.
Keywords: Dietary antioxidants, insulin deficiency, oxidative stress, type 2 diabetes mellitus
|How to cite this article:|
Waly MI, Guizani N, Suresh S, Rahman MS. Ginger extract attenuates preliminary steps of streptozotocin-mediated oxidative stress in diabetic rats. Int J Nutr Pharmacol Neurol Dis 2015;5:151-8
|How to cite this URL:|
Waly MI, Guizani N, Suresh S, Rahman MS. Ginger extract attenuates preliminary steps of streptozotocin-mediated oxidative stress in diabetic rats. Int J Nutr Pharmacol Neurol Dis [serial online] 2015 [cited 2020 Nov 28];5:151-8. Available from: https://www.ijnpnd.com/text.asp?2015/5/4/151/167503
| Introduction|| |
Experimental and clinical studies indicate that oxidative stress is involved in the pathogenesis of type 2 diabetes mellitus (T2DM).,, It is well documented that oxidative stress is a condition in which there is an accumulation of reactive oxygen species (ROS), and a decline in the cellular antioxidant defense mechanisms. Oxidative stress in experimental animals results in the impairment of different organs' biological functions by mechanisms that include lipid peroxidation of cell membranes, oxidative damage of cellular organelles, and autoxidation of biological proteins.,, ROS-mediated signaling pathways are associated with the development of two etiological factors for T2DM: Insulin resistance and hyperglycemia.,, T2DM acts as a risk factor for comorbidities and mortality among adult populations, and the World Health Organization (WHO) has stated that T2DM will be a global health threat over the next 15 years with a worldwide increase in the number of diagnosed cases from 171 million in 2014 to 366 million in 2030. Exposure to oxidative stress-inducing agents and low intake of dietary antioxidants are the two major environmental risk factors contributing to an increasing incidence of T2DM; meanwhile, urbanization and sedentary lifestyles are considered as the behavioral risk factors.,,
Ginger is considered a bioactive agent useful for combating oxidative stress as it is rich in antioxidant nutrients and phytonutrients, and these compounds are thought to be of medicinal value in the treatment of T2DM.,, Experimental diabetes mellitus has been induced in laboratory animals by diabetogenic drugs such as streptozotocin (STZ), which caused degeneration of the Langerhans islet β-cells in the pancreas. STZ is an N-nitroso compound that acts as a nitric oxide donor in pancreatic islets, induces the death of insulin-secreting cells, and produces an animal model of diabetes that is characterized by hyperglycemia and insulin deficiency.
Although the role of STZ in the pathogenesis of diabetes in rats has been well investigated as evidenced by several citations, no study had been carried out to examine the presteps of STZ-mediated oxidative stress in pancreatic rat tissues.
Therefore, the aim of the present study was to evaluate the potential antioxidant properties of ginger against STZ-induced oxidative stress in rats.
| Materials and Methods|| |
Preparation of ginger extract
Ginger (Zingiber officinale) was cut into small pieces and homogenized with distilled water (15 g dry solids/100 mL; dry solids were estimated from the moisture content of the sample and water was estimated based on added water and water in the sample) in an electric mixer until smooth. Ginger homogenate was allowed to mix separately on a magnetic stirrer for 3 h at medium speed in the absence of light. The homogenate was then centrifuged separately at 6000 rcf for 30 min at 4°C using a Harrier 18/80 refrigerated centrifuge (Sanyo, Lower Sydenham, London, SE26 5AZ, United Kingdom). The extract was then filtered by Whatman filter paper # 1 (150 mm) ), Sigma-Aldrich product number Z274852 and stored at -40°C for later experiments. The dose of ginger was calculated as an extract from 0.1 g dry-solids/kg body weight (1.5 mL/kg body weight per day) for each rat.
Flavonoids and polyphenol contents
Total polyphenol content was analyzed using the Folin-Ciocalteu method. An appropriate calibration curve was prepared using standard solutions of gallic acid. Polyphenols were expressed as mg gallic acid equivalents (GA)/g dry solids of fresh ginger. The total flavonoid content of the freeze-dried peel extract was determined, as previously reported. An appropriate calibration curve was prepared using different concentrations of catechin solutions. The final flavonoid contents were expressed as mg catechin equivalent (CE)/g dry solids of fresh ginger.
The protocol used in this study was approved by the Animal Ethics Committee at Sultan Qaboos University (SQU/AEC/2010-11/1). Forty-eight male Sprague-Dawley rats weighing 250-300 g were obtained from the animal house facility, SQU, Muscat, Oman. The animals were housed in an air-conditioned room at 23 ± 1°C on a 12:12 h light-dark cycle, and all rats were initially fed a standard chow diet and given water ad libitum.
Diabetes induction experimental design
Diabetes was induced in each rat in the diabetic group by a single intraperitoneal injection of STZ (Sigma, St. Louis, MO, USA) dissolved in 0.1 M citrate buffer (pH 4.5) at 60 mg/kg body weight. The normal control rats received a single intraperitoneal injection of 0.1 M citrate buffer solution. After 72 h of the STZ injection, only rats with blood glucose level over 300 mg/dL were considered to be diabetic and included in the study. The blood glucose levels were measured from the tail vein, using a portable glucose meter (One Touch II; Johnson and Johnson, Milpitas, CA, USA). The distal part of the tail was gently snipped; the first blood drop was discarded and the second was absorbed by a test strip inserted in the glucose meter. Blood glucose levels were measured twice per week and always measured at 8 AM.
As represented in [Figure 1], 24 diabetic rats were randomly divided into two groups (n = 12 rats/group): Diabetic rats (DM), and diabetic rats treated with an oral dose of 1.5 mL extract/kg body weight per day (DM + GE). Another 24 normal, nondiabetic, rats were allocated into two groups (n = 12 rats/group): Control (C) rats, and a control group that received oral feeding of 1.5 mL GE extract/kg body weight per day (GE). GE was started at 1 week after STZ injection and was administered once daily by oral gavage for 12 weeks. The normal control (C) and diabetic control (DM) rats were treated with the same volume of saline within the same time.
Animal sacrifice and sample collection
At the end of the 12 weeks' treatment, animals were anesthetized with a lethal dose of a cocktail containing ketamine (1mg), xylazine (5mg), and acepromazine (0.2 mg). Blood was collected from the heart and the serum was separated by centrifugation, then stored at -80°C. The serum was used later for the following biochemical analyses: Glucose, insulin, and oxidative stress indices [advanced oxidation protein products (AOPP), protein carbonyls, and nitrates plus nitrites]. The pancreas tissue of each rat (~50 mg) was immediately homogenized in 5 mL of 100 mM potassium phosphate buffer (pH 7.2) in a glass-Teflon homogenizer with an ice-cold jacket and centrifuged at 10,000 × g at 4°C for 60 min. The resulting supernatant was used for protein content measurement, and for cellular antioxidant markers [glutathione (GSH) and total antioxidant capacity (TAC)].
Analysis of protein content
The protein content of pancreas tissue homogenates was assayed by Lowry's method using bovine serum albumin as standard, and protein content was expressed as mg/mL of sample.
Serum glucose and insulin measurements
Serum glucose was measured by the High Sensitivity Glucose Assay Kit Glucose Assay Kit (Sigma-Aldrich, product number MAK181).; meanwhile serum insulin was measured by the enzyme-linked immunosorbent assay (ELISA)-based technique, the Human Insulin ELISA Kit (RAB0327-1KT, Sigma).
Serum oxidative stress indices
Serum in the total quantity 200 µL, diluted 1:5 in phosphate-buffered saline was added into each well of a 96-well plate, followed by 20 µL of acetic acid. For the standard, 10 µL of 1.16 M potassium iodide (Sigma, St. Louis, MO, USA) was added to 200 µL of chloramine-T solution (0-100 µmol/L) (Sigma, St. Louis, MO, USA) in a well and then 20 µL of acetic acid. The absorbance of the reaction mixture was immediately read at 340 nm against a blank consisting of 200 µL of phosphate-buffered saline, 10 µL of 1.16 M potassium iodide, and 20 µL of acetic acid. AOPP concentrations are expressed as µmoles/L of chloramine-T equivalents.
A total quantity of 200 µL serum was treated with 200 µL of 10 mM 2,4-dinitrophenylhydrazine (DNPH) in 2 M hydrogen chloride (HCl), or with 0.5 mL 2 M HCl alone for the blank. Samples were incubated for 1 h at room temperature in the dark, and then treated with 10% trichloroacetic acid and centrifuged. The pellet was washed three times in ethanol/ethyl acetate and solubilized in 1 mL of 6 M guanidine in 20 mM potassium phosphate, then adjusted to pH 2.3 with trifluoracetic acid; the resulting solution was incubated at 37° C for 15 min. The carbonyl concentration was determined from the difference in absorbance at 370 nm between DNPH-treated and HCl-treated samples; the carbonyl content was expressed as nanomoles of carbonyl/mg protein.
Nitrates plus nitrites
The nitrate concentration was determined with a spectrophotometric assay using oxidation catalyzed by cadmium metal, which converts nitrates to nitrites. The total nitrites thus determined correspond to nitrate production. Proteins were first removed from the serum by incubation with zinc sulfate. Cadmium metal was then added and the medium incubated overnight with shaking in the dark. The next day, the solution was mixed, and sulfanilamide and N-1 naphthylethylenediamine were added. These compounds form a colored complex with nitrites, which can be assayed by spectrophotometry.
Aliquots of pancreas tissue supernatant (100 µL) were transferred to fresh Eppendorf Microcentrifuge tube (Sigma-Aldrich, product number Z334006). and 2 µL of monochlorobimane (25 mmol/L) and 2 µL of glutathione S-transferase reagent were added, as provided by a commercial kit (Catalog # K251, Biovision, Mountain View, CA, USA). After 30 min of incubation at 37°C, the samples and standards were read in a fluorescence plate reader at 380/460 nm. GSH content was determined by comparison with values from a standard curve using freshly prepared GSH and normalized to the protein content of the assayed pancreas tissue homogenates.
A colorimetric method using the Randox Assay Kit (Sanyo, Lower Sydenham, London, SE26 5AZ, United Kingdom) was used to measure the TAC. The assay is based on the incubation of samples with 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate (6)] (ABTS) diammonium salt with a peroxidase (metmyoglobin) and hydrogen peroxide to produce the radical cation ABTS +, which has a relatively stable blue-green color that is measured at 600 nm. Antioxidants present in the assayed pancreas tissue homogenates inhibit the oxidation of ABTS to ABTS + (causing suppression of the color production) to a degree that is proportional to their concentration. The capacity of the assayed sample antioxidants was compared with that of standard 6-hydroxy-2, 5, 7, 8-tetramethylchroman-2-carboxylic acid (Trolox), a water-soluble tocopherol analogue, which is widely used as a traditional standard for TAC measurement assays. The assay results are normalized to the protein content of the assayed pancreas tissue homogenates.
The data were expressed as mean ± standard deviation (SD). GraphPad Prism software (version 5.03; GraphPad Software Inc., San Diego, CA, USA) was used to calculate one-way analysis of variance (ANOVA), followed by Tukey's test and Student's t-test for means comparisons. A P value of <0.05 was considered to be significant.
| Results|| |
Polyphenols and flavonoids
The polyphenol and flavonoid contents of GE were determined as 5.9 mg GA/g dry-solids 0.71 mg CE/g dry-solids, respectively.
Body weight gain of animals
No mortality occurred in any experimental group. The daily consumption of water and food in control nondiabetic (C) rats were measured as 25 ± 4 ml, and 10 ± 2 g, respectively. The same pattern was observed for GE-supplemented groups, indicating that GE had no negative interactive effects. Meanwhile, in diabetic rats, the daily consumption of water and food were increased as 165 ± 5 mL and 40 ± 4 gm, respectively. However, the GE supplementation for diabetic rats caused a reduction in water and food consumption. All control nondiabetic (C), and GE-supplemented rats grew at a similar rate, and the average weight gain was 10 g per week. In diabetic rats, there was a time-dependent loss of weight between the initial body weight (250-300 g) and the final body weight (100-120 g) at the end of the experiment. A remarkable observation was that the GE supplementation for diabetic rats caused a recovery in the final body weight (200-240 g) at the end of the experiment.
Effects of GE and STZ on serum glucose and insulin levels
Control nondiabetic rats (C) had serum glucose and insulin concentrations as 128 ± 6 mg/L and 3 ± 0.2 mIU/L, respectively. In diabetic rats (DM), STZ injection caused a significant increase in serum glucose, 480 ± 16 mg/dL, and a significant decrease in serum insulin level as 1.5 ± 0.1 mIU/L, according to t-test, P < 0.05.
As shown in [Figure 2], the serum glucose level was significantly higher in STZ-induced diabetic rats as compared with nondiabetic control animals. However, treatment with GE caused a significant reduction in the STZ-induced increase in serum glucose, P < 0.05. It was observed that treatment with GE significantly restored the STZ-induced insulin deficiency to levels that are comparable to the nondiabetic control group, P < 0.05 [Figure 3]. STZ is a diabetogenic agent that alters the insulin secretion by the pancreatic islet insulin-producing β-cells. It is a glucosamine-nitrosourea compound that acts as a DNA-alkylating agent and enters pancreatic cells exclusively via the glucose transporter 2 (GLUT2) glucose transport protein.
|Figure 2: GE suppressed the STZ-induced hyperglycemia in diabetic rats (DM). *Significantly higher as compared to control non-diabetic rats (C). #DM plus GE supplementation is significantly lower than DM group, P < 0.05|
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|Figure 3: GE suppressed the STZ-induced insulin deficiency in diabetic rats (DM). *Significantly lower as compared to control nondiabetic rats (C). #DM plus GE supplementation is significantly higher than DM group,P<0.05|
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There was no significant differences in either serum glucose levels or insulin levels between control, nondiabetic (C), and GE-supplemented rats, P > 0.05.
Effects of GE on STZ-induced oxidative stress
[Table 1] demonstrates that the concentrations of serum oxidative stress indices (AOPP, protein carbonyls, and nitrates plus nitrites) were significantly higher in diabetic rats than in the control, nondiabetic (C) group, P < 0.05. GE supplementation in diabetic rats was significantly effective in ameliorating the observed STZ-induced oxidative stress, P < 0.05. Concentrations of serum oxidative stress indices were not different between nondiabetic rats and GE-supplemented rats, P > 0.05.
Protective effect of GE against STZ-induced GSH-depletion in pancreas tissue
[Figure 4] shows the intracellular GSH levels in pancreas tissue homogenates of various experimental groups. The GSH levels for nondiabetic and GE-supplemented groups were comparable with no statistical significant difference (25.18±6.1and 25.22±4.7, respectively), P > 0.05. On the other hand the STZ-injected group showed a significant reduction in the GSH levels (6.78 ± 0.87 nmol/mg protein) as compared to nondiabetic rats, P < 0.05. It was observed that the diabetic rats fed GE supplementation had recovered the GSH level so that it was comparable to the control nondiabetic rats, 24.91 ± 2.32 nmol/mg protein.
|Figure 4: GE prevents the STZ-induced GSH depletion in pancreatic cells of diabetic rats (DM). *Significantly lower as compared to control nondiabetic rats (C).#DM plus GE-supplemented group is significantly higher than DM group,P<0.05|
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Impact of GE and STZ-induced impairment of TAC in pancreas tissue
The mean TAC values among the control and GE-supplemented groups were comparable with no statistically significant difference: 155.65 ± 11.82 µmol/mg protein and 155.21 ± 12.31 µmol/mg protein, respectively; P > 0.05. The lowest TAC value (61.25 ± 9.82 µmol/mg protein) was observed for the STZ-injected group, while the GE supplementation for the diabetic group caused a significant improvement in the measured TAC level, 140.87 ± 13.86 µmol/mg protein [Figure 5].
|Figure 5: GE attenuates the STZ-induced TAC impairment in pancreatic cells of diabetic rats (DM). *Significantly lower as compared to control nondiabetic rats (C).#DM plus GE-supplemented group is significantly higher than DM group,P<0.05|
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| Discussion|| |
T2DM is a multifactorial disorder in which oxidative stress synergizes with dietary, environmental, and genetic predisposition for the disease pathogenesis. In human cells the production of radical reactive species and the antioxidant defense systems are approximately balanced. When ROS combats the antioxidant defense systems, this situation is called oxidative stress. Examples of ROS include charged species such as superoxide and hydroxyl radicals, and uncharged species such as hydrogen peroxide; the oxidative damage from these species to biomolecules such as DNA, lipids, and protein is involved in the pathogenesis of T2DM.
Ginger is a good a source of antioxidants, which have been the basis of numerous studies in the last decade in relation to T2DM. The cellular antioxidant capacity includes enzymatic and nonenzymatic molecules that are located in the intracellular and extracellular milieu, and they are responsible for detoxifying or scavenging the free radical insults generated by various oxidizing agents. In this context our data suggest that the diabetogenic drug STZ acts as an oxidizing agent and causes oxidative damage to the pancreas tissue, as evidenced by low GSH, impaired TAC, and release of serum oxidative stress indices (AOPP, protein carbonyls, and nitrates plus nitrites). However this STZ-mediated oxidative stress was abrogated by GE supplementation.
Oxidation alters the structure and activity of serum proteins. The prolonged half-life of AOPP and carbonyl groups allows an indirect reflection of the intensity of the oxidative stress. Serum protein oxidation products are elevated in several conditions in humans, including diabetes mellitus. Examination of individual serum proteins may be useful in determining specific pathways of oxidative stress in vivo and the possible functional consequences of oxidative stress exposure. In this study, we determined the impact of redox imbalance on biomolecules by evaluating direct oxidative indices such as total protein carbonyl groups and AOPP.,
Total nitrates plus nitrites levels are used as markers of the activity of nitric oxide synthase and the production of nitric oxide radicals. We found an increase in serum total nitrates plus nitrites in the sera of nondiabetic and diabetic rats. This result suggests that nitric oxide plays a role in STZ-induced oxidative stress. GE supplementation was effective in restoring the STZ-induced diabetes markers (hyperglycemia and insulin deficiency), which suggested that GE supplementation was potentially beneficial in the control of T2DM, and that it may also prevent pancreas damage. The data on the antioxidant and antidiabetic properties of GE supplementation against the STZ insult in our study are novel and consistent with the recent research studies reported in a rat model: The long-term ingestion of GE suppressed the ROS-mediated pathogenesis through an antioxidative effect.
As presented in [Figure 6], our data suggest that the STZ injection in rats created an oxidative stress milieu in pancreatic cells by depleting the intracellular GSH. However, this GSH depletion could be restored by GE supplementation in the diabetic rats and can reach a comparable GSH level as observed in the nondiabetic groups. The data from this study lead us to hypothesize that dietary GE supplementation can restore the pancreatic cells' antioxidant capacity under STZ insult. The molecular mechanisms underlying the modulation of GE-mediated intermediates of pancreatic cells under the effect of STZ treatment need to be further investigated.
|Figure 6: The mechanism of action of GE supplementation against STZ-induced oxidative stress in the diabetic rats|
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| Conclusion|| |
In summary, the STZ-injected rats fed on GE supplementation showed significantly higher GSH and TAC levels and lower oxidative stress indices as compared to diabetic groups. These findings indicate that a supplementary GE dietary regimen by STZ injection can attenuate the STZ suppression effect on GSH and TAC levels in pancreatic cells. The observed highest GSH and TAC values in GE supplementation for diabetic rats indicate the efficacy of GE in counterbalancing and scavenging the ROS and its related oxidative damage in pancreas tissue. Our observation opens new avenues toward the primary prevention of T2DM.
Authors 'contributions: Mostafa Waly, Nejib Guizani, and Mohammad Shafiur Rahman contributed equally in the designing of the experiments ad writing of the manuscript. Sithara Suresh carried the analysis of phenols and flavonoid measurements.
This research was supported by the internal grant fund (IG/AGR/FOOD/13/05 and IG/AGR/FOOD/14/2) and His Majesty's Strategic Fund (SR/AGR/FOOD/11/01) offered by SQU though the College of Agricultural and Marine Sciences. The authors acknowledge the assistance of Mr. Sultan Al-Maskari and Mrs. Nasneen Thrikkannoor Kolathod at the animal unit at SQU for their participation in the animal care component of this study.
Financial support and sponsorship
Conflicts of interest
There are no conflicts of interest.
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[Figure 1], [Figure 2], [Figure 3], [Figure 4], [Figure 5], [Figure 6]