Daryoush Babazadeh1*, Ali Shabestari Asl2, Alireza Sadeghi3, Muhammad Saeed4, and Arman Moshavery5
- School of Veterinary Medicine, Shiraz University, Shiraz, Iran
- Department of Clinical Science, Faculty of Veterinary Medicine, Tabriz Branch, Islamic Azad University, Tabriz, Iran
- Doctor of Veterinary Medicine, Faculty of Veterinary Medicine, Tabriz Branch, Islamic Azad University, Tabriz, Iran
- Department of Pharmacy, University of Peshawar, 25120, Peshawar, Pakistan
- Doctor of Veterinary Medicine, Faculty of Veterinary Medicine, Karaj Branch, Islamic Azad University, Karaj, Iran
* Corresponding author: Daryoush Babazadeh, School of Veterinary Medicine, Shiraz University, Shiraz, Iran. Email: daryoush.babazadeh@shirazu.ac.ir
Comparative Histopathologic Evaluation of the Effects of Portulaca oleracea, Omega-3, and Combination of Sodium Selenite and Vitamin E on Hepatic enzymes of Experimental Diabetic Rats
ABSTRACT
Introduction: Portulaca oleracea (PO) plant, Omega 3, and Sodium Selenite plus Vitamin E have antidiabetic effects by compensating for the deficiency in insulin release and enhancing antioxidant status. The purpose of the present study was to comparatively assess the effect of Portulaca oleracea, omega-3, and a combination of Sodium Selenite and Vitamin E on hepatic enzymes activities in streptozotocin-induced diabetic Rats.
Materials and methods: A total of 48 adult male Wistar rats (weighing approximately 220 ± 10 g) were injected by a single intraperitoneal injection of streptozotocin (60 mg/kg body weight.) and were randomly assigned to 4 groups and 4 replicates for each group. Group 1 served as diabetic control, groups 2, 3, and 4 received Portulaca oleracea extract (1.5 mg/kg/day, orally), Omega-3 (500 mg/kg/day, orally), and Sodium Selenite (0.5 mg/kg/day, orally) plus Vitamin E (400 Iu/kg/day, orally), respectively, for 28 days. At the end of the study, blood samples were taken for biochemical investigations.
Results: The levels of blood glucose, AST, ALP, and GGT enzymes in all treatment groups were less than those of the control group. The ALT enzyme activity in rats treated with Portulaca oleracea and Vitamin E plus Selenium was less than control and omega-3 treatment groups.
Conclusion: Results indicated that Portulaca oleracea is more effective on hepatic enzyme activities of diabetic rats, compared to other treatment groups.
Keywords: Diabetes, Hepatic enzymes, Omega-3, Portulaca oleracea, Vitamin E
Introduction
Diabetes mellitus is a pathologic condition that causes extensive and non-physiological metabolic imbalance disorders, including an increase in blood glucose, and changes in carbohydrate, lipid, and protein metabolism in different body tissues, such as liver, and pancreas1,2. An increase in blood glucose initiates a series of cascade reactions, which leads to an increase in the production of free radicals (including oxygen free radicals) in various body tissues3,4. The high potency of these compounds for chemical reactions damages cells and tissues. Several reports have been published concerning the involvement of Reactive Oxygen Species (ROS) in the tissue damages5 among which the high level of ROS in pancreatic islets and changes in oxidative stress markers in laboratory animals can be noted6. Aerobic cells can be protected against free radicals particularly ROS by antioxidants compounds, such as glutathione, Vitamins E and C, as well as super Oxide Dismutase (SOD), glutathione Peroxidase (GPx), and catalase enzymes7,8. On the other hand, studies have also shown a significant decline in both non-enzymatic antioxidants (including rehabilitated glutathione (GSH) and Vitamin E) and enzymatic antioxidants (such as SOD, catalase, and GPx in diabetic rats)9,10. It has been also indicated that the free radicals can cause diabetic damages in different organs, such as the pancreas and liver, by declining SOD, catalase, and antioxidants activities10,11.
Free radicals can also damage the unsaturated fatty acid in cell membranes12. The combination of fatty acids in cell membranes can affect cell membrane-related phenomena, such as the interaction between insulin and its receptors13. In addition, it has been indicated that the fatty acid composition of membrane phospholipids in insulin targets tissues, such as the liver and skeletal muscles, affecting both insulin secretion and its biological activity14. Red blood cells are also susceptible to oxidative damage, due to the presence of fatty acid in their membrane and high concentration of oxygen and hemoglobin11. Hence, it is beneficial to use antioxidants compounds (particularly natural antioxidants) and omega3 fatty acids to prevent oxidative damage.
Vitamin E plus Selenium is one of the important food compounds which not only have high antioxidant properties but it can also affect different biological processes of the body. Shamsi et al15 has also shown that Vitamin E decreases blood glucose in diabetic rats and reduces diabetic disorders. It has been reported that Vitamin E declines Malondialdehyde (MDA) and increases GSH and SOD in diabetic rats16.
Vitamin E prevents lipid peroxidation and protects cells against peroxide radicals thus it is the most important antioxidant in the biological membrane which can neutralize free radicals17. Selenium is the only trace element that enters the genetic code as selenocysteine. This element can be extensively found in selenoproteins, namely the GPx enzyme, through which the Selenium antioxidant effect can be activated18. Reports available on the efficacy of Selenium in diabetes have indicated a decline in the effectiveness of streptozotocin (STZ) and enhancement of positive effects on GPX enzyme activity in laboratory rats19,20.
Portulaca oleracea is a rich source of omega-3 polyunsaturated fatty acids (alpha-linolenic acid), different vitamins (A, C, and E), and minerals which has different pharmacological (such as antioxidant, anticancer, anti-inflammatory, and antimicrobial) properties21. However, bioactive compounds of Portulaca oleracea can have beneficial effects against diabetes22. There are few studies addressing the anti-diabetic effects of Portulaca oleracea in previous years23,24. Thus, the aim of the present study was to compare the effect of Portulaca oleracea, omega-3, and Sodium Selenite plus Vitamin E on hepatic enzymes activities in streptozotocin-induced diabetic Rats.
Materials and Methods
Ethical approval
All procedures were approved by the Animal Care Committee of Veterinary Medicine, Islamic Azad University, Tabriz Branch, Iran. The principles of laboratory animal care were followed, and specific international laws were observed.
Animals
A total of 48 male Wistar rats aged 2-3 months, with an average weight of 220 g were bought from Razi institute, Iran, and kept in laboratory conditions with ad libitum water and food intake. Experimental animals have been kept in standard cages with a minimum of 50% humidity, 24°C temperature, and 12 hours dark/light cycle with appropriate ventilation in a particular cage. The rats were divided into 4 main groups of 10 and 8 rats randomly remained as a control group. The groups contained the control group of diabetic rats, which received the standard ration daily, the second group of diabetic rats were fed standard ration plus Portulaca oleracea extract (1.5 mg/kg/day) via gastric feeding tube daily, the third group of diabetic rats was fed standard ration plus omega 3 (500 mg/kg/day) via gastric feeding tube daily, the fourth group of diabetic rats was fed standard plus Vitamin E (400 iu/kg/day) and Selenium (0. 5 mg/kg/day) via gastric feeding tube daily.
Extraction of Portulaca oleracea
The aforementioned atmospheric parts of Portulaca oleracea were prepared from the farm of Islamic Azad University, Tabriz, Iran, and kept in a dark glass bottle at 10°C temperature away from direct sunlight. Then, 250 g of the intended powder was extracted by ethanol-water solvent (70% ethanol-30% water) three times at normal laboratory temperature based on the method of Abdullah and Kusumaningtyas25. The extracts were mixed and condensed with reduced pressure so that their volume reached 500 ml, which was equal to 0. 5 g of the powder per milliliter. For further investigation, the extract was divided into equal volumes (25 ml) and stored at -20°C temperature.
Diabetes infusion
The rats were diabetic via IP Injection of STZ solution at a dosage of 60 mg/kg dissolved in buffer citrate 0.1 at pH=4.5. On the second day, blood samples were collected through a tail vein from animals, under anesthesia with chloroform. Rats with fasting blood glucose higher than 250 mg/dl were considered diabetic and were used in the present study.
Blood sample
One day after the last gavage, blood samples were taken from all mice through a tail vein under chloroform anesthesia conditions once before receiving the medication (fasting) and once 1 hour after receiving the medication and serum was extracted by centrifugation device for 15 minutes at a speed of 3000 rpm. Then, serum levels of blood glucose and liver enzymes, such as alanine aminotransferase (ALT), aspartate aminotransferase (AST), alkaline phosphatase (ALP), and alpha-glutamyl transpeptidase (GGT) were measured and recorded by standard kits of Pars Azmoon Company (Iran).
Statistical analysis
The obtained data were collected and recorded in Excel software. The statistical analysis was performed with SAS version 19 using the mean comparison with Duncan’s multi-domain test with 95% confidence level (p < 0.05)26.
Results
Fasting blood glucose levels
Blood glucose levels in all treatment groups were significantly lower than the diabetic control group (p < 0.05), but indicated a higher blood glucose level than the control group (p < 0.05, Table 1). The lowest blood glucose levels were observed in the omega-3 (244 mg/dl), Portulaca oleracea (253 mg/dl), and Vitamin E + Selenium (283 mg/dl) groups, respectively, although the difference was not significant (p > 0.05).
Blood glucose levels one hour after the last treatment
Blood glucose levels in all treatment groups were significantly lower than the diabetic control group (p < 0.05), but showed higher blood glucose levels than the control group. The obtained results indicated a significant difference between the Portulaca oleracea group and the control group (p < 0.05, Table 1). The lowest blood glucose levels were observed among the treatment groups as 136, 146, and 163 mg/dl in Vitamin E + Selenium, omega-3, and Portulaca oleracea groups, respectively, although the difference between them was not significant (p > 0.05).
AST activity levels
The activity of AST enzyme in all treatment groups was significantly lower than the diabetic control group (p < 0.05), but still showed a higher difference in enzyme activity than the control group (p < 0.05, Table 2). The lowest AST enzyme activity among the treatment groups was observed in the groups of Portulaca oleracea (164 U/L), Vitamin E + Selenium (183 U / L), and omega-3 (236 U / L), respectively. However, this enzymatic activity in the groups consuming Portulaca oleracea and Vitamin E + Selenium showed a significant decrease compared to the group consuming omega-3 fatty acids (p <0.05).
ALT activity levels
ALT enzyme activity was significantly lower in the groups treated with Portulaca oleracea and Vitamin E + Selenium and also in the healthy control group than the diabetic control group and omega-3 consuming group (p < 0.05, Table 2). The lowest ALT enzyme activity was observed among the treated groups in the groups consuming Vitamin E + Selenium (83 U/L), Portulaca oleracea (85 U/L), and omega-3 (117 U/L), respectively. Portulaca oleracea and Vitamin E + Selenium showed enzymatic activity close to the healthy control group.
ALP activity levels
Alkaline phosphatase activity in all treated groups was significantly lower than the diabetic control group, and the Vitamin E + Selenium group showed a significant decrease, compared to the omega-3 group (p < 0.05, Table 2). The lowest ALP enzyme activity was observed among the treatment groups in the groups consuming Vitamin E + Selenium (129 U/L), Portulaca oleracea (145 U/L), and omega-3 (184 U / L), respectively. Portulaca oleracea and Vitamin E + Selenium showed enzymatic activity close to the healthy control group.
GGT activity levels
GGT activity was significantly lower in all treatment groups than in diabetic and control groups (p < 0.05, Table 2). The lowest activity of the GGT enzyme among the treatment groups was observed in the groups of Portulaca oleracea (14 U/L), Vitamin E + Selenium (16 U/L), and omega-3 (21 U/L), respectively. However, this enzymatic activity did not show a significant difference among the groups (p > 0.05).
Discussion
Portulaca oleracea extract contains pharmacological active agents, such as alkaloids, glycosides, terpenoids, sterols, and flavonoids. It may be stated that some of these compounds can reduce the severity of autoimmune reactions and the inflammation process to the extent that leads to the destruction of beta cells. Consequently, the destruction of the remaining cells is prevented which provides ample opportunity for the proliferation of these cells and the regeneration of the pancreatic islets. It was found that the consumption of Portulaca oleracea extract caused the regeneration of pancreatic islets in diabetic rats with STZ due to the presence of flavonoids, such as quercetin, existing in the aerial parts of the plant, which can release insulin by changes in Ca++ metabolism27.