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Volume 16, Issue 1 (January 2022)                   IJT 2022, 16(1): 43-50 | Back to browse issues page


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Gazor R, Niknezhad F, Yousefbeyk F, Aghajany-Nasab M, Mohammadghasemi F. The Extract of Viola odorata Flowers Improves the Biochemical, Pancreas Histological, and Insulin Resistance Parameters in an Animal Model of Diabetes. IJT 2022; 16 (1) :43-50
URL: http://ijt.arakmu.ac.ir/article-1-1019-en.html
1- Department of Anatomy, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran.
2- Student Research Center, Department of Anatomy, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran.
3- Department of Pharmacognosy, School of Pharmacy, Guilan University of Medical Sciences, Rasht, Iran.
4- Cellular & Molecular Research Center, Department of Biochemistry, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran.
5- Cellular & Molecular Research Center, Department of Anatomy, School of Medicine, Guilan University of Medical Sciences, Rasht, Iran. , parsahistolab@gmail.com
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Introduction
Type 2 Diabetes Mellitus (T2DM) is a metabolic and multifactorial disorder. Various conditions, such as lifestyle, family inheritance, obesity, hypertension, aging, and other risk factors lead to diabetes, in which insulin secretion or action is deficient [1]. Following diabetes, hyperglycemia occurs and the metabolism of electrolytes, lipids and proteins are disturbed [1]. If left untreated, diabetes may lead to cardiovascular, kidney and neurological complications and may ultimately result in death [2]. The prevalence of T2DM is rising globally and the number of adult diabetes may probably reach 592 million in 2035 [1]. This disorder can be successfully managed with proper diet, exercise and therapeutic drugs [3]. Diabetes is associated with oxidative stress, a prevalent cause of which is glucose autoxidation [4]. Persistent hyperglycemia disturbs the antioxidants and leads to oxidative stress and inflammation, which may cause the death of the pancreas β-cells [4]. The antioxidant defense in pancreas is usually low if the cells are exposed to toxic substances daily or frequently. Therefore, the cells are damaged due to the oxidative stress. 
In addition to the endocrine secretions by the Langerhans islands, pancreas has exocrine secretions from its acinar cells. These cells secrete digestive enzymes, including proteases, lipase and amylase [5]. Since the acinar and Langerhans island cells are in close proximity to each other, defect in insulin secretion may have adverse effects on the acinar cells’ function [6]. Both insulin deficiency in diabetes type 1 and 2, and insulin resistance in obesity and metabolic syndrome lead to low serum amylase [6, 7]. The serum amylase and lipase levels are important for determining the pathologic mechanisms in pancreas [7]. In pancreatitis, pancreatic cancer and pancreatic duct obstruction, the lipase and amylase secretions increase [6]. Treatment with STZ in neonate rats reduces the pancreatic amylase without affecting its lipase and trypsin secretions after 16 days [8]. 
In experimental animal models, Streptozotocin (STZ), is usually used to induce diabetes. This compound interacts with the pancreatic beta-cells via glucose transporter-2 receptors and causes the production of Reactive Oxygen Species (ROS) [9]. Currently, the use of drugs such as insulin or its sensitizers or insulin secretory drugs are the main treatment for diabetes. However, hypoglycemia and gastrointestinal discomforts are known to be the side effects of these drugs. Therefore, there is a need for safer and more effective drugs for the management of diabetes [1]. Nowadays, the application of traditional medicine is growing in the management of many 
chronic conditions, including diabetes. Traditional medicine has been used widely in developing countries and about two-thirds of the people use traditional medicine for health care purposes globally [10]. Traditional medicines are cheaper, more accessible and patients have better tolerance using them [3]. They may have better and useful effects on diabetes due to their antioxidant properties [4].
Various substances with antioxidant properties have been identified in various parts of plants such as fruits, stem, root, leaves, flowers, and seeds [4]. Viola odorata L. or sweet violet from the Violaceae family of plants, has traditionally been used for the management of hypertension, anxiety, mouth infection, sleep disorders and fever in children [11]. This plant product has anti-inflammatory, antibacterial, antioxidant and anticancer properties [12, 13]. In addition, this product protects against conditions that adversely affect the liver, vasculature, and the antioxidative systems [14]. The Viola blossoms powder protects both liver and kidney functions in rats treated with carbon tetrachloride (CCl4) by reduction of malondialdehyde [15]. The Viola flowers also reduce lipid peroxidation and have protective effects against rat’s kidney damages induced by STZ [11]. Studies on the effects of Viola odorata on the endocrine and exocrine functions of the pancreas in diabetic animal models are lacking. Thus, the present study was conducted to evaluate the therapeutic and ameliorative effects of the extract of Viola odorata flowers on the pancreas histology and function in streptozotocin-induced diabetes in rats. 

Materials and Methods
Extraction procedure: Viola odorata (VO) flowers were collected from the plant in Mazandaran province, Iran. The plant was identified and given herbarium specimen number (HGUM-302) by a botanist at Pharmacology Department, Guilan University of Medical Sciences. The flowers were powdered, using an electric mill, and dissolved in 70% ethanol for 72hr in darkness. Then, the solution was filtered through a sterile paper filter and dried by evaporating the alcohol. The prepared powder was then stored in a refrigerator at 4°C [11, 17]. 
Animal study: The study protocol was reviewed and approved by the Laboratory Animals Ethics Committee of Guilan University of Medical Sciences (Ethics Code:1395.226). A total of forty adult Wistar male rats, weighing 250±10 g each, were used in this study. They were divided equally into five groups of eight rats each, and were kept under 12hr of light and dark cycles at 23°C temperature. They had free access to food pellets and tap water ad libitum. 
Animals were divided in groups as follows: A: the control group was treated with normal saline; B: the diabetic group treated with STZ; C, D, and E: the experimental groups received STZ (45 mg/kg) and the following doses of VOE at 100, 200, and 400 mg/kg, respectively. To induce diabetes in rats, a single dose of STZ (45 mg/kg) was injected Intraperitoneally (IP). On the 3rd day after the STZ injection, all animals were checked for their serum glucose levels. Blood samples were collected from their tails and the rats with the glucose levels higher than 250 mg/dl were considered as diabetic [18, 19]. Three days after the induction of diabetes, the VOE was administered IP to the rats daily, for the next 30 days. On day 31, the animals were anesthetized with 50 mg/kg ketamine and 22 mg/kg xylazine. The pancreas tissue was removed surgically from each animal for the subsequent histopathological examinations. Blood samples were also collected from the rats’ inferior vena cava. 
Biochemical assays: The sera were obtained by centrifuging the blood samples at 4000 rpm for 10 min and stored at -20˚C for further analysis. The serum glucose, amylase and lipase levels were measured using the respective kits (Parse Azmun, Tehran, Iran) for spectrophotometric assays. The kits’ sensitivity levels were 5mg/dl (glucose) and 3 IU/L for lipase and amylase. Insulin was measured by a rat ELISA kit (Demeditec, Germany) with a sensitivity of 0.1ng/ml. The Total Antioxidant Capacity (TAC) was measured by the specific kit (Zell-Bio, Germany) at 0.1 mM sensitivity. All procedures were performed by adhering to the kits’ instructions. 
The homeostatic model of assessing insulin resistance: The Homeostatic Model of Assessing Insulin Resistance (HOMA-IR) was determined based on the flowing formula: HOMA-IR=Insulin×Glucose/22.5 [20]. 
Histopathological analyses: The pancreas tissue samples were fixed in 10% formaldehyde for 72 hours, then dehydrated using graded concentration of ethanol. Xylene was also used as a transitional solution. The histology samples were embedded in melted paraffin and sectioned in 5-micrometer thick tissue slices, using a rotary microtome (Leitz, Germany). The slides were stained with hematoxylin and eosin (H&E) and examined under a light microscope (Olympus, Japan). The morphology of the Langerhans islets and the acinar cells were examined microscopically at 400 X magnification. The islet cells were counted from 50 microscopic fields in each group. The areas of islets were measured using a computer software (Digimizer Version 11). To determine the beta cell numbers, slides were stained with aldehyde fuchsin. For each rat, 10 Langerhans islets were examined and the beta cell numbers were counted and divided by the sum of the beta cells versus none beta cell. The answer in each case was multiplied by 100 and recorded as the percentage. 
Statistical analyses: The statistical analysis of the data was performed on SPSS version 16 and Graph Pad Prism version 7. Analysis of Variance (ANOVA) and Tuckey’s tests were used to compare the data among the groups. The data were reported as the means and standard error. The value of P<0.05 was considered as a statistically significant difference.

Results
Biochemical parameters: As reflected by the data in Table 1, the administration of VOE at 100, 200 and 400mg/kg reduced the serum glucose and amylase levels significantly compared to that of the STZ and control groups, respectively (P=0.001).

Doses of VOE reduced the serum amylase compared to that of the diabetic rats (P<0.05), (Table 1). The serum lipase was insignificantly lower in STZ group than the controls, and there were no significant differences in the serum lipase levels among all groups. Also, an insignificant reduction the serum insulin was detected in the STZ group compared to that of the controls. There were no significant differences in the insulin levels among all of the experimental groups (Table 1). A lower serum TAC was present in the STZ group compared to that of the controls. The administration of VOE increased the TAC compared to that of the STZ groups (P=0.001) (Table 1).
HOMA-IR: Within the STZ group, the HOMA-IR was significantly higher compared to that of the controls (P=0.001). However, the treatment with VOE at all doses significantly lowered HOMA IR compared to that of the STZ group (P=0.001).
Histological findings: The histologic findings revealed a severe atrophy in both the islets and non-islets of the pancreas samples. There were no clear morphological differences among the groups with respect to the exocrine tissues. A significant reduction in the pancreatic islet areas was observed in the STZ group compared to that of the controls (P=0.001). Treatment with doses of VOE increased the pancreatic islet areas significantly (P=0.01). The mean number of beta cells in the pancreatic islets reduced significantly in the STZ group compared to that of the controls (P=0.001). There were no significant differences in the number of beta cells between the STZ group and VOE-treated groups. An insignificant reduction in the Islets’ numbers was observed within the STZ group compared to that of the controls. The VOE at varying doses increased the Islets numbers insignificantly. See details in Figures 1 and 2, and Table 2. 

Discussion
This study demonstrated that the VOE prevented the hyperglycemia and ameliorated the side effects induced by STZ treatment in rat pancreas. The administration of VOE at 100, 200 and 400mg/kg reduced the serum glucose and amylase in rats. The findings also confirmed that the VOE reduced the HOMA-IR, and that STZ reduced the numbers of beta cells in the pancreatic islets.
Toxic mechanisms: The exact mechanisms by which STZ leads to a reduction in the pancreatic islet cells is not fully clear. One possibility may be the reduction in the Nnicotinamide Adenine Dinucleotide (NAD), which plays multiple roles in cellular metabolism. It may also be due to the methylation of DNA in the pancreatic islet cells [21, 22]. It is also known that STZ increases the generation of free radicals in cells. In this context, the adverse effects of STZ on glucose and HOMA-IR supports its toxicity against the beta cells. In this respect, it has been demonstrated that no correlation exists between the glycemic control and ROS generation in diabetes [4]. Indeed, the generation of ROS in diabetes is multifactorial [4]. Multiple variables are involved in the generation of ROS [4]. They include glucose autoxidation, increased level of ferritin and homocysteine as pro-oxidants, glycation of protein and increased level of Advanced Glycation End products (AGEs), and imbalance in free radicals and other antioxidants [4]. 
Antioxidant effects: This study showed that the Total Antioxidant Capacity (TAC) declined in STZ groups and the VOE at varying doses increased that capacity, confirming the anti-oxidative properties of the extract. The anti-oxidant activity of both hydroalcoholic and water extract of VOE has been demonstrated in previous studies [12, 23]. In this context, it has been shown that VOE possesses cyclotides, violacein A [24], which gives it various metabolites, with anti-bacterial, anti-cancer and immune-stimulant properties [24]. About 30 cyclotides have extracted from the roots of V. odorata plant. Phenolic and flavonoid compounds have been isolated from the V. odorata leaves [12, 23]. The antioxidant property arises from its constituents, such as alkaloids, steroids, tannins, phenolics, flavonoids glycoside, flavonoids, saponins, methyl salicylate, mucilage and vitamin C [13]. The current study demonstrated a significant decrease in serum amylase and along with an insignificant decrease in lipase compared to those measured for the STZ-treated group. This finding may be due to the altered cell function in the exocrine parts of the damaged pancreatic islets. 
Effect of phenolics: Dietary phenolic compounds have inhibitory effects on α‐amylase. Therefore, they can be potential candidates for the management of Type 2 diabetes. Alpha‐amylase enzyme catalyzes the breakdown of starch to malto-oligosaccharides, which are degraded to glucose and lead to hyperglycemia and insulinemic changes [25]. Both α-amylase and α- glucosidase enzymes are inhibited by polyphenols of plants origin [26]. The reduced amylase level in rats treated with VOE may be due to the inhibitory activities of polyphenols present in the extract. As demonstrated by the current study, following amylase reduction, glucose was also reduced due to the hypoglycemic effect of the VOE. Acute pancreatitis, pancreas tumors, diabetic ketoacidosis and renal dysfunction may be associated with a rise in serum amylase [7]. Insulin deficiency in type-1 and some cases of type 2 diabetes, and insulin resistance may be associated with low serum amylase [7]. Low serum amylase and lipase is associated with diabetes and metabolic syndrome [6]. 
HOMA-IR: Our findings showed that a rise in HOMA-IR occurs in STZ-treated rats, and the VO extract was able to reduce the HOMA-IR compared to that of the STZ-treated group. Probably, low amylase in rats treated with the VOE in this study is due to a low HOMA-IR and glucose levels that we achieved. Insulin, regulates amylase secretion through its receptors on exocrine cells. However, similar mechanism of action about lipase is not known in details in diabetic patients [22]. Clinical studies have shown low amylase and lipase can occur in type 1 or type 2 diabetes [6, 7, 27]. Similarly, a recent experimental study has reported that 66% and 43% reductions, respectively, occur in amylase and lipase secretion by the pancreas in diabetic rats [28]. However, there are conflicting reports about the serum amylase and lipase levels in diabetic patients [29, 30]. Other factors responsible for the exocrine insufficiency of pancreas may be the overexpression of TGFβ-1 in pancreas, aging and reduction of free calcium in the cytosol of the pancreatic cells [6]. The exact mechanism of action of the VOE on pancreas tissue is not fully understood and needs further investigations.

Conclusions
This study provided evidence regarding the beneficial effects of VOE on the pancreas tissue in STZ-treated rats. The VOE at doses of 200 and 400 mg/kg improved both the histologic structures and function of the pancreas. These effects are likely due to reductions in HOMA-IR and/or amylase activity and the increase in total antioxidant capacity that may be linked to the phenolic characteristics of the VOE.
Limitations of the study: One of the limitations was using the rat model. Although there are many physiological similarities between rats and humans, obviously, they are not identical in all respects. Another limitation was the STZ treatment, which may have toxic effects on other organs. Lack of laboratory resources to quantify the VOE in the blood and pancreas tissues of the treated rats were other limitations.
Recommendations for future research: The evaluation of cellular and molecular mechanisms and histological examination of the pancreatic also ultrastructure following treatment with VOE and STZ are recommended in future studies.

Ethical Considerations
Compliance with ethical guidelines

Sampling and protocols were approved by the ethics committee of Guilan University of Medical Sciences (Code: IR.GUMS.REC.1395.226).

Funding
This study is supported by the Vice-Chancellor and Research Technology Guilan University of Medical Sciences (No: 95071709). 

Authors' contributions
Conceptualization and supervision: Rouhollah Gazor and Fahimeh Mohammadghasemi; Methodology: Fatemeh Niknezhad; Investigation, writing - original draft, and writing - review & editing: All authors.

Conflict of interest
The authors declared no conflict of interest.

Acknowledgments
We wish to thank the vice-chancellor and research technology of the Guilan University of Medical Sciences for their supports.


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Type of Study: Research | Subject: Special

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