1. Introduction:

Diabetes mellitus (DM) is a chronic metabolic disorder of the endocrine system and caused by a confluence of two main factors: improper insulin secretion by pancreatic beta-cells and improper insulin response in insulin-sensitive organs (Galicia-Garcia et al., 2020; Roden & Shulman, 2019). The International Diabetes Federation (IDF) predicted that by 2030, there will be 643 million cases, and by 2045, there will be 783 million cases of diabetes (International Diabetes Federation, IDF Diabetes Atlas-10th Edition). Diabetes patients may not properly produce or use insulin in their bodies and therefore, they have a high blood glucose level and various issues with the metabolism of fats, proteins, and carbohydrates (Schinner et al., 2005). More than 90% of diabetic patients have type 2 DM, according to statistical data and it is mostly caused by obesity and insulin resistance, and increases the risk of cardiovascular diseases. Exercise and anti-diabetic drugs are prioritized in the initial treatment of Type 2 diabetes (DeFronzo & Abdul-Ghani, 2011; DeFronzo et al., 2015; Garvey et al., 2014). A number of fatal consequences, including cardiovascular disease, nephropathy, neurodegeneration, atherosclerosis and retinopathy, may result from persistent failure to maintain normoglycemia (Sarwar et al., 2010; Veiseh et al., 2015). Additionally, hyperglycemia is known to produce reactive oxygen species (ROS), which exacerbate diabetes mellitus and cause both micro and macrovascular problems and these complications result in the patients' deaths (Giacco, 2011; Priyanka et al., 2019; Singhal et al., 2019; Senduran et al., 2020). Controlling blood glucose levels in Type 1 DM and advanced Type 2 DM requires subcutaneous insulin therapy. Tight glycemic control, however, is still a persistent problem for patients with diabetes, and frequent subcutaneous injections may lead to poor patient adherence (Mathieu et al., 2021). As a result, alternative therapies for the treatment of DM have been studied for a long time and numerous bioactive substances produced from plants, animals, and fungi have been demonstrated to have hypoglycemia or anti-diabetic properties (Sarkar et al., 2020; Zhou et al., 2021; Zahnit et al., 2022; Benchikha et al., 2022; Rahman et al., 2022).

Chitin, the second most prevalent natural biopolymer in the world is a polymer that resembles cellulose and can be found in the exoskeletons of crustaceans and molluscan organs, fungi, insects, and yeasts (Tharanathan and Kittur, 2003; Kou et al., 2021). Chitosan (CS), a polysaccharide with cationic structure, is one of the most popular drug carriers and has gained a major attention in diverse biomedical applications, because of its distinctive properties, including low toxicity, biocompatibility, biodegradability, and mucoadhesion (Riva et al., 2011; Shalaby & El-Refaie, 2018; Ahmadifard et al., 2020). Chitosan is a random copolymer of N-acetyl-D-glucosamine and d-glucosamine units that results from the partial degradation of chitin by N-deacetylation in an alkaline environment (Song and Ratner, 2012) connected with β-(1-4) linkage with varying degree of deacetylation.

Chitosan oligosaccharide (COS), which is formed by the depolymerization of chitosan via acid hydrolysis, physical hydrolysis, and enzymatic degradation, is a low-molecular-weight form of chitosan and a nontoxic polyglucosamine (Lodhi et al., 2014; Swiatkiewicz et al., 2015). Chitosan and its oligosaccharide derivatives differ from chitin in that they have antimicrobial (Zheng and Zhu, 2003; Holappa et al., 2006), anti-inflammatory (Yoon et al., 2007; Ma et al., 2011), anti-oxidative (Kim and Thomas, 2007; Yen et al., 2008), and hypocholesterolemic (Liu et al., 2008) properties since they contain reactive, functional groups, such as amino acids and hydroxyl groups.

Chitosan and its derivatives have gained increasing interest recently due to their potential health benefits in the development and management of diabetes, rather than their use as materials in biomedical applications (Katiyar 2011 et al.; Guo et al. 2020). Since prebiotic and biopreservative oligosaccharides are noncariogenic, nondigestible (NDO), and low calorie substances that promote the growth and development of gastrointestinal microflora, there has been an increase in interest in their use in the food industry. In neonatal streptozotocin (STZ)-induced diabetic mice, chitosan is found to have hypoglycemic effects (Miura et al., 1995). According to research by Liu et al. (2007), COS has been shown to increased glucose tolerance and insulin secretion, enhance the impaired pancreatic function in STZ-induced diabetic mice and speed up the proliferation of β-cells in vitro, improving insulin release and glycemic management. COS is typically thought to be biologically more active than chitosan, in addition to sharing some of its properties (Tzeng et al., 2022). COS possesses many functional activities, including the ability to scavenge free radicals, as well as soluble and absorbable qualities whereas in vivo, chitosan has a weak solubility and low absorbability.

However, the efficacy of dietary chitosan oligosaccharides for reducing the incidence of diabetes and cardiovascular diseases- the burning public health issues in Bangladesh yet to be explored. Therefore, the research was designed to examine the efficacy of chitosan oligosaccharides (COS) on the maintenance of health physiology in diabetic mice. The study was conducted to assess the effects of COS on growth rate in diabetic mice, to analyze the efficacy of COS on biochemical profile and to evaluate the role of COS on pancreatic islets in diabetic mice.

2. Materials and Methods

2.1 Selection of Materials

For induction of diabetes, the alloxan was chosen because it preferentially kills pancreatic cells through a variety of mechanisms. Chitosan oligosaccharide (COS), an oligomer of chitosan prepared by the deacetylation of chitin and the second most abundant polymer in nature was selected for this experiment to study the efficacy of dietary chitosan oligosaccharides for reducing the incidence of diabetes and cardiovascular diseases. 2% commercially available powdered chitosan oligosaccharides were used in this regards.

2.2 Evaluation of antidiabetic effects

The chitosan oligosaccharide (COS) was tested for antidiabetic effects. The study was approved by The Animal Welfare and Experimentation Ethics Committee, Bangladesh Agricultural University, Mymensingh, Bangladesh (AWEEC/BAU/19/41) and was carried out according to the current guidelines and developed criteria about study ethics on laboratory animals.

2.2.1. Experimental animals and diets

A total of fifty 8 weeks of old male mice have been collected from ICDDRB, Dhaka. During the 4-week experimental period, all mice have been allowed to access to standard pellets as a basal diets and water ad libitum. Mice were housed in individual cages at 22 ± 3^o C and 50 ± 10 % humidity. After 1-week adaptation period, among fifty mice 10 were grouped as- 1) Normal control group (NC), non-diabetic mice; and remaining 40 mice were used to induce diabetes.

2.2.2. Induction, confirmation and validation of diabetes mellitus model 

Diabetes was induced by using alloxan @ 110 mg/Kg body weight in mice by single intraperitoneal injection after overnight fasting. Using a Fora Comfort plus mini G71 glucometer made by the ForaCare Switzerland Company, the postprandial glycemia was measured from blood samples (0.5 μl) taken in the morning after 2 h of food intake in overnight fasted mice on the days 1 (24 h after alloxan administration), 2nd, 4th, and 7th. For the following stage of the experiment, mice with blood glucose levels greater than 15 mg/dL were chosen.

2.2.3. Evaluation of antidiabetic efficacy of COS in diabetic mice

The diabetic mice were divided into 4 groups as: 1) diabetic control (DC), without COS supplementation; 2) diabetic mice supplied with 0.5% of COS (T1); 3) diabetic mice supplied with 1% COS (T2); 4) diabetic mice supplied with 2% COS in drinking water (T3). Along with these groups, the normal control (NC) group consisted of 10 healthy mice is also used. To measure the serum glucose level, blood was withdrawn from a tail vein to minimize stress or injury on the days 0, 7, 14, 21 using a Fora Comfort plus mini G71 glucometer made by the ForaCare Switzerland Company.

2.3 Sampling and blood collection

At the end of the experimental period all mice were fasted for 12 hrs, and blood was collected once from orbital sinus of eyes under anesthetic condition to analyze the serum biochemical profile. Before sampling, the body weight of mice was measured using a weighing balance and deeply euthanized within a vacuum glass jar using chloroform.

2.4 Serum preparation and biochemistry

For serum biochemistry, at the next day of blood collection, test tubes containing blood samples were removed from the refrigerator and placed in slanting condition at room temperature for 30 minutes. Blood samples were centrifuged at 3,000×g (Centrifuge 5415C; Eppendorf, Hamburg, Germany) for 15 min to extract serum, and all serum samples were preserved at −80 o C in a deep freezer.

2.5 Collection of pancreas for histopathology

At necropsy, after ventral thoraco-abdominal dissection, pancreas was collected. All visceral organs were examined very carefully for gross changes and recorded carefully. Immediate after gross examination, a piece (1 cubic cm, or suitable size) of collected pancreas was transferred into 10% formalin to fix for histopathology. Plastic cassettes with 10% formalin were used for fixation of samples, overnight. Then the samples went through dehydration and clearing steps and infiltrated in paraffin wax. After trimming, the paraffin blocks were sectioned at 4-6 μm thickness using a rotary microtome (Leica RM 2155, Japan) and collected on glass slides. At next step, the slides were stained with routine stain (haematoxylin and eosin), mounted and finally examined under light microscopes.

2.6 Photography and illustration

Necessary photo of animal rearing during study period and sampling (gross morphometry) were taken using the digital camera (Canon®). The histological pictures of different organs were captured using the digital camera (Canon®). The necessary illustration and contrast adjustment was done by using adobe Photoshop 2019 software.

2.7. Statistical analysis

Shapiro-Wilk test was carried out to evaluate the normality of the data set. During the course of the experiment, data were collected and subjected to statistical analysis using one way of ANOVA approach with post hoc Tukey’s multiple comparisons test with a completely randomized design (CRD) using Graph Pad Prism (version 9.0). Significant means were separated using multiple comparisons at 5% level of significance. All data points (n-numbers) are plotted in each bar graph (three independent experiments).

3. Results

3.1. Effect of COS on body weight

The mean body weight of mice of various groups on different day of the experiment is shown in Figure 1. The mean body weight was increased with the growing age of the mice in the normal control group (NC) and decreased in the diabetic control group (DC), whereas the COS treated groups (T1, T2, T3) showed a gradual increase in body weight with the increased concentration of COS which is statistically significant.

Figure 1: Effects of COS on body weight of mice at different concentration and different time periods (Mean ± SEM). DC = control, T1 = 0.5% COS, T2 = 1% COS, T3 = 2% COS with basal diet. Significance was considered at the level of 5% (P < 0.05). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001, ns = non-significant.

There were non-significant differences among the control and the highest dosage treatment group (T3) on different days of experiment that reveals the propitious improvement of the final body weight of mice in group T3.

3.2. Effects of COS on blood glucose concentration

The concentration of blood glucose of mice of various groups on different day of the experiment is shown in Figure 2. The result showed that the blood glucose concentration increased very little with the growing age of the mice in normal control group (NC) which is physiologically congruent, but increased significantly in diabetic control group (DC) compared to the normal control group. The COS treated groups (T1, T2, T3) showed a gradual decrease in blood glucose concentration with the increased concentration of COS which is statistically significant compared to diabetic control group (DC). The significant difference was also present among COS treated groups and the lowest value of blood glucose concentration was found in group T3 at day 21 which was not significant with normal control group which indicates the positive effects of high dose of COS.

Figure 2: Effects of COS on blood glucose of mice at different concentration and different time periods (Mean ± SEM). DC = control, T1 = 0.5% COS, T2 = 1% COS, T3 = 2% COS with basal diet. Significance was considered at the level of 5% (P < 0.05). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001, ns = non-significant.

3. Effects of COS on ALT and AST

The results of serum ALT and AST of normal control (NC), diabetic control (DC), COS treated groups (T1, T2, T3) of mice on different day of the experiment are revealed in Figure 3 and Figure4. At Day 0, the result showed that the serum ALT and AST were significantly higher in diabetic control group (DC) and COS treated groups (T1, T2, T3) compared to the normal control group (NC). But with advancement of the experiment it was found that a gradual decrease in the serum ALT and AST was evident in COS treated groups (T1, T2, T3) at day 7, 14, 21 which is statistically significant compared to diabetic control group (DC). The values of serum ALT and AST were increased in diabetic control group (DC) with the passage of time but the treatment with COS showed lowering of these values and most propitious and auspicious result was found in group T3 at day 21 which is almost near to normal control group (NC).

Figure 3: Effects of COS on serum ALT of mice at different concentration and different time periods (Mean ± SEM). DC = control, T1 = 0.5% COS, T2 = 1% COS, T3 = 2% COS with basal diet. Significance was considered at the level of 5% (P < 0.05). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001, ns = non-significant.

Figure 4: Effects of COS on serum AST of mice at different concentration and different time periods (Mean ± SEM). DC = control, T1 = 0.5% COS, T2 = 1% COS, T3 = 2% COS with basal diet. Significance was considered at the level of 5% (P < 0.05). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001, ns = non-significant.

4. Effects of COS on serum creatinine

The results of serum creatinine of normal control (NC), diabetic control (DC), COS treated groups (T1, T2, T3) of mice on different day of the experiment are revealed in Figure 5. At the beginning of the study, the value of creatinine in normal control group is significantly lower compared to diabetic control group (DC) and COS treated groups (T1, T2, T3). With the passage of time the level of creatinine in diabetic control group (DC) was increased gradually but in COS treated groups (T1, T2, T3) a gradual decrease was found at day 7, 14, 21. The treatment with COS showed returning of creatinine level in normal range and most excellent result was found in group T3 at day 21. There is no significant difference between normal control (NC) and T3 group.

Figure 5: Effects of COS on creatinine of mice at different concentration and different time periods (Mean ± SEM). DC = control, T1 = 0.5% COS, T2 = 1% COS, T3 = 2% COS with basal diet. Significance was considered at the level of 5% (P < 0.05). *P ≤ 0.05, **P ≤ 0.01, ***P ≤ 0.005, ****P ≤ 0.001, ns = non-significant.

​​​​​​​5. Effects of COS on pancreatic islets

In normal control group (NC), the pancreatic acini and pancreatic islets showed normal histological architecture (Figure 6, NC). In diabetic control group, the pancreatic islets showed a great loss and degeneration of cells (Figure. 6, DC). After the ending of the experiment, the COS treated groups showed a variety level of recovery of the pancreatic islets which were affected by diabetes with increasing concentration of COS (Figure. 6, T1, T2, T3). In group T1 (0.5% COS), the cells of pancreatic islets did not show much improvement and the cells were degenerated and necrosed, but regeneration of cells was clearly visible in group T2 (1% COS). The regeneration of cells of pancreatic islets in group T3 (2% COS) was at the greater extend and the histoarchitecture was almost near to the normal control group (NC) which indicates the recovery of diabetic pancreas with COS treatment at a higher concentration.

Figure 6: Effects of COS on pancreatic islets of mice of different groups. Asterisks indicate pancreatic islets (20X). NC = Normal control, DC = Diabetic control, T1 = 0.5% COS, T2 = 1% COS, T3 = 2% COS.