Newly synthesised chalcone derivative attenuates hyperglycaemia by regulating blood glucose and serum biochemical profile in streptozotocin-induced rats

INTRODUCTION

Diabetes has emerged as a silent killer worldwide, irrespective of gender, age and other environmental factors. The continuous efforts and contributions of the researchers have made this war easier and convenient for all of us, which might cause the end of this disease. In 2000, India (31.7 million) ranked one in the world with the highest number of people having diabetes mellitus, followed by China (20.8 million) and then the United States (17.7 million) in second and third place, respectively. According to the experts, diabetes mellitus is expected to impact up to 79.4 million people in India by 2030, with considerable rises in the number of people afflicted in China (42.3 million) and the USA (30.3 million).^[1,2] Majorly, there are two types of diabetes, categorised as type I diabetes, a chronic metabolic disorder with high blood sugar level in the body; it is occurred mainly due to the lack of insulin production (in the beta cell of the pancreas), and the type II diabetes mellitus is categorised as per the poor utilisation of insulin in the body.^[3] Like any other maladies, diabetes comes with different types of clinical complications, including microvascular and macrovascular endpoints, among which microvascular multi-system complications are considered as retinopathy, nephropathy and neuropathy and the macrovascular complications, which include ischemic heart disease, stroke and peripheral vascular diseases. It has been observed that, along with other reasons, the prevalence of diabetes has increased rapidly in the last few decades, along with changes in our lifestyle.^[4] As the disease is increasing in prevalence so are many options available for the treatment of hyperglycaemia, but unfortunately most of them shows severe toxic effects, such as the insulin therapies which are available, include different types of adverse conditions and complication, one of the most prevalent adverse effects is weight gain and hypoglycaemia if the dosage is not properly maintained along with intake of food. Weight gaining is also a major impact on the patients of uncontrolled diabetes, resulting in truncal fat and muscle bulk due to the energy loss through glycosuria.^[5-8] Besides Insulin treatment, one of the common treatments for diabetes is the sulfonylurea group of drugs. According to researchers, in most cases, sulphonylurea requires a functional pancreas to show the hypoglycaemic activity.^[9] Among the different drawbacks of the available treatments, most of them are prescribed throughout the life and the chances for toxicity increase. Combining all of these reasons, the researchers concluded to go for the research providing better treatment with very less adverse effects. In this context, the plant-based research grabbed the attention of the researchers for its comprehensive nature. According to the different studies, the phytochemicals are in the spotlight in the field of pharmacology. Being a derivative of a flavonoid molecule, the chalcones are also categorised as an anti-diabetic agent which protects our body from radicals and other oxidative compounds, and that is one of the reasons that flavonoid-containing foods reduce the chances of diabetes.^[10-12] In some studies, about chalcone, terpenoid chalcones were found to be effective in the treatment of diabetes due to their excellent blood glucose reduction activity.^[13] It has also been researched as nuclear factor erythroid factor 2-related factor 2 activator, which potentiates the glutathione activity for the treatment of diseases like diabetic neuropathy, generates excessive oxidative stress.^[14]

In this study, a novel chalcone derivative has been synthesised, and the antihyperglycaemic activity has been assessed in diabetic rats. Moreover, an in silico study was carried out to determine the physicochemical and pharmacokinetic properties of the chalcone derivative. The molecular docking was also performed to rule out potential targets for the newly synthesised molecule with regard to its antidiabetic activity.

MATERIALS AND METHODS

RESULTS

OGTT

The post-prandial glucose-lowering activity of the novel chalcone derivative after a glucose load was evaluated by OGTT, and the results have been depicted in Figure 1. The results demonstrated that the test compound significantly (P < 0.05) reduces the BGL at both 10 and 20 mg/kg doses compared with vehicle control [Figure 1a] at different time intervals. Moreover, the percentage reduction of blood glucose was also determined [Figure 1b] at various time intervals. Results demonstrated that, in comparison with standard control (43.14%), the test compound had been associated with a substantial (P < 0.05) percentage decrease in blood glucose at both 10 mg/kg (46.78%) and 20 mg/kg (58.68%) doses.

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Figure 1:

Oral glucose tolerance test. (a) Effect of novel chalcone derivative on post-prandial blood glucose level after glucose load. (b) Percentage reduction of blood glucose level. (c) Body weight and (d) Water intake of rats at every experimental week. Haematological and serum biochemical parameter of rat throughout the experimental weeks. (e) Glycated haemoglobin. (f) Blood glucose (mg/dL). (g) Blood urea nitrogen (mg/dL). (h) Serum triglycerides (mg/dL), (i) High-density lipoprotein (mg/dL), (J) Low-density lipoprotein (mg/dL), (k) Creatinine (mg/dL). The data has been presented as mean ± standard deviation (n = 6). *p < 0.05 as compared to vehicle control group. #p < 0.05 as compared to diabetic control group. $p < 0.05 as compared to 10 mg/kg chalcone treated group. αp < 0.05 as compared to 20 mg/kg chalcone treated group. The levels of proinflammatory cytokines (l) tumour necrosis factor-alpha (TNF-α) and (m) interleukin-6 (IL-6) were detected using enzyme-linked immunosorbent assay. The data have been presented as mean ± standard deviation (n = 3). Statistical analysis was carried out by one-way analysis of variance followed by Tukey’s post hoc multiple comparison test (*P < 0.05, **P < 0.01, ****P < 0.0001).

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Histological analysis

Histopathological section of pancreas depicted normal cellular morphology in the normal CG [Figure 2a], which includes islets of Langerhans cells (il), acinar cells (ac), interlobular duct (d) and intralobular duct (id). Whereas, diabetic CG [Figure 2b and c] showed severe degeneration of islets of Langerhans (il), ductal and vascular degeneration and low b cell count, ductal hyperplasia, nuclear pyknosis and nuclear fragmentation. In this case, cellular degeneration was still visible in the 10 mg/kg of chalcone-treated group [Figure 2d], despite a slight improvement in cell structure. The number of Langerhans cells (il) significantly increased, but failed to maintain cell size. The 20 mg/kg of chalcone-treated group [Figure 2e] showed less cellular deterioration, significant granulation of β-cells and increased Langerhans cell count. However, the pancreatic portion of the rats receiving 100 mg/kg/day of metformin showed nearly normal cellular morphology [Figure 2f].

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Figure 2:

Photomicrograph of histopathological pancreatic sections of control and experimental groups. (a) The normal control group depicted islets of Langerhans cells (il), acinar cells (ac), interlobular duct (d) and intralobular duct (id). Represented as black arrows. (b) Diabetic control group showed cellular degeneration in Langerhans cells (il) and ducts. (c) Hyperplasia (arrowhead) was noted around the ducts with nuclear pyknosis (arrow head) and nuclear fragmentation (arrow). (d) 10 mg/kg chalcone group showed minor restoration of pancreatic architecture with minimal increase in Langerhans cells. However, islet size was not maintained and cellular degeneration was slightly less compared to untreated diabetic rats. (e) 20 mg/kg chalcone group decreased the granulation, and restored cell size and number of β cells (arrowhead). (f) Vascular degeneration was also reversed which restored normal architecture metformin 100 mg/kg/day group showed better restoration of pancreatic architecture.

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DISCUSSION

In the 21^st century, the chalcones have readily proved the interest among the researchers due to their unique structural alignment, amenable synthesis protocols and a vast pharmacological activity, including anti-diabetic activity.^[16] It has been determined that chalcones are progenitors of isoflavonoids and flavonoids. It could be developed by Claisen-Schmidt condensation reaction between acetophenone and benzaldehyde in the presence of 40% sodium hydroxide solution. It is composed of a benzylideneacetophenone scaffold with two aromatic nuclei associated with a 3-carbon a, β-unsaturated carbonyl bridge.^[17,18] In addition, a wide range of novel techniques, including irradiation with domestic microwave, could be employed for producing a large number of chalcone derivatives. ^[19]

The pharmacophore analysis has been performed to determine the physicochemical and pharmacokinetic properties of this chalcone derivative. In addition, the molecular docking was performed to investigate the binding efficacy and affinity of the ligand towards the specific target proteins. Since nuclear peroxisome proliferator-activated receptors (PPARs) regulate cellular differentiation, development and metabolism of proteins, lipids and carbohydrates in humans, they have been identified as an essential target protein in this instance.^[20] The PPAR-retinoid X receptor (RXR) complex binds with specific DNA binding sites and regulates transcription of insulin-responsive genes^[21] which, in turn, reduces BGL, suppresses gluconeogenesis and increases lipid storage in the muscle tissues.^[22] Previous studies demonstrated that the chalcone derivatives have excellent agonistic activity towards PPAR-gamma (PPAR-g) with great hyperglycaemic activity.^[23] On that basis, the molecular docking study revealed that this chalcone derivative has significant binding affinity for human glucokinase receptor and PPARg-RXR-alpha nuclear receptor complex with docking scores of −8.6 and −7.4, respectively, which suggested an excellent binding of the ligand with the target proteins for associated pharmacological events, including antihyperglycaemic activity.

In this research, the novel chalcone derivative, namely, 1-{3-[3-(4’-methoxy phenyl) prop-2-enoyl] phenyl} thioureas, has been synthesised by classic Claisen-Schmidt condensation reaction and characterised by different analytical techniques. To investigate the antihyperglycaemic activity of the novel chalcone derivative, a safe dose for the compound was determined by an acute oral toxicity study. LD₅₀ dose for the compound was then found to be 50 mg/kg body weight, and subsequently, the therapeutic doses for the in vivo anti diabetic study were determined to be 10 and 20 mg/kg body weight, which were completely nontoxic to the experimental animals. The in vivo anti-diabetic activity was carried out on male SD rats by inducing type II diabetes through the administration of 50 mg/kg STZ intravenously, which showed progressive disease symptoms resembling the human type II diabetic model with hyperinsulinemic dysglycaemia, pancreatic β-cell dysfunction, late-stage hyperglycaemia, dyslipidaemia and renal dysfunction.^[24]

The primary strategy for treatment of type II diabetes is regulation of blood glucose level. Thus, OGTT has been performed to investigate the effect of this chalcone derivative on the sudden upregulation of BGL, followed by a glucose load. The result depicted that administration of 10 and 20 mg/kg chalcone significantly reduced the BGL with a percentage reduction of 46.78% and 58.68%, respectively, as compared to standard control (43.14%), which suggested the potential role of the chalcone derivative in the regulation of post-prandial BGL.

Significant muscular degeneration and protein degradation have been observed in STZ-induced diabetic rats, resulting in a significant reduction in body weight.^[25] Furthermore, STZ-induced diabetic rats also exhibit increased symptoms of polydipsia, that is increased rate of water intake.^[26] In this study, the STZ-treated group showed severe loss of body weight and increased water intake rate. However, the treatment with chalcone derivative significantly reduced the water intake and restored the normal body weight of the chalcone-treated rats. These outcomes signified the decrease in BGL, which, in turn, increased the glucose intake in the muscles and decreased the polyuria that prevented tissue dehydration-mediated polydipsia.^[27] Consistent with the previous studies, the STZ-induced diabetic rats also exhibit increased levels of blood HbA1c due to haemoglobin damage, which confirms the induction of type II diabetes in the rats.^[28] The treatment with chalcone derivative significantly reduced the blood HbA1c level when compared to diabetic CG and showed a similar result as that of standard CG, which signified that this novel chalcone derivative can successfully prevent diabetes and regulate the BGL in the long-term goal.

In addition, the effect of this chalcone derivative on the serum biological parameters was evaluated to investigate the function of lipid and the kidney. The STZ-induced diabetes is associated with alterations of metabolic and regulatory mechanisms, leading to lipid accumulation and the development of hyperlipidaemia.^[29] In association with this, increased proteolysis and reduced protein synthesis are also shown in STZ-induced diabetes due to alteration of distinct renal metabolism.^[30] In this current study, the STZ-induced diabetic CG showed increased levels of BUN, LDL and creatinine with decreased HDL levels. Conversely, the chalcone-treated group significantly reduced BUN, LDL and creatinine levels and increased HDL levels as compared to diabetic CG. Thus, the chalcone derivative restored normal serum biological parameters of lipid and kidney in the STZ-induced diabetic model of rats.

TNF-α performs an essential function in the maintenance of intestinal homeostasis, and alteration of this signalling pathway may often lead to severe diseases, including diabetes.^[31] An increased level of TNF-α has been found in the diabetic condition, which is correlated with the development of diabetic nephropathy due to ROS-mediated cytotoxicity of the kidney.^[32,33] Moreover, TNF-α is directly associated with the destruction of β-cells in isolated islets.^[34] The increased level of IL-6 has also been depicted in diabetes^[35] which provoked ROS-mediated oxidative stress and damaged lipids and proteins that disrupt normal cellular function in hyperglycaemic conditions.^[36] In this study, the diabetic control rats exhibited increased expression of TNF-α and IL-6, whereas the treatment with chalcone derivative significantly downregulated the expression of TNF-α and IL-6 and prevented the pancreatic β-cells from inflammatory cytokine-mediated oxidative damage.

The histopathological analysis of the pancreas was carried out to investigate the regenerative effect of this chalcone derivative against STZ-induced diabetes. In addition to vascular degradation in islets and a reduction in the quantity of β-cells, diabetic CG had degranulation and degeneration of Langerhans cells. Furthermore, the pancreatic tissue additionally demonstrated hyperplasia and degradations of islet cells with nuclear pyknosis and nuclear fragmentation. Conversely, treatment with chalcone derivative restored the normal morphological architecture of the pancreas and decreased the degeneration of islet cells. Thus, chalcone therapy attenuated the STZ-induced destruction of β-cells and increased β-cell mass by preventing the proinflammatory cytokine-mediated oxidative injury and apoptotic death of β-cells in hyperglycaemia. In addition, this study also deals with some limitations, such as the absence of power analysis for sample size calculation and the lack of confirmatory experimental studies. Keeping things in mind, further in-depth experimentation is required to establish the antihyperglycaemic effect of this novel chalcone derivative.

CONCLUSION

The present study demonstrated the potential antihyperglycaemic effect of a novel chalcone derivative against STZ-induced type II diabetes through the regulation of BGL, HbA1c and serum biochemical parameters. In addition, the chalcone treatment restored the pancreatic β-cell mass and prevented degeneration of islet cells through downregulating the expression of proinflammatory cytokines and elevating the antioxidant levels in the pancreas.