Evaluation of Antidiabetic Activity of Oxadiazole Derivative in Rats

The oxadiazole ring has long been used for the treatment of several diseases. This study aimed to analyze the antihyperglycemic and antioxidant roles of the 1,3,4-oxadiazole derivative with its toxicity. Diabetes was induced through intraperitoneal administration of alloxan monohydrate at 150 mg/kg in rats. Glimepiride and acarbose were used as standards. Rats were divided into groups of normal control, disease control, standard, and diabetic rats (treated with 5, 10, and 15 mg/kg of 1,3,4-oxadiazole derivative). After 14 days of oral administration of 1,3,4-oxadiazole derivatives (5, 10, and 15 mg/kg) to the diabetic group, the blood glucose level, body weight, glycated hemoglobin (HbA1c), insulin level, antioxidant effect, and histopathology of the pancreas were performed. The toxicity was measured by estimating liver enzyme, renal function, lipid profile, antioxidative effect, and liver and kidney histopathological study. The blood glucose and body weight were measured before and after treatment. Alloxan significantly increased blood glucose levels, HbA1c, alanine transaminase, aspartate aminotransferase, urea, cholesterol, triglycerides, and creatinine. In contrast, body weight, insulin level, and antioxidant factors were reduced compared to the normal control group. Treatment with oxadiazole derivatives showed a significant reduction in blood glucose levels, HbA1c, alanine transaminase, aspartate aminotransferase, urea, cholesterol, triglycerides, and creatinine as compared to the disease control group. The 1,3,4-oxadiazole derivative significantly improved body weight, insulin level, and antioxidant factors compared to the disease control group. In conclusion, the oxadiazole derivative showed potential antidiabetic activity and indicated its potential as a therapeutic.


Introduction
Diabetes mellitus (DM) is often simply referred as a group of metabolic diseases in which a person has high blood sugar as well as altered fat, carbohydrate, and protein metabolism because either the body does not produce enough insulin (Type I) or cells do not respond to the insulin (Type II) [1]. Tis high blood sugar produces the classical symptoms of polyuria (frequent urination), polydipsia (increased thirst), and polyphagia (increased hunger) [2]. Type 1 diabetes occurs due to the selective destruction of beta cells of the pancreas [3]. Te outcome of this destruction is the decreased production of insulin and its secretion from pancreatic cells [4]. Both environmental and genetic factors are involved in the pathogenesis of Type 2 DM. Among the diabetic subjects, about 75% of patients are afected by Type 2 DM. Tus, this type of diabetes is more prevalent than Type 1 diabetes [5].
Although the future cannot be predicted with certainty, it is surely possible that an extensive epidemic of resistance to antidiabetic drugs could potentially harm millions of people. Given that it takes more than 10 years to establish the efcacy and safety of the new compound, there is an urgent need to restock the antidiabetic agents. Only a few new antidiabetic compounds have received approval from the US Food and Drug Administration [6]. As we learn more about fundamental pathophysiological pathways, one of these is a lack of glucose stimulated insulin sensitivity as well as the prominence of lipotoxicity as a possible reason for the hepatic and muscular resistance to the efects of insulin on glucose metabolism [7].
1,3,4-oxadiazole derivative exerts its efects on diabetes by inhibiting the carbohydrate hydrolyzing enzymes, i.e., α-amylase and α-glucosidase, while preventing oxidative stress is deemed as a practicable strategy for regulating postprandial glucose levels in diabetic patients [11]. 1,3,4oxadiazole derivative reduces hydrolytic activity and enzyme substrate complex to induce a slower release of the hydrolysed product, either by orthosteric inhibition or allosteric inhibitors which have been reported as α-glucosidase inhibitor in many molecular hybrid designs [12]. Conceivably, this can result in decreased systemic glucose concentration, thus alleviating postprandial hyperglycemia and its associated complications. Te association between 1,3,4-oxidazole derivative and Nrf2 (nuclear factor E2related factor or nuclear factor erythroid 2) enhances antioxidant enzyme activity, which suppresses free radical levels and promotes the pancreas to produce insulin and reduce blood glucose levels [13].
Oxadiazole occurs in four feasible interpretations of isomers, but 1,3,4-oxadiazole is broadly researched for diferent functions [14]. Oxadiazole rings were launched for various purposes in drug discovery development programs [15]. Tese were used as an essential member of the pharmacophore in some situations, thus leading to the ligand binding [16]. Various methods were reported in the literature for the synthesis of 1,3,4-oxadiazole and its derivatives [17]. Te most widely applicable route to the synthesis of 1,3,4-oxadiazole and its 2,5-disubstituted derivatives is the thermal, acid, and base-catalyzed cyclisation of their corresponding carbonyl hydrazides. Preparation of 2,5dialkyl(aryl)-1,3,4-oxadiazoles from acid hydrazide is shown in Figure 2 [18].
Te ring of 1,3,4-oxadiazole is attached with the phenol and sulfanyl hydroxyethyl group (Figure 3). In view of the certain biological traits of 1,3,4-oxadiazole derivatives reported in the past, the present study was investigated to undertake further attempt towards the assessment of the antidiabetic role of 1,3,4-oxadiazole derivatives in rats.

Experimental Animals.
Studies on albino Wister rats (weighing 150-200 g) of both sexes were performed. All rats were kept at room temperature (25 ± 1°C) under normal laboratory conditions for 12 h light-dark period with a humidity of 45-55%. A standard diet of pellets and water was fed before and during the experimental rats. All methods were issued by the Research Ethical Committee (REC/RIPS-LHR/011) of Riphah International University, Lahore, Pakistan.

Experimental Diabetes Induction.
In this study, a single intraperitoneal injection (i.p.) of alloxan (150 mg/kg in 0.9% NaCl solution) was used to induce diabetes [19]. Te intraperitoneal cavity of a 250 g rat could intake 2.5 mL of fuid [20]. Te experimental animals were fasted for 18 h before injecting alloxan [21].  Blood glucose levels were determined using a glucometer before diabetes induction and during treatment time. Te blood samples were collected on basal, zero, 5 th , 10 th , and 14 th day from the tail tip using a glass capillary tube. Basal blood glucose is the normal blood glucose content attained before inducing diabetes, while the day on which diabetes is completely induced in rats before starting the treatment is called zeroday [19].

Glycated Haemoglobin Estimation.
Te level of HbA1c in blood represents the average blood glucose level over the last 2-3 months. When diabetes is not well regulated, blood glucose levels will also be elevated, which will cause higher levels of HbA1c. Glycosylated hemoglobin is measured by the Nycocard reader by glycosylated hemoglobin kits (Axis shield, Norway).

Serum Insulin Level.
All animals were sacrifced on the 15 th day of treatment after anaesthetizing with isofurane (diluted with 2% oxygen), and their blood samples were taken from cardiac puncture. Te blood was then centrifuged for 15 min at 2500 rpm to extract blood serum.

Estimation of Protein.
Tissues were adequately diluted with phosphate bufer for protein estimation, and 5 mL of copper sulfate reagent comprising 1% Na 2 CO 3 , 2% sodium potassium tartrate, and 1% CuSO 4 was added. Te mixture containing 0.5 mL of Folin-Ciocalteau phenol reagent was incubated for 10 min. Te sample was incubated for 30 min before reading the absorbance at 620 nm [22]. Te protein level was determined using the following equation:  Evidence-Based Complementary and Alternative Medicine 3 spectrophotometrically reported at 240 nm every 5 sec. Te CAT activity was expressed as units per milligram of protein as compared to the standard [23].
CAT activity � δOD E × Vol.of the sample(mL) × mg of protein, where δ OD is the change in absorbance per min and E is extinction coefcient (0.071 mmol·cm − 1) of H 2 O 2 [24].

MDA Determination.
Te rates of MDA, an indicator of lipid peroxidation, were calculated using the double heating method. Te procedure is based on spectrometric measurement of violet colour produced by the TBA reaction. For this purpose, 2.5 mL of TCA solution was added to each centrifuge tube containing 0.5 mL of the supernatant of the tissue preparation. Tubes were incubated in a boiling water bath for 15 min. Upon cooling to ambient temperature, the tubes were centrifuged for 10 min and 2 mL of each supernatant sample was mixed to 1 mL of TBA solution. Each tube was then placed for 15 min in a boiling bath. Te absorbance was measured at 532 nm [25].
where VT is total mixture volume (4 mL), 1.56 × 10 5 is the coefcient of molar extinction, WT is the weight of the dissected brain (1 g), and VU is volume of aliquot (1 mL) [26].

Estimation of GSH.
For the calculation of reduced glutathione, 1 mL of homogenous tissue with 1 mL of 10% TCA was precipitated. Ten, 4 mL of phosphate solution and 0.5 mL of DTNB reagent were applied to the supernatant aliquot, and the absorbance was read at 412 nm.
where Y is the absorbance at 412 nm of tissue homogenate, DF is the dilution factor (1), BT is brain homogeneous tissue (1 mL), and VU is volume of aliquot (1 mL).

Histological Studies.
Te pancreas was blotted free of mucus from laboratory rats, washed in normal saline, and preserved in 10% of formaldehyde for 24 h, followed by implanting in parafn. Te kidney and liver were collected after dissection and fxed in the blocks of parafn wax, followed by storing in 10% formalin solution. Te pancreas, kidney, and liver were stained with hematoxylin-eosin for histopathological study.

Toxicological Study.
Te toxicological study of 1,3,4oxadiazole derivative was observed by contrasting the degree of diferent parameters of biochemistry between the test group, control group, disease group, and normal group. Te method of collecting blood and separating serum from blood has already been described earlier.
Preparation of the supernatant from tissue is also described.

Biochemical Metabolic Parameters.
Liver function tests viz. ALT and AST were analyzed by Crescent Diagnostics CZ 902L and Crescent Diagnostic CZ 904L kit, respectively. Te kidney function test, i.e., the urea level was measured by Crescent Diagnostic CS612 kit, and the creatinine level was measured by Crescent Diagnostic CS604 kit. Triglyceride and cholesterol were analyzed by Crescent Diagnostic CS611 and Crescent Diagnostic CS603, respectively.
2.11. Antioxidant Activity. Superoxide dismutase was calculated according to the method of Kim et al. [27]. GSH and MDA activities were analysed by Bhangale and Acharya [26]. CAT was measured by Kaur [24], and protein content was estimated by Lowry et al. [22].

Histological Studies.
Te kidney and liver were collected and fxed in the blocks of parafn wax, followed by saving in 10% formalin solution. Te kidney and liver were stained with hematoxylin-eosin for histopathological observations. 4 Evidence-Based Complementary and Alternative Medicine

Statistical Analysis.
Results were presented as the mean ± SEM (standard error mean). For graphical interpretation, GraphPad prism was applied. Te test of two-way ANOVA (analysis of variance) was applied. Te results were intimated as moderately signifcant (P < 0.05 and * * P < 0.01) and as highly signifcant ( * * * P < 0.001).

1,3,4-Oxadiazole Derivative Efect on the Blood Glucose
Level. 1,3,4-oxadiazole derivative did not show a dosedependent reduction in the glucose level, i.e., the degree of response was the same at all dose levels, indicating that maximum response can be achieved at the lowest dose, i.e., 5 mg/kg ( Figure 4). However, the glucose reduction efect of all 1,3,4oxadiazole derivative groups was found duration-dependent, indicating that a maximum efect was observed after the 14 th day of treatment. Te blood glucose level of 1,3,4-oxadiazole derivative at 5, 10, and 15 mg/kg doses was reduced from 236 ± 14.7, 235 ± 9.9, 275 ± 17.9 mg/dL to 142 ± 2.8, 125 ± 3.2, 118 ± 4.9 mg/dL, respectively, from 0 to the 14 th day of treatment.

Histopathological Study of Pancreas.
Histology of the pancreas demonstrated normal acini and cellularity in the normal control group. In diseased animals, extensive damage to islet of Langerhans and hemorrhagic condition can be seen clearly in Figure 6. On the other hand, 15 mg/kg of 1,3,4-oxadiazole derivative showed hypertrophy and vacuolations of β cells of Langerhans.  (Figure 8). Figure 9 illustrates the treatment outcomes of diferent doses of 1,3,4-oxadiazole derivative, alloxan, glimepiride, and acarbose on the creatinine level of test animals. Te diseased control group exhibited high creatinine levels when compared to the normal control. Te results of the study exhibited that treatment with 1,3,4-oxadiazole derivative at a dose level of 5, 10, and 15 mg/kg, glimepiride, and acarbose reduced the level of creatinine as compared to the disease control animals.

Antioxidant Activity of 1,3,4-Oxadiazole Derivative on
Oxidative Biomarkers in Liver and Kidney Tissue. Table 3 illustrates

Histopathological Study of the Liver.
Hepatic microscopic examination of the normal group showed healthy liver cells with a well-preserved cytoplasm, nucleus, core nuclei, and major vein ( Figure 10). Te disease group displayed a necrotic region where blood drained from a ruptured blood vessel and obvious fatty impairment. Te main vein detrimentally clogged up. Focal hemorrhage areas were also established. Te fat change was evident. Kupfer cell activation and dilation of the central vein can be seen in the liver treated with 15 mg/kg of 1,3,4-oxadiazole derivative.

Histological Study of the Kidney.
In normal animals, the histology of the glomeruli and the Bowman capsule is appropriate. When lipid is deposited in the glomeruli of diseased rats, the basement membrane of the arterioles of the glomeruli becomes slightly thicker. Tis condition is known as glomerular lipidosis, and it includes fbrosis and cellular proliferation in the mesangial as well as congestion in the Bowman's as shown in Figure 11. After administration of 15 mg/kg of 1,3,4-oxadiazole derivative, the glomerulus and tubules were well rejuvenated and entirely free of congestion.
Evidence-Based Complementary and Alternative Medicine 7 trillion [29]. Alloxan is reduced to form diluric acid by binding with the SH sugar-binding site of glucokinase, an enzyme which is essential for glucose-induced insulin secretion causing hyperglycemia [30]. In this study, the administration of 1,3,4-oxadiazole derivative at a dose level of 5, 10, and 15 mg/kg for 14 days led to decrease in blood glucose levels, relative to the group of disease control. Alloxan-induced diabetes is distinguished by a massive weight loss due to the muscle loss and tissue protein catabolism, as seen at day zero [31]. Treatment with the 1,3,4oxadiazole derivative, however, represented a signifcant gain in body weight as compared to the disease control group. Glycated hemoglobin is an extensively accepted parameter for the identifcation of plasma glycemic levels in a diabetic patient [32]. Protein glycation involves a series of complex reactions that occur between monosaccharides (glucose and fructose) and amino acids or proteins, producing an unstable Schif base and then forming Amadori products such as fructosamine [33]. Alloxan has two distinct pathological efects, it selectively inhibits glucose induced insulin secretion through specifc inhibition of glucokinase, the glucose sensor of the beta cell, and it causes a state of insulin-dependent diabetes through its ability to induce ROS formation. ROS causes the selective necrosis of beta cells leading to the increase in protein glycation, three days just after alloxan injection [34]. Tese two efects can be assigned to the specifc chemical properties of alloxan, the common denominator being selective cellular uptake and accumulation of alloxan by the beta cell [35]. In this investigation, diabetic rats showed higher levels of Amadori product, suggesting their impaired glycemic control [36]. Te dose level at 5, 10, and 15 mg/kg of 1,3,4-oxadiazole derivative showed a concentration-dependent reduction in Amadori product within 14 days, by activating the glucokinase enzyme and reduction in ROS production [37].
Insulin is a hormone made by the pancreas that binds to the glycoprotein receptor on the cell membrane of the cell and stimulates the transportation of glucose across the cell membrane by glucose transporter GLUT4 [38]. In diabetic rats treated with 1,3,4-oxadiazole derivative as compared to  Evidence-Based Complementary and Alternative Medicine the diseased, an increase in plasma insulin levels was observed. Tis could be due to the potential of insulin sensitivity on the cell membrane [39]. Improving insulin sensitivity is the ultimate physiological efect of oxadiazole derivative in improving the homeostasis of glucose [40].
Diluric acid is oxidized to alloxan to generate the superoxide radical [41]. Te SOD plays an important role in the metabolism of oxygen by reducing superoxide anion free radical (O 2 − ) to nonreactive H 2 O 2 and molecular oxygen [42]. At a dose level of 15 mg/kg, 1,3,4-oxadiazole derivative showed a signifcant (P < 0.001) rise in the SOD level when compared to the diabetic group. Catalase performs its activity to convert H 2 O 2 into water and O 2 and protect the cell from oxidative stress [43]. However, unlike superoxide, H 2 O 2 can rapidly difuse across cell membranes, and in the presence of transition metal ions, it can be converted to hydroxyl radicals via Fenton chemistry [44]. Highly reactive hydroxyl radicals are then formed in the presence of Fe 2 + and H 2 O 2 according to the Fenton reaction. Te imbalance ratio of the antioxidant enzymes and oxidants (ROS) results in the emergence of illness [45]. Glutathione peroxidases present in cytosol and mitochondria have a major role in converting hydroxyl radical (OH − ) into water. In this mechanism, hydroxyl production is afected [46]. Our results assessed that the reduced levels of GSH were restored to normal when diabetic animals were treated with 15 mg/kg of pancreatic 1,3,4-oxadiazole derivative. In liver, 10 and 15 mg/kg of 1,3,4-oxadiazole derivative showed a signifcant   Data are presented as mean ± SEM (n � 6). * * * P < 0.001, * * P < 0.01, and * P < 0.05 represent signifcantly diferent values as compared to the disease group. Note. OXD: 1,3,4-oxadiazole derivative.
(P < 0.05) increase in the GSH level. Similarly, 10 and 15 mg/ kg of 1,3,4-oxadiazole derivative showed a signifcant (P < 0.01) increase in the GSH level in the kidney as compared to the diabetic group. Te current data revealed that consistent hyperglycemia via alloxan-generated ROS caused marked oxidizing efects, as evidenced by an increase in diabetic animal MDA rates compared to the nondiabetic animals [47]. High levels of MDA were reduced to normal values by 1,3,4-oxadiazole derivative treatment. Te 1,3,4-oxadiazole derivative at a dose of 15 mg/kg showed a highly signifcant reduction in the MDA serum level in the liver (P < 0.001) and kidney (P < 0.01), whereas nonsignifcant reduction was reported in the pancreas as compared to the diabetic group. Te protein level was increased in 1,3,4-oxadiazole derivative as compared to the diabetic groups. MDA is the mutagenic in bacteria and mammalian cells [48].
Histology of the pancreas demonstrates normal acini and normal cellularity in the control islets of Langerhans. In diabetic animals, extensive damage to Langerhans islets was treated and reduced islet dimensions were observed too. On the other hand, at a dose level of 15 mg/kg, 1,3,4-oxadiazole derivative showed hypertrophy and vacuolations of Langerhans β cells.
In the serum of diabetic rats, hepatic enzymes have increased [49]. Tis may mainly be due to the leakage of these enzymes from the liver cytosol to the bloodstream as a result of the hepatotoxic alloxan efect. 1,3,4-oxadiazole derivative reduced the serum ALT and AST levels at all doses that showed a protective efect and normal liver function in restoring impairment to organs due to diabetes.
Cholesterol and triglyceride play a major role in the pathogenesis of DM-related complications. Te unusually high serum cholesterol and triglyceride content in diabetics were primarily due to the increased production of free fatty acids from peripheral fat depots. As lipase is suppressed by insulin, insulin insufciency or resistance may result in dyslipidemia [50]. Te group treated with 1,3,4-oxadiazole derivative showed changes in the cholesterol and triglyceride profle in contrast to the diseased one. Diabetic hyperglycemia contributes to an increase in urea and creatinine plasma levels, which are regarded as important markers of renal dysfunction [51]. With respect to the control group, the fndings showed an increase in plasma levels of urea and creatinine in the diseased group. Tese fndings suggested that diabetes may cause renal dysfunction. When compared to the diseased group, treatment with an 1,3,4-oxadiazole derivative signifcantly (P < 0.001) decreased the plasma urea level. Similarly, the level of creatinine was decreased (P < 0.001) after the administration of 1,3,4-oxadiazole derivative in comparison to the diseased group.
Hepatic photomicrographs displayed the normal liver cells through a well-preserved cytoplasm, nucleus, core nuclei, and main vein in the normal group. Te standard lobular structure was conserved in diabetic rats. Te main vein detrimentally clogged up. Focal hemorrhage was also established. Te accumulation of fat was noticeable. Kupfer cell activation and congestion of the central vein can be seen in the liver treated with 15 mg/kg of 1,3,4-oxadiazole derivative. In normal animals, the histology of the glomeruli and the Bowman capsule is appropriate. For diabetic rats, the basement membrane of the arterioles of glomeruli is slightly thickened, including a slight change in mesangial mesangium density. Epithelial proteins were deposited in the lumen of the renal tubules after 15 mg/kg of oxadiazole derivative treatment.
Te potential mechanism for oxadiazole derivative action was found to be a high-afnity ligand of the receptoractivated peroxisome proliferator, a member of the superfamily of nuclear receptors that cause transcription of insulin-responsive genes [52]. Te glucose-lowering efect of oxadiazole derivative is mediated through the improvement of insulin sensitivity on the cell membrane, causing an increased uptake of glucose into the cell for energy [40]. In addition, oxadiazole derivative caused regranulation of pancreatic beta cells in pancreatectomised rats [53].
Numerous studies have been conducted on Nrf2 as a potential antioxidant signaling mechanism [54]. Nrf2 acts as an inactive dimer due to its cytoplasmic association with the Kelch-like ECH-associated protein 1. Nrf2 activation is attenuated by Keap1, and its nuclear translocation is prevented. Te complex splits whenever a stress signal is detected, permitting Nrf2 to move freely into the nucleus and activates the translocation, where it triggers the antioxidant enzymes. Nrf2 binds to antioxidant response elements in the nucleus and promotes the transcription of endogenous defensive enzymes [55]. As the previous results shows that the quantity of antioxidant enzymes was increased by treating the rat with oxadiazole derivative, so the mechanistic studies showed that 1,3,4-oxadiazole derivative could trigger Nrf2 nuclear translocation, subsequently resulting in increased expression of Nrf2 target gene. Meanwhile, oxadiazole derivative suppressed the increase of the ROS level [56].

Conclusions
It can be concluded that the imbalance between oxidants and antioxidant enzymes play a pivotal role in development and progression of diabetes. In this study, the 1,3,4-oxadiazole derivative produced pronounced antidiabetic activity at a dose of 15 mg/kg, showing substantial decrease in the blood glucose level, body weight, and Hb1Ac level. Other biochemical metabolic parameters including ALT, AST, urea, creatinine, triglyceride, and cholesterol were also reduced by 1,3,4-oxadiazole derivative. Te antioxidant enzyme activity was signifcantly increased by 1,3,4-oxadiazole derivative. Te exact mechanism by which 1,3,4-oxadiazole derivative decreases the level of blood glucose in diabetic rats requires further analysis. Moreover, further studies are needed to explore the pharmacological profle of 1,3,4oxadiazole derivative.

Data Availability
Te data used to support the fndings of this study are included within the article.

Conflicts of Interest
Te authors declare that they have no conficts of interest.