Evaluation of Alpha-Amylase Inhibitory and Antidiabetic Potential of Novel 1,3,4-Oxadiazole Derivatives

Diabetes mellitus (DM) is one of the oldest endocrine disorders, which is most chronic, multifactorial, incurable metabolic disorder characterized by severe hyperglycaemia. Over 3000 years ago, the first case was reported in Egyptian manuscript and currently 100 million populations across the globe are suffering with diabetes. It has been marked by the lack of insulin or the action of insulin on its target site and interruption in the metabolism of carbohydrates, lipid, and protein. The numerous factors are involved in the cause of disorder, among them the highlighted is elevated basal metabolic rate, high oxidative stress, and deficiency and fault in reactive oxygen species scavenging enzymes bring about damage to pancreatic cells.¹ As per International Diabetes Federation (IDF), approximately 463 million adults (20-79 years) affected with diabetes, by 2045 this will rise to 700 million, every 1 in 11 living with diabetes. The diabetics persistent with hyperglycemia coupled with long-term complication which dysfunctions the vital organs include retinopathy, nephropathy, neuropathy and angiopathy. The risk factors associated with diabetes are genetics, family history diabetes, old age, dietary deficiency, incomplete digestion, fatness, sedentary lifestyle, stress, drug-induced, infection in pancreas, hypertension, high serum lipid and lipoproteins, less glucose utilization and other factors.² In the year of 1980, WHO classified diabetes mellitus into Type 1, which is also called as Insulin-dependent diabetes mellitus(IDDM). It has been characterised as deficiency of insulin due to loss of insulin-producing beta cells of the islets of pancreas and it is an idiopathic. On the flip side, wide prevalent category, Type 2 DM, known as Non-insulin-dependent diabetes mellitus (NIDDM), results from an imbalance between insulin sensitivity and insulin secretion. Type 2 DM goes with insulin resistance in the skeletal muscle and liver which results of hyperinsulinemia, and depletion of insulin making by the pancreas genetic susceptibility and several environmental factors contribute for Type 2 DM.³ 1,3,4-Oxadiazole, a heterocyclic molecule has occupied and attracted a wide attention in medicinal chemistry of its broad horizon of biological activities. Oxadiazole is obtained by furan substitution of two methylene groups (=CH) with two pyridine type nitrogen atoms (-N=). The scientists have been reported three isomers such as 1, 2, 4-oxadiazole, 1, 2, 3-oxadiazole and 1, 2, 5-oxadiazole.^4-5 1, 3, 4-oxadiazole and 1, 2, 4-oxadiazole are well known and extensively studied, because of their wide chemical and biological properties. Amid heterocyclic compounds, 1,3,4-oxadiazole has become the key molecule for the building up of new therapeutic agents. Literature survey have been revealed, 1,3,4-oxadiazole has wide pharmacological actions including antibacterial, antifungal, analgesic, anti-inflammatory, antiviral, anticancer, antihypertensive, anticonvulsant, and anti-diabetic properties.^6-8 Apart from biolological uses, it has drawn an attention as surrogates (bioisosteres) for carboxylic acids, esters and carboxamides in medicinal chemistry. Two examples of compounds containing the 1,3,4-oxadiazole unit currently used in clinical medicine are Raltegravir an antiretroviral drug and Zibotentan an anticancer agent.^9-10

Materials and Methods

Experimental Design Procurement of synthetic derivatives:

The novel synthesized substituted 1,3,4-oxadiazole were procured from Department of Pharmaceutical Chemistry, SCS College of Pharmacy, Harapanahalli, India.

Experimental animals

Swiss albino mice (20-25 g) and Wistar rats (150–200 g) of either sex were procured from Venkateshwara enterprises, Bangalore. Animals were quarantined in standard laboratory conditions (temperature 26 + 2°C and humidity 65 ± 10% with 12 h day: night cycle) for one week. All animals received standard laboratory diet and water ad libitum. The animal clearance was approved from Institutional Animal Ethical Committee with registration number SCSCOP/01/17 of SCS College of Pharmacy, Harapanahalli, India. 

Determination of acute toxicity

According to OECD guidelines no 425 (Annexure-2d) of CPCSEA, the toxicity study was carried on albino female mice.¹¹ 

α- amylase inhibitory assay

The management of blood glucose level is a critical strategy in the control of diabetes complications. Inhibitors of carbohydrate hydrolyzing enzymes (such as α-amylase and α-glucosidase) have been useful for the control of hyperglycemia. Inhibition of these factors plays an important role in management of diabetes.¹² This study was performed by a modified starch iodine protocol.¹³ In short 1 mL of 1,3,4-oxadiazole derivatives of different concentrations (0.5, 1, 1.5, 2, 2.5, 3, 3.5 mg/ml) were taken in pre-labelled test tubes. A volume of 20 µL of 1% w/v α -amylase was added to each test tube and incubated for 10 min at 37 °C. After the incubation 200 µL of 1% starch solution was added to each test tube and the mixture was re-incubated for 1 h at 37 °C. Then 200 µL of 1% iodine solution was added to each test tube and after that, 4 mL distilled water was added. Absorbance was measured at 565 nm in UV-Visible spectroscopy. Sample, substrate and α-amylase blank were undertaken under the same conditions. Each experiment was done in triplicate. IC₅₀ value was calculated using regression analysis.¹⁴

Inhibition of Amylase(%) = Absorbance sample - Absorbance control / Absorbance control x 100

Alloxan induced diabetic rat model

Alloxan monohydrate is a diabetogenic agent, which is often used to induce hyperglycaemia in rats by a single injection (120 mg/kg) intraperitonially. Subsequently, the rats were allowed to hyperglycemia (glucose level >250 mg/dL), for 5 days and divided into different groups, comprising six rats each. On the same day, the treatment was started on the diabetic rats for a period of 10 days; except normal and diabetic control rats. On the 4^th, 7^th and 10^th day, the biomarkers such as body weight and blood sugar level were estimated and on 10th day, under light ether anesthesia, the blood samples were collected from overnight fasted rats by cardiac puncture for biochemical estimation of fasting blood glucose and lipid profiles. Later, the pancreas were isolated from the rats and stored in 10% formalin solution for histopathological examination.¹⁵

Group I - Received normal saline (i.p) + vehicle (p.o).

Group II - Received alloxan monohydrate 120 mg/kg (i.p) + normal saline (p.o).

Group III - Alloxan + Glipizide (10 mg/kg P.O.) served as standard.

Group IV-VIII - Alloxan monohydrate + compound 1 to 5, respectively.

Results

Determination of acute toxicity

SOZDs were studied for acute toxicity upto the dose of 2000 mg/kg by oral route. The SOZDs were found to be devoid of morbidity and mortality at 2000 mg/kg. Thus, synthesized substituted oxadiazole derivatives are non-toxic upto the dose of 2000 mg/kg.

In vitro antidiabetic activity

α-amylase inhibition method

The in vitro alpha amylase inhibitory activity of SOZD’s exhibited significant activity. The percent inhibition ranging from 1000 μg/mL to 7000 μg/mL concentration of SOZDs show concentration dependent (Table 1 and Figure 1). The results revealed that SOZD1, SODZ2, SOZD3, SOZD4 and SOZD5 at a concentration of 7000 μg/mL showed a maximum per cent inhibition 55.55, 46.50, 80.64, 78.32 and 88.08 respectively whereas, in standard acarbose 0.969. The IC50 value of SOZD 4 and SOZD 5 were found as 2.84 and 3.12 whereas, standard was found to be 4.76 μg/mL.

In vivo antidiabetic activity

Alloxan induced diabetic rats

The changes in body weight during the period (10 days) of study are given in Table 2. This shows an increase in the mean body weight of normal rats which is an indication of gained body weight. Similarly, the treatments in diabetic control group of rats have shown a change in body weight from a mean 238.8±9.656 g to 190±9.789 g.

The glipizide (10 mg/ kg) treated group showed a mean body weight 262.5±21.26 g in initial day and it was found to have been increased to 280.0±21.70 g on 10^th day. This is an indication that the glipizide has protected losing body weight in a significant manner when compared with control group. Similarly, the SOZDs treated diabetic rats show reduction in body weight during the treatment period. Group IV, Group V shown a mean (± SEM) body weight of 230.8±17.34 g, 217.5±9.287 g, respectively in initial day, weight got increased to 224.7±16.13 g, 220.8±9.610 g in 10^th day. In these groups, Group IV, V, reduction of weight is less compared to positive control group. This shows that SOZDs treated has protected diabetic rats from loosing body weight in a significant manner when compared to control group.

Effect of SOZDs biochemical parameters in alloxan induced diabetic rats:

The biochemical parameters are significantly increased in positive control compared to negative control, but the serum protein and hepatic glycogen concentration gets reduced in positive control. These parameters in other groups (STD, IV and V) show significant control compared to positive control group with the mean (± SEM) as shown in Table 4.

Discussion

Oxadiazoles are the class of heterocyclic aromatic chemical compounds of the azole family has wide biological actions. Any compounds holdding 1,3,4-oxadiazole have an extensive pharmacological potentials.^24-25 Very interestingly, the different substituted oxadiazole groups possessing extended analgesic, anticonvulsant activities, ^26-27 antitubercular,¹³ and anti-hepatitis B viral activities.¹⁴ α-amylase and α-glucosidase are the key enzymes play an important role in the digestion of carbohydrates and starch to produce glucose and maltose in intestine for absorption to blood stream. α-Amylase, splits the internal α-1,4-glycosidic linkages starch, converting into low-molecular-weight products such as glucose, maltose, and maltotriose units.²⁸ In the present study, α-amylase inhibition assay reported its higher activity at concentration of 7000 μg/mL and shows reduction in blood sugar levels. These findings are indication to decrease glucose availability from the intestine from digestible carbohydrates, hence are used as an oral anti-hyperglycaemic agent. In diabetic patient, protein synthesis is reduced in all tissues and muscle. A proteolytic activity enhancement causes, a greater influx of amino acids to the liver, elevating serum levels of urea and creatinine, and thereby increasing renal work excretion.²⁹ Treatment with SOZDs are effective in the protection of renal functions and reduces the work excretion, by diminishing the urea and creatinine levels in diabetic rats. Diabetes mellitus is frequently associated with dyslipidemia, with high levels of lipid profiles and it is found that it has been reversed by SOZDs. Moreover, the pancreas histopathology findings revealed that alpha and beta cells normal proportion restored by SOZDs in diabetic rats.

Conclusion

The findings of our study delineated SOZD4 and SOZD5 could be considered as potential drugs for the treatment of diabetes. However, further studies could be recommended to elucidate the exact mechanism of action of SOZD derivatives as potential antidiabetic drugs. 

Acknowledgement

The authors express deep sense of gratitude to the management and Principal, TMAE’s SCS College of Pharmacy, Harapanahalli.

Declaration of interest: none.

Funding sources: nil.