INTRODUCTION:

Diabetes is defined as a state in which carbohydrate and lipid metabolism is disordered and improperly regulated by insulin. This mainly results in raised fasting and postprandial blood glucose levels. If this imbalanced homeostasis does not return to routine and continues for a prolonged period of time, it leads to hyperglycemia that in due course turns into a syndrome called Diabetes¹. Type II DM is prevailing worldwide moving nearly 6 % of the population. It is one amongst the quickest growing health issues worldwide and will have an effect on 366 million individuals within the next thirty years, if correct preventive measures don't seem to be enforced within the immediate future². The clinical symptoms include weight loss without any workout, recurrent infections, slow wound healing process, rashes around neck or armpit and being cranky. Whenever left untreated, diabetes can cause numerous entanglements.

Intense difficulties can include diabetic ketoacidosis, nonketotic hyperosmolar trance like state, or passing. Genuine long-haul entanglements include coronary illness, stroke, constant kidney disappointment, foot ulcers, and harm to the eyes^3-4. Moreover, diabetic patients experience a two-to-three-fold increase in cardiovascular morbidity and mortality in comparison to non-diabetics⁵. With a serious complication often resulting in high death rate, the treatment of diabetes includes medicines, diets, physical training and so on in all countries. A search for a new class of compounds is essential to overcome diabetic problems⁶, as oral hypoglycemic agents used in clinical practices have serious side effects like hematological effects, coma, disturbances of liver and kidney⁷.

A primary therapeutic approach to treat diabetes is to decrease postprandial hyperglycemia⁸. This can be accomplished by the inhibition of carbohydrate hydrolyzing enzymes like alpha amylase and alpha glucosidase⁹. Inhibition of alpha amylase can retard carbohydrate digestion and entrapment of the digested glucose within the intestine for a longer duration in diabetic patients would cause a reduction in the rate of glucose absorption into the blood¹⁰. Alpha glucosidase is involved in break down of starch and disaccharides to glucose. It has been recognized that alpha glucosidase inhibitors can also be used to prevent some disorders such as diabetes, obesity, hyperlipidaemia and hyperlipoproteinemia and also show anti-HIV activity¹¹. Thus, Alpha amylase and alpha glucosidase inhibitors are the potential targets in the development of lead compounds for the treatment of diabetes¹². Higher plants, animals and microorganisms also produce a huge number of diverse protein inhibitors of which regulate the activity of these enzymes¹³. Some of these enzyme inhibitors act by directly blocking the active centre of the enzyme at various local sites¹⁴. In animals, alpha amylase inhibitors decrease the high glucose levels that can occur after a meal by reducing the conversion speed with which alpha amylase convert starch into simple sugars¹⁵. This is of importance in diabetic people where low insulin levels prevent the fast clearing of extracellular glucose from the blood¹⁶. Diabetics show low alpha amylase levels in order to keep their glucose levels low. Plants also use alpha amylase inhibition as a defense mechanism for protection against insects. These inhibitors alter and inhibit the digestive action of alpha amylases and proteinases in the gut of insects and inhibit their normal feeding activities. Therefore, alpha amylase inhibitors have key roles in controlling blood sugar control and crop protection¹⁷. Alpha glucosidase inhibitors act as competitive inhibitors of alpha glucosidase enzyme needed to digest these carbohydrates. The intestinal alpha glucosidases hydrolyze complex carbohydrates to glucose and other monosaccharides in the small intestine. Inhibition of these enzyme systems helps to reduce the rate of digestion of carbohydrates¹⁸. Less amount of glucose is absorbed because the carbohydrates are not broken down into glucose molecules. In diabetics, the short-term treatment of these inhibitors can reduce high blood glucose levels. Thus, lowering hyperglycaemia is the most current and common therapeutic strategy for type 2 diabetes. Mellitus.

Quinazolinone derivatives have been reported to possess varied biological and pharmacological properties. They are found to be useful as antibacterial, antifungal and anticancer agents¹⁹. SAR study of different Quinazoline derivatives has revealed that the substitution at 2,3- position has a significant role as hypotensive. Quinazoline and condensed quinazoline exhibit potent central nervous system (CNS) activities like anti-anxiety, analgesic, anti-inflammatory and convulsant. Quinazoline-4-ones with 2,3-di substitution is reported to possess significant analgesic, anti-inflammatory, and anticonvulsant activities²⁰. We have synthesized compounds which act on metabolic disorders and possess anti-diabetic activity by different mechanisms. They slow down the digestion of starch or glucose absorption. Inhibition of the mammalian alpha amylase enzyme in the intestine will prevent starch and oligosaccharides from being broken down into monosaccharides and ingested. This would minimise glucose absorption and, as a result, postprandial blood glucose levels. Therefore, screening of newly synthesized quinazolinone compounds has received attention.

MATERIAL AND METHODS:

Chemicals and reagents:

0.02 M sodium phosphate buffer (Astron Chemicals), alpha amylase enzyme (Sigma Aldrich), 1% starch suspension, 3,5-dinitrosalicylic acid (DNSA), Dimethyl sulfoxide (DMSO), 4-Nitrophenyl beta-D-glucoside (PNPG), alpha glucosidase solution, Sodium Carbonate, Acarbose (Standard) (Samex Overseas).

Instrumentation:

UV Spectrophotometer (Simadzu), Micro plate Reader

In-vitro alpha amylase inhibitory activity²¹:

The assay mixture was prepared to contain 0.02M sodium phosphate buffer (200μL), a-amylase enzyme (20μL 2unit/mL) together with 30 synthesized test compounds (PM1 to PM30) in the concentration range of 20-100μg/mL. Then, it was incubated for 10 min at room temperature followed by the addition of 200μL of 1% starch suspension to all the tubes containing reaction mixture. The reaction was later stopped by the addition of 400μL of 3,5-dinitrosalicylic acid (DNSA) colour reagent dropwise. All tubes were kept in boiling water bath for 5 minutes, cooled down to room temperature and diluted with 15mL of distilled water. The absorbance of each reaction mixture was measured at 540nm. Control was also prepared accordingly without addition of test sample under investigation and were compared with the test samples containing concentration of synthesized compounds (PM1-PM30) (20-100 μg/mL) freshly prepared in DMSO solvent. The results were indicated as inhibition activity (%) using the following formula:

Abs (Control) - Abs (Sample)

Inhibition activity (%) = --------------------------- X 100

Abs (Control)

Where Abs (control) is the absorbance of the control reaction (containing all reagents except the test sample) and Abs (sample) is the absorbance of different synthesized compounds (PM1-PM30)

The IC₅₀ values (inhibitory concentration which will produce 50% inhibition of the enzyme activity) of the synthesized compounds (PM1-PM30) were calculated. Acarbose which is a well-known anti-diabetic drug used to treat T2DM, was applied as a positive control in the concentrations ranged from 20 to 100μg/mL. Experiments were achieved in triplicate.

In-vitro alpha glucosidase inhibitory activity²²:

The reaction mixture was prepared by addition of 50μL of 0.1M phosphate buffer (pH 7.0) and 25μL of 0.5mM PNPG (dissolved in 0.1M phosphate buffer, pH 7.0). All Synthesized compounds (PM1-PM30) (10μL), Acarbose (0.02 to 3mg/mL) and α-glucosidase solution (25μL) (a stock solution of 1mg/mL in 0.01 M phosphate buffer, pH 7.0, was diluted to 0.1U/mL with the same buffer, pH 7.0, just before the assay) were added to the reaction mixture. It was then incubated at 37°C for 30 min. The reaction was then terminated by the addition of 100μL of 0.2M sodium carbonate solution. The enzymatic hydrolysis of the substrate was monitored based on the amount of p-nitrophenol released in the reaction mixture by observation at 410nm using a microplate reader. All experiments were carried out in triplicate and the results are expressed as the mean±S.D. of three determinations.

RESULTS:

In-vitro alpha amylase inhibitory activity is summarized in Table 1 as IC₅₀ values. IC₅₀ Values were expressed as mean±SEM for compounds (PM1 to PM30) and standard acarbose. Standard drug acarbose showed an IC₅₀value of 48.96±0.95μg/mL, whereas IC₅₀ value of synthesized compounds PM20, PM7, PM28 and PM29 were found to be 49.05±2.64, 50.03±0.63, 50.89±1.48 and 50.98±1.39μg/mL respectively.

Table 1: In-vitro alpha amylase inhibitory activity

Sample No.	IC₅₀ (μg/mL) (Mean ± SEM)	Sample No.	IC₅₀ (μg/mL) (Mean ± SEM)
PM1	53.21±2.12	PM17	61.08±3.89
PM2	75.68 ±2.54	PM18	52.91±0.89
PM3	52.64± 1.68	PM19	74.13±1.24
PM4	50.95± 3.65	PM20	49.05±2.64
PM5	81.07± 1.26	PM21	70.75±1.44
PM6	51.48±1.32	PM22	74.20±0.86
PM7	50.03±0.63	PM23	59.44±1.18
PM8	82.63±0.74	PM24	52.85±2.82
PM9	53.49± 1.35	PM25	99.27±0.68
PM10	55.75± 2.08	PM26	61.67±1.17
PM11	53.68±4.15	PM27	55.20±0.87
PM12	64.00±1.04	PM28	50.89±1.48
PM13	59.98± 1.26	PM29	50.98±1.39
PM14	64.79±1.23	PM30	59.39±1.73
PM15	55.90±1.78	Acarbose	48.96±0.95
PM16	63.80±2.05

SEM= standard error of the mean

Significant differences were observed between IC₅₀values of all synthesized compounds and standard drug acarbose. However, compounds PM20, PM 7, PM 28 and PM29 were comparable to acarbose and have significant anti diabetic activity as alpha amylase inhibitor.

In-vitro alpha glucosidase inhibitory activity is summarized in Table 2 as IC₅₀ values. IC₅₀ Values were expressed as mean±SD for compounds (PM1 to PM30) and standard Acarbose. All synthesized compounds showed concentration dependent inhibition of alpha glucosidase in comparison with standard antidiabetic drug acarbose.

Table 2: In-vitro alpha glucosidase inhibitory activity

Sample No.	IC₅₀ (μg/mL) (Mean ± SD)	Sample No.	IC₅₀ (μg/mL) (Mean ± SD)
PM1	51.20±0.75	PM17	31.11±0.86
PM2	28.72±1.25	PM18	40.35± 2.45
PM3	38.24± 0.68	PM19	39.29±0.91
PM4	21.04± 0.94	PM20	28.13±1.24
PM5	23.56± 0.72	PM21	63.79±1.82
PM6	25.69±084	PM22	42.17±2.86
PM7	29.53±1.45	PM23	32.78±0.86
PM8	34.75±0.84	PM24	39.15±1.25
PM9	26.56±1.45	PM25	40.36±0.97
PM10	36.04± 1.51	PM26	47.38±1.54
PM11	32.01± 1.35	PM27	35.53±0.78
PM12	90.59±0.67	PM28	19.84±0.97
PM13	107.28± 1.34	PM29	20.11±1.08
PM14	21.52± 1.61	PM30	18.87±0.73
PM15	35.66±1.87	Acarbose	17.08±0.54
PM16	85.91±1.47

SD= standard deviation

Acarbose showed an IC₅₀ value of 17.08±0.54μg/mL whereas IC₅₀Value of compounds PM 30, PM28, PM29 and PM4 were found to be 18.87±0.73, 19.84±0.97, 20.11±1.08 and 21.04±0.94 respectively and therefore are considered as promising alpha glucosidase inhibitors.

DISCUSSION:

Diabetes Mellitus is well known metabolic disorder which is characterized by elevated blood glucose level and the reason for this condition is due to loss of production of insulin by the pancreas or reduced response by cells to produce insulin or both controlling the post prandial hyperglycaemia of prime importance in management of type II diabetes mellitus.

Alpha amylase inhibitors are also known as starch blockers as they prevent or slow down the absorption of starch into body mainly by blocking the hydrolysis of 1-4 glycosidic linkage of starch and other oligosaccharides mainly into maltose, maltase and other simple sugar. From result indicates that PM 20 could be beneficial in reducing absorption of starch into the body and help to improve Diabetes. Alpha glucosidase plays a vital role in digestion of complex carbohydrates to glucose. Hence its inhibition is considered as one of the targets for managing the post prandial hyperglycaemia. Acarbose is well-known alpha glucosidase inhibitor drug but has a complication of gastric problems (ulceration, diarrhoea, bloating and flatulence)^23-25, we need to advance other inhibitors.

CONCLUSION:

This study gives an ingenuity that the synthesized quinazolinone compounds must be explored and developed as inhibitors which are as effective as marketed drug but have lesser complication. They can be used to develop lead compound for designing a potent anti-diabetic drug which can be used for effective treatment and management of metabolic disorder of diabetes mellitus.

ACKNOWLEDGEMENT:

The authors deeply appreciate the assistance of the Department of Pharmacology, A One Pharmacy College, Ahmedabad, Gujarat, India for carrying out screening activity of synthesized compounds.

CONFLICT OF INTEREST:

The authors declare no conflict of interest.

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Received on 09.04.2021 Modified on 08.10.2021

Research J. Pharm. and Tech 2022; 15(9):4226-4229.

DOI: 10.52711/0974-360X.2022.00710