SYNTHESIS AND EVALUATION OF α-AMYLASE AND LIPASE INHIBITORY ACTIVITIES OF SYNTHETIC COUMARIN DERIVATIVES
This research work involved synthesis of nine Coumarin-hydrazone derivatives and study of their enzyme inhibition activity. The purpose of the study was to investigate the α-amylase and lipase inhibition activity of the synthesized coumarin derivatives. The experiment was performed by taking 5, 10, 50,100 µg/mL of each of the compounds and Acarbose and Orlistat was taken as a positive control. The studies showed that compound 5d (120.6 µM), 5e (111.5 µM) and 5h (124.0 µM) was showing better inhibition activity against α-amylase enzyme, in comparison with the standard (IC50=81.70 µM). The lipase enzyme inhibition revealed that the compounds, 5c (142.3 µM), 5d (82.3 µM) and 5i (93.6 µM) showed better inhibition compared to the standard (IC50=236.4±2.83 µM). The enzyme kinetics study also revealed good results and it has been discussed in the results section.
Introduction<!>Materials and methods<!>General<!>Synthesis of 2-((4-methyl-2-oxo-2H-chromen-7-yl) oxy) propanehydrazide(4)<!>Synthesis of substituted -2-((4-methyl-2-oxo-2H-chromen-7-yl)oxy) propane hydrazide(5a-i)<!>Analysis of α-Amylase inhibitory activity 2.3.1 Determination of percentage of inhibition<!>Type of inhibition analysis<!>Analysis of Lipase inhibitory activity 2.4.1 Determination of percentage of inhibition<!>Type of inhibition analysis<!>Inhibition study of α-amylase<!>Inhibition study of lipase<!>Conclusion
<p>Coumarin is a naturally occurring organic compound and best known as aromatic lactones. For the first time, in the year 1820, Vogel (1820) reported the isolation of coumarin. Since its discovery in 1820, coumarins have been extracted from many plants, notably tonka bean, apricots, cinnamon, lavender, licorice, strawberries, sweet clover and Woodruff [1,2]. It has attracted synthetic chemists mainly because of the easy acceptability of the coumarin ring system by the biological system. Coumarins are extensively used in the preparation of pesticides, cosmetics, food additives and sweetener [3]. Coumarin ring derivatives are also used as commercially significant aromatic fluorescent materials due to their extensive spectral range, high emission quantum yields and photostability [4]. Coumarins are known to possess unique biological activities [5]. The natural role of coumarin is to inhibit germination and subsequent root growth of plants. On the other hand, it is also likely that coumarins can even act as growth regulators and induce intracellular growth mechanisms in plants. On a larger scale, coumarins have gathered medical interests are owing to its distinctive biological properties like anti-bacterial, anticoagulants, antibiotic, antifungal, anticancer and anti-inflammatory [6]. Since the discovery of coumarins, several methods have been developed for its synthesis. Some of them are Pechmann, Perkin, Knoevenagel, Witting, and Reformatsky [7]. Most of these methods have been further modified by the different generations of chemists.</p><p>At present the synthetic chemicals mimicking natural properties are attaining more significance.</p><p>So the present study is approaching therapeutic role of synthesized coumarin derivative. Nowadays obesity and diabetic cases are increasing significantly worldwide as most prone diseases.</p><p>Adipose triglyceride lipase (ATGL) which catalyzes the breaking down of triacylglycerol and intensely decides the amount of fatty acids in our body. The high content of fatty acid levels are directly related to the development of insulin resistance and reduced glucose tolerance, which ultimately leads to type-2 diabetes [8].</p><p>The mounting rate of type-2 diabetes mellitus (T2DM) is very high through-out of the world. The latest statistical report achieved by International Diabetes Federation showed that almost 380 million people are right now suffering by this disease [9], and the estimated figure may be exceed to 592 million by 2035, due to an intense boost in T2DM. Diabetes disease also causes some other health related complications like nephropathy, retinopathy, liver damage, stroke, coronary heart disease and peripheral-nephropathy. α-amylase is an enzyme that attacked starch which accelerates the internal a-1, 4-glucan linkage inside the polysaccharides via hydrolysis, consist of three or more d-glucose units of alpha-1, 4-linked, which offered a glucose and maltose mixture respectively [10]. α-amylase instantly transforms starch-substrate to glucose in human bodies and the patient with diabetes disorder did not properly consumed glucose content in their body.</p><p>Therefore it is crucial to control the lipase and α-amylse enzymes to reduce the obesity and diabetes in our body [11]. Various heterocyclic compounds were used as a drug to inhibit these enzymes.</p><p>The standard drugs available in the market to control lipase enzyme is orlistat, and for α-amylase inhibition [12], Acarbose is the marketed drug. They have various side-effects like liquid stools, steatorrhea, abdominal cramping and fat-soluble vitamin deficiencies, fecal urgency, incontinence, flatulence [10]. Therefore it is imperative to find new drugs which are very active which comes with minimal side-effects [13].</p><!><p>The starting materials and reagents were commercially obtained. Freshly distilled solvents were used for the reaction. TLC was performed using pre-coated silica gel plates and UV absorption/iodine chamber used for observation. Proton and carbon-13 NMR data were obtained from Bruker Ascend 400 MHz device. The internal standard was tetramethylsilane, and the chemical shift values were recorded. Shimadzu FTIR-8400S instrument was used to record IR spectra.</p><!><p>The starting compound, 7-hydroxy-4-methyl coumarin (5.7 mmol) was taken in dry acetone (10 ml), potassium carbonate (anhydrous) (5.7 mmol) was added and kept stirring for several minutes.</p><p>Ethyl 4-Bromo butyrate (5.7 mmol) added and the resultant solution was refluxed for 10 hrs. The reaction was monitored by TLC using hexane: ethyl acetate (1:2) as the mobile phase. The completed reaction mixture was by poured to ice-cold water and product was filtered out. The solid product was dried and recrystallized using ethanol [13].</p><!><p>A suitable amount of compound 3 (3.0 mmol) was taken in ethanol (10 ml), and hydrazine hydrate (9.0 mmol) was dropwise added. The resultant clear solution was kept refluxing for six hrs. The reaction was monitored by TLC using hexane: ethyl acetate (1:2) as the mobile phase. The precipitate obtained was filtered, dried, and recrystallized using ethanol [13].</p><!><p>A mixture of compound 4(3.0 mmol) and aromatic aldehydes (3.0 mmol) was taken in ethanol (10 ml). Few drops of glacial acetic acid added and the mixture was kept for refluxing for 4 hrs. The precipitate was filtered, washed with water and dried. The dried sample was recrystallized using ethanol [13].</p><!><p>The 5, 10, 50 and 100 μg/ml of the test extract was made to react with 200 μl of α-amylase enzyme and 100 μl of 2 mM of phosphate buffer (pH 6.9). It was kept for incubation for 20 min, 100 μl of 1% starch solution was added. The procedure was repeated for the control where 200 μl of the enzyme was replaced by the buffer [14]. The working standard for enzyme was prepared by dissolving 1 mg of amylase in 10 ml of phosphate buffer (pH 6.9). After incubation for 5 min, 500 μl of dinitro salicylic acid reagent was added to both control and tests [15]. They were kept in boiling water bath for 5 min. The absorbance was recorded at 540 nm using a spectrophotometer, and percentage inhibition of α-amylase enzyme was calculated using the formula:</p><p>Suitable reagent blank and inhibitor controls were simultaneously carried out and subtracted.</p><!><p>A 0.5 ml of amylase was taken in a set of test tubes and incubated with 0.5 ml of inhibitor samples separately and incubated for 5 min at room temperature. Starch solution (substrate) was added in the range of 0.1 to 1.0 ml to all the tubes having enzyme and inhibitor sample. A set of control was maintained with enzyme and starch but without inhibitor sample. All the tubes were then incubated for 5 min at room temperature, and 1 ml DNS (dinitrosalicylic acid) reagent was added and boiled for 5 min to stop the reaction and to estimate reducing sugar generated as a product from enzyme action on starch. Optical density was read, and conc. of reducing sugar was calculated which is used to determine the activity of amylase [11,12].</p><p>The enzyme activity was expressed in international units. One international unit is the amount of enzyme required to convert one micromole of the substrate into the product at optimum condition.</p><p>Lineweaver-Burk plot was prepared with and without inhibitor. The plot provides a useful graphical method for analysis of the Michalis-Menten equation:</p><p>Taking the reciprocal gives,</p><p>Where V is the reaction velocity (the enzyme activity), Km is the Michalis-Menten constant, Vmax is the maximum reaction velocity, and [S] is the substrate concentration.</p><!><p>Lipase inhibitory activity of the methanol extract was tested by mixing 5,10,50,100 μg/ml of each concentration of methanol extract, 8ml of oil emulsion and 1ml of chicken pancreatic lipase followed by incubation of 60 minutes. The reaction was stopped by adding 1.5 ml of a mixture solution containing acetone and 95% ethanol (1:1). The liberated fatty acids were determined by titrating the solution against 0.02M NaOH (standardized by 0.01M oxalic acid) using phenolphthalein as an indicator [11].</p><p>Percentage inhibition of lipase activity was calculated using the formula:</p><p>Where A is lipase activity, B is the activity of lipase when incubated with the extract.</p><!><p>Lipase inhibitory activity of the different sample was tested by mixing 100μg of each with 1ml of pancreatic lipase and incubated for 10 min at room temperature, and 8 ml olive oil (substrate) emulsion was added to the mixture followed by incubation of 60 minutes. Different substrate concentration in the range of 1µM to 10 µM was maintained for the entire samples. The reaction was stopped by adding 1.5 ml of a mixture solution containing acetone and 95% ethanol (1:1). The liberated fatty acids were determined by titrating the solution against 0.01M NaOH (standardized by 0.01M oxalic acid) using phenolphthalein as an indicator. A solvent (DMSO) control was also maintained for each of the sample used to compensate for the effect of the solvent as an inhibitor [16].</p><p>One unit of lipase activity was defined as one µmol of fatty acid liberated from olive oil per minute by 1.0 ml of the sample under the standard assay conditions. The activity of lipase was carried out by the titrimetric method and calculated by the formula. Lineweaver-Burk plot was prepared with and without inhibitor. The plot provides a useful graphical method for analysis of the Michalis-Menten equation.</p><!><p>The percentage of inhibition of α-amylase was determined at 5,10,50 and 100 µM concentrations.</p><p>Then the IC50 values were calculated by the graphical method. The IC50 (µM) data were compared with the standard drug Acarbose (Table 2). Acarbose is the reference drug; data are expressed as mean ±S.E.M. (n = 3).</p><!><p>The percentage of inhibition of lipase was determined at 5, 10, 50 and 100 µM concentrations.</p><p>Then the IC50 values were calculated by the graphical method. The IC50 (µM) data were compared with the standard drug Orlistat.</p><p>-     Acarbose is a well-known drug for α-amylase inhibition. Acarbose (at a concentrations 5-100 μg/ml) showed α-amylase inhibitory activity from 9 to 60% with an IC50 value 126.5 ± 1.72 µM.</p><p>in Table 1, the compounds 5d (120.6 µM), 5e (111.5 µM) and 5h (124.0 µM) showed good IC50 values.</p><p>Orlistat is a commercial drug for lipase inhibition. It showed lipase inhibitory activity with an IC50 value 236.4±2.83 µM (Table 4). Orlistat is a hydrogenated derivative of lipstatin, and it is obtained from Streptomyces toxytricini. It forms a covalent bond with the active serine site of lipases and thus inactivates them to hydrolyze dietary fat. In the series of compounds synthesized, most of the molecules showed similar IC50 values as the standard, and few molecules such as 5c (142.3 µM), 5d (82.3 µM) and 5i (93.6 µM) showed better inhibition against lipase enzyme.</p><p>In a valuable discussion of how antagonists effects are analyzed [17], it was pointed out that antagonists (synthesized compound used in the present study) can only be studied in the presence of agonists (in the present study starch and olive oil for α-amylase and lipase enzyme used respectively). Also, it was argued that this made it natural to express an antagonist effect as a percentage reduction in the corresponding agonist effect. There is an obvious parallel here with the effects of enzyme inhibitors, which are likewise manifest only in the presence of substrates, and much of the analysis is likewise parallel [10].</p><p>The analysis we have presented shows first how IC50 can be determined and secondly how the resulting estimate can be used to characterize the inhibition. In this way, we hope to have brought   As it can be seen that several molecules in each scheme has shown better Km value than the standard drug (Table 8). This means the binding capacity with the substrate is good for the most of the molecules. Hence very small concentration is needed to achieve half of Vmax which will be beneficial as a drug.</p><!><p>All the synthesized coumarin derivatives were subjected to enzyme inhibition studies. Alphaamylase and lipase enzymes were selected for the study as they are the major cause of diabetes and obesity disease. The commercial drugs which are used to inhibit α-amylase enzyme are</p><p>Acarbose and for the lipase enzyme inhibition, Orlistat.</p><p>The percentage of inhibition in different concentrations (5, 10, 50,100 µM) were checked and IC50 values were calculated. It is revealed that scheme 23 and 24 compounds shown better inhibition (good IC50) against two enzymes. This may be due to the less flexible alkyl chain between hydrazone and coumarin moiety. Molecules with electron withdrawing functional groups like 'F', and 'Cl' (halogens) attached to the para position of benzene ring showed better inhibition among the series of compounds. Presence of hydrazone moiety in the derivative also may play in firm binding to enzyme and leading to better inhibition.</p>