Amylase & Lipase Inhibitory Effects and Antioxidant Effects of Novel Oxazolines

Oxazoline play a major role in medicinal chemistry. These are five membered heterocycle with one double bond and oxygen and nitrogen as hetero atoms. There are many natural and synthetic molecules which contain oxazoline nucleus having biological significance. The nitrogen present in Oxazoline is basic in nature. The synthesis, structures and biological activities of oxazoline derivatives have long been focused of research interest of organic chemists in the field of medicine due to the potential biological activities exhibited by them [1]. Alpha-amylases hydrolyze complex polysaccharides to produce oligosaccharides and disaccharides which are then hydrolyzed by α-glucosidase to monosaccharides which are then absorbed through the small intestines into the hepatic portal vein. Inhibitors of α-amylase and/or α-glucosidase such as acarbose, miglitol and voglibose [2] are known to lower postprandial hyperglycemia (PPHG) in type 2 diabetic patients by delaying digestion and subsequent absorption of carbohydrates from the gut. Newer approaches for the treatment of obesity have involved inhibition of dietary triglyceride absorption via inhibition of pancreatic lipase.


Introduction
Oxazoline play a major role in medicinal chemistry. These are five membered heterocycle with one double bond and oxygen and nitrogen as hetero atoms. There are many natural and synthetic molecules which contain oxazoline nucleus having biological significance. The nitrogen present in Oxazoline is basic in nature. The synthesis, structures and biological activities of oxazoline derivatives have long been focused of research interest of organic chemists in the field of medicine due to the potential biological activities exhibited by them [1]. Alpha-amylases hydrolyze complex polysaccharides to produce oligosaccharides and disaccharides which are then hydrolyzed by α-glucosidase to monosaccharides which are then absorbed through the small intestines into the hepatic portal vein. Inhibitors of α-amylase and/or α-glucosidase such as acarbose, miglitol and voglibose [2] are known to lower postprandial hyperglycemia (PPHG) in type 2 diabetic patients by delaying digestion and subsequent absorption of carbohydrates from the gut. Newer approaches for the treatment of obesity have involved inhibition of dietary triglyceride absorption via inhibition of pancreatic lipase.
Prevention of digestion of digestive lipids could be an effective strategy for preventing systemic absorption of lipids. Any imbalance between the free radicals and antioxidants leads to produce a condition known as "oxidative stress" that results in the development of pathological condition among which one is diabetes. Most of the studies reveal the inference of oxidative stress in diabetes pathogenesis by the alteration in enzymatic systems, lipid peroxidation, impaired Glutathione metabolism and decreased Vitamin C levels. Lipids, proteins, DNA damage, Glutathione, catalane and superoxide dismutase are various biomarkers of oxidative stress in diabetes mellitus. Reactive oxygen (ROS) and nitrogen (RNS) species include superoxide (O2•−), hydrogen peroxide (H 2 O 2 ), hypochlorite (ClO−), hydroxyl radical (OH•), nitric oxide (NO), and peroxynitrite (ONOO−) [3]. In physiological conditions, mitochondria are the major site of intracellular ROS production, due to electron leakage along the respiratory chain; nevertheless, they can also arise from plasma membrane systems, endoplasmic reticulum, lysosomes, peroxisomes and cytosolic enzymes.
At low concentrations, ROS/RNS exert a multitude of biological effects, including immune-mediated defense against pathogenic microorganisms and intracellular signaling; conversely, high levels of these extremely reactive species can damage DNA, lipids, and proteins, thus leading to tissue injury and cell death Endogenous antioxidant compounds are urate, glutathione, ubiquinone, and thioredoxin; furthermore, some proteins (ferritin, transferrin, lactoferrin, caeruloplasmin) act as antioxidants, as they bind and sequester transition metals that may start oxidative reactions. Antioxidant enzymes are superoxide dismutase (SOD), glutathione peroxidase (GPx), glutathione reductase, glutathione S-transferase, catalase, thioredoxin reductase, peroxiredoxins (Prx), and NAD(P) H:ubiquinone oxidoreductase (NQO1). Obesity may develop oxidative stress and it is the major cause of metabolic syndrome.
Here we are making an attempt to find a lead molecule with desired effects like anti-amylase, anti-lypase and anti-oxidant effects.

Preparation of sample
Proposed compounds prepared by making reaction between substituted aromatic aldehydes and dimethylamine in presence of Pyridinium Hydrobromide PerBromide. The resultant mixture is washed with sodiumthiosulphate and successively with sodium chloride. The resultant product recrystallised from ethanol. The structure of obtained compounds characterized and screened for anti-amylase, anti-lipase and anti-oxidant effects.

Evaluation of Lipase Inhibitory Activity
Pancreatic lipase inhibitory properties have been extensively examined for the determination of the potential effect of these oxazoline derivatives as antiobesity agents. Acinar cells of pancrease secrete an enzyme pancreatic lipase and it releases fatty acids from the triglyceride skeleton at the C-1 and C-3 position. These fatty acids are incorporated into bile acidphospholipid micelles and further absorbed at the small intestine, and finally enter to the peripheral circulation as chylomicrons.
Interference with fat hydrolysis leads to decreased utilization of ingested lipids; hence, lipase inhibition reduces fat absorption. Lipase activity was assayed by turbidimetric method using olive oil-ethanol suspension. This suspension was prepared by mixing 1 ml of olive oil to 100 ml of ethanol and shaken vigorously. 1mL of this olive oil-ethanol suspension was added to 9 mL of 0.05 M Tris-HCl buffer, pH 8.0 containing 0.025M sodium deoxycholate. This emulsion was used as substrate. Reaction mixture containing enzyme and inhibitor (in requisite amount) was incubated at room temperature for 10 min. Reaction was started by addition of 1 ml of substrate. Incubated for 10 min at 37°C. The decrease in turbidity was measured at 660 nm. Inhibitors present in the reaction prevented the decrease of turbidity of the mixture. Suitable 'control' tubes were run parallel [4].

Evaluation of anti oxidant activity
Human body is rich in free radical. The ability of oxazoline derivatives to scavenge hydrogen peroxide was determined according to the method of Ruch. A solution of 40 mM hydrogen peroxide was prepared in phosphate buffer (pH 7.4).100 μg / ml prepared compound in distilled water were added to a hydrogen peroxide solution (0.6 mL, 40mM). Absorbance of hydrogen peroxide at 230 nm was determined after 10 minutes against a blank solution containing the phosphate buffer without hydrogen peroxide [5].

% Scavenged [H 2 O 2 ] = [(A C -A S )/AC] x 100
Here AC and AS are the absorbance of control and absorbance of test/standard respectively.

In -Vitro Alpha-amylase inhibitory assay
Alpha amylase and alpha glucosidases are responsible for the breakdown of oligosaccharides/disaccharides to monosaccharide. So inhibitor of this enzyme delay carbohydrate metabolism which results in marked decrease in glucose absorption. To 500 μl of (100μg/ml) test compounds, 500 μl of starch in phosphate buffer (pH 6.9) containing 6.7mM of sodium chloride was added. The reaction was initiated by adding 500 μl porcine pancreatic amylase and incubated at 37ºc. To the above mixture add 1ml of DNSA (1g of DNSA, 30g of sodium potassium tartarate and 20 mL of 2N sodium hydroxide) was added and made up to a final volume of 100 mL with distilled water and kept it in boiling water bath for 5 minutes and cooled to room temperature. The reaction mixture diluted with 10 ml of distilled water and absorbance was read at 540 nm. Blank tubes were prepared by replacing the enzyme solution with 500 μL in distilled water. Control, representing 100% enzyme activity was prepared in a similar manner, without sample. Maltose is used as standard [6]. All results were expressed as mean ± standard deviation (n=3). Significance of difference from the control was determined by dunnet test (one way ANOVA), Twenty five Oxazoline derivatives are prepared, characterized and among the tested compounds LJ 11 , LJ 12, LJ 15 , LJ 16 LJ17, LJ 22 , LJ 23 found to have maximum Amylase inhibitory effects (Figure 1 & 2). LJ2, LJ4, LJ8, LJ10, LJ11, LJ12, LJ15, LJ16, LJ17, LJ22, LJ23, LJ24 found to have maximum lipase inhibitory activity (Figure 3). LJ1, LJ7, LJ8, LJ10, LJ11, LJ12, LJ14, LJ15, LJ16, LJ18, LJ20, LJ23 (Figure 4) found to have maximum anti oxidant activity. Amylase inhibitory effects, that may be attributed to the presence of hydroxyl/ methoxyl/halogen/ nitro/dimethylamino functional groups group at 3rd/4th position in phenyl ring. The compounds with more lipase inhibitory effects didn't show any specific pattern of presence of certain functional groups. In the case of antioxidant activity screening phenyl ring attached with halogenated/ methylated/ hydroxylated showed promising activities.

Conclusion
There are many natural and synthetic molecules which contain oxazoline nucleus having biological significance. The synthesis, structures and biological activities of oxazoline derivatives have long been focused of research interest of organic chemists in the field of medicine. Due to the potential biological activities exhibited by them. There is some sort of association observed between Anti-oxidant and Lipase inhibitory effect. Among the substituent's Dimethoxyl, Dimethylamino and halogens enhanced Amylase inhibitory effect.