Evaluation of In Vitro Anti-inflammatory Activity of Azomethines of Aryl Oxazoles

Ability to inhibit erythrocyte hemolysis is often used as a characteristic of the membrane stabilising action of chemical compounds. Azomethines of aryl oxazoles were evaluated for anti-inflammatory by in vitro hemolytic membrane stabilising study. The effect of inflammation condition was studied on erythrocyte exposed to hypotonic solution. In this in vitro method the membrane stabilising action leads to anti-inflammatory activity and was compared with that produced by diclofenac sodium as the reference standard. Results of the evaluation indicate that the synthesised compounds found to exhibit membrane stabilising activity.


Introduction
Compounds with membrane stabilising properties are well known for their ability to interfere with the release of phospholipases that trigger the formation of inflammatory mediators likes prostaglandins and leukotrienes etc. through COX (cyclooxygenase) and LOX (lipoxygenase) pathway Membrane stabilisers are chemical compounds which depress excitability in nerve and muscle without causing obvious changes in the resting membrane potential 1 .These compounds protect erythrocytes against hypotonic hemolysis and stabilise the lysosomes and cell organelles under in vitro conditions.The literature review suggests that the heterocyclic compounds are known to possess anti inflammatory activity [2][3][4] , hence the antiinflammatory potential of the synthesised compounds were evaluated by erythrocyte membrane stabilising method.The effect of inflammation condition was studied by erythrocyte exposing to hypotonic solution, the results were compared with that of the standard diclofenac sodium.The synthesis, experimental and spectral data are already sent for publication.The structure of the synthesised compounds are as follows.

Experimental
Erythrocyte suspension [5][6][7][8] Blood was collected from median cubital vein of healthy volunteers.The blood was mixed with isosaline and centrifuged at 3000 rpm.The packed cells were further washed with isosaline and the sedimented erythrocytes were collected.A suspension of 2%v/v of erythrocyte in isosaline was prepared.

Procedure (hypotonic solution-induced haemolysis)
This test was done by the following method 9,10 with slight modification.Blood was collected from median cubital vein of healthy volunteers.The blood was mixed with isosaline and centrifuged at 3000 rpm.The packed cells were further washed with isosaline and the sedimented erythrocytes were collected.A suspension of 2%v/v in isosaline was prepared.
The assay mixture comprises of hyposaline (2 mL), sodium phosphate buffer at pH 7.4 (1 mL), varying volumes of drugs in the concentration ranging from 88.88-444.44 µg/mL and erythrocyte suspension (0.5 mL) were made up with isosaline to give a total assay volume of 4.5 mL.The control was prepared as above by omitting the drug sample.The reaction mixtures were incubated at 56 0 C for 30 minutes.The tubes were cooled under running water followed by centrifugation at 5000 rpm.The supernatant was collected and the absorbance of the supernatant liquid at 560 nm.The results are given in the Table 1.

Calculation
The percentage of membrane stability was estimated using the expression: Percentage inhibition of hemolysis = C-T/C * 100, Where, C -absorbance of control, T -absorbance of test sample

Results and Discussion
The activity and the viability of the cell depend upon the integrity of the cell membrane.Exposure of erythrocyte cells to injurious substances such as hypotonic medium results in the lysis of membrane associated with hemolysis and oxidation of haemoglobin.The hemolysis effect of hypotonic solution is related to excessive accumulation of fluid within the cells and damaging of cell membranes.The damage of erythrocytes will further destruct the cell and make it vulnerable to secondary damage through free radical induced lipid peroxidation.It is therefore expected that compounds with membrane stabilising properties should offer significant protection of cell membrane.
In the study of membrane stabilization by hemolytic method the synthesised compounds at concentration range of 88.88-444.44 µg/mL were found to protect significantly the erythrocyte membrane against lysis induced by hypotonic solution.The compounds 7a,7b, 7c exhibited significant membrane significant activity when compared that of the reference; the other compounds 7d, 7e had moderate activity.It was also observed that all the compounds showed dose dependent inhibition of hemolysis.Hence all the synthesised compounds are potential to anti inflammatory activity by erythrocyte membrane stabilisation method.