Evaluation of (In-Vitro) Anti-Inflammatory Activity of Azomethines of Aryl Thiazoles

 

NiraimathiV*, Jerad Suresh A and Kokil A.R

Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College, Chennai –3. 

*Corresponding Author E-mail: vnm_anr@yahoo.co.in.

 

ABSTRACT:

The anti-inflammatory activity of azomethines of aryl thiazoles synthesised was evaluated by in-vitro membrane stabilisation study. The effect on inflammatory condition was studied on erythrocytes exposed to hypotonic solution. The anti-inflammatory activity was compared with diclofenac sodium as reference standard against the in-vitro model. Results of the evaluation revealed that the synthesised compounds exhibited membrane stabilising action leading to anti-inflammatory activity.

 

KEYWORDS: Azomethines, Thiazoles, Anti-inflammatory activity, membrane stabilising activity.

 

 


 

INTRODUCTION:

The membrane stabilizers interfere with the release of Phospholipases that trigger the formation of inflammatory mediators like Prostaglandin and Leukotriene etc through COX (cyclooxygenase) and LOX (lipooxygenase) pathway.

 

The term 'membrane stabilizer' was used by Shanes (1958) to designate chemical compounds  which reduce excitability in nerves and muscle without causing obvious changes in the resting membrane potential. These compounds also protect erythrocytes against hypotonic haemolysis and stabilize the lysosomes and other cell organelles under in -vitro conditions. The literature review indicates thiazoles and their Schiffˈs base are known to possess anti-inflammatory activity2. Hence azomethines of aryl thiazoles were evaluated for their anti-inflammatory activity by the in-vitro membrane stabilisation method. The effect of synthesised compounds on inflammatory conditions was studied on red blood cells exposed to hypotonic solution. The results were compared with diclofenac sodium as standard. The synthesis, experimental data, and the spectral data are already sent for publication.

 

The hetero compound synthesised includes

N4-(2-methylphenyl)-N2-[(Z)-phenylmethylidene]-1, 3-thiazole-2, 4-diamine (K1),

N2-[(Z)-(4-chlorophenyl)methylidene]-N4-(2-Methylphenyl)-1,3-thiazole-2,4-diamine(K2),

N4-(2-methylphenyl)-N2-[(Z)-phenylmethylidene]-1, 3-Thiazole-2, 4-Diamine (K3),

 

N4-(2-methylphenyl)-N2-[(Z)-(3-nitrophenyl) methylidene]-1, 3-thiazole-2, 4-diamine(K4),

N2-[(Z)-(4-methoxyphenyl)methylidene]-N4-(2-methylphenyl)-1,3-thiazole-2,4diamine(K5).

 

Structures of the synthesised compounds are as follows:

 

 


Table I:

Percentage inhibition of haemolytic activity ( membrane stabilisation)

Conc in µg/mL of std and test compounds

STD

K1

K2

K3

K4

K5

88.88

54.24

48.91

40.63

24.46

29.29

27.84

177.77

66.83

58.26

49.64

31.48

36.99

30.31

266.66

76.27

61.74

65.38

37.29

50.61

43.09

355.55

79.67

67.07

68.52

47.94

61.99

47.69

444.44

80.87

72.15

77.23

57.62

70.46

55.59

 


 

EXPERIMENTAL:

Requirements:

·         Hyposaline – 2mL

·         Sodium phosphate buffer – 1mL(pH 7.4)

·         Erythrocyte suspension – 0.5mL

·         Varying concentrations of drug – (88.88-                444.44µg/mL).

 

Erythrocyte Suspension:

Blood from the median cubital vein of healthy volunteers was collected and mixed with isotonic saline which was centrifuged at 3000 rpm. The packed cells were further washed with isotonic saline and the sedimented erythrocytes were collected. A suspension of 2%v/v in isotonic saline was prepared.

Procedure:4

The assay mixture comprises of hyposaline (2mL), sodium phosphate buffer at pH 7.4 (1mL) and varying volumes of drugs in the concentration ranging from 88.88µg/mL to 444.44µg/mL. To the mixture erythrocyte suspension (0.5mL) was added and made up with isotonic saline to give a total assay volume of 4.5mL.The control was prepared as above omitting the drug sample. The reaction mixtures were incubated at 56şC for 30 minutes. The tubes were cooled under running water followed by centrifugation at 5000rpm. The supernatant liquid was collected and the absorbance of the supernatant liquid was measured at 560nm.

 

The percentage of inhibition of haemolysis was estimated using the following expression and the results are given in Table I:

 

The percentage inhibition of haemolysis = C-T/C*100

Where,

C - Absorbance of Control

T – Absorbance of Test Sample

 

RESULTS AND DISCUSSION:

The activity and viability of cells depends on the integrity of the cell membrane, exposure of erythrocytes to injurious substances such as hypotonic medium results in lysis of membrane accompanied by haemolysis and oxidation of haemoglobin. The haemolytic effect of hypotonic solution is related to excessive accumulation of fluid within the cell resulting in the rupture of cell membrane. Such injury to erythrocytic membrane will further render the cell more susceptible to secondary damage through free radical induced lipid per oxidation. It is therefore expected that compounds with membrane-stabilizing properties, should offer significant protection of cell membrane against injury.

 

The percentage inhibition of the haemolytic activity (membrane stabilizing action) of the synthesized compounds is presented in Table (I). The compounds K2, K1, K4 were found to have significant activity when compared with the standard. The compound K3 and K5 shows moderate activity when compared to that of the standard. The results showed that the synthesized compounds are potential anti-inflammatory agents.

 

ACKNOWLEDGEMENTS:

The authors are sincerely thankful to Department of Pharmaceutical Chemistry, College of Pharmacy, Madras Medical College, Chennai-3.

 

REFERENCES:

1)        Hiroshi Kato,Kazuo Tanaka,Masaki Ohta.Bulletin of the chemical society of Japan. Vol.39; 1966:1248-1253.

2)        Satsangi, R.K., Zaidi, S.M., MisraV.S. 1-(4-Substitutedthiazol-2-yl) hydantoins as anti-inflammatory and CNS-active agents. Pharmazie.38; 1983:341±342.

3)        Alagarsamy et al.Reported the synthesis, anti-tubercular,antibacterial and antifungal activities of 3-(pyridyl-4-carbonylamino)spiro(3H-indol-3,2-thiazolidine)-2,4’(1H)-dione.

4)        Chinnasamy velmurugan, Balasubramanian ,vivek et al.Evaluation of anti-inflammatory activity of ethanolic and acetone extracts of Bauhina Variegate.Int J Pharm Sci Bio. 1(3); 2010:138-140.

5)          Augusto O, Kunze KL, Montellano PR.N. phenylprotoporphyrin formation in the haemogolobinphenylhydrazine reaction.J Biol Chem1982:257:6231-41.

6)          Ferrali M, Signorni C, Ciccoli L, Comporti M. Iron release and membrane damage in erythrocytes exposed to oxidizing agents, phenylhydrazine, divicine and isouramil. Biochem J 1992; 285:295-301.

7)        Aitadafoun M, Mounieri C et al. 4-Alkoxybenzamides as new potentphosholipase A2 inhibitors. Biochem Pharmacol.1996; 51:737-42.

8)        Hilmi orhan,Gonal sahin,In-vitro effects of NSAID and paracetamol on oxidative stress related parameters of human erythrocytes, Exp.toxic.pathol.2001;53:133-140

 

 

 

 

Received on 19.02.2011          Modified on 12.03.2011

Accepted on 21.03.2011         © RJPT All right reserved

Research J. Pharm. and Tech. 4(6): June 2011; Page 942-943