Evaluation of antioxidant potential of pyrazolone derivatives.

In this article the antioxidant property of pyrazolones derivatives (PYZ1 to PYZ10) are reported. It was assessed by estimation of Malonaldehyde (MDA) and 4-Hydroxyl-2-noneal (4-HNE) as lipid peroxidation markers in myocardial ischemic reperfusion injury. The inhibition of lipid peroxidation was compared with the standard ascorbic acid. Among synthesized derivatives PYZ2, PYZ3, PYZ7, PYZ8, PYZ9, and PYZ10 were found to have potent antioxidant effect against MDA marker. In case of 4-HNE, PYZ4, PYZ5, PYZ6, PYZ7, PYZ8, PYZ9 and PYZ10 were found to have effective antioxidant activity and the rest of the compounds are moderately active. Comparatively PYZ7, PYZ8, PYZ9 and PYZ10 are having effective role to control both MDA and 4-HNE generation. All the experimental data were statistically significant at p< 0.05 level. Interestingly, beyond its NSAID property, this study explores the protective role of pyrazolone derivatives in ischemic heart injury.

INTRODUCTION

Pyrazolone derivatives have been used in patients with acute brain infarction since April 2001 in Japan[1]. These derivatives have been shown to be effective against brain edema after ischemia and reperfusion injury in animal models[2] and in stroke patients[3]. Moreover, it has been shown that these molecules have preventive effects on myocardial injury following ischemia and reperfusion in the rat heart[4] and in patients with acute myocardial infarction[5]. Several lines of evidence have demonstrated that oxidative stress plays an important role in the pathogenesis and development of cardiovascular diseases, including hypertension, hypercholesterolemia, diabetes mellitus, atherosclerosis, and myocardial infarction, angina pectoris, and heart failure[6]. Lipid peroxidation is found to cause formation of atherosclerotic plaques, neurological disorders, cancer, diabetes mellitus, myocardial infarction[7] and ageing. Lipid peroxidation is associated with ischaemia-reperfusion injury and hyperoxic lung injury. The peroxides derived from lipid peroxidation such as MDA (TBARS) and 4-HNE have been strongly associated with myocardial ischemic reperfusion injury[89]. It is expected that pyrazolone derivatives have beneficial effects on coronary artery and myocardial cells after ischemic and post ischemic myocardial injury in patients with ischemic heart diseases, including acute myocardial infarction and angina pectoris. Some animal studies using acute myocardial ischemic-reperfusion models have suggested the protective effects of pyrazolone derivatives on myocardial damage. By taking this research finding as evidence, the present study is focused to explore the antioxidant potential of pyrazolone derivatives.

MATERIALS AND METHODS

Pyrazolone derivatives were synthesized in our laboratory by reported procedure[10], and used as test drugs in the experiments at 100 mg/ kg body weight. The physical data of the synthesized compounds have been given in Table. 1. Thiobarbituric acid (TBA) was obtained from Loba Chemie, India. 2, 4-dinitrophenyl hydrazine (DNPH) and 1, 1, 3, 3-tetramethoxy propane (TMP) were obtained from Sigma Chemicals, USA. Ferrous sulphate, trichloroacetic acid (TCA), hydrogen peroxide, ascorbic acid, potassium dihydrogen phosphate, potassium hydroxide, hexane, methanol and HCl were of analytical grade and obtained from Ranbaxy Fine Chemicals. 4-HNE (4- Hydroxy- 2- Noneal) was obtained from Ranbaxy Ltd. As a gift sample. All other chemicals and reagents used were of analytical grade.

Table 1

Physical data of the synthesized compounds

Experimental animals

Toxicological studies of the pyrazolone derivatives (as suspension in 0.5% w/v carboxy methylcellulose) were carried out by standard method in oral dose of 100 to 1500 mg/ kg body weight in albino mice. The LD50 of the test compounds was found at 1000 mg/kg.b.w. The one tenth of the LD50 (100mg/ kg) was considered to be the dose of test compounds. Male albino rats, weighing between 150-200gm were included in the study. Rats were housed in the departmental animal house at an ambient temperature of 25°C, under a 12 h dark -12 h light, cycle, for the whole period of the study. Experiments were carried out according to the guidelines of the animal ethics committee of the institute. Animals were fasted overnight and were divided in to twelve group's i.e. control, standard and different test groups each consisting of three animals. Rats in the control group received the vehicle solution without drugs, rats in the standard group received the standard ascorbic acid (100mg/ kg p. o) and the pyrazolone derivatives were administered orally to the test group of rats.

In vitro myocardial ischemic-reperfusion injury[11]

After 48 h the rats were anaesthetized with ether, the chest opened and the heart along with one cm of ascending aorta attached was quickly removed and dipped in ice-cold saline. The hearts were then mounted on Langendorff's apparatus and perfused with Krebs's Hensleit (K-H) buffer at a constant pressure of 60-70mm Hg at 3TC, and aerated with a mixture of O2 (95%) and CO2 (5%). Following an initial period of 5 min of stabilization, the flow was stopped for 9 min (ischemia) followed by perfusion with K-H buffer for 12 minutes (reperfusion). Then the heart was removed from the apparatus and subjected for the biochemical estimation.

In vitro antioxidant activity of pyrazolone derivatives Estimation of TBARS[12]

TBARS activity in the myocardium was determined by a modified version of the method described by Okhawa et.al. 1979. Hearts were homogenized in 10% trichloroacetic acid at 4°C. 0.2 ml homogenate was pippeted in to a test tube followed by the addition of 0.2 ml of 8.1% sodiumdodecyl sulphate (SDS), 1.5 ml of 20% acetic acid (pH 3.5) and 1.5 ml of 0.8% TBA. All tubes were boiled for 60min at 90°C and then cooled on ice. 1.0 ml double distilled water and Sml of n-Butanol: pyridine (15:1v/v) mixture was added to the tubes and centrifuged at 4000 rpm for 10 minute. The absorbance of developed colour in organic layer was measured at 532nm. TBARS activity was determined, from the standard curve of TBA adduct formation when various concentration of commercially available 1, 1, 3, 3--tetramethoxypropane was subjected to the above procedure (Fig. 1 and 2). The concentration of MDA was expressed in nM.

Fig. 1

Standard calibration curve of MDA estimation

Fig. 2

Antioxidant activity of Pyrazolone derivatives on MDA suppression model C-Control, Std-Ascorbic acid

Estimation of 4-HNE[13]

The heart homogenate was prepared as the procedure mentioned above. 2ml of filtrate was taken in a 13 x 100mm glass tube with cap. 1ml of DNPH was added to all the tubes containing heart homogenate, mixed thoroughly and set aside for lh to react with 4-HNE. Then the formed adduct of 4-HNE and DNPH was extracted by hexane, which was evaporated under argon at 40°C. After cooling, 2 ml of methanol was added to all the samples and the absorbance was measured at 350 nm in the spectrophotometer. The quantity of 4-HNE was calculated by linear regression analysis. The cone. Of 4-HNE present in myocardial tissues was expressed in nM (Fig. 3 and 4).

Fig. 3

Standard calibration curve of 4-HNE estimation

Fig. 4

Antioxidant activity of Pyrazolone derivatives on 4-HNE suppression model C-Control, Std-Ascorbic acid

RESULTS AND DISCUSSION

The experimental study was based on the estimation of MDA and 4-HNE and their suppression by the pyrazolone derivatives are presented in Table-2. From the experimental results, it has been proved that the pyrazolone derivatives have significant antioxidant activity. The quantification of MDA and 4-HNE can be directly correlated with the lipid peroxidation inhibition capacity of the pyrazolone derivatives. The toxic radicals’ quantification is also an indicator to monitor the overall progress of lipid peroxidation which is associated with myocardial ischemic reperfusion injury. The antioxidant activity of pyrazolone derivative was compared with standard antioxidant (ascorbic acid). The results were analyzed statistically and found significant at P<0.05 level.

Table 2

In vitro antioxidant activity of pyrazolone derivatives by MDA and 4-HNE model