A CASE CONTROL STUDY OF OXIDATIVE STRESS IN TUNISIAN PATIENTS WITH ISCHEMIC HEART DISEASE

A CASE CONTROL STUDY OF OXIDATIVE STRESS IN TUNISIAN PATIENTS WITH ISCHEMIC HEART DISEASE

1. Khelil ^*1, M. Ben-Hadj-Mohamed ¹, L. Khlifi ¹, M. Ben Dbibis ¹, H. Chahed ¹, S. Ferchichi¹, S. Ernez ² and A. Miled ¹

Biochemistry Laboratory ¹, Cardiologylogy Department ², Farhat Hached hospital, Sousse, Tunisia.

ABSTRACT: Ischemic heart disease (IHD) is the major cause of morbid-mortality in most countries. The involvement of oxidative stress (OS) in the development of atherosclerosis, ischemic cardiopathy triggering phenomen, has been investigated. Serum total homocysteine (Hcy) concentration was measured by fluorescence polarization immunoessay. Plasma levels of substances reacting with thiobarbituric acid (TBARS) were carried out by fluorimetric method (Yagi). Erythrocyte activity of superoxide dismutase (SOD) and plasma total antioxidant status (TAS) were determined by a colorimetric method at 505nm and 600nm, respectively. Erythrocyte activity of catalase (CAT) was measured by colorimetric assay (Góth). Glutathione peroxidase (GPx) and the glutathione reductase (GR) erythrocyte activities were determined by a colorimetric method at 340 nm. Our study showed significant elevation of Hcy and TBARS levels in patients compared to controls. While we noted significant decrease in TAS levels, SOD, CAT, GPx and GR activities in patients compared to the control group. We also noted positive correlations between: Hcy-TBARS, SOD-TAS, CAT-TAS, GR-TAS, GPx-TAS, SOD-CAT and GPx-GR. However negative correlations between: Hcy-SOD, Hcy-CAT, Hcy-GPx, Hcy-GR, Hcy-TAS, TBARS-SOD, TBARS-CAT, TBARS-GPx, TBARS-GR and TBARS-TAS were founded. This unsuitability, elevated pro-oxidant rate parameters and decrease of antioxidant parameters in patients compared to controls allows supporting the hypothesis that OS is involved in the genesis and progression of atherosclerosis.

Hcy, TBARS, SOD, CAT, GPx, GR, TAS, Ischemic heart disease

INTRODUCTION: Inappropriate oxidation of biomolecules is a hazard associated with all aerobic life. Harmful oxidation is often mediated by reactive oxygen species (ROS) that are generated by a wide range of biological processes, including mitochondrial respiration and both enzymatic and non-enzymatic chemical reactions ¹. The fine balance between ROS and antioxidants - that “neutralize” ROS before their react with cellular components and alter their structure or function ² is disturbed when excessive amounts of free radicals (FR) are produced or antioxidant capacity is decreased ³.

There are two types of antioxidant substances, endogenous and exogenous; Endogenous antioxidants include superoxide dismutase (SOD), catalase (CAT), glutathione enzymes and uric acid. There are three forms of glutathione dependent antioxidant enzyme: GPX, GR and glutathione transferase, out of which assessed the activities of GPX and GR ⁴. Exogenous antioxidants witch derived from diet include vitamins A, C, and E ¹. The total antioxidant status (TAS) is potential activity mirror’s of the antioxidant system because effects of antioxidant components are additive ⁵. Pro-oxidant substances can be described as either FR species or non-radical species that mediate oxidation of several substances, such as homocysteine (Hcy) or by Substances reacting with thiobarbituric acid (TBARS) known as products of lipid peroxidation. The two major sub-groups of FR are ROS and reactive nitrogen species (RNS) ¹. This disturbance plays an important role in cardiac pathophysiology ³, one of the major health problems of developing countries of the world ⁶.

Ischemic heart disease (IHD) results from the interaction between two primary mechanisms: atherosclerosis and thrombosis ⁷.

Currently, an increasing number of studies suggest that levels of oxidative markers in body fluids correlate with atherosclerotic disease ³ and it’s a critical key in most steps of the atherogenosis ⁸.

Several studies support the hypothesis that OS was intimately linked with the pathogenesis of oxidative myocardial damage with consequential IHD ^{9, 10, 11}.

Our study attempted to assess OS in atherosclerosis and its contribution to the IHD, wich was accomplished by the evaluation of levels and correlations between Hcy, TBARS, SOD, CAT, GPX, GR and TAS.

 MATERIALS & METHODS:

Study population:

The present study had involved 298 patients (mean-age: 61±12 years) recruited, from the Cardiology Department of the University Hospital, Farhat Hached, Sousse-Tunisia. All cases fulfilled these inclusion criteria: presenting symptoms and signs suggestive of IHD such as chest pain and dyspnea supported by electrocardiogram and cardiac markers who presented within six hours. Patients having revascularization, Infection, cerebrovascular accident, transplantation, liver disease, renal failure, Cancer, splenomegaly, intermittent claudication and those with other major illness were excluded from the study. The control group consisted of 176 healthy volunteer subjects (mean-age: 46±7 years).

All patients and controls gave informed consent to participate in this study which was approved by the National Medical and Research Ethics Committee. The methods carried out in the study are in accordance with the approved relevant guidelines and regulations.

 Blood samples processing:

Venous blood samples from patients and healthy volunteers were collected in fluoride, heparinized and without anti-coagulant tubes.

Plasma glucose levels, renal profile (urea, creatinin, uric acid) levels and serum total cholesterol (TC), triglycerides (TG), high density lipoprotein cholesterol (C_HDL)  were measured with a colorimetric assay using an automated system (Cx5 and Cx9 Pro-Bechman Coulter-Fuller-Ton CA). Low density lipoprotein cholesterol (C_LDL) was calculated using Friedwald’s formula: C_LDL=TC–(C_HDL+TG/2.2) ¹².

Serum was used to determine hs CRP according to the instructions of the manufacturer using microparticular immunoenzymatic method (COBAS-INTEGRA 400, Roche) and Hcy levels by fluorescence polarization immunoassay (FPIA) from AxYM Abbott diagnostics.

Lipid peroxidation was estimated by the determination of plasma TBARS levels, according to the fluorimetric method of Yagi¹³.

The heparinized whole blood was used to determine the erythrocyte activities of SOD at 505 nm and GPX and GR at 340 nm (RANDOX Kits) by a colorimetric method, in Daytona – analyser.

Erythrocyte CAT activity was measured using the molybdate colorimetric method described by Góth et al.1991¹⁴.

SOD, GPx, GR and CAT activities were expressed in kilo unit per gram of hemoglobin (Hb).

Hb was determined using the spectrophotomertic method of David L. Drabkin 1932¹⁵ by (Beckman coulter LH750) Analyser.

TAS levels were determined in plasma by colorimetric method at 600 nm according to the method of Miller et al ¹⁶ (RANDOX Kits) in Daytona–analyser.

 Statistical Analysis:

Database management and statistical analyses were performed by the SPSS (Statistical Package for the Sociological Sciences) software, version 20. The Student’s test was used for differentiating averages and Pearson’s correlation coefficient to determine the relationships between parameters.  P values less than 0.05 were considered as significant.

RESULTS:

Anthropometric characteristics of the study groups are presented in Table 1.

The mean age’s±SD of the IHD patients and controls was significantly different. However, the body mass index (BMI) was significantly higher in subjects compared to the controls.

 TABLE 1: RISK FACTOR DISTRIBUTION IN THE POPULATION.

N: number of subjects, x: mean, SD: Standard Deviation, BMI:  Body Mass Index.

The comparison of biochemical parameter variations between IHD cases and controls is shown in Table 2.  Patients had significantly higher glucose, urea, creatinin, uric acid, CT, TG and C_LDL levels than the controls, while no significant differences in C_HDL levels compared to controls.

TABLE 2: BIOLOGICAL PARAMETER VARIABILITY IN PATIENTS AND CONTROLS.

N: number of subjects, x: mean, SD: Standard Deviation, p: significance (2-tailed), CT:  cholesterol, HDL: High Density Lipoprotein, LDL: Low Density Lipoprotein.

Hcy and TBARS rates were elevated in patients compared to controls. While we noted a significant decrease in SOD, CAT, GPx and GR activities and TAS levels in patients compared to the control group (Table 3).

TABLE 3: OXIDATIVE STRESS PARAMETER VARIABILITY IN PATIENTS COMPARED TO CONTROLS.

N: number of subjects, IQR: inter-quartil range, x: mean, SD: Standard Deviation, p: significance (2-tailed), TBARS: Substances reacting with thiobarbituric acid, SOD: superoxide dismutase, CAT: catalase, GPx: glutathione peroxydase, GR: glutathione reductase, TAS: Total antioxidant status.

Our study showed significant positive correlations between Hcy-TBAR: r=0.925;p<10^-3, SOD-TAS: r=0.658; p<10^-3, CAT-TAS: r=0.898; p<10^-3, GPx-TAS: r=0.408; p<10^-3, GR-TAS: r=0.278; p<10^-3, SOD-CAT: r=0.763; p<10^-3 and GPx-GR: r=0.860; p<10^-3 represented in respectively Fig. 1, 2 and 3.

FIG. 1: POSITIVE CORRELATION AMONG IHD PATIENTS BETWEEN HCY AND TBARS.

“It is a bottom line that represents the relationship between; Hcy (µmol/l) and TBARS (µmol/l) rates. Hcy: homocysteine, TBARS: substances reacting with thiobarbituric acid”.

However, significant negative correlations between Hcy-SOD, Hcy-CAT, Hcy-GPx, Hcy-GR, Hcy-TAS, TBARS-SOD, TBARS-CAT, TBARS-GPx, TBARS-GR and TBARS-TAS were observed (Table 4).

FIG. 2: POSITIVE CORRELATIONS AMONG IHD PATIENTS (SOD-TAS, CAT-TAS, GPX-TAS AND GR-TAS).

 “It is a bottom line that represents the relationship between; (a): SOD activity (U/g Hb) and TAS level (mmol/), (b): CAT activity (U/g Hb) and TAS level (mmol/l), (c): GPx activity (U/g Hb) and TAS level (mmol/l), (d): GR activity (U/g Hb) and TAS level (mmol/l). SOD: superoxide dismutase, CAT: catalase, GPX: Glutathione peroxydase, GR: Glutathion peroxydase, TAS: Total antioxidant status”.

FIG. 3: POSITIVE CORRELATIONS AMONG IHD PATIENTS (SOD- CAT AND GPx-GR).

 “It is a bottom line that represents the relationship between; (a): SOD and CAT activities (U/g Hb) and (b): GPx and GR activities (U/g Hb), SOD: superoxide dismutase, CAT: catalase, GPx: glutathione peroxydase, GR: glutathione reductase”.

 TABLE 4: NEGATIVE CORRELATIONS AMONG PATIENT GROUP.

r: Pearson correlation coefficient, p: significance (2-tailed), Hcy: homocysteine, TBARS: Substances reacting with thiobarbituric acid, SOD: superoxide dismutase, CAT: catalase, GPx: glutathione peroxydas, GR: glutathione reductase, TAS: Total antioxidant status.

 DISCUSSION: Increased serum Hcy levels in IHD patients compared to control a subject is in close conformity with other findings ¹¹.

Hcy are associated with risk accenting and increased mortality in the general population. The published literature indicates that Hcy is an independent atherosclerotic disease risk factor (RF) in coronary, cerebral and peripheral arteries and show a correlation between hyper homocysteinaemia and several atherosclerosis RF (hypertension, diabetes, smoking, dislepidemia ^{14, 17}.

Hcy can act either independently of atherosclerosis and thrombosis or by increase thrombosis ^{7, 18}.

Other studies support the hypothesis that Hcy is a prooxydant marker. In fact, the sulfhydryl groups in Hcy were oxidized to a disulfide, catalyzed by the transition metals which product several Oxygen free radicals (OFR) such as hydroperoxides (H₂O₂), and initiates lipid peroxidation responsible of endothelial injury ^{19, 20}.

Our findings show a significant increase in plasma TBARS levels. This is in accordance with those of khatibi F et al. High TBARS levels may be resulted from increase lipid peroxidation that’s marker of OS^{21, 22}.

Lipid peroxidation may increase endothelial permeability of triglycerides, which cause increased blood viscosity and possibly through oxidative damage of erythrocytes. Lipid peroxidation may also promote acute-phase reactions by increasing endothelial/monocyte interactions and release cytokines, which are mediator’s key of inflammatory reactions ^{23, 24}.

Positive correlation between Hcy and TBARS may be explained by the adverse effects of hcy witch involve oxidative damage to vascular endothelial cells and oxidative modification of low density lipoprotein, all there lead to atherosclerosis ^{19, 20}.

OFR such as superoxide anions (O^-) and hydroxyl radicals (OH^-), produced by reduction of oxygen, have been implicated in cardiac ischemic injury. In normal circumstances, they were removed from the different scavenger systems present in blood and tissues. In case of myocardial ischemia, which can lead to myocardial infarction, unstable angina and heart failure, excessive OFR may be generated ²⁵.

Erythrocyte SOD activity was significantly decreased in IHD cases which is in agreement with several studies such as those reported by Almzaiel A et al.^{11, 26, 27, 28}. SOD, FR scavenging enzyme, is the first line of cellular defense against oxidative injury ²¹. SOD is responsible for converting the superoxide radical to H₂O₂ ⁶ before interacting to form more reactive hydroxyl radical (SOH). This enzyme protects the red cells against O^- and OH^- mediated lipid peroxidation. Decrease in the activity of SOD could be due to inactivation of the enzyme by cross linking or due to exhaustion of the enzymes by increased peroxidation ²¹.

Thus, low level of SOD activity could reflect either a possible accumula­tion of H₂O₂ which has the potential to bring about oxidative tissue damage, or low capacity of defense against it. High OS status has been reported in IHD patients, even in stable cases ²⁵.

We have also observed a significant decrease in the activity of CAT in patients with IHD, as compared to controls. CAT is enzymes that catalyze the conversion of H₂O₂ to H₂O and O₂, using either an iron or manganese cofactor ²⁹. CAT is inactivated when counteracting these FR³⁰.

Moreover, treatment with antioxidant enzymes, SOD plus CAT, in the heart, protects it against these changes ^{31, 32, 33}.

Erythrocytes GPx and GR activities were significantly lowers in the patient’s compared to controls, which is in accordance with previous researches.

GPx, one of the major antioxidant enzymes, being added in both antioxidant enzymes indicated on the folding screen (SOD and CAT), plays a significant role in the H₂O₂ scavenging mechanism, and in maintaining the functional integration of the cell membranes. The rise in the activity of GPX could be due to its induction to counter the effect of increased OS ³⁴.

GR catalyses the reduction of oxidized glutathione (GSSG) to reduced glutathione form, (GSH). Cysteinyl residue of GSH offers a nucleophilic thiol, which is important in the detoxification of electrophilic mobilities and metabolically produced oxidizing agents and GSH is a substrate for the enzyme GPX and Glutathione transferase.

GSH is converted to GSSG is reduced back to GSH by an NADPH dependent enzyme glutathione reductase. In the present study GR levels were found to be significantly decreased in all the groups when compared with normal, which is concurrent with Jawalekar SL et al. A positive correlation is observed between GPX and SOD. GPx is negatively correlated with TBARS and Hcy ⁴.

However, it is known that GPx, as well as GR, can be inactivated by oxidant species. For instance, GPx is particularly susceptible to inactivation by myeloperoxidase-derived hypochlorous acid. Remarkably, the presence of myeloperoxidase as an oxidation catalyst and of hypochlorous acid modified proteins has been proven in human atherosclerotic lesions. Peroxides are cytotoxic for vascular cells, especially in the presence of redox-active transition metals, which are available in a catalytically active form in human atherosclerotic plaques.

The pivotal role of GPx in vascular antioxidant protection is further pointed out by the findings that CAT activity is lacking in human vascular cells and that SOD is poorly effective against human cell oxidant damage. Thus, the deficient GPx and glutathione redox cycle status of the atherosclerotic tissue may significantly weaken its antioxidant potential favoring oxidative stress and atherogenic processes, even in the presence of an apparently adequate property of low molecular-weight scavenging antioxidants. Consistently, our data indicate that the lack of GPx activity in atherosclerotic lesions may be associated with a more severe expression of atherosclerosis in humans ^{35, 36}.

The present study revealed a significant decrease in the levels of TAS. These results are in agreement with those reported by khatibi F et al ²².

One possible explanation of these findings is depletion of the antioxidant barrier as an effect of the long term OS ^{27, 28, 37}.

This study has some limitations which have to be pointed out. Our study did not compare results of non enzymatic antioxidant (ceruloplasmin, transferin). We did not assess the protein peroxidation by measuring carbonyl proteins to show another pro-oxidant effect. However, this will be the subject of ongoing studies.

 CONCLUSION: Our study supports the hypothesis that the generation of ROS is intimately linked with the initiation and progression of atherosclerotic plaque  which can lead to myocardial infarction, Unstable Angina or heart failure.

 CONFLICT OF INTERESTS: The authors declare that they have no competing interests.

 ACKNOWLEDGEMENTS: The authors are grateful to the patients and volunteers for their collaboration, to Dr Samia Ernez Hajri (Cardiology Department, Farhat Hached hospital, Sousse, Tunisia) and all the team for collecting blood samples from IHD patients and the staff of the Biochemistry Laboratory (Farhat Hached University Hospital, Sousse, Tunisia) for their precious technical support.

 REFERENCES:

1. Goszcz K, Deakin SJ and Duthie GG: Antioxidants in cardiovascular therapy: panacea or false hope? Frontiers in Cardiovascular Medicine 2015; 2:29.
2. Dey S, Sidor A and O'Rourke B: Compartment-specific Control of Reactive Oxygen Species Scavenging by Antioxidant Pathway Enzymes.  The Journal of Biological Chemistry 2016; 291: 11185-97.

3. Vichova T and Motovska Z: Oxidative stress: Predictive marker for coronary artery disease. Experimental & Clinical Cardiology2013; 18: 88-91.
4. Jawalekar S, Kulkarni U and Surve V: Role of oxidants and anti oxidants in patients with cardiovascular diseases. Asian Journal of Medical Sciences 2010; 2: 181-184.
5. Li Y, Browne RW and Bonner MR: Positive relationship between total antioxidant status and chemokines observed in adults. Oxidative Medicine and Cellular Longevity 2014; 6: 1-6.
6. Finegold JA, Asaria P and Francis DP: Mortality from ischaemic heart disease by country, region, and age: statistics from World Health Organisationand United Nations. European Journal of Cardiovascular Prevention & Rehabilitation 2013; 30: 168: 934-45.
7. Ben Alaya N, Ben Romdhane H and Delpeuch F: Modèle causal des cardiopathies ischémiques en Tunisie, CIHEAM 2002; 9: 5-118.
8. Pashkow FJ: Oxidative Stress and Inflammation in Heart Disease: Do Antioxidants Have a Role in Treatment and/or Prevention? International Journal of Inflammation 2011; 9: 1-9.
9. Octavia Y, Brunner-La Rocca HP and Moens AL: NADPH oxidase-dependent oxidative stress in the failing heart: From pathogenic roles to therapeutic approach. Journal of Free Radicals in Biology & Medicine  2012; 2: 91-297.
10. Almzaiel AJT: Oxidative stress and inflammation in ischemic heart disease: role of trace elements, oxidants and antioxidants. International Journal of Medical Sciences 2015; 2: 18-22.

11. Houston MC: New Concepts in the Diagnosis and Non-Surgical Treatment of Cardiovascular Disease. Internal Medicine 2014; 11: 1-8.
12. Friewald WT, Levy RL and Fredrickson DS: Estimation of the concentration of low density lipoprotein cholesterol in plasma without the use of the preparative ultracentrifuge. Clinical chemistry 1972; 18: 499−502.
13. Yagi K: Simple assay for the level of total lipid peroxides in serum or plasma. Methods in  molecular biology 1998; 108: 101-6.

14. Goth L: A simple method for determination of serum catalase activity and revision of reference range. Clinical Chimica Acta 1991; 196: 143-51.
15. Drabkin DL and Austin JH. Spectrophotometric studies I: Spectrophotometric constants for common hemoglobin derivatives in human, dog, and rabbit blood. The Journal of Biological Chemistry 1932; 98: 719-733.
16.  Miller NJ, Rice-Evans C and Davies MJ: A novel method for measuring antioxidant capacity and its application to monitoring the antioxidant status in premature neonates. Clinical Science London 1993; 84: 407-12.

17. Ganguly P and Alam SF: Role of homocysteine in the development of cardiovascular disease. Nutrition Journal 2015; 14: 1-6.
18. Tayal D, Goswami B and Koner BC: Role of homocysteine and lipoprotein (A) in atherosclerosis: An update. Biomedical Research 2011; 4: 391-40.
19. Mirdamadi A, Farzamnia H and Varzandeh P: Association between serum homocysteine concentration with coronary artery disease in Iranian patients. ARYA Atherosclerosis Journal 2011; 2: 63-67.
20. Guney T, Alisik M and Akinci S: Evaluation of oxidant and antioxidant status in patients with vitamin B12 deficiency. Turkish Journal of Medical Sciences 2015; 45: 1280-1284.
21. Verma A, Soni Y and Bishnoi L: C-reactive protein and homocysteine risk prediction in coronary artery disease. International Journal of Medical and Health Science 2015; 5: 39 – 415.
22. Khatibi F, Yaghoubi A and Rahbani M: Study of antioxidant enzymes, lipid peroxidation, lipid profile and Immunologic factor in coronary artery disease in East Azarbijan. International Journal of Medicine & Biomedical Research 2012; 2:147-152.
23. Kashinakunti SV, Kollur P and Kallaganada GS: Comparative study of serum MDA and vitamin C levels in non-smokers, chronic smokers and chronic smokers with acute myocardial infarction in men. Journal of research in medical sciences 2011; 8: 993-8.
24. Castellon X and Bogdanova V: Chronic Inflammatory Diseases and Endothelial Dysfunction. Aging and Disease 2016; 7: 81-9.
25. Rodrigo R, Libuy M and Feliu F: Molecular Basis of Cardioprotective Effect of Antioxidant Vitamins in Myocardial Infarction. BioMed Research International 2013; 2013:437613.
26. Gawron-Skarbek A, Chrzczanowicz J and Kostka J: Cardiovascular Risk Factors and Total Serum Antioxidant Capacity in Healthy Men and in Men with Coronary Heart Disease. BioMed Research International 2014; 2014:216964.
27. Jha S, Parchwani H and Ahmad: Antioxidant status and level of oxidants in patients of coronary heart disease. International Journal of Biomedical and Advance Research 2013; 04: 823-826.
28. Souiden Y, Mallouli H and Meskhi S: MnSOD and GPx1 polymorphism relationship with coronary heart disease risk and severity.Biol Res2016; 49: 22.
29. Kumar SV, Saritha G and Fareedullah Md: Role of antioxidants and oxidative stress in cardiovascular diseases. Annals of Biological Research 2010; 3: 158-173.
30.  Schenkel PC, Fernandes RO and Viegas VU: Catalase influence in the regulation of coronary resistance by estrogen: joint action of nitric oxide and hydrogen peroxide. Oxidative Medicine and Cellular Longevity 2014, 2014: 159852.
31. Dhalla NS, Golfman L and Takeda S: Evidence for the role of oxidative stress in acute ischemic heart disease: a brief review. Canadian Journal of Cardiology 1999; 15: 587-593.

32. Dhalla NS, Elmoselhi AB and Hata T: Status of myocardial antioxidants in ischemia–reperfusion injury. Cardiovascular Research 2000; 47: 446-456.
33. Nimse SB and Pal D: Free radicals, natural antioxidants, and their reaction mechanisms. RSC Advances 2015; 5: 27986-28006.
34. Priya V and Surapaneni K: Erythrocyte lipid peroxidation glutathione, ascorbic acid, vitamin e, antioxidant enzymes and serum homocysteine levels in patients with coronary artery disease. Journal of Clinical and Diagnostic Research 2008; 2: 1180-1185.
35. De Gioia L, Ciofani G and Mezzetti A: Glutathione-related antioxidant defenses in human atherosclerotic plaques. Circulation 1998; 97: 1930-1934.
36. Metta S, Basalingappa DR and Uppala S: Erythrocyte antioxidant defenses against cigarette smoking in ischemic heart disease. Journal of Clinical and Diagnostic Research 2015; 9: 08-11.

37. Lorente L, Martin MM and Perez-Cejas A: Association between total antioxidant capacity and mortality in ischemic stroke patients. Annals of Intensive Care 2016; 6: 39.
    

    ---

    How to cite this article:

    Khelil S, Ben-Hadj-Mohamed M, Khlifi L, Ben Dbibis M, Chahed H, Ferchichi S, Ernez S and Miled A: A case control study of oxidative stress in tunisian patients with ischemic heart disease. Int J Pharm Sci Res 2016; 7(11): 4449-55.doi: 10.13040/IJPSR.0975-8232.7(11).4449-55.

    ---

    All © 2013 are reserved by International Journal of Pharmaceutical Sciences and Research. This Journal licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 3.0 Unported License.

    ---