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Title of Thesis
Oxidative Stress Status Evaluation at Chemical and Biochemical Levels using Novel/Improved and Contemporary Methods

Muhammad Nadeem Asghar
Institute/University/Department Details
GC University, Lahore, Pakistan
Number of Pages
Keywords (Extracted from title, table of contents and abstract of thesis)
N,N-dimethyl-p-phenylenediamine (DMPD), potassium persulfate, DMPD decolorization, citrullus colocynthis

A number of analytical methods exist for the evaluation of oxidative stress. Some of these directly measure the accumulation of oxidizing species or of oxidized products, while other methods measure the defence capacity of the organism against oxidation, which is referred to as the specific or the total antioxidant capacity (TAC). Amongst these TAC methods, TEAC (Trolox Equivalent Antioxidant Capacity) assays which involve the use of radicals of compounds such as N,N-dimethyl-p-phenylenediamine (DMPD) or 2,2’-azinobis(3- ethylbenzothiazoline-6-sulphonic acid) (ABTS), have attracted the attention of biochemists and analysts due to their operational simplicity. These methods are based upon the spectrophotometric measurement of the antioxidant activity of body fluids, herbal extracts, fruits, vegetables and other food samples and have certain limitations with regard to their application range, stability and environmental fate. In the present study we have improved upon the validity/applicability of two already existing methods and explored two new and affordable decolorization assays with greater reproducibility and application range. At the cellular level, the effect of xenobiotics on E.coli, used as a model organism, has also been studied.

An improved N,N-dimethyl-p-phenylenediamine (DMPD) radical cation decolorization method was developed for the measurement of the antioxidant activity in food and biological samples. The radical cation (DMPD.+) is generated through reaction between DMPD and potassium persulfate and is subsequently reduced in the presence of hydrogen-donating antioxidants. This assay has a clear advantage over the currently used version of the DMPD assay (the DMPD/FeCl3 assay) on a number of counts. The chemistry involves the generation of a more stable DMPD radical monocation by a reaction with potassium persulfate. It does not involve Fe(II) ions for the generation of the radical cations, which through Fenton’s Reaction can cause negative deviations in the measurement of the antioxidant activity of biological samples. The new experimental procedure is fast, inexpensive and ensures sensitivity along with reproducibility in the measurement of antioxidant activity of hydrophilic and lipophilic compounds and thus promises use in the screening of large numbers of fruit samples. The effectiveness of the new DMPD decolorization assay was verified by evaluation of antioxidant capacity of different citrus fruits.

It was applied to the measurement of the antioxidant activity of a variety of extracts of fresh and stored seeds, peel and pulp of Grewia asiatica. The results were compared with those obtained by the ABTS radical cation decolorization assay. It was observed that the antioxidant activity of G. asiatica extracts is significantly affected when the samples are stored at 0 oC for the duration of one month. This may possibly be due to degradation and/or transformation of phenolic constituents of the extracts into other products. High TEAC values of fresh samples indicate potent in vitro antioxidant activity of G. asiatica extracts, which may also be related to its use in the traditional medicinal system against diseases such as diabetes mellitus, hepatitis etc. Statistically a positive correlation was found between quantity of phenolic contents and the antioxidant activity.

The well known ABTS (2.2’-azinobis(3-ethyl benzothiozoline-6-sulfonic acid) radical cation decolorization assay, reported in hundreds of research papers, has been applied under different pH conditions for the determination of the TEAC of biological and food samples. Due to the innate chemical nature of ABTS·+, it is unstable at physiological pH. Further, its sensitivity is lost in an acidic medium. No single method was claimed to cover both the drawbacks of the ABTS procedure. An improved ABTS decolorization method was developed for the measurement of antioxidant activity of human plasma and herbal extracts with greater stability and sufficient sensitivity. A relatively stable ABTS radical cation was generated through reaction between ABTS and potassium persulfate in sodium acetate buffered solution at pH 6.5 (20 mM). The developed method was successfully applied towards the measurement of the antioxidant activity of plasma samples and extracts of Terminalia arjuna and Cuscuta reflexa. The modified assay proved to be highly reproducible. TEAC values for plasma samples ranged between 1.410-2.025 mM with a percent relative standard deviation (% RSD) of 1.64 and 1.72-1.98 and a mean % RSD of 1.27 for the already reported and the modified versions of the ABTS assay respectively.

A novel, rapid and cost-effective Trifluoperazine dihydrochloride (TFPH) decolorization assay was developed for the measurement of the antioxidant activity of fruits that naturally exhibit a low pH. A chromogenic reaction between TFPH and potassium persulfate at low pH produced an orange-red radical cation with a maximum absorption at 502 nm in its first order derivative spectrum. A linear inhibition of color production was observed with linearly increasing amounts of antioxidants, with correlation coefficients (R2) ranging from 0.999 to 0.983. The antioxidant capacities of synthetic solutions of standard antioxidants were evaluated by comparison with the standard inhibition curve using Trolox as the standard. Comparison of the antioxidant capacities determined using this newly developed TFPH assay and the ABTS-Persulfate decolorization assay not only proved the efficacy and sensitivity of the developed procedure but also yielded promising results for the determination of the antioxidant activity of a range of antioxidants found at a low pH. The newly developed assay is many times more cost effective and requires less time for the preparation of working solution than the ABTS assay. This suggests that the antioxidant activity of samples with a naturally low pH should preferably be evaluated through an assay in which the radical cation itself has been stabilized at low pH.

Another new, quick and economical decolorization assay based upon the generation of a radical cation made from promathazine hydrochloride (PMZH) is described for screening of antioxidant activity of plants and herbal extracts. PMZH radical cations, which were produced through a reaction between PMZH and potassium persulfate in phosphoric acid medium, have a maximum absorption at 515 nm in their first order derivative spectrum. The concentrations of chromagen and potassium persulfate were optimized (final concentration of PMZH and K2S2O8 were 0.166 mM and 0.11 mM respectively) for better stability and sensitivity of the radical cation produced. A good linear correlation was found between the inhibition of color formation and the increasing amounts of standard antioxidants, with correlation coefficients ranging from 0.989 to 0.999. The newly developed assay was employed to evaluate the antioxidant capacity of Citrullus colocynthis L. and Artemisia absinthium extracts. The proposed assay involved a more stable radical cation and required only 1 hr for preparation of a working solution as compared to the ABTS radical cation decolorizaion assay which was reported to be less sensitive at low pH and required 12-16 hours for preparation of the radical cation solution. Other assays also employed to evaluate the antioxidant potential and radical scavenging capacities of the extracts were the ferric reducing antioxidant power, 2,2’-diphenyl-1-picrylhydrazil radical scavenging, total phenolic contents assay, the total flavonoid contents and metal chelating activity assays, and the lipid peroxidation value in linoleic acid emulsion systems.

Extracts of medicinal plants have been reported to be very effective in the control of chronic diseases due to their high contents of antioxidants. Litchi chinensis Sonn. is used for its hypoglycemic activity in the traditional medicinal system of the Indian subcontinent. The antioxidant activity of the aqueous and organic extracts of leaves, stem and fruit pulp of L. chinensis was investigated using the 2,2’-azinobis(3-ethylbenzothiazoline-6-sulpohonic acid) (ABTS) decolorization assay, the ferric reducing antioxidant power (FRAP) assay, the 2.2’- Diphenyl-1-picrylhydrazil (DPPH) assay, the total phenolic content (TPC) assay and the total antioxidant activity assay.

In the second part of research work, the actual response of cellular systems towards an artificially created stress environment was studied. E.coli was taken as a model organism and pesticides were used as the stress inducing compounds. Logarithmically growing batch cultures of Escherichia coli were exposed to sub-lethal concentrations of pyrethroid and carbamate pesticides of four different technical grades. This induced the formation of 17 to 20 stress proteins, as observed by two demensional polyacrylamide gel electrophoresis. Two dimensional polyacrylamide gel electrophoresis was performed to resolve the proteins. Visualization of the protein spots by rapid silver staining resolved these 17-20 stress proteins which were absent in the standard protein profile of E.coli. On average 29% of these stress proteins were unique to each of the stress inducing agents, while the remaining number overlapped with those of other pesticides. The iso-electric points (PIs) and molecular weights of the proteins were determined by comparison with protein markers of known PIs and molecular weights. Furthermore, upon comparing the pesticide-induced proteins within the same class and between the two different classes (pyrethroid and carbamate), it was apparent that the gene or set of genes responsible for stress expression are also the same, irrespective of the chemical nature of the substituents of the pesticides. The analysis of stress proteins will aid the development of more sensitive techniques for pollutant analysis. The unique proteins could be purified to enable quick and sensitive detection of xenobiotics in the environment with an antibody based strip method.

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S. No. Chapter Title of the Chapters Page Size (KB)
1 1 Introduction 1  




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  1.1 What are Oxidative and Nitrosative Stresses? 1
  1.2 Terms Involved in Oxidative Stress Chemistry 2
  1.3 Characterization of an Antioxidant 5
  1.4 Reactive Oxygen and Nitrogen Species 6
  1.5 Chemical and Biological Effects of Oxidative Stress 8
  1.6 The Radical Nature of Oxygen 9
  1.7 Fundamentals of Oxidative Stress 11
  1.8 Diseases Associated with Oxidative Stress 14
  1.9 Antioxidant Defence Systems 15
  1.10 Antioxidants as Supplements 20
  1.11 Food Sources of Antioxidants 20
  1.12 Metal Catalysts 21
  1.13 Non-Metal Redox Catalysts 21
  1.14 Immune Defence 22
  1.15 Measurement of Oxidative and Nitrosative Stresses 22
  1.16 Oxidative Stress Status Evaluation at the Chemical and Biochemical Levels 26
  1.17 Plan of Work 42
2 2 Literature Survey 45
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3 3 Experimental 79  

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  3.1 Chemicals and Equipment 79
  3.2 Sample Preparation 80
  3.3 Procedures of Total Antioxidant Capacity Assays 86
  3.4 Semi-lethal Concentrations of E.coli for Different Pesticides and Preparation of Protein Lysate 94
  3.5 Two Dimensional Polyacrylamide Gel Electrophoresis 95
4 4 Results and Discussion 97  



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  4.1 Improved DMPD Radical Cation Decolorization Assay 97
  4.2 In Vitro Antioxidant Activity of Grewia asiatica using Improved DMPD•+ Decolorization Method and Standard ABTS•+ Decolorization Method 107
  4.3 Development of Trifluoperazine Dihydrochloride Radical Cation Decolorization Assay 115
  4.4 Improvement of ABTS Radical Cation Decolorization Assay 128
  4.5 Promethazine Hydrochloride Radical Cation Decolorization Assay 145
  4.6 In Vitro Radical Scavenging Activity and Antioxidant Potential of Aqueous and Organic Extracts of C. colocynthis and A. absinthium 153
  4.7 In Vitro Radical Scavenging Activity and Antioxidant Potential of Aqueous and Organic Extracts of Aerial Parts of Litchi chinensis Sonn 169
  4.8 Analysis of Pesticide-Induced Stress Proteins of E.coli 178
  5 References 188
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