faty
Get Complete Project Material File(s) Now! »
Antioxidants and their role in chemoprevention
Many mutagens act through generation of reactive oxygen species (ROS) which induce oxidative stress in living cells. Many mutations related to oxidative stress, or DNA damage and repair, have been identified in human disease syndromes (Beckman and Ames, 1998). Oxidative stress is involved in more than 100 common diseases including cancer, all inflammatory diseases (arthritis, vasculitis lupus etc.), autoimmune diseases, diabetes, emphysema, catactogenesis and macular degeneration, gastric ulcers, hemochromatosis, hypertension, heart diseases, and neurologic diseases (multiple sclerosis, Alzheimer‟s disease, Parkinson‟s disease, amyotrophic lateral sclerosis, muscular dystrophy etc.) (Sies, 1998, Schafer and Buettner, 2001). Oxygen free radicals or more generally reactive oxygen species (ROS) and reactive nitrogen species (RNS) are products of cellular metabolism and are common in biological systems. ROS and RNS harms living systems by inducing oxidative damage to cell structures and biomolecules such as lipids, nucleic acids and proteins. Normally there is a balance between the amount of free radicals generated in the body and the defence systems that scavenge or quench these free radicals, preventing them from causing deleterious effects in the body. When there is a shift or imbalance in the pro-oxidation and antioxidation homeostatic phenomena resulting from excessively high levels of these oxidative species in the body, either due to environmental conditions or being produced within the body, these free radicals, increase the burden in the body leading to oxidative stress which results in tissue injury and subsequent diseases (Finkel and Holbrook, 2000, Castro and Freeman, 2001). In recent years, there has been an increased interest in the application of antioxidants in the health sector (Adams and Adams, 2002). Widespread attention is currently being given to the identification of novel potent antioxidant compounds. Antioxidants are chemical substances that are capable of slowing or preventing the oxidation of other molecules. They have a wide application in the health sector due to the pathological role of free radicals in a variety of diseases and in the food sector because free radicals result in deterioration of food products (Benzie and Strain, 1999). Antioxidants are widely used as ingredients in dietary supplements in the hope of preventing diseases such as cancer and coronary heart disease. Prevention of cancer and cardiovascular disease has been linked to the intake of vegetables, fruits and teas rich in natural antioxidants (Johnson, 2001).
Alkaline single-cell gel electrophoresis/Comet assay
The protocol of Singh et al. (1988) was followed to evaluate the DNA damaging and protective effects of the four plant extracts. Microscope slides were pre-coated by spreading 300 µl 1% normal melting point (NMP) agarose in water evenly over the slides and allowing the agarose to harden. C3A cells at a density of 200000 cells/ml were treated with different concentrations of the test sample in 24 well plates and incubated for 24 hours at 37°C in a 5% carbon dioxide incubator. Based on cytotoxicity results in the neutral red uptake assay (NRU), 500 µg/ml was the highest sample concentration tested. The plant extracts were tested at concentrations of 500, 250, 125 and 62.5 µg/ml. Ethyl methane-sulfonate (EMS) at 1 mM was used as a positive control/mutagen. For mutagenicity testing, the cells were exposed to plant extracts alone and for antimutagenicity testing, the cells were exposed to a combination of the plant extracts and 1 mM EMS. After incubation, cells were trypsinised and 10 µl of a 10 000 cell suspension was added to 300 µl of 0.8% low melting point (LMP) agarose at 37°C. The mixture was spread on the precoated slides and allowed to harden under a coverslip on ice. Once the agarose had been prepared, the coverslips were removed and the microscope slides placed in lysis buffer overnight. Denaturation was conducted using the electrophoresis buffer at 17°C for 40 minutes. Electrophoresis was conducted using the same solution at 25V, current adjusted to 300 mA for 20 minutes. After electrophoresis, neutralization of the microscope slides was carried out in Tris buffer (pH 7.5) and dried. The slides were then placed in ice cold ethanol for 10 minutes and allowed to dry at room temperature. The gels were stained with 100 µl of 20 µg/ml ethidium bromide, left for 10 minutes and rinsed in distilled water. The slides were analysed using a fluorescence microscope supplied with a camera. The tail length, % DNA in tail and tail moment were determined using the PC image-analysis programme TriTek CometScoreTM. This programme allows measurement of tail length, percentage DNA in tail and tail moment as parameters to measure DNA damage in the comet assay. For mutagenicity testing, differences in parameters used to measure DNA damage (i.e. tail length, percentage DNA in tail and tail moment) were compared between sample concentration and solvent blank (negative control). For antimutagenicity testing, to measure antimutagenicity, the same parameters used for mutagenicity testing were used. In this case, the measurements in the test samples were compared to the positive control.
CHAPTER 1 Introduction
1.1. Background
1.2. Literature review
1.3. Aims and objectives
1.4. Hypothesis and Justification
1.5. Structure of the thesis
CHAPTER 2 The antioxidant activity and total phenolic content of 120 south african plant species as a preliminary step in identifying antimutagenic plant species
2.1. Introduction
2.2. Materials and methods
2.3. Results and discussion
2.4. Conclusion
CHAPTER 3 The mutagenic, antimutagenic and cytotoxic activities of 31 plant species with high antioxidant activity
3.1. Introduction
3.2. Materials and methods
3.3. Results and discussion
3.4. Conclusions
CHAPTER 4 Genotoxic and antigenotoxic activity of Combretum microphyllum, Leucospermum erubescens, Thespesia acutiloba and Kirkia wilmsii against 4-NQO, MMC and EMS
4.1. Introduction
4.2. Materials and methods
4.3. Results and discussion
4.5. Conclusions
CHAPTER 5 Isolation and chemical characterization of antimutagenic compounds from Combretum microphyllum
5.1. Introduction
5.2. Materials and Methods
5.3. Results and discussion
5.4. Structure elucidation of compounds isolated from C. microphyllum
5.5. Conclusion
CHAPTER 6 Antimutagenicity, cytotoxicity and antioxidant activity of n-tetracosanol, eicosanoic acid and arjunolic acid; compounds isolated from Combretum microphyllum
6.1. Introduction
6.2. Materials and methods
6.3. Results and discussion
6.4. Conclusions
CHAPTER 7
General conclusions
