Relevance of reactive oxygen species and antioxidants in wound heaing

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General introduction

Wounds are physical injuries that result when the skin is broken or an opening occurs, with subsequent disturbance of its anatomy and function.The result is a loss of continuity of epithelium with or without the loss of underlying connective tissue. Badly treated or untreated wounds can become chronic and represent a significant burden in patients due to cost and duration of treatment.

Microbial infection

The wound forms a fertile ground for growth of microorganisms such as bacteria. Several microbial organisms, including Bacillus subtilis, Enterococcus faecalis, Escherichia coli, Klebsiella pneumonia, Pseudomonas aeruginosa, and Staphylococcus aureus have been isolated from various wounds.These microorganisms hinder wound healing by mainly prolonging theinflammatory process.

Inflammatory response

As stated earlier, the inflammatory response starts immediately after injury has occurred, with the infiltration of immune cells into the injured area. A normal inflammatory response is an acute response that resolves after removal of the inciting stimulus. However, when the normal inflammatory response progresses to a chronic response because of long-term inappropriate responses to a stimuli or the inability to remove offending agents, tissue damage or disease can occur. Apart  from the potential of chronic inflammation to promote extended wound healing, usually
due to chronic infections, it is also responsible for a number of inflammatory conditions such as rheumatoid arthritis, asthma and inflammatory bowel disease.

Models for assessing wound healing activity

Several models have been used to measure the efficacy of various wound healing compounds in a scientific manner. These models have been classified into in vivo and in vitro methods which are discussed below.

Declaration
Acknowledgements
Abstract
Chapter 1: General introduction
1.1. The burden and pathophysiology of wounds
1.2. Phases of wound healing
1.2.1. Haemostasis
1.2.2. Inflammatory phase
1.2.3. Proliferative phase
1.2.4. Remodelling phase
1.3. Barriers to wound healing
1.3.1. Oxidative stress
1.3.2. Microbial infection
1.3.3. Inflammatory response
1.4. Treatment of wounds
1.4.1. Pharmacologic treatment of wounds
1.4.2. Ethnomedicines
1.5. Models for assessing wound healing activity
1.5.1. In vivo models
1.5.2. In vitro models
1.6. Ghanaian medicinal plants selected for the study
1.6.1. Ethnomedicinal relevance of Aspilia africana
1.6.2. Ethnomedicinal relevance of Boerhavia diffusa
1.6.3. Ethnomedicinal relevance of Erythrina senegalensis
1.7. Aim of the study
1.8. Objectives
Chapter 2: Plant collection, preparation of extracts, and phytochemical analysis
2.1. Introduction
2.1.1. Usefulness of plants in healthcare
2.1.2. Methods for the extraction of phytochemical components
2.1.3. Phytochemical screening of plant extracts
2.1.4. Separation methods
2.1.5. Methods of identification
2.2. Aim and objectives of chapter
2.3. Materials and Methods
2.3.1. Plant material
2.3.1.1. Plant collection
2.3.1.2. Preparation of extracts
2.3.2. Phytochemical analysis
2.3.3. High performance liquid chromatography fingerprinting
2.4. Statistical analysis
2.5. Results and discussion
2.5.1. Yield of plant extracts
2.5.2. Phytochemical analysis
2.5.2.1. Aspilia africana
2.5.2.2. Boerhavia diffusa
2.5.2.3. Erythrina senegalensis.
2.5.3. UPLC-MS fingerprinting
2.5.3.1. Aspilia africana
2.5.3.2. Boerhavia diffusa
2.5.3.3. Erythrina senegalensis.
2.6. Conclusion
Chapter 3: Cytotoxicity evaluation
3.1. Introduction
3.1.1. Relevance of cytotoxicity evaluations
3.1.2. In vitro methods for assessing cytotoxicity
3.1.3. Cytotoxicity assays based on cellular activity
3.1.3.1. Measurement of DNA synthesis
3.1.3.2. Measurement of metabolic activity
3.1.3.3. ATP measurement
3.1.3.4. Measurement of proliferation markers
3.1.3.5. Cellular protein content
3.2. Aim and objectives of chapter
3.3. Methodology
3.3.1. Culture, maintenance and seeding of cells
3.3.2. Differentiation of THP-1 monocytic cells
3.3.3. Cytotoxicity determination
3.3.3.1. Cytotoxicity evaluation with SRB
3.3.3.2. Morphological observation
3.3.3.2.1. Light microscopy
3.3.3.2.2. Live-dead staining
3.4. Statistical analysis
3.5. Results and discussion
3.5.2. Boerhavia diffusa
3.5.3. Erythrina senegalensis
3.6. Conclusion
Chapter 4: Antioxidant and anti-inflammatory activity of extracts
4.1. Introduction
4.1.1. Relevance of reactive oxygen species and antioxidants in wound heaing
4.1.2. Methods for determining antioxidant activity
4.1.3. Inflammation and wounds
4.1.4. Relationship between xanthine oxidase and inflammation
4.2. Aim and objectives of chapter
4.3. Methodology
4.3.1. Antioxidant activity determination
4.3.1.1. ABTS radical scavenging activity
4.3.1.2. DPPH radical scavenging activity
4.3.1.3. Determination of effect on oxidative stress
4.3.2. Evaluation of xanthine oxidase activity
4.4. Statistical analysis
4.5. Results and discussion
4.5.1. Antioxidant activity
4.5.2. Effect on AAPH-induced oxidative stress
4.5.3. Xanthine oxidase inhibitory effect
4.6. Conclusion
Chapter 5: Effect of extracts on cellular migration
5.1. Introduction
5.1.1. Cells involved in wound healing
5.1.2. Cell migration models
5.1.3. Two-dimensional models
5.1.3.1. Cell exclusion assays
5.1.3.2. Chemotactic assays
5.1.4. Three-dimensional models
5.2. Aim and objectives of chapter
5.3. Methodology
5.3.1. Cell culture and maintenance
5.3.2. Scratch wound assay
5.3.3. Data acquisition
5.4. Statistical analysis
5.5. Results and discussion
5.5.1. Aspilia africana
5.5.2. Boerhavia diffusa
5.5.3. Erythrina senegalensis
5.6. Conclusion
Chapter 6: Antimicrobial effect of extracts
6.1. Introduction
6.1.1. Impact of infection on wound chronicity
6.1.2. Biofilms and resistance to treatment
6.1.3. Medicinal plants as sources of antimicrobials
6.2. Aim and objectives of chapter
6.3. Methodology
6.3.1. Microorganisms
6.3.2. Determination of zone of inhibition
6.3.3. Minimum inhibitory concentration
6.3.4. Minimum bactericidal concentration
6.3.5. Determination of effect on biofilms
6.3.5.1. Biofilm culture
6.3.5.2. Biofilm inhibition assay.
6.4. Statistical analysis
6.5. Results and discussion
6.5.1. Aspilia africana
6.5.2. Boerhavia diffusa
6.5.3. Erythrina senegalensis.
6.5.4. Antibiofilm activity
6.6. Conclusion
Chapter 7: General conclusions, limitations, and recommendations
7.1. Conclusions
7.2. Study limitations and recommendations
References

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