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Bioassays and viability/cytotoxicity profiling
Bioassays are used in the investigation of natural products as part of the screening methods in drug development. The main objective of bioassays is to determine whether samples under study have any desired (or detrimental) biological activity. Screening with bioassays helps in the selection of plant materials for secondary testing and they are also incorporated in guided fractionation of crude extracts in order to isolate the bioactive component (Tringali, 2001).
The tetrazolium dye assay 3-(4,5-dimethylthiazol-2-yl)-2-5-diphenyltetrazolium bromide, commonly referred to as MTT, is one of the most popular assay used to measure cytotoxicity and cell proliferation. This assay is dependent on the ability of the mitochondrial enzymes in live cells to reduce the water soluble tetrazolium salt into a water insoluble purple formazan product. The healthy cells absorb the purple formazan and the absorption of light is done at wavelengths ranging from 540-570nm (Abd-Elazem et al., 2002; Kamatou et al., 2008; Liu and Nair, 2010; Twentyman and Luscombe, 1987).
MTT reduction is associated with mitochondria, cytoplasm and with non-mitochondrial membranes including the endosome/lysosome compartment and the plasma membrane (Berridge et al., 2005). The net positive charge on tetrazolium salts like MTT appears to be the predominant factor involved in their cellular uptake via the plasma membrane potential (Berridge et al., 2005). However, a second generation of tetrazolium dyes (MTS, XTT and WST-1) forms water-soluble formazans and require an intermediate electron acceptor for reduction which is characterised by a net negative charge and is therefore largely cell-impermeable (Berridge et al., 2005). In other words, MTT reduction occurs inside the cells (cytoplasm) while that of second generation dyes occurs outside the cell via plasma membrane electron transport.
Screening strategy
The overall methodology for this study is summarized in the schematic illustration shown in Figure 2.13. In the figure, sixteen plants were collected from the herbarium of the University of Pretoria and all voucher specimens deposited in the herbarium.
Organic extraction was carried out on fresh leaves of the plants to produce crude extracts and detailed explanations of the extraction procedure are provided in chapter 3 and appendix (Figure A3.1). Extracts were first screened for inhibitory properties against HIV-1 enzymes in direct enzyme assays. Only extracts from two plants, P. barbatus (chapter 4) and O. labiatum (chapter 5), inhibited HIV-1 protease (PR) by more than 50%. These two plants were further studied for other properties such as antiinflammatory and antioxidant behavior. Of the two plants that inhibited HIV-1 PR well, O. labiatum demonstrated the strongest inhibition and was selected for bioassay-guided purification. Purification yielded compounds that inhibited HIV-1 PR (detail in chapter 5 and section 10.2 of the appendix), activated latent HIV-1 (detail in chapter 6 and section 10.3 of the appendix) and demonstrated anti-inflammatory properties (chapter 7). Information on the binding kinetics of pheophytin-a to HIV-1 PR is provided in the appendix (Figure A5.14). The remaining fourteen plants had moderate to weak inhibitory properties in the direct enzyme assays (PR and RT) but three of these plants demonstrated promising inhibition against HIV-1 expression (p24 antigen) in a chronically infected HIV cell line. Extracts were also tested for activity against HIV-1 integrase (IN) and two plant extracts showed moderate inhibition (detail in chapter 8 and section 10.1.2 of the appendix).
CHAPTER 1
INTRODUCTION
CHAPTER 2
LITERATURE REVIEW
2.1 HIV/AIDS drug development
2.2 Latent HIV-1: a major hurdle in HIV treatment
2.3 Lamiaceae plants as sources of alternative anti-HIV agents
2.4 Status of plant products for treating HIV/AIDS
2.5 Natural products and the alleviation of oxidative stress in HIV/AIDS
2.6 Role of natural products in inflammation and immunomodulation
2.7 Common drug discovery techniques
2.8 Hypothesis
2.9 Purpose and objective of the study
2.10 Screening strategy
2.11 Outputs
CHAPTER 3
Lamiaceae plant extracts suppress HIV-1 expression in chronically infected monocytic cells
3.1 Introduction
3.2 Methods
3.3 Statistical analysis
3.4 Results
3.5 Discussion
3.6 Conclusion
3.7 Authors‟ contributions
CHAPTER 4
Inhibition of HIV-1 enzymes, antioxidant and anti-inflammatory activities of Plectranthus barbatus
4.1 Introduction
4.2 Material and methods
4.3 Results and discussion
CHAPTER 5
Anti-HIV activity of Ocimum labiatum extract and isolated
pheophytin-a
5.1 Introduction
5.2 Methods
5.3 Results
5.4 Discussion
CHAPTER 6
Triterpenoids from Ocimum labiatum activates latent HIV-1 expression: potential for use in adjuvant therapy
6.1 Introduction
6.2 Methods
6.3 Results
6.4 Discussion
6.5 Conclusion
CHAPTER 7
Ocimum labiatum demonstrates anti-inflammatory and antioxidant activities
7.1 Background
7.2 Methods
7.3 Results
7.4 Discussion
7.5 Conclusion
7.6 Authors‟ contributions
CHAPTER 8
CONCLUDING DISCUSSION AND FUTURE WORK
8. OVERVIEW
8.1 Chapter 3: Crude extracts inhibited HIV-1 replication
8.2 Chapter 4: Inhibition of HIV-1 protease, antioxidant and anti-inflammatory activities of Plectranthus barbatus (published in Journal of Ethnopharmacology)
8.3 Chapter 5: Potent HIV-1 inhibitor from Ocimum labiatum
8.4 Chapter 6: Latent HIV-1 activated by triterpenoids from O. labiatum
8.5 Chapter 7: Anti-inflammatory and antioxidant properties of Ocimum labiatum
8.6 Revisiting hypothesis
8.7 Hypothesis accepted
8.8 Other questions
8.9 Conclusion
CHAPTER 9
REFERENCES
CHAPTER 10
