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Preparation of plant extracts
The extraction yield of the extracts from plant species highly depend on the solvent polarity, which determines both qualitatively and quantitatively the extracted compounds. The highest yields are usually achieved with ethanol, methanol and their mixtures with water, although other solvents (acetone or ethyl acetate) have been widely used for the extraction of polyphenols from plants. Ethanol and water are the most widely used solvents because of their low toxicity and high extraction yield with the advantage of modulating the polarity of the solvent by using ethanol / water mixtures at different ratios (Sineiro et al., 2008).
In the present study only ethanol was selected because of its similarity with water, especially with regard to polarity. Hundred and fifty grams of each plant part were extracted with two successive 500.0 mL portions of ethanol for 24 hours at room temperature. The extracts were concentrated to dryness at reduced pressure with a rotary evaporator at 40oC.
Antimycobacterial activity on M. smegmatis using the agar method
All the ethanol plant extracts were dissolved in 10% dimethyl sulphoxide (DMSO) in sterile Middlebrook 7H9 broth (Sigma-Aldrich, South Africa), to obtain a concentration of 500.0 mg/mL. The bacteria were carefully scraped and transferred into a sterilized glass tube containing a few glass beads (2 mm in diameter) and 50.0 mL of 7H9 broth base was added to the culture and then recovered for testing by growth in 7H9 broth base for 24 hours at 37oC.
Before streaking, the culture was adjusted to an optical density (OD) of 0.2 log-phase (an optical density value which would ensure that the bacteria was at the start of the log phase when the test commenced) at 550 nm using spectrophotometer, yielding 1.26 x 108 colony-forming units per millilitre (CFU/mL) (Salie et al., 1996; Newton et al., 2002).
The MIC of the ethanol extracts was determined by incorporating various amounts (5.0 – 200.0 mg/mL) of each into petri dishes containing the culture media. Before congealing, 5.0 mL of 7H11 agar medium containing the plant extract was added aseptically to each petri dish and swirled carefully until the agar solidified. The bacteria was streaked in radial patterns on the 7H11 agar plates containing the plant extracts, before incubating at 37oC for 24 hours (Mitscher et al., 1972). All extracts were tested at 200.0, 100.0, 50.0, 25.0, 10.0 and 5.0 mg/mL. Ciprofloxacin (Sigma- Aldrich, South Africa) added to 7H11 agar medium at final concentrations of 0.5, 0.01 and 0.05 mg/mL served as positive control. Three blank plates containing only 7H11 agar medium and three with 10% DMSO without plant extracts served as negative controls. The MIC was regarded as the lowest concentration of the extracts that did not permit any visible growth of M. smegmatis. Tests were done in triplicates.
Microplate susceptibility testing against M. smegmatis
All extracts were tested against M. smegmatis using the microplate dilution method (Newton et al., 2002). The MIC and the bacterial effect (minimum bactericidal concentration, MBC) were determined according to the methods described by Salie et al., 1996. The ethanol extracts were dissolved in 10% DMSO in sterile Middlebrook 7H9 broth base to obtain a stock concentration of 100.0 mg/mL. Serial two-fold dilutions of each sample to be evaluated were made with 7H9 broth to yield volumes of 100.0 μL/well with final concentrations ranging from 6.25 to 0.09 mg/mL.
Ciprofloxacin served as the positive drug control. 100.0 μL of M. smegmatis suspension (0.2 log-phase, yielding 1.26 x 108 CFU/mL) was also added to each well containing the samples and mixed thoroughly to give a final volume of 200.0 μL/well.
The solvent control DMSO at 2.5% v/v or less, in each well did not show inhibitory effects on the growth of M. smegmatis. Tests were done in triplicate.
The cultured microplates were sealed with parafilm and incubated at 370C for 24 hours. The MIC of the samples was detected following the addition (40.0 μL) of 0.2 mg/mL p-iodonitrotetrazolium violet (INT, Sigma-Aldrich, South Africa) and incubated at 370C for 30 minutes (Eloff, 1998). Viable bacteria reduced the yellow dye to a pink colour. The MIC was defined as the lowest sample concentration that prevented this change and exhibited complete inhibition of bacterial growth. The MBC was determined by adding 50.0 μL aliquots of the preparations to 150.0 μL of 7H9 broth in all the wells. These preparations were incubated at 37oC for 48 hours.
The MBC was regarded as the lowest concentration of extract which did not produce an absorbance at 550 nm using an ELISA plate reader (Salie et al., 1996).
Chapter 1: Literature Review: Introduction
1.1 Introduction
1.2 Medicinal plants with antimycobacterial activity
1.3 Scope of thesis
1.4 Structure of thesis
Chapter 2: Epidemiology, prevention, infection and treatment of tuberculosis
2.1 Introduction
2.2 Targets, mode of action of first-line TB drugs
2.3 Why are new TB drugs needed?
2.4 New TB drugs in the pipeline
2.5 Discussion and Conclusion
Chapter 3: Phytochemistry and medicinal uses of selected medicinal plants
3.1 Introduction
3.2 Plants selected
3.3 Discussion and Conclusion
Chapter 4: Antituberculosis activity of selected South African medicinal plants against Mycobacterium smegmatis and M. tuberculosis
4.1 Introduction
4.2 Materials and Methods
4.3 Results
4.4 Discussion and Conclusion
Chapter 5: Cytotoxicity activity of selected South African medicinal plants against Vero cells
5.1 Introduction
5.2 Materials and Methods
5.3 Cell culture
5.4 Cytotoxicity assay
5.5 Results and Discussion
5.6 Conclusio
Chapter 6: Bioassay guided fractionation of G. africana L. var. africana
6.1 Introduction
6.2 Bioassay guided fractionation
6.3 Results and Discussion
6.4 Conclusion
Chapter 7: Determination of the antimycobacterial activity of the fractions and the isolated compounds from the roots of G. africana L. var. africana
7.1 Introduction
7.2 Materials and methods
7.4 Conclusion
Chapter 8: Synergistic effect, cytotoxic and intracellular activity against M. tuberculosis
8.1 Introduction
8.2 Materials and methods
8.3 Results and Discussion
8.4 Conclusion
Chapter 9: Overall discussion and conclusion
Chapter 10: References
Chapter 11: Acknowledgements
Chapter 12: Appendices – Publications
12.1 Publications resulting from this thesis
12.2 Article in preparation
12.3 Chapter to be submitted in a book
12.4 Conference presentation.
