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Bioautography
Bioautograms were made of the different extractants and bacteria to confirm MIC values, as well as to see if the same compounds (Rf values) were responsible for antibacterial activity for the different bacteria (Figures 3.6, 3.7 and 3.8). In each of Figures 3.6-3.8, the top row is the plant material of P. myrtifolia leaves, the middle row is P. myrtifolia fruit and bottom row is Q. littoria leaves. In Figures 3.6-3.8, the white areas indicate growth inhibition of bacteria and thus antibacterial activity of plant substance on the specific area (Rf ) of the TLC.
Areas of growth inhibition were not the same for all bacteria. In figure 3.6 the Rf (of the areas of growth inhibition (1-4)) value in brackets behind each bacterium’s name, for the hexane extract of P. myrtifolia leaves, differed for the bacteria S. aureus ((1) no Rf ), E. faecalis ((2a) 0.04 and (2b) 0.28), P. aeruginosa ((3) 0.28), and E. coli ((4) 0.58). This indicated that (except for some similarities) different compounds were responsible for antibacterial activity for the different bacteria. In cases where the Rf values are the same, it would indicate one compound having a broad antibacterial spectrum. It could also be seen that the n-hexane extract of leaf material (Pteleopsis and Quisqualis) showed no or slight inhibition of growth of S. aureus and the water extract of Pteleopsis leaves and fruit showed no or slight inhibition of growth of P. aeruginosa. For all plant material types, the CEF eluent system indicated areas of bacterial growth inhibition the clearest. In some cases (e.g. E. coli with P. myrtifolia) there is a good correlation between low MIC and clear areas of inhibition. In other cases (e.g. E. coli with Q. littorea) however, the clear area of inhibition on the bioautogram, is not as large as one would expect from a low MIC value. This can be explained if the active compounds are volatile, then they may have evaporated from the overnight drying of the chromatograms before treatment with bacteria.
The bacterium S. aureus also indicated areas (on all three of the eluent systems, clearer with BEA and EMW (Figure 3.6 and 3.8)) of growth promotion (areas that stained darker red than the rest of the plate) and all plant materials. For S. aureus the MIC values obtained may be an average of the growth inhibitory and growth promoting areas. Above-mentioned areas of bacterial growth inhibition (clear, but show white, as the TLC plate is white) may not be the only ones. Brown areas formed after application (on the baseline of the plates) of the more polar extracts like tetrahydrofuran, ethyl acetate, acetone, ethanol, methanol and water or 50% water and 50% acetone. In Figure 3.7 it can be seen that they were not in all cases (like with E. faecalis) clearly covered by a red formazan colour after sprayed with INT, and they probably contribute to the extracts’ antibacterial activity.
Areas of antibacterial inhibition were tabulated according to Rf values. While tabulated, it was almost impossible to make a reasonable interpretation about the Rf values. The Rf values were therefore plotted in graphs to find visual presentations of antibacterial activity. Figures 3.9 and 3.10 are visual presentations of some Rf values found for the CEF and EMW respectively. For the CEF system the Rf values of the methylene dichloride, acetone and ethyl acetate extracts are represented in Figure 3.9, and for the EMW system the di-isopropyl ether, acetone and methanol extracts are represented in Figure 3.10. Similar Rf values (within one eluent system), indicate which extracts isolated the same compound, for example, the methylene dichloride, acetone and ethyl acetate extractants all isolated the compounds ‘ 1 ‘ and ‘ 2 ‘ in Figure 3.9, and compounds ‘ 3 ‘ and ‘ 4 ‘ in Figure 3.10. Compounds with similar Rf values that inhibited more than one bacterium, indicate compounds with broad antibacterial spectra, for example, “a” and “e” from Pteleopsis leaves, “b” and “c” from Pteleopsis fruit, and “d” from Quisqualis leaves (Figures 3.9 and 3.10). In Figure 3.9, only methylene dichloride isolated a compound indicated by “ f ”, from Pteleopsis leaves o which S. aureus was sensitive, and acetone and ethyl acetate did not. Similarly, only the acetone and methanol extracts isolated a compound indicated by “ g ”, from Quisqualis leaves to which E. faecalis was sensitive, and the di-isopropyl ether did (Figure 3.10).
Stability of extracts over time
MIC values of P. myrtifolia leaf extracts that were determined when freshly prepared, were compared to leaf extracts extracted and redissolved in acetone 8 months previously. The amount extracted was taken into account and total activity was calculated. For the extractants investigated: tetrahydrofuran, ethyl acetate, ethanol and methanol, the total activity for S. aureus was reduced within 8 months. The total activity for E. faecalis, P. aeruginosa and E. coli increased for tetrahydrofuran, ethyl acetate, acetone and ethanol after an 8-month period (Figure 3.11).
The loss of activity of certain extracts after storing in the cold could have been due to chemical modification of active compounds or to their precipitation over time. Since the extracts were kept in screw-capped containers and the volume was checked and evaporation losses corrected before testing their antibacterial activity, the surprising increased antibacterial activity could be explained if some inhibitory compounds were volatile or unstable over time, and this would explain the increased activity. The mechanism of the enhancement of potency and subsequent stability should e investigated (Eloff, 1999).
Chapter 1: Introduction, hypotheses, aims and objectives
1.1 Introduction
1.2 Hypothesis, aim and objectives of this study
1.3 Schematic representation of research’s methodology
1.4 Envisaged contributions of this study
1.5 Statistical considerations
1.6 Literature references
Chapter 2: Extraction of plant material
Abstract
2.1 Introduction
2.2 Material and Methods
2.3 Results and Discussion
2.4 Conclusions
2.5 Literature references
Chapter 3: Antibacterial activity of different extracts of Pteleopsis myrtifolia leaves and fruit and Quisqualis littorea leaves.
Abstract
3.1 Introduction
3.2 Material and Methods
3.3 Results and Discussion
3.4 Conclusions
3.5 Literature references
Chapter 4: Solvent-solvent separation
Abstract
4.1 Introduction
4.2 Material and Methods
4.3 Results and Discussion
4.4 Conclusions
4.5 Literature References
Chapter 5: Cytotoxic activity of Pteleopsis myrtifolia leaf extracts
Abstract
5.1 Introduction
5.2 Materials and Methods
5.3 Results and discussion
5.4 Conclusions
5.5 Literature references
Chapter 6: Antioxidant activity of Pteleopsis myrtifolia leaf extracts
Abstract
6.1 Introduction
6.2 Material and methods
6.3 Results and discussion
6.4 Conclusions
6.5 Literature references
Chapter 7: Column chromatography and isolation of pure compounds from Pteleopsis myrtifolia
Abstract
7.1 Introduction
7.2 Materials and Methods
7.3 Results and Discussion
7.4 Conclusions
7.5 Literature References
Chapter 8: Antibacterial, antioxidant and cytotoxic activity of taraxerol, a pentacyclic triterpenoid, isolated from Pteleopsis myrtifolia leaves
Abstract
8.1 Introduction
8.2 Materials and Methods
8.3 Results and Discussion
8.4 Summary and comparison of taraxerol’s activity
8.5 Conclusions
8.6 Literature references
Chapter 9 General discussion, conclusions and recommendations
9.1 Less known genera of the Combretaceae
9.2 Antibacterial activity of extracts from P. myrtifolia and Q. littorea
9.3 Growth inhibition effect of P. myrtifolia leaf extracts on human cell lines
9.4 Antioxidant activity of P. myrtifolia leaf extracts
9.5 Solvent-solvent separation to find best fraction for purification
9.6 Pure compounds isolated from P. myrtifolia leaves
9.7 Bioactivity of taraxerol
9.8 Recommendations for future
9.9 Literature references
