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TLC fingerprinting analysis
Chromatograms of 14 plant species extracted with solvents of different polarities hexane, acetone, methanol and water, and developed in BEA, CEF, EMW and FAWE solvent systems are shown in Fig.4.1.The number of different coloured bands observed on the TLC plates show the diversity of the compounds present in the plant extracts.
Pascaline and co-workers report that the different colours of the fluorescence rings are due to different atoms present in the compound having different wavelengths. When atoms are excited to a higher energy level, they may fall back to their original position using the same or a different wavelength resulting to the emission of different colours (Pascaline et al., 2011). Some of the compounds showed same colour and Rf values in the same solvent system but in different extracting solvents, this may suggest that the separated compounds are of similar nature.
Only 582 compounds were separated by the four solvent systems because most of the plant extracts did not separate well, they remained at the baseline of the TLC plate, this might be attributed to too much plant extracts applied to the baseline or compounds in the plant extracts were too polar to be eluted because normal phase silica of TLC plate retains polar components (Fried and Serma, 1999). Comparing the four solvent systems, BEA separated compounds in the plant extracts more efficiently than CEF, EMW and FAWE, representing 38% of the separated compounds, followed by EMW with 29%, CEF with 26% and the least was FAWE with only 7%. Our group has also observed the separation of more bands in the BEA solvent system than in CEF and EMW solvent systems (Masoko et al., 2008, Eloff et al., 2005). This indicated that most of the separated compounds were non-polar.
TLC bioautography of fungal species
The results of bioautography of plant extracts against C. albicans are shown in Fig.4.3 and their corresponding Rf values are shown in Table 4.1. Both BEA, CEF and EMW solvent systems had few and less visible clear bands of inhibition against C. albicans. This might be an indication of antifungal compounds present in low concentrations or that they do not react with the p-Iodonitrotetrazolium violet (INT) (Eloff et al., 2005). Aqueous plant extracts developed in FAWE solvent system had poor activity against C. albicans since no bands of inhibition were observed (results no shown) and this is in line with the previous findings (Eloff et al., 2005). This shows that water did not extract inhibiting compounds from the dried ground leaves, possibly because the inhibiting compounds were made unavailable by lipid soluble membranes (Eloff et al., 2008). Our group has acknowledged the difficulties of getting good bioautograms against fungi (Suleiman et al., 2010). However, number of active compounds against C. albicans was 53 representing only 9 % of all the components separated by BEA, CEF, EMW and FAWE solvent systems.
Amongst the 53 active compounds, 72% (38) were relatively polar compounds separated by EMW solvent system with Rf values ranging from 0.16 to 0.82, followed by 21% (11) components having intermediate polarity separated by CEF solvent system with Rf values ranging from 0.23 to 0.94 and the least 8% (4) non-polar components separated by BEA solvent system and their Rf values ranged from 0.11 to 0.34. Out of the fourteen plant species tested. F.saligna and R. brasiliensis had 8 active compounds each while O. lanceolata, A. caffra and C. gratissimus had 1 active component each. C. glabrum had 8 active compounds against C. albicans, 5 of them were observed in the hexane extract, 1 compound was observed in acetone extract, 2 components observed in methanol extract had the same Rf values as those observed in the hexane extract, 0.16 and 0.17 and this is indicative of similar compounds.
Therefore, hexane extract of C. glabrum was targeted for the isolation of antifungal compounds and even its MIC value was 0.06 mg/ml in the previous experiments. The experience in our group has shown that most of the antimicrobial activity is found highly in non-polar compounds (Eloff, 2001).
TLC bioautography of bacterial species
In the bioautography of bacterial species only acetone plant extracts of the selected plant species were tested because acetone is known to extract compounds with a wide variety of polarity from plants, easy removal of solvent after extraction and safety to microorganisms in bioassays (Eloff, 1998). The results of bioautograms of S. aureus are shown in Fig.4.4 and their corresponding Rf values are shown in Table 4.2. The bioautograms of E.faecalis were not shown because they didn‘t come out well after several attempts. Bands of inhibition of active compounds against S. aureus were prominent in both BEA, CEF and EMW solvent systems. These findings are supported by the results obtained by Martin and Eloff (1998) and Suleimann et al. (2010) indicating that S. aureus has the highest number of inhibition bands. In the BEA solvent system clear bands of inhibition were near the base line, suggesting that the active compounds were relatively polar and EMW solvent system showed most inhibition bands near the solvent front, suggesting that the active components were relatively non-polar. Mdee et al. (2009) also observed the same trend in the investigation of the activity of extracts of seven common invasive plant species on fungal phytopathogens.
Total number of active compounds against S. aureus was 103, 36 compounds were separated by BEA solvent system, followed by CEF solvent system with 35 compounds and the least was EMW solvent system with 32 compounds. The use of solvent systems which differ in polarity, BEA (non-polar), CEF (intermediate) and EMW (polar) has managed to separate an average of 34 active compounds which were non-polar, intermediate and polar.
This is supported by Eloff (1998), when he asserted that the larger the variety of compounds that are extracted by the extractant, the better the chance that biologically active components will also be extracted if a specific class of chemical component is not targeted.
Chapter 1. Introduction
1.1. Statement of the problem
1.2. Hypothesis
1.3. Aim
Chapter 2. Literature review
2.1. Importance of fungal diseases
2.2. The human immune system in the fight against fungal infections
2.3. Opportunistic organisms
2.4. Plants as potential therapeutic agents
Chapter 3. Ethnobotanical use of plants to manage candidiasis and related infections in Venda, South Africa.
3.1.Introduction
3.2. Study site
3.3. Materials and Methods
3.4. Results and Discussion
3.5. Conclusion
Chapter 4. Phytochemical and bioautographic investigation of the selected South African medicinal plant species
4.1. Introduction
4.2. Materials and Methods
4.3. Results and discussion
4.4. Conclusion:
Chapter 5. Antifungal and antibacterial activity of some South African medicinal plants used traditionally to treat candidiasis.
5.1. Introduction
5.2. Materials and methods
5.3. Results and discussion
5.4. Conclusion
Chapter 6. Evaluating cytotoxicity and antioxidant activity of the selected South African medicinal plant species
6.1. Introduction
6.2. Materials and methods
6.3. Results and Discussion
6.4. Conclusion
Chapter 7. Clerodendrumic acid, a new triterpenoid from Clerodendrum glabrum (Verbenaceae) and antimicrobial activity of fractions and constituents
7.1. Introduction
7.2. Results and Discussion
7.3. Conclusion
7.4. General experimental procedures
Chapter 8. Summary and conclusions
8.1. Objectives
Chapter 9. References
