The Brine Shrimp Bioassay was used to determine the cytotoxic potential of the crude extracts. TLC and column chromatography were performed to evaluate the chemical composition of the Bulbine species. 11 Activity of the crude organic root extracts against S typhi 48 12 Activity of the crude organic root extracts against S.
21 Activity of the organic root extracts against S epidermidis 56 22 Activity of the crude organic root extracts against P. Representative TLC fingerprints of the stem extracts of the Bulbine species using 3 different solvent systems. Representative TLC fingerprints of the leaf extracts of the Bulbine species using 3 different solvent systems.
Representative TLC fingerprints of the root extracts of the Bulbine species using 3 different solvent systems.
CHAPTER ONE
INTRODUCTION AND
LITERATURE REVIEW
INTRODUCTION
- Background and Motivation for the study
- Aims
The ancient Babylonians, Egyptians and Greeks were also aware of the medicinal value of a number of plants. The ancient Egyptians possessed an understanding of human anatomy as well as knowledge of the medicinal uses of plants and animals. In traditional African medicine, various Bulbine species are used to treat a variety of conditions, including sexually transmitted diseases (STDs), wound infections, dysentery, and urinary tract infections (UTIsf). Bulbine species belong to the Asphodelaceae family and there are over fifty Bulbine of South Africa species.
Furthermore, with the emergence of bacterial resistance to antimicrobials, it is necessary to find alternative therapies. Although Bulbine species are widely used for a variety of infectious diseases, there is very little documented information regarding their cytotoxic potential, antimicrobial activity and chemical composition. Extensive, controlled and standardized studies into both the safety and efficacy of the Bulbine strain were therefore imperative.
The aim of this study was to investigate the antimicrobial activity and chemical composition of selected Bulbin species.
Literature Review
- Bacteriology
- Size and Shape of Bacteria
- Structure of Bacteria
- The Bacterial Cell Wall
- Mycology
- Traditional Medicine in South Africa
- Traditional Healers and reasons for consultation
- Advantages and Disadvantages of consulting traditional healers
- Useful Medicinal Plants
- Adverse effects of plant used medicinally
- The Bulbine species
The outer membrane of the Gram-negative cell wall contributes an additional barrier and this makes Gram-negative bacteria resistant to some antimicrobial agents. Between seventy and ninety percent of the population of rural Africa is not covered by health care. Due to the severe shortage of trained professional personnel in the field of health care, it was recommended to provide existing traditional health care services.
The program is sensitive to both the practitioner's needs and the demands of the traditional healthcare market4. Despite the enormous advances in medicine, there are a number of diseases for which modern medicine has no cure44. This is complicated by the lack of knowledge about what many of the herbal remedies contain63.
The following is a brief botanical description of the Bulbine species with particular emphasis on the plants used in this study.
CHAPTER TWO
MATERIALS
AND METHODS
- PREPARATION OF THE CRUDE EXTRACTS
- Collection of the Plant Material
- Organic Extraction
- Aqueous Extraction
- ANTIBACTERIAL ACTIVITIES
- Bacteriology
- Preparation of the Bacterial Cultures
- Preparation of the agar plates
- Preparation of the crude extracts
- Disk Diffusion Method 14
- Bore Well Method 15
- Mycology
- Fungi used in this study
- Preparation of the fungal spores
- Preparation of C albicans
- Antifungal activity using the TLC Bioassay16
- Antifungal activity of C albicans
- Cytotoxic Activity
- The Brine Shrimp Method
- Preparation of the crude extracts
- Preparation of the brine shrimps
- Methodology
- Chemical Analysis
- Chemotaxonomy
- Column Chromatography
The chemistry of Bulbine species is largely unknown and not many documented studies have been conducted to determine their antimicrobial activity. Recent literature indicates a move away from the traditional differential solvent extraction method as well as the use of a Soxhlet apparatus in the extraction procedure due to the potential for curative compounds to be destroyed by heating. In addition, drying may affect the constituents found in the aqueous fraction of fleshy leaves.
The aqueous evaporation was carried out to see if there were any differences in the antimicrobial activities of the extracts using the two different extraction solvents. The plants used were collected with the help of the curator of the herbarium at the University of Durban-Westville (UDW). This study investigated the activity of the Bulbine species against some of the pathogens that occur in the conditions mentioned in Table 1, viz.
To evaluate spore counts using this technique, serial dilutions of the harvested spores were made with distilled water. The crude extract solutions were prepared by dissolving the crude extract (50 mg) in methanol: dichloromethane (1:1, v/v) (1 mL). Under adverse conditions such as tank evaporation and unfavorable temperatures, they produce resistant eggs that can restart the population when suitable conditions return89.
To determine the cytotoxicity of crude organic extracts, the Brine Shrimp Bioassay of Meyer et al.17 was used. In addition to Rf comparisons, the most important diagnostic information in this technique is provided by the variations or similarities in the color of the bands that provide fingerprints for the particular species8. The separation of a mixture results from the differential adsorption of the components on the surface of the solid.
The column was washed with additional amounts of ethyl acetate, after which the liquid level was brought to the top of the suspension. This solution was applied to the top of the column (silica gel 60 column height was 27 cm) with a Pasteur pipette as a narrow band.
CHAPTER THREE
RESULTS AND DISCUSSION
Yield of Crude Extract
- Results
- Results
- Discussion
Tables 8 and 9 show the antibacterial activity of the crude organic solvent and aqueous extracts of the leaves, roots and stems of B. Tables 10 and 11 show the antibacterial activity of the crude organic and aqueous extracts of the leaves, stems and roots of B. The aqueous extracts of the stems had less activity than the extracts of the stems with organic solvents (Table 8).
It is interesting to note the activity of the aqueous extracts of the leaves against S. With the crude aqueous extracts, K pneumoniae was inhibited by six of the extracts (Table 8 and Figures 18 and 20). It is interesting to note that two of the crude organic extracts had activity against C.
Cytotoxic Activity
- Results
- Discussion
- TLC Fingerprinting
- Results
- Column Chromatography
- Results
Most of the crude organic extracts were not cytotoxic at dilution 2, which had a concentration of 0.5 mg/ml, and none of the crude organic extracts were cytotoxic at dilution 3, which had a concentration of 0.05 mg/ml. Further cytotoxicity tests were performed with 3 crude organic extracts that were not cytotoxic at dilution 1, i.e. root extracts of B. After 24 h incubation, the number of surviving shrimps in each of the tests was 10.
Figures 33, 34 and 35 represent TLC fingerprints of stem, leaf and root extracts of five different Bulbine species used in this study using three different solvent systems. They differed in appearance from other leaves in that they were strap-shaped. Chromatograms of leaf extracts (Figure 34c) showed that TLC fingerprinting of leaves can be a useful tool for the identification of different species.
The chromatograms using solvent system 2 (Figure 34b) and solvent system 1 (Figure 34a) showed many similarities between the five plants. Solvent system 2 showed many similarities between the plants and from Figure 34b it could be seen that B. From Figures 33a, 33b and 33c it was clear that the TLC fingerprints of the stem extract of B.
The chromatogram obtained using solvent system 3 (Figure 33c) showed that the strain fingerprints have distinctly different components than the other two strains. From these chromatograms it was clear that all three species were related, they also had characteristic differences and that these strain fingerprints could be a tool to identify the different species. The chromatograms obtained using solvent system 3 (Figure 3Sc) showed that each of the root extracts had a characteristic fingerprint.
However, the root extracts shared many components and these included the pink bands shown by (g) and (h). TLC using solvent system 1 did not show the differences between species as clearly as the other two solvent systems (Figure 3Sa).
CHAPTER FOUR
Conclusions, Limitations and
Recommendations and Limitations
- Limitations
- Recommendations
The other 2 plants were purchased from the nursery and a sufficient quantity of plants was not available to obtain a sufficient yield to perform the antimicrobial tests. Therefore, the aqueous extraction was performed only with roots, stems and leaves of B. These two plants were readily available as they grew in the garden at UDW.
The aqueous extraction should be performed with both hot and cold water treatments to ascertain any differences in their activities. The aqueous extract must be concentrated to obtain a higher concentration of crude extract and the amount of crude aqueous extract must be quantified to assess the possible antimicrobial concentrations. Instead of making dilutions of the crude extracts with seawater, the crude extracts should first be inoculated onto filter paper discs, dried and then added to the seawater.
This way the liquid will not be dark and cloudy and higher concentrations of crude extracts can be tested. It is also recommended that tests be performed using aqueous extracts in the same manner as crude organic extracts. Crude organic and aqueous extracts should be tested against STD-causing bacteria, e.g. Neisseria gonohorroea and Treponema pallidum to determine potential antibacterial activity. N gonorrhea is anaerobic and requires special incubation conditions that were not available to the researcher.
Therefore, antimicrobial activity studies with different root and stem fractions should be conducted to identify potential similarities in their activities. It is also recommended that all other crude organic extracts be fractionated and antimicrobial studies performed. Further studies, preferably doctoral studies, should be carried out using the results already obtained to further elucidate the antimicrobial activity, cytotoxicity and chemical composition of this genus.
Aspects of clinical practice and traditional organization of indigenous healers in South Africa. Social Science and Medicine 15B: 361-365. Health care planning in South Africa - Is there a role for traditional healers? Social Sciences and Medicine. Availability, accessibility, acceptability and adaptability: Four attributes of African ethno-medicine. Social Sciences and Medicine.
Can collaborative programs between biomedical and African indigenous medicine practitioners succeed? Social Sciences and Medicine. 1998. In vitro antiviral activity of seven Psiadia species, Asteraceae: isolation of two antipoliovirus flavonoids from Psiadia dentata. A study on diterpenes from the leaves of Rhabdosia tricliocarpa and their antimicrobial activity against oral microorganisms.
APPENDICES
APPENDIX ONE
MATERIALS FOR ANTIMICROBIAL TESTING
Nutrient Agar (Oxoid) Lab Lemco Powder
Nutrient Broth (Oxoid) Lab Lemco Powder
Potato Dextrose Agar (Oxoid) Potato Extract
APPENDIX TWO
