- Title
- Screening medical plants for potential immunomodulatory action on macrophages in the fight against mycobacterium tuberculosis.
- Creator
- Shauli, Mathulo Mthabiso
- Date Issued
- 2023-12
- Date
- 2023-12
- Type
- Doctoral theses
- Type
- text
- Identifier
- http://hdl.handle.net/11260/12744
- Identifier
- vital:76161
- Description
- Pulmonary tuberculosis remains the leading cause of death in developing countries, it is an ancient disease that was discovered in the eighteenth centuries. The World Health Organization (WHO) tuberculosis (TB) regimen entails the first line drugs, rifampicin, isoniazid, ethambutol, and pyrazinamide. These drugs are taken for long periods and the load is high, they carry some side effects like liver damage and peripheral neuropathies, hence pyridoxine that is administered with TB treatment to mitigate the toxic effects to peripheral nerves. Drug resistance has emerged because of unbearable side effects of TB treatment in TB patients. In view of all these collective challenges of TB drugs, this study draws its motivation towards exploring natural products in the form of medicinal plants toward inventing new therapies that could be easily available and safer, and the macrophages were considered the therapeutic target for this study as they are the primary cells that get infected by Mycobacterium in the lung. Therefore, the study aimed at screening medicinal plants used by traditional healers and herbalists for the management and control of signs and symptoms related to pulmonary tuberculosis, for potential immunomodulatory activity on RAW 264.7 cells, and the objectives were; to obtain information on indigenous plants used to treat pulmonary tuberculosis (PTB) from traditional healers and herbalist, to test the cytotoxicity of crude extracts on macrophages and hepatocytes (C3A cells), to determine the polarizing effects of crude extracts on macrophages, to determine the polarizing effect of the crude extract fractions on macrophages and to finally isolate and characterize the compounds in active fraction. vii All medicinal plants investigated in this study were collected through ethnobotanical survey, authenticated in Botany Department, dried, and extracted in 70% ethanol solvent. The dried crude extracts were screened for toxicity using 3-4,5-dimethylthiazole-24-25 diphenyl tetrazolium bromide (MTT) technique on RAW264.7 macrophages and dual fluorescence staining technique on C3A cells at concentration 50, 100 and 200μg/ml in cell culture system. Seven medicinal plants displayed dose dependency toxicity, while three plants did not show any signs of toxicity with cell viability maintained at 100%. The three medicinal plants (Anthrixia phylicoides, Lippia javanica and Sanicula elata) were further investigated for immunomodulatory activity (Griess method) and antioxidant activity (CellROX® Orange and Hoechst 33342). Lippia and Anthrixia were found to be anti-inflammatory with low amounts of nitric oxide (NO) production, while Sanicula displayed a considerable amount of nitric oxide when compared to the lipopolysaccharides (LPS) control. NO was the biological marker measured that demonstrated the immunomodulatory effect of plants on macrophages. Furthermore, the two plants showed antioxidant activity on liver cells at higher concentration (100 and 200μM) while Sanicula exhibited antioxidant activity across all tested concentrations. Compound profiling for Sanicula was therefore determined by UPLC-MS hyphenation technique and the major compounds profiled were phenolic compounds. The crude extracts of Sanicula were further partitioned into five fractions using partition solvent technique (n-hexane, Dichloromethane, ethyl acetate, N-butanol, and water). Anti-inflammatory activity was repeated on the five fractions, and Dichloromethane fraction and water fraction drove the macrophage switching towards the M1 phenotype viii with considerable amount of NO produced. Compound isolation and identification was conducted through chromatography techniques, and ten samples (S1-S10) were isolated. The samples were further subjected to NMR-Spectrometry analysis for elucidation and characterization of isolated compounds, and S1, S7, S9, S10 were final compounds. RAW 264.7 cells were again treated with the four compounds and results indicated absence of NO production, the opposite of the two previous outcomes where there was clear evidence of immunomodulation. The results obtained from the compounds has indicated that drugs work in synergy, and in combination, like TB treatment which is taken as combination of all drugs. We therefore conclude that indeed Sanicula immunomodulated RAW264.7 macrophages, and we present a strong observation of macrophage polarization from M0 phenotype to M1 phenotype which is capable of degrading and destroying Mycobacterium, and the antioxidant activity of the crude extracts of this medicinal plant. We therefore recommend further studies in the animal disease model.
- Description
- Thesis (D.Ed) -- Faculty of Medicine and Health Sciences, 2023
- Format
- computer
- Format
- online resource
- Format
- application/pdf
- Format
- 1 online resource (195 leaves)
- Format
- Publisher
- Walter Sisulu University
- Publisher
- Faculty of Medicine and Health Sciences
- Language
- English
- Rights
- Faculty of Medicine and Health Sciences
- Rights
- All Rights Reserved
- Rights
- Open Access
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| Thumbnail | File | Description | Size | Format | |||
|---|---|---|---|---|---|---|---|
| View Details Download | SOURCE1 | SHAULI MM (1).pdf | 6 MB | Adobe Acrobat PDF | View Details Download |