Artemisia afra is one of the oldest, most well known and widely used traditional medicinal plants in South Africa. It is used to treat many different medical conditions, particularly respiratory and inflammatory ailments. There is no reported evidence of its use for the treatment of cancer but due to its reported cytotoxicity, an investigation of the mode of cell death induced by an ethanol A. afra extract using two cancer cell lines was done. IC50 values of 18.21 and 31.88 μg/mL of ethanol extracts were determined against U937 and HeLa cancer cells, respectively. An IC50 value of the aqueous extract was greater than 250 μg/mL. The ethanol extract was not cytotoxic against confluent control cell lines, Chang Liver and Vero cells. The effect of the cytotoxic ethanol A. afra extract on U937 and HeLa cells and their progression through the cell cycle, apoptosis and mitochondrial membrane potential was investigated. After 12 hours of treatment with A. afra a delay in G2/M phase of the cell cycle was evident. Apoptosis was confirmed using the TUNEL assay for DNA fragmentation, as well as fluorescent staining with annexin V-FITC. Apoptosis was evident with the positive control and A. afra treatment at 24 and 48 hours. JC-1 staining showed a decrease in mitochondrial membrane potential at 24 hours. It was deduced that A. afra ethanol extract induces caspase-dependent apoptosis in a mitochondrial dependent manner. Plants harbour many compounds that are not only useful to the plants but also to mankind. Many metabolites have been isolated from A. afra and their biological activity characterised. Due to observed apoptosis induction, isolation of cytotoxic compounds was done and a new sesquiterpene lactone from A. afra was isolated. Structural elucidation of the compound was done by IR, 1D and 2D NMR, CD and mass spectrometry and it was identified as isoalantolactone. HeLa cancer cells were treated with isoalantolactone and cytotoxicity was exhibited in a dose-dependent manner. A low IC50 value of 8.15 ± 1.16 μM was achieved. This study showed that isoalantolactone is partly responsible for the observed A. afra cytotoxicity. Due to the evidence of G2/M arrest, the anti-mitotic potential and the possible onset of mitotic catastrophe by A. afra and isoalantolactone was investigated. It was evident from various flow cytometric analysis of cyclin B1 and phospho-H3 and confocal microscopy that A. afra does possess anti-mitotic activity by causing hyperpolymerisation of tubulin and cells progress into the mitotic phase where M arrest is experienced. The anti-inflammatory activity of sesquiterpene lactones is well documented; however, the anti-inflammatory activity of A. afra is not. Here, it is reported that the production of NO and COX-2 protein levels in RAW 264.7 cells decrease in the presence of A. afra and isoalantolactone after stimulation with LPS. The activated NF-κB subunit, p65 was also investigated. The results suggest that A. afra and isoalantolactone inhibit p65 activation as a decrease in the activated subunit was evident. Thus, the results indicate that exposure to A. afra and isoalantolactone induces an anti-inflammatory response. In conclusion, this study shows, for the first time, the mechanism of induced apoptosis, the anti-mitotic and anti-inflammatory activity of A. afra and its isolated compound, isoalantolactone. It also proves that although extensive research may have been done on a particular plant, as with A. afra, more can be discovered leading to the identification of new compounds and integration of signalling pathways that can be exploited for the treatment of various diseases and ailments.
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:nmmu/vital:10363 |
| Date | January 2014 |
| Creators | Venables, Luanne |
| Publisher | Nelson Mandela Metropolitan University, Faculty of Science |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis, Doctoral, PhD |
| Format | xxx, 228 leaves, pdf |
| Rights | Nelson Mandela Metropolitan University |
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