Thesis (M.Sc. (Biochemistry)) -- University of Limpopo, 2021 / Lung cancer is the most diagnosed cancer with an estimated 3 million deaths expected
by 2035. Bioactive phytochemicals present in plants are preferred as anticancer
therapeutic agents, due to their ability to differentiate between cancerous and normal
cells. One such plant, Senna italica, is traditionally used to treat diabetes, malaria,
constipation, jaundice, fever and sexually transmitted diseases. Several studies have
reported on its anti-proliferative potential against different types of cancers. However,
there is scanty information regarding its molecular mechanism of action against
different types of cancers, more especially lung cancer. This study, therefore, aims to
determine the differential expression profiles of apoptotic genes in lung A549 cancer
cells induced by treatment with S. italica leaf and root extracts in an attempt to
understand its purported anticancer molecular mechanism of action.
The leaves and roots of S. italica were dried in the dark and extracted with ethyl acetate
and methanol. Screening for the presence of secondary metabolites was performed
using thin layer chromatography and various standard chemical-based tests. The total
phenolic and flavonoid compounds were evaluated using gallic acid and quercetin
equivalence assays. The antioxidant activity of S. italica extracts was determined using
DPPH free radical scavenging and ferric ion reducing power assays. The cytotoxicity of
both leaf and root extracts on lung A549 cancer cells was evaluated using 3-(4,5-
dimethylthiazol-2-yl)-2-5-diphenyl tetrazolium bromide (MTT) and further confirmed by
Muse cell count and cell viability assays. The proliferation of cells, after treatment with
different concentrations of the extracts, was examined using the Ki67 proliferation
assay. Genotoxicity was determined to assess the potential damage caused by the
extracts on the DNA using a MUSETM multicolour DNA damage kit following
manufacturer’s protocol. The morphological change of cells treated with different
concentrations of S. italica ethyl acetate root extract was analysed using acridine
orange/ ethidium bromide (AO/EB) dual staining assay and examined under
fluorescent light. The total number of cells undergoing apoptosis was also determined
using the Annexin V assay. The expression of 84 key genes, involved in programmed
cell death or apoptosis, was determined using the Human Apoptosis RT² Profiler PCR
array kit.
Senna italica methanol extract had a high content of plant materials in both leaves and
roots compared to the ethyl acetate extract. A higher phenolic content was observedxii
mainly in the leaf extract and a higher flavonoid content was observed in the root
extract. Phytochemicals, such as phenols, tannins, flavonoids, terpenoids and
steroids, which are known to exhibit anti-cancer activity against cancerous cells were
abundant in the ethyl acetate leaf and root extracts as compared to the methanol leaf
and root extracts. Additionally, the ethyl acetate root extract exhibited more
antioxidants and radical scavenging activity in comparison to the methanol root extract.
The IC50 of ethyl acetate root extract was determined to be 200µg/ml. Both methanol
and ethyl acetate root extracts had little to no effect on the viability of lung A549 lung
cancer cells. The results were confirmed by cell count and viability assay results. The
cytotoxicity of ethyl acetate root extract was also evaluated against the normal kidney
HEK-293 cells, which displayed little cytotoxic effect. The proliferation results indicated
that S. italica ethyl acetate root extract has the potential to reduce the proliferation of
lung A549 cancer cells. The ethyl acetate root extract was found to induce late
apoptosis in A549 cells, but the genotoxicity data indicated that the DNA double strand
breaks (DSBs) were repairable. The results further showed an expression of different
genes that inhibit apoptosis, such as XIAP in lung A549 cells, following treatment with
S. italica ethyl acetate root extract. In conclusion, the ethyl acetate root extract
displayed a promising anti-cancer therapeutic potential, and thus warrants further
investigation to elucidate the identity of the inherent chemical components that are
responsible for the observed biological activity. / University of Limpopo and
SAMRC
| Identifer | oai:union.ndltd.org:netd.ac.za/oai:union.ndltd.org:ul/oai:ulspace.ul.ac.za:10386/3698 |
| Date | January 2021 |
| Creators | Moloantoa, Malose Ivan |
| Contributors | Mampuru, L. J., Mokgotho, M. P., Poopedi, K. W. |
| Source Sets | South African National ETD Portal |
| Language | English |
| Detected Language | English |
| Type | Thesis |
| Format | xii, 70 leaves |
| Relation |
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