- Title
- Phytochemicals derived from Australian eucalypts as anticancer agents for pancreatic malignancies
- Creator
- Bhuyan, Deep Jyoti
- Relation
- University of Newcastle Research Higher Degree Thesis
- Resource Type
- thesis
- Date
- 2018
- Description
- Research Doctorate - Doctor of Philosophy (PhD)
- Description
- The poorest prognostic outcome for pancreatic cancer (PC) patients, among all gastrointestinal malignancies, can be attributed to the molecular heterogeneity and lack of specific therapeutic strategies. The emergence of resistance against the common chemotherapeutic drug gemcitabine has also been widely reported. Several studies have demonstrated improved efficacy using gemcitabine in conjunction with plant polyphenolics and antioxidants for PC treatment. This suggests that plant secondary metabolites should be investigated further in a search for adjuncts to current PC treatments. Moreover, plant-derived bioactive compounds have played a key role in the development of anticancer drugs over many decades. Eucalypts dominate the Australian landscape with over 800 distinct species. Eucalypt-derived phytochemicals have been associated with a wide range of bioactivity, both in traditional indigenous Australian bush medicine and in the scientific literature. However, a few eucalypt species and their essential oils have to date been exploited for their anticancer properties. An extensive review (Chapter 1) confirmed that more research was required to gain an improved understanding of the anticancer potential of Australian eucalypt phytochemicals with activity specific to PC. Therefore, the research reported herein was designed to address two main aspects, namely; (1) determining the optimal extraction conditions for phenolics and antioxidants from eucalypts, and (2) assessing their antiproliferative activity against PC cells including the delineation of potential molecular mechanisms of action responsible for this activity. Conventional extraction with water was employed to prepare crude extracts from eight different eucalypt species and was shown to be the most efficient method for extracting phenolics and antioxidants when compared to microwave-assisted (MAE) and ultrasound-assisted extractions (UAE) (Chapter 2). Crude extracts derived from Angophora floribunda, Angophora hispida and Eucalyptus microcorys were demonstrated to possess the most potent phytochemical profile, exhibiting statistically similar cytotoxicities against MIA PaCa-2 cells as discussed in Chapter 3. In addition, E. microcorys crude extracts exerted significantly greater cytotoxicity against glioblastoma, neuroblastoma and lung cancer cells than the other extracts. In MIA PaCa-2 cells, E. microcorys crude extracts induced caspase 3/7-mediated apoptosis. Therefore, the aqueous E. microcorys extract was subjected to further investigation to obtain a greater depth of understanding of their bioactivity. Chapter 4 focuses on the significant antioxidant, antifungal and antibacterial properties of aqueous E. microcorys extract. Subsequent bioassay-guided fractionation of E. microcorys aqueous crude extract using semi-preparative Reversed-Phase (RP) HPLC revealed that fraction-1 was significantly more efficacious in terms of its antioxidant and antiproliferative activity against MIA PaCa-2 cells in comparison to other four fractions, as stated in Chapter 5. Flow cytometry analyses validated that the cytotoxicity was mediated by induction of apoptosis and abrogation of the cell cycle in the G2/M phase. Western blot analysis showed that the active fraction significantly downregulated the antiapoptotic protein B-cell lymphoma 2 (Bcl-2) and upregulated the proapoptotic proteins Bcl-2 homologous antagonist killer (Bak) and Bcl-2-associated protein (Bax) and cleaved Poly (ADP-ribose) polymerase (PARP) in MIA PaCa-2 cells. Combination treatment of the active fraction with gemcitabine increased apoptosis and cell cycle abrogation of MIA PaCa-2 greater than either mono treatment, indicating a potential additive/synergistic effect against the PC cells. Untargeted metabolomics using High performance/pressure liquid chromatography/electrospray ionisation/mass spectroscopy/mass spectroscopy (HPLC-ESI/MS/MS) revealed the tentative identities of the phytochemicals in the active fraction to be mostly phenolic compounds, of which several have previously been described to possess antipancreatic cancer activity. The findings presented in this thesis provide further scientific evidence of the antipancreatic cancer activity of extracts from Australian eucalypts. This is the first report to optimise the MAE and UAE techniques and parameters for extracting phenolic compounds and antioxidants from Eucalyptus robusta and establish the antiproliferative activity of species belonging to all three main genera of Australian eucalypts against the PC cells. Bioassay-guided fractionation of E. microcorys aqueous crude extract, investigation of bioactive compounds in the most potent fraction by liquid chromatography-mass spectroscopy (LC-MS) based-metabolomics and studies to obtain a mechanistic explanation of antiproliferative activity against PC cells are other key contributions of this project.
- Subject
- pancreatic cancer; Australian eucalypts; LC-MS; cytoxicity; antiproliferation; anticancer; antimicrobia; MAE; UAE; conventional extraction; RSM; Eucalyptus robusta; Eucalyptus; Eucalyptus saligna; Eucalyptus globulus; Corymbia citriodora; Corymbia maculata; Angophora hispida; Angophora floribunda; PDAC; gemcitabine; antifungal; antibacterial; MIA PaCa-2; aqueous extract; HPLC; RP-HPLC; HPLC-ESI/MS/MS; thesis by publication; Eucalyptus microcorys; bioassay-guided fractionation; apoptosis; cell cycle arrest; phenolic compounds; antioxidants
- Identifier
- http://hdl.handle.net/1959.13/1388005
- Identifier
- uon:32711
- Rights
- Copyright 2018 Deep Jyoti Bhuyan
- Language
- eng
- Full Text
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| View Details Download | ATTACHMENT01 | Thesis | 15 MB | Adobe Acrobat PDF | View Details Download | ||
| View Details Download | ATTACHMENT02 | Abstract | 296 KB | Adobe Acrobat PDF | View Details Download |