Thesis

Phytochemical, anti-cancer, anti-inflammatory and neuroprotective studies on compounds isolated from Malaysian plants, Aquilaria malaccensis and Hopea dryobalanoides

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Awarding institution
  • University of Strathclyde
Date of award
  • 2020
Thesis identifier
  • T15819
Person Identifier (Local)
  • 201652435
Qualification Level
Qualification Name
Department, School or Faculty
Abstract
  • Natural products have been used for medicinal purposes for centuries and remain the main source of new drugs to treat diseases due to their biological activities such as anticancer, anti-inflammatory and neuroprotective effects. This thesis aimed to : 1) isolate bioactive compounds from two Malaysian plants which are: Aquilaria malaccensis and Hopea dryobalanoides using column chromatography and characterised them by nuclear magnetic resonance (NMR); 2) assess the anti-cancer activity of the isolated compounds through various in vitro assays and metabolomics profiling; 3) investigate the anti-inflammatory properties of the compounds by inhibition of the pro-inflammatory cytokines (tumour necrosis factor-α (TNF-α), interleukin-1β (IL-1β) and interleukin-6 (IL-6)) levels in response to lipopolysaccharide (LPS) stimulation in macrophage cells and identify the effects of the compound treatments on the gene expression against the inflammatory response caused by LPS-stimulated macrophage cells; 4) examine the protective effects of the compounds against oxidative stress induced in neuron cells.1) Plants extracts, investigated phytochemically, resulted in the isolation of 7-methoxyacacetin (7-aca) from the hexane extract of A. malaccensis twigs for the first time, a mixture of two compounds quercitrin and afzelin (Q/A) from the methanol extract of A. malaccensis twigs and two compounds named balanocarpol (bala) and heimiol A from the ethyl acetate extract of H. dryobalanoides bark. There are limited reports on the biological activity of 7-aca, however a similar compound called acacetin (aca) has been reported to have a range of bioactivates including anti-cancer, anti-inflammatory and protective effects. Therefore, aca was purchased commercially for comparison purposes.;2) Cytotoxicity screening of 7-aca, aca, Q/A and bala resulted in decreased viability of A2780 (ovarian) and ZR-75-1 (breast) cells in a concentration dependant manner. 7-Aca exhibited a more potent cytotoxicity against A2780 and ZR-75-1 cells (IC50=7.9µM and 8.5µM, respectively) compared to aca (IC50= 19.5µM and 17.5µM,respectively). Heimiol A did not show toxic effects against both cancer cells even at the highest concentration (150µM). All compounds showed selective toxicity toward the cancers cells, as they did not cause toxicity against normal PNT2A (prostate) cells. Further investigation behind the cytotoxic effect of the compounds revealed that 7-aca,aca, Q/A and bala caused the production of ROS, depletion of mitochondria membrane potential (ΔΨM) and an increase in caspase-3/7 levels in A2780 and ZR-75-1 cells. Many studies have reported that an excessive increase in ROS in cancer cells disrupts the mitochondrial membrane thus causing the loss of ΔΨM that leads to activation of effectors such as caspase-3, which eventually result in apoptotic cell death. Moreover,7-aca showed the strongest anti-metastatic effects through the inhibition of adhesion of cancer cells to the ECM protein fibronectin as well as inhibiting the migration and invasion of A2780 and ZR-75-1 cells by approximately 50-70%. The overall results revealed that 7-aca showed a stronger anti-cancer effects towards both cancer cells compared to aca and bala. Due to the more potent activity shown by 7-aca, A2780 and ZR-75-1 cells treated with 7-aca was chosen for metabolite analysis of cell lysates and aca was used as comparison. Several biomarker metabolites were decreased significantly (P<0.05) by the compounds, including ATP and NAD+ in the oxidative phosphorylation (OXPHOS) pathway and glucose-6-phosphate dehydrogenase, glutathione disulphide and NADPH in the pentose phosphate pathway (PPP). 7-Acashowed to cause greater reduction in all these metabolites compared to aca. These findings showed promising results for the compounds to be used as anti-cancer agents.;3) Anti-inflammatory assessment of 7-aca, aca and bala demonstrated the ability of these compounds to inhibit production of the pro-inflammatory cytokines TNF-α, IL1β and IL-6 by LPS-stimulated THP-1 macrophage cells. Bala at 10µM showed the strongest inhibition of TNF-α, IL-1β and IL-6 by 75%, 68% and 67%, respectively. 7-aca and aca showed similar activity by causing approximately 40-50% inhibition of all the cytokines. Due to the potent activity shown by bala, this compound was selected to carry out a retrospective investigation into any gene changes. RNA-sequencing analysis on RNA isolated from THP-1 macrophage cells treated with LPS or combination treatment of LPS and bala (LPS/bala) revealed that 27 genes associated with the cytokine-cytokine interaction pathway including TNF-α and IL-6 were downregulated by LPS/Bala treatment compared to LPS treatment alone. Furthermore, another two significant (P<0.05) pathways were also affected; the TNF-α and JAKSTAT signalling pathways. The genes associated with these pathways were shown to be significantly (P<0.05) down-regulated by LPS/bala treatment compared to LPS treatment alone. 4) 7-aca, aca and bala significantly (P<0.01) protected SH-SY5Y cells(neuroblastoma) from ROS generated by tert-butyl hydroperoxide (TBPH). Furthermore, these compounds showed their protective effects from TBPH-induced toxicity/oxidative stress through an increase in ΔΨM and decrease in caspase 3/7 levels in SH-SY5Y cells. Glutathione (GSH) levels were significantly (P<0.01) increased by all compounds which confirmed their potential antioxidant activity.;Bala showed to be the most potent antioxidant by increasing GSH level by 66.1%, decrease ROS level by 69% and inhibiting DPPH radical (IC50 = 6.2 µM). 7-Aca and aca showed similar antioxidant activity and neuroprotective effect. These findings suggest that compounds from A. malaccensis and H. dryobalanoides can provide excellent sources of natural raw material to isolate bioactive agents. In addition, the data also showed that the compounds isolated could possibly be used as anti-cancer, anti-inflammatory and neuroprotective agents in the future, based on the positive results obtained from the in vitro assays in this study.
Advisor / supervisor
  • Young, Louise
  • Ferro, Valerie A.
Resource Type
DOI
Date Created
  • 2020
Former identifier
  • 9912965993302996

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