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Adomako-Bonsu, Amma Gyapomah (2016) Protection against oxidative stress in hepatic and pancreatic cells by selected plant-derived chemicals. PhD thesis, University of Nottingham.

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Abstract

Persistent accumulation of free radicals in cells leads to oxidative stress, which plays a causative role in the induction and progression of various chronic diseases. Therapeutic focus has therefore shifted towards the use of antioxidants, with recent interest in those of plant origin. This study investigated radical scavenging and cytoprotective activities of phytochemicals (quercetin, curcumin, sulforaphane, rosmarinic acid, caffeic acid, danshensu (3,4-dihydroxyphenyllactic acid), ferulic acid and m-coumaric acid) against DPPH free radical in a non-cellular assay, and oxidative damage in hepatic (HepG2) and pancreatic (1.1B4) cells, elicited by an organic hydroperoxide (tert-butylhydroperoxide - tBHP) and a more physiologically relevant stressor (palmitate). Direct and indirect cytoprotective activities were assessed by neutral red viability assay after 5 h co-exposure and 20 h pre-exposure conditions, respectively.

Radical scavenging activities of three well-known phytochemicals - quercetin, curcumin and sulforaphane - were initially validated against DPPH (non-cellular assay), where quercetin was shown to be more potent than curcumin; sulforaphane was without effect. With quercetin as positive control, radical scavenging activities of rosmarinic acid and three of its principal metabolites (caffeic acid, danshensu and ferulic acid) were comparable, while m-coumaric acid lacked antiradical activity against DPPH radical.

Subsequently in HepG2 hepatoma cells, quercetin and curcumin were confirmed to possess direct and indirect cytoprotective acitivities against 0.5 mM tBHP while, sulforaphane only had indirect cytoprotective acitivities. Additionally, co-treatment of HepG2 cells with low concentrations of quercetin and curcumin (used together) exhibited direct cytoprotective activities against tBHP. However, direct cytoprotective potencies of rosmarinic acid and caffeic acid were less than quercetin. Similar pattern was observed for indirect cytoprotective activities; with danshensu, ferulic acid and m-coumaric acid lacking hepatoprotective activity in co-exposure and pre-exposure conditions. These results highlight the discrepancy between non-cellular and cellular antioxidant activities, which could be accounted to the poor lipophilicity profiles of rosmarinic acid and its principal metabolites.

Cytotoxicity assay in 1.1B4 human pancreatic β-cells revealed that these cells were more vulnerable to tBHP-induced oxidative damage than HepG2 cells. An investigation of selected phytochemicals in 1.1B4 cells produced novel findings, with quercetin exhibiting direct and indirect cytoprotective activities against tBHP (0.125 mM and 0.5 mM). Curcumin and caffeic acid were also cytoprotective against 0.125mM tBHP but only exhibited direct cytoprotection against 0.5mM tBHP. Sulforaphane lacked both direct and indirect cytoprotective activities in 1.1B4 cells, exhibiting marked cytotoxic effects in both conditions.

Further analysis in both HepG2 and 1.1B4 cells proved that indirect cytoprotective activities of selected phytochemicals were not dependent on pro-proliferative activities of quercetin, curcumin, caffeic acid and sulforaphane. Moreover, it was observed that high concentrations of curcumin and sulforaphane caused necrosis in both cell types, rather than apoptosis; caffeic acid also produced necrotic effect in 1.1B4 cells.

Whilst prolonged exposure of HepG2 and 1.1B4 cells to high glucose concentrations failed to elicit any evidence of glucotoxicity, sodium palmitate caused concentration-dependent cytotoxicity after short-term (5 h) and long-term (20 h) exposure to both cell types. Overall, selected phytochemicals caused additive cytotoxicity in the presence of palmitate, although quercetin demonstrated direct cytoprotection alone in HepG2 cells.

Using Western blot, curcumin, caffeic acid and sulforaphane did not upregulate NQO1, but 20 h exposure to 0.1 mM quercetin resulted in upregulation in HepG2 cells, amidst high basal levels of NQO1 in this cell type. However, both basal and inductive expression of NQO1 has not been observed in 1.1B4 cells.

Thus, although rosmarinic acid, danshensu, caffeic acid and ferulic acid may possess good intrinsic antioxidant properties, their physicochemical properties may limit pharmacological activities at the cellular level. Moreover, the additive cytotoxicity resulting from treatment with selected phytochemicals and sodium palmitate highlights a discrepancy between mechanisms of cytotoxicities by tBHP and palmitate.

Item Type:	Thesis (University of Nottingham only) (PhD)
Supervisors:	Chan, S.L.F. Fry, J.R. Pratten, M.
Keywords:	Oxidative stress, Cell survival, Plant antioxidants, Phytochemicals, Cytoprotection, Quercetin, Curcumin, Sulforaphane
Subjects:	QS-QZ Preclinical sciences (NLM Classification) > QZ Pathology R Medicine > RB Pathology
Faculties/Schools:	UK Campuses > Faculty of Medicine and Health Sciences > School of Life Sciences
Item ID:	37172
Depositing User:	Adomako-Bonsu, Amma
Date Deposited:	15 Dec 2016 06:40
Last Modified:	16 Oct 2017 08:36
URI:	https://eprints.nottingham.ac.uk/id/eprint/37172

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