The Role of Dietary Phenolic Antioxidants in Health and Disease
(You are what you eatand excrete?)
Background: There is currently enormous interest in the human diet – with “healthy” diets
reportedly offering protection against disease, while “bad” diets are being blamed for
increasing prevalence of obesity and related diseases such as diabetes and coronary heart
disease. Within a healthy diet, one class of compounds has been singled out for special
attention – phenolic antioxidants or biophenols. These are powerful antioxidants that occur
naturally in plants and are associated with consumption of fruits, vegetables, wine, tea and
olive oil. Our group has had an interest in biophenols for more than 10 years, but recently our
attention has focussed specifically on diet. Despite efforts by many groups around the world
big questions remain: how are biophenols absorbed, and what effect do they have on
metabolic processes that may be associated with disease prevention? This project is aimed at
working towards providing answers to these questions.
Project outline: During a recent Honours project on olive leaf extract we looked at urinary
biomarkers of biophenol ingestion and antioxidant activity. To our surprise we found that
urinary profiles seem highly specific to an individual and difficult to standardise. This makes
it incredibly challenging to define a “normal” profile, let alone one that may show evidence of
disease, or evidence of a successful dietary intervention. Therefore, one of the first aims of
this project will be to look at the relationship between diet and urinary profile, in a small
number of individuals, to attain a steady baseline of metabolic markers. Once this is achieved,
various acute dietary interventions will be trialled e.g. wine, olive leaf extract, tea, etc.
Urinary profiles will be examined to determine biomarkers that can be reliably attributed to
acute intake of the various biophenolic dietary interventions. Significant new knowledge will
be gained by this unique approach to diet and health.
This project will make extensive use of HPLC coupled to diode array and mass spectrometric
detection to profile metabolites in urine. It is expected that new metabolites will be discovered
that will require full structural characterisation, possibly by NMR through collaboration with
other researchers. This offers the candidate exposure to the burgeoning field of
“metabolomics”, which together with the other “-omics” i.e. genomics and proteomics,
promises much in improving our understanding of disease. Further, the candidate will gain
experience in diet and health related research, specifically related to biophenolic antioxidants.
Possible extensions to this project may involve a feeding trial involving a healthcompromised cohort (e.g. coronary heart disease patients) and the collection of other health
related data (apart from urinary profiles) such as blood-pressure (BP) of peripheral vascular
function (PVF). The opportunity may exist to correlate levels of biophenolic antioxidants with
improvements in these health measures, as well as changes in urinary profiles. This offers the
prospect of simultaneously measuring macroscale changes in health (BP or PVF) together
with molecular markers associated with the change (i.e. metabolites in the urine).
Contact: Dr Danielle Ryan (dryan@csu.edu.au) or Dr Paul Prenzler (pprenzler@csu.edu.au)
Analysis of Trace Organic Compounds in Bottled Water.
Background: Semi-volatile and volatile organic compounds (SV&VOC) are ubiquitous in
nature and range from natural products such as aroma compounds through to synthetic
pesticides such as DDT. To our surprise we discovered through a recent PhD project that
water from the Murrumbidgee River had extremely low levels of these compounds compared
to nanopure water that had been stored in plastic containers. This raises the question of
whether plastic containers (such as those used for bottled water) leach SV&VOC and if so,
whether these compounds have implications for human health.
Project outline: The project will involve determination of SV&VOC by solid-phase
microextraction (SPME) and gas chromatography (GC). In the first instance, several brands of
bottled water will be analysed qualitatively to see the types of compounds commonly
encountered. These samples will be compared to tap water, nanopure water, and distilled
water to gauge the range of compounds encountered in nominally pure water. Identification of
compounds will be done by tentative assignment using mass spectrometry (GC-MS) with a
limited number of compounds confirmed with standards. Quantitative determinations of
SV&VOC will then be made following choice of suitable internal standards. In the second
stage of the project, different types of plastic (e.g. from different types of drink bottles) will
be surveyed for their ability to leach compounds. Modelling experiments will be performed to
determine if factors such as heat and light affect leaching. Chemical hazard databases will be
consulted to ascertain what risk, if any, the presence of the SV&VOC compounds pose to
human health.
Contact: Dr Paul Prenzler (pprenzler@csu.edu.au) or Dr Alek Zander (azander@csu.edu.au)
Chemical characterisation of off-flavours
produced in grape berries following fungal infection
Background: Grape vines are prone to attack by a range of fungal pathogens; the mature
berries are particularly susceptible in climates that experience summer rainfall during the
ripening period. Diseases of the grape berry are refereed to collectively as ‘bunch rots’. There
are a range of organisms involved in bunch rot of grapes, some act as pathogens while others
grow as secondary invaders. All impact on grape and wine quality, producing off-flavours,
colour reduction (in the case of red wines) and depending on the pathogen problems in juice
handling due to the formation of viscous polysaccharides. The chemical nature of the offflavours and aromas produced in bunch rot affected grapes is poorly understood and what is
known relates largely to Botrytis cinerea infections (grey mould). For example wine made
from Botrytis affected fruit has musty and earthy aromas, attributed to a range of compounds
but in particular 1-octen-3-ol (Genovese, Gambuti et al. 2007; La Guerche, Dauphin et al.
2006)
Project outline: Established knowledge on the chemical composition of Botrytis-affected
grape berries will be used as a starting point for this work. Grape berries will be inoculated in
the laboratory with a range of berry pathogens and then analysed at various time points
following inoculation using analytical techniques (e.g. HPLC, GC-MS) to investigate the
chemical profile of the grapes. The aim will be to see if compounds such as 1-octen-3-ol are
produced in grapes following fungal infection by fungi other than Botrytis. A further aim is to
determine if it is possible to detect off-flavours in grapes prior to the appearance of visible
symptoms of berry rot. Separate to this project a series of wines were made from fruit with
varying degrees of ripe rot (Colletotrichum acutatum) infection. These wines will also be
available for the honours student to analyse to determine if the compounds detected in ripe rot
affected grapes are the same as those found in ripe rot affected wine.
This project is a multi-disciplinary one with the opportunity for an honours student to gain
experience in plant pathology and analytical chemistry techniques.
Contact: Prof. Chris Steel (csteel@csu.edu.au) or Dr Paul Prenzler (pprenzler@csu.edu.au)
Development of a Rapid, Robust Method to Measure Antioxidant Activity in a Lipid
System
Background: Lipid oxidation is problem both in the food industry (rancidity) and in human
health (involvement in atherosclerosis). There is currently enormous interest in antioxidants
from natural sources that may protect lipids from oxidative damage. The TBARS
(ThioBarbituric Reactive Substances) assay is one of very few assays designed to measure the
effectiveness of an antioxidant in protecting a lipid. However, there are numerous drawbacks
in using the TBARS assay including: lack of reproducibility, lack of precision, slow, not
amenable to multiple samples etc. Therefore there is a need to develop alternatives to the
TBARS assay that will be robust and rapid in assessing antioxidant activity in a lipid system.
Project outline: Beginning with the TBARS assay, the student will undertake a systematic
study of the reaction in order to determine the stages of the reaction where reproducibility and
precision are compromised. Once the method is made more robust, various reaction
parameters will be altered to speed up the reaction. In order to validate the developed method,
the student will need to characterise the products of lipid oxidation under carefully controlled
conditions. This project will thus involve a range of modern analytical techniques: GC,
HPLC, GC-MS, LC-MS, SPME, etc. Further, the student will gain a firm grounding in the
chemistry of lipid oxidation and the measurement of antioxidant activity relevant to the food
industry and human health.
Contact: Dr Paul Prenzler (pprenzler@csu.edu.au)