Natural Products Chemistry Group Annual One

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Natural Products Chemistry Group
Annual One-day Symposium
Charles Sturt University
Convention Centre
Wagga Wagga
rd
Friday, 3 October 2014
i
NATURAL PRODUCTS CHEMISTRY GROUP
ANNUAL ONE-DAY SYMPOSIUM
Charles Sturt University Convention Centre, Wagga Wagga 2678
Friday, 3rd October 2014
8:30-8:50
Coffee, Registration and Poster Set Up
8:55
Welcome
Leslie A. Weston, Jane C. Quinn,
Naresh Kumar
Metabolomics and Metabolic Profiling of Natural Products
9:00-9:30
Keynote Speaker
Metabolomics approaches in plant systems
biology
9:30-9:55
9:55-10:15
Simone Rochfort
Centre for AgriBio Science, DEPI, VIC
Dairy Futures Co-operative Research
Centre, Australia
La Trobe University, VIC
Invited Speaker
Application of Mass Profiler Professional in
disease treatment discovery: A statistical
analysis & visualization software tool in
metabolomics
James S. Pyke
Agilent Technologies, Mulgrave VIC
From leaf metabolome to in vivo testing:
Identifying new vertebrate antifeedant
compounds for ecological studies of
marsupial diets
K. J. Marsh
Research School of Biology, ANU,
Canberra
10:15-10:40
Invited Speaker
Wine metabolomics: combining sensory and
instrumental measures in the multiblock
framework – A Case of Chardonnay
Leigh M. Schmidtke
National Wine and Grape Industry
Centre, CSU
10:40-11:00
Small-scale metabolic profiling of secondary
compounds in Echium plantagineum and E.
vulgare
Dominik Skoneczny
School of Agricultural and Wine
Sciences, CSU
11:00-11:25
Tea Break
Activity of Natural Products—Signalling, Toxicity and Therapeutics
11:25-11:50
Invited Speaker
Antileukemic properties of chemical
constituents isolated from Indonesian
medicinal plants
Mamoru Koketsu
Department of Chemistry and
Biomolecular Science, Faculty of
Engineering, Gifu University, Japan
RACI NPG One Day Symposium 2014
ii
11:50-12:10
Invited Speaker
Perennial rye grass toxicosis; investigating
the complex biochemistry of shaky sheep
Jane C. Quinn
School of Animal and Veterinary
Sciences, CSU;
Graham Centre for Agricultural
Innovation
12:10-12:30
Antioxidant, anti-diabetic and
neuroprotective activities of four Australian
Acacia species
Nusrat Subhan
School of Biomedical Sciences, CSU
12:30-12:50
Discovery of the FFAs from the mushroom,
Fulaga dive (Amanitaceae) used traditionally
in Papua New Guinea
Edwin Castillo Martinez
Research School of Chemistry,
Australian National University,
Canberra
12:50-1:10
Olive Biophenols: A Natural Weapon Against
Alzheimer’s Disease
Syed H. Omar
School of Biomedical Sciences and
Graham Centre for Agricultural
Innovation, CSU
1:10-2:00
Lunch Break and Poster Session Visitation
2:00-2:20
Lead Compounds Targeting Hedgehog
Signalling Pathway from Natural Products
Yusnita Rifai
Hasanuddin University
Food Chemistry
2:20-2:45
Genetic and Environmental Effects on
Canola Oil Quality Traits, Tocopherols and
Carotenoids
Clare L. Flakelar
School of Agricultural and Wine
Sciences, CSU
Graham Centre for Agricultural
Innovation
2:45-3:00
Effects of canola proteins and hydrolysates
on adipogenic differentiation of C3H10T/2
mesenchymal stem cells
Adeola M. Alashi
Graham Centre for Agricultural
Innovation
School of Agricultural and Wine
Sciences, CSU
3:00-3:15
Understanding oxidation and antioxidant
activity in linoleic acid (LA) emulsion
through lipid-based antioxidant assay
M. A. Ghani
Graham Centre for Agricultural
Innovation, CSU
School of Agricultural and Wine
Sciences, CSU
3:15-3:30
Bioactive compounds in canola meal
Saira Hussain
Graham Centre for Agricultural
Innovation, CSU
School of Biomedical Sciences, CSU
ARC Industrial Transformation
Training Centre for Functional
Grains, CSU
3:30-3:55
Tea Break
RACI NPG One Day Symposium 2014
iii
Secondary Product Identification and Natural Product Synthesis
3:55-4:15
Invited Speaker
Novel Grass Cell Wall Phenolic Acids
Wade J. Mace
AgResearch Ltd, Grasslands Research
Centre, Palmerston North, New
Zealand
4:15-4:35
Identification and localization of
Xiaocheng Zhu
isohexenylnaphthazarins in mature roots and Graham Centre for Agricultural
Innovation, CSU
seedlings of Paterson’s curse (Echium
plantagineum)
4:35-4:55
Multiple roles of flavonoids in plant-rhizobia
symbioses
Samira Hassan
Department of Plant Sciences,
Research School of Biology, The
Australian National University,
Canberra
4:55-5:15
Sample Handling and Plant Phenols: Too
Important to Ignore
5:15-5:35
Backbone-fluorinated amino acids: synthesis Luke Hunter
School of Chemistry, UNSW Australia
and applications
5:40-6:15
Optional walking tour of CSU National Life Sciences Hub facility
6:00-7:00
Cocktail hour
Dinner
$45.00 per person, CSU convention centre, choice of entrees, 3 course meal, RSVP
to [email protected] by 28 September 2014. Featuring local produce and CSU
vintage wines.
Organizing committee: Prof Leslie A. Weston
Hassan K. Obied
Graham Centre for Agricultural
Innovation, and School of Biomedical
Sciences, CSU
[email protected]
Dr Jane C. Quinn
[email protected]
Dr Xiaocheng Zhu (Diego)
[email protected]
Symposium Managers:
W. http://www.csu.edu.au/research/grahamcentre/people/plant-and-animal-toxicologygroup/index.htm
RACI NPG One Day Symposium 2014
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We extend our sincere thanks to our corporate sponsors and exhibitors!
RACI NPG One Day Symposium 2014
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NATURAL PRODUCTS CHEMISTRY GROUP
ANNUAL ONE-DAY SYMPOSIUM
Charles Sturt University Convention Centre, Wagga Wagga 2678
Friday, 3rd October 2014
Table of Contents
Symposium Schedule
RACI NPG One Day Symposium
2014
Our sponsors and Exhibitors
i
iv
Metabolomics approaches in plant systems
biology
Simone Rochfort
1
Application of Mass Profiler Professional in
disease treatment Discovery: A statistical analysis
& visualization software tool in metabolomics
James S. Pyke, Malcolm J.
McConville, James McCarthy
2
From leaf metabolome to in vivo testing:
Identifying
new
vertebrate
antifeedant
compounds for ecological studies of marsupial
diets
K. J. Marsh, J. Au, B. Yin, I-P. Singh,
D. J. Tucker and W. J. Foley
3
Wine metabolomics: combining sensory and
instrumental measures in the multiblock
framework – A Case of Chardonnay
Leigh M. Schmidtke, John W.
Blackman, Andrew C. Clark, Paris
Grant-Preece, Hildegarde
Heymann, Douglas Rutledge
4
Small-scale metabolic profiling of secondary
compounds in Echium plantagineum and E.
vulgare
Dominik Skoneczny, Paul A.
Weston, Xiaocheng Zhu, Geoff M.
Gurr and Leslie A. Weston
5
Antileukemic properties of chemical constituents
isolated from Indonesian medicinal plants
Mamoru Koketsu
6
RACI NPG One Day Symposium 2014
vi
Perennial rye grass toxicosis; investigating the
complex biochemistry of shaky sheep
Martin A. Combs, Paul A. Weston,
Leslie A. Weston and Jane C. Quinn
7
Antioxidant, anti-diabetic and
neuroprotective activities of four Australian
Acacia species
Nusrat Subhan, Hassan K. Obied,
Philip Kerr, Geoffrey Burrows
8
Discovery of the FFAs from the mushroom, Fulaga
Dive (Amanitaceae) used traditionally in Papua
New Guinea
Edwin Castillo Martinez, Stewart W.
Wossa, Oromo Kevo & Russell A
Barrow
9
Olive Biophenols: A Natural Weapon Against
Alzheimer’s Disease
Syed H. Omar, Christopher J.
Scott, Adam Hamlin, Hassan K.
Obied
10
Yusnita Rifai, Midori A. Arai, Samir
K. Sadhu, Masami Ishibashi
11
Genetic and Environmental Effects on Canola Oil
Quality Traits, Tocopherols and Carotenoids
Clare L. Flakelar, David J. Luckett,
Julia A. Howitt, Gregory Doran, Paul
D. Prenzler
14
Effects of canola proteins and hydrolysates on
adipogenic differentiation of C3H10T/2
mesenchymal stem cells
Adeola M. Alashi, Christopher L.
Blanchard, Rodney J. Mailer,
Samson O. Agboola, A. John
Mawson, Rotimi E. Aluko and
Padraig Strappe
15
Understanding oxidation and antioxidant activity
in linoleic acid (LA) emulsion through lipid-based
antioxidant assay
M. A. Ghani, C. Barril, D. R.
Bedgood, P. D. Prenzler
16
Bioactive compounds in canola meal
Saira Hussain, Ata-Ur-Rehman,
David J. Luckett, Padraig Strappe
and Christopher L. Blanchard
17
Novel Grass Cell Wall Phenolic Acids
Wade J. Mace, Marty Faville,
Xuezhao Sun, Casey Flay, and
Michelle Turner
18
Identification
and
localization
of
isohexenylnaphthazarins in mature roots and
seedlings
of
Paterson’s
curse
(Echium
plantagineum)
Xiaocheng Zhu, Brigette Ryan,
Dmitry V. Sokolov, Geoff M. Gurr
and Leslie A. Weston
19
Multiple roles of flavonoids in plant-rhizobia
symbioses
Samira Hassan, Anton Wasson and
Ulrike Mathesius
20
Sample Handling and Plant Phenols: Too
Important to Ignore
Hassan K. Obied
21
Lead Compounds Targeting Hedgehog Signaling
Pathway from Natural Products
RACI NPG One Day Symposium 2014
vii
Backbone-fluorinated amino acids: synthesis
and applications
Luke Hunter
22
To conduct biological screening, isolate and
structurally elucidate bioactive compounds from
medicinal plants traditionally used by Indigenous
people
Kaisarun Akter, Joanne Jamie,
Subramanyam Vemulpad, David
Harrington
23
Amaranthus toxicity in production livestock
Emily Birckhead, Cecile Bouveret,
Dominik Skoneczny, Allan E.
Kessell, Leslie A. Weston and Jane
C. Quinn
24
Understanding photosensitization in grazing
animals caused by ingestion of the pasture
legume Biserrula pelecinus
Cecile Bouveret, Xiaocheng Zhu,
Jane C. Quinn, and Leslie.A. Weston
25
Acid Catalyzed Dimerization of Chromene Type
Natural Products
Kenneth Kam-Chung Hong, David
StC Black, Graham Ball, Naresh
Kumar
26
The genoprotecitve effects of faba beans (Vicia
faba)
Emma Kalle, Wouter Kalle, Hassan
Obeid, Christopher L. Blanchard
27
The Development of Isatin-based Prosthetic
Groups for Peptide Radiopharmaceuticals
S. K. Lim, N. Kumar, A. Katsifis
28
Pharmacokinetic Study of a Standardised
Herbal Extract of Diospyros kaki Leaves in Rats
Mitchell N. Low, Dennis Chang,
Cheang Khoo, Chun G. Li, Kelvin
Chan, Manilar Nang and Alan
Bensoussan
29
Changes in the composition of wine during
ethanol reduction processing
L. Manera, H. Ghantous B. Saha, A.
Deloire, L. Schmidtke, & P. Torley
30
Synthesis and Biological Activity of Novel Bisindole Inhibitors of Bacterial Transcription
Initiation Complex Formation
M. Mielczarek, D. StC. Black, R.
Griffith, P. J. Lewis, N. Kumar
31
Alkaloids in Australia: analysis of selected fungally
produced alkaloids in fifteen geographically
distinct annual ryegrass ecotypes
Joseph R. Moore, James E. Pratley,
Leslie A. Weston and Wade J. Mace
33
Phytochemical characterisation and analysis of
Curcuma xanthorrhiza extracts
Jarryd Pearson, Cheang Khoo, Alan
Bensoussan
34
RACI NPG One Day Symposium 2014
viii
Optimisation of small molecules targeting
childhood cancers
Hoang An Phan, Naresh Kumar and
Belamy-Bin Cheung
35
Pomegranates as a possible source of toxic illness
in cattle
Elizabeth Read, Myrna A. Deseo,
Mark Hawes and Simone Rochfort
36
Development of high throughput screening assays
for determining endophyte associated toxins
Priyanka Reddy, Myrna A. Deseo,
John Forster and Simone Rochfort
37
The role of leaf surface chemistry and morphology
in plant defense of two related invasive weeds
Echium vulgare and Echium plantagineum
Brigette Ryan, Dominik Skoneczny,
Xiaocheng Zhu, Paul A. Weston,
Jane C. Quinn and Leslie A. Weston
38
Impact of Paterson’s curse (Echium plantagineum
L.) establishment on species richness in invaded
and native range
Dominik Skoneczny, Xiaocheng Zhu,
Ragan M. Callaway and Leslie A.
Weston
39
Determination of nitric oxide inhibitory activities
of selected raw and processed Chinese materia
medica
John Truong, Xian Zhou, Valentina
Razmovski-Naumovski, Cheang
Khoo, Kelvin Chan
40
Flavonoids as potential scaffold for hybrid drug
molecules
Yee M. H. E, Kumar N.
41
Aqueous extracts of Danshen-Sanqi herb-pair
inhibit lipopolysaccharide- induced nitric oxide
release in RAW264.7 macrophage cells through
the PI3K pathway
Xian Zhou, Antony Kam, Valentina
Razmovski-Naumovski, Kelvin Chan
42
RACI NPG One Day Symposium 2014
1
 Poster
 Oral Presentation
(please tick)
Metabolomics approaches in plant systems biology
Simone Rochfort1,2,3
1
Centre for AgriBio Science, Biosciences Research Division, Department of
Environment and Primary Industries, Bundoora, VIC
2
Dairy Futures Co-operative Research Centre, Australia
3
La Trobe University, Bundoora, VIC
Corresponding author email address: [email protected]
Perennial ryegrass Lolium perenne L., is an important forage grass in temperate grazing
systems. The grass can harbour fungal endophytes of the Neotyphodium genus. The
endophyte produces a suite of secondary metabolites that convey competitive advantage to
the grass in terms of both biotic and abiotic stress resistance. In farming systems this
translates as a more persistent and resilient pasture and the symbiota are used extensively
in grazing and dairy systems in Southern Australia and New Zealand. Many of these
metabolites convey insect resistance to the plant however, others, in particular the ergot
alkaloids and lolitrem metabolites can cause serious detrimental effects to grazing animals
including neurological disorders that are commonly termed rye grass staggers. Metabolomic
analysis has been applied to investigate novel rye grass cultivar – endophyte interactions
uncovering a strong genome-by-genome interaction. Metabolomic applications in optimising
symbiota, alkaloid pathway analysis and validation of gene knock down events will be
discussed.
RACI NPG One Day Symposium 2014
2
 Poster
 Oral Presentation
(please tick)
Application of Mass Profiler Professional in disease treatment Discovery: A
statistical analysis & visualization software tool in metabolomics
James S. Pyke1, Malcolm J. McConville2,3, James McCarthy4
1
2
3
Agilent Technologies, Mulgrave VIC 3170, Australia
Metabolomics Australia, The University of Melbourne, VIC 3010, Australia
Bio21 Molecular Science and Biotechnology Institute, The University of Melbourne,
Parkville, VIC 3010, Australia
4
Queensland Institute of Medical Research
Corresponding author email address: [email protected]
Metabolomic approaches are increasingly used to understand fundamental aspects of cell
metabolism and physiology, to identify biomarkers of different physiological states and the
effects on metabolism due to disease and drug administration, to name a few biomedical
applications. A major goal of metabolomics techniques is to provide a quantitative analysis of
all low molecular weight compounds in a biological extract and Mass Spectrometry (MS)
based differential metabolite profiling is just one way of monitoring metabolic actives in a
biological system over time. Advances continue to be made in the development of new
analytical techniques and instrumentation, which aid in a wider coverage of the metabolome
of a biological system. However, as advances in analytical techniques increase the coverage
of the metabolome, the data complexity, analysis and finally biological interpretation is more
and more reliant on efficient software tools to process and ultimately interpret the biology
from complex data sets.
The overall experimental design, data acquisition, data processing, and statistical analysis
workflow of an experiment searching for the metabolic effects of a disease and subsequent
treatment will be used to exemplify advances in some analytical software tools. In particular,
the benefits of MassHunter Profiler for the untargeted molecular feature extraction across an
entire batch of LC-QTOF data generated from this experiment will show the impacts of data
extraction techniques in subsequent statistical analysis tools, such as MassHunter Profiler
Professional (MPP). The genotypic, phenotypic variations over time are statistically analysed
in MPP by clustering the replicates of samples according to the extracted molecular features.
The combination of these tools improves the ability to answer defined biological questions
that are answerable within the experiments design. The overall workflow focuses on the
significant molecular features of interest found, reducing the complexity of the analysis and
allowing focused identification and biological interpretation against known metabolic
pathways within the organism.
RACI NPG One Day Symposium 2014
3
 Poster
 Oral Presentation
(please tick)
From leaf metabolome to in vivo testing: Identifying new vertebrate antifeedant
compounds for ecological studies of marsupial diets
K. J. Marsh1*, J. Au1, B. Yin1, I-P. Singh2, D. J. Tucker3 and W. J. Foley1
1
Research School of Biology, Australian National University, Canberra 0200
2
3
National Institute of Pharmaceutical Education & Research, Nagar, India
School of Science & Technology, University of New England, Armidale 2351
*
Corresponding author email address: [email protected]
Different folivorous marsupials (koalas, greater gliders and ringtail and brushtail possums)
select their food from different subgenera of Eucalyptus, but the choices cannot be explained
by known antifeedants, such as formylated phloroglucinol compounds or tannins, or by
nutritional quality. Therefore, we used a metabolomic approach in which we employed 1H
nuclear magnetic resonance spectroscopy to identify chemicals that consistently differ
between representatives of the two subgenera. We observed1 that dichloromethane extracts
of leaves from most species in the subgenus Monocalyptus differ from those in subgenus
Symphyomyrtus by the presence of free flavanones, with no substitution in ring B. Given the
broad biological effects of these compounds, we hypothesised that they exert an antifeedant
effect against marsupials.
Extraction of Eucalyptus sieberi foliage provided about 9.8 g of pinocembrin of good purity.
We compared the antifeedant effect of pinocembrin and three other flavonoids with varying
substitution in the B ring via in vivo experiments with common brushtail possums
(Trichosurus vulpecula). We found that flavanones with un-substituted B rings (pinocembrin
and flavanone) deterred possums from eating a palatable artificial diet, whereas a flavanone
with a substituted B ring (naringenin) and a flavone (chrysin) did not. We conclude that unsubstituted B ring flavanones are likely major influences on the variable palatability of
Monocalyptus foliage. We are developing a HPLC assay to enable us to seek correlations
between the concentration of un-substituted B ring flavanones in Monocalyptus leaves and
the food intake of marsupial folivores.
--------------------References
1. Tucker et al (2010) J. Chem Ecol 36:727
RACI NPG One Day Symposium 2014
4
 Poster
 Oral Presentation
(please tick)
Wine metabolomics: combining sensory and instrumental measures in the
multiblock framework –
A Case of Chardonnay
Leigh M. Schmidtke1*, John W. Blackman1, Andrew C. Clark1, Paris Grant-Preece1,
Hildegarde Heymann2, Douglas Rutledge3
1
2
National Wine and Grape Industry Centre, Charles Sturt University, Locked Bag
588, Wagga Wagga, New South Wales, 2678, AUSTRALIA
Department of Viticulture and Enology, University of California, Davis, CA 95616,
USA
3
laboratoire de Chimie Analytique, AgroParisTech, Paris, France
*Corresponding author email address: [email protected]
Phone Int + 61 2 69334025
Multiple instrumental measures of wine composition may enable insight to the contribution
and synergistic effects of volatile and non volatile components to the overall sensory profiles
of wines. Employing numerous analytical techniques for the determination of wine
composition generates large volumes of data, comprising several data matrices, and requires
multivariate multiblock data analysis to elucidate the importance of wine components to the
overall sensory profile. In this investigation, multi-instrumental measure of wine composition,
targeting the volatile and non-volatile wine fractions was undertaken simultaneously during
full descriptive assessment of 21 Australian Chardonnay wines selected to represent
important commercial styles. Descriptive sensory assessment of each wine was undertaken
in triplicate using a fully trained panel of assessors. The relative concentrations of wine
components were obtained from the extracted peak areas of chromatographic data. A
multivariate curve resolution alternating least squares (MCR-ALS) approach was used to
obtain peak area tables and compound spectral profiles from GC-MS and UHPLC-PDA
chromatograms. These two respective data tables provided a profile of the volatile and
phenolic composition of the wines. The relative concentrations of wine organic acids were
estimated from HPLC chromatograms with detection at 210nm, whereas carbohydrate and
ethanol profiles were estimates using HPLC with refractive index. All analytical measures
were conducted on the day of descriptive sensory analysis. Common components and
specific weights analysis of the data matrices is used to illustrate the importance and
contribution of the measured components to the sensory profiles of the wines.
RACI NPG One Day Symposium 2014
5
 Poster
 Oral Presentation
(please tick)
Small-scale metabolic profiling of secondary compounds in Echium
plantagineum and E. vulgare
Dominik Skoneczny1, Paul A. Weston2, Xiaocheng Zhu2, Geoff M. Gurr3 and
Leslie A. Weston2*
1
Charles Sturt University, School of Agricultural and Wine Sciences, Life Sciences
Building Wagga Wagga, NSW 2678, Australia
2
Charles Sturt University, Graham Centre, Life Sciences Building, Wagga Wagga,
NSW 2678, Australia
3
Charles Sturt University, School of Agricultural and Wine Sciences, Orange, NSW
2800, Australia
*Corresponding author email address: [email protected]
Understanding the role of environment and genetics in accumulation of plant defense
secondary metabolites may provide clues towards improved understanding of factors
influencing successful invasion by noxious weedy species. Twenty-two Australian
populations of Paterson’s curse (Echium plantagineum L., Boraginaceae) and 4 populations
of Viper’s bugloss (E. vulgare) were screened by UHPLC-ESI-Q-ToF. Two families of key
bioactive compounds isohexenylnaphthazarins (IHNs) and pyrrolizidine alkaloids (PAs)
were evaluated. IHNs occur as red pigments in roots periderm; in contrast PAs accumulate
in foliar and floral tissues. Ethanolic extracts of root periderm and methanolic leaf extracts
were evaluated qualitatively and quantitatively. Small-scale metabolic profiling allows for
monitoring of selected groups of compounds in complex matrices such as plant root or
shoot extracts. PCDL Manager software (Agilent) was utilised to create databases of 14
PAs and 24 putative IHNs. Untargeted analyses were also performed and compared to
results of targeted analyses. Interestingly, we found several differences and similarities in
constituents associated with plant defense among both species; selected secondary plant
products can be potentially utilized as biomarkers. Metabolic profiles of plants grown in
controlled conditions revealed considerable differences in comparison to field grown plants.
Identifying patterns of abundance among key constituents in Echium spp, including native
Spanish genotypes, will provide crucial information to improve our understanding of plant
invasion mechanism(s).
Keywords: metabolic profiling, plant invasions, E. plantagineum, E. vulgare,
isohexenylnaphthazarins, pyrrolizidine alkaloids, mass spectrometry
RACI NPG One Day Symposium 2014
6
 Poster
 Oral Presentation
(please tick)
Antileukemic properties of chemical constituents isolated from Indonesian
medicinal plants
Mamoru Koketsu1*
1
Department of Chemistry and Biomolecular Science, Faculty of Engineering, Gifu
University, 1-1 Yanagido, Gifu 501-1193, Japan
*
Corresponding author email address: [email protected]
Plant constituents have proven to be the most reliable source of innovative therapeutic
agents for various conditions. Exploration of natural active compounds from medicinal plants
for treatment of broad range of diseases has attracted substantial attention worldwide.
Herein, I would like to introduce our investigations including structural analysis and
antileukemic properties of chemical constituents from Indonesian medicinal plants. The
following is table of contents of my presentation.
1. Sentulic acid: A cytotoxic ring A-seco triterpenoid from Sandoricum koetjape Merr1
A new ring A-seco triterpenoid, sentulic acid, along with a known oleanane-type triterpenoid,
3-oxoolean-12-en-27-oic acid, were isolated from the Indonesian plant S. koetjape Merr. In
addition, the results of bioassays showed that these compounds were able to induce
cytotoxicity in these cells. Furthermore, morphological analysis indicated that their cytotoxic
effects were mediated by apoptosis.
New compound
Sentulic acid 50 mM
2. Phytochemical analysis and antileukemic activity of polyphenolic constituents of
2
Toona sinensis
The aim of this study was to identify the potential compounds
responsible for anticancer activity of T. sinensis. Our
phytochemical research of these extracts led to the isolation of
various polyphenolic constituents. Among the isolates, gallic
acid and loropetalin D showed the inhibition of cell
Loropetalin D
proliferation and possible induction of apoptosis in these cells.
In addition, an analysis of structure-activity relationship
indicated that the number of galloyl groups affects their antileukemic potency.
--------------------References
1. M. Efdi, M. Ninomiya, E. Suryani, K. Tanaka, S. Ibrahim, K. Watanabe, and M. Koketsu, Bioorg.
Med. Chem. Lett., 2012, 22, 4242.
2. A. Kakumu, M. Ninomiya, M. Efdi, M. Adfa, M. Hayashi, K. Tanaka, and M. Koketsu, Bioorg. Med.
Chem. Lett., 2014, 24, 4286.
RACI NPG One Day Symposium 2014
7
 Poster
 Oral Presentation
(please tick)
Perennial rye grass toxicosis; investigating the complex biochemistry of shaky
sheep
Martin A. Combs1,3, Paul A. Weston2, Leslie A. Weston3 and Jane C. Quinn1,3
1
School of Animal and Veterinary Sciences Charles Sturt University, Wagga Wagga
NSW
2
School of Agriculture and Wine Sciences Charles Sturt University, Wagga Wagga
NSW
3
Graham Centre for Agricultural Innovation, (Charles Sturt University and NSW
Department of Primary Industries), Wagga Wagga NSW
Corresponding author email address: [email protected]
Perennial Ryegrass Toxicosis (PRGT) is a common disease entity in Australian livestock
systems. Clinical signs of PRGT include alterations in normal behaviour, ataxia (‘staggers’),
ill thrift, electrolyte disturbances and gastrointestinal dysfunction (scours). The clinical signs
associated with ingestion of toxic perennial rye grass are caused by the neurotoxin Lolitrem
B, an indole–diterpenoid alkaloid produced by the endophytic fungus Neotyphodium lolii, a
symbiont of perennial ryegrass. Perennial ryegrass is a pasture species with widespread
prevalence across south-east Australia, Tasmania and New Zealand. Cases of PRGT can
range in severity; animals can exhibit mild gait abnormalities and a general failure to thrive,
to those who present with severe movement abnormalities, seizures, lateral recumbency and
death. Mild outbreaks of PRGT result mainly in subclinical production losses, management
challenges and animal welfare issues for affected stock whilst severe outbreaks can result in
significant stock losses. In either scenario there are significant economic losses for the
affected producer.
Lolitrem B has a widespread activity in the central nervous system and is a potent inhibitor of
large conductance Ca+ activated potassium (BK) channels, causing direct effects on
neuroexcitation and neuronal function. To better understand the role of this toxin in the
pathogenesis of PRGT we have examined its effects in both small and large animal models
of the disease, showing that the clinical signs associated with intoxication can be accurately
recapitulated in our model systems. To better define the systemic activity of Lolitrem B, we
have also developed simplified and efficient methods for recovery of toxins from intoxicated
animals, with up to 1000-fold greater sensitivity in detecting these analytes. Recovery of the
compounds from mammalian tissue samples were found to be greatly facilitated using
extraction by grinding in liquid nitrogen (Bligh-Dyer) with improved quantitation using
reversed-phase HPLC coupled with mass-spectrometry (LC-MS). This could also be
achieved using standard solvents (e.g. acetonitrile and water) and columns (e.g. C18),
negating the use of halogenated solvents and columns previously described in the literature.
This increased sensitivity has allowed identification of Lolitrem B toxin in the kidney, a
previously unconfirmed target of the toxin. Together, these studies will allow a greater
understanding of the systemic effects of lolitrem B in perennial rye grass toxicosis, as well as
having potential applicability to other complex neurotoxicities affecting domestic livestock.
RACI NPG One Day Symposium 2014
8
 Poster
 Oral Presentation
(please tick)
Antioxidant, anti-diabetic and neuroprotective activities of four Australian
Acacia species
Nusrat Subhan1*, Hassan K. Obied1, Philip Kerr1, Geoffrey Burrows2
1
School of Biomedical Sciences and 2 School of Agricultural and Wine Sciences,
Charles Sturt University, Wagga Wagga, Australia
*
Corresponding author email address: [email protected]
The genus Acacia is large with a worldwide spread. Approximately 1400 species are native
to Australia. Many species of Acacia have been used in folk medicine for the treatment of
various pathophysiological conditions such as diabetes, toothache, gastro-intestinal,
respiratory, eye and skin disorders. Four Australian Acacia species, viz Acacia. deanei, A.
implexa, A. pycnantha and A. verniciflua were selected as local representative examples
used by the Wiradjui people. Leaves and phyllodes were collected, freeze-dried and ground.
The powdered leaves were sequentially extracted using hexane, dichloromethane, methanol
and water. Antioxidant activities were measured using the DPPH radical and ABTS+ radical
scavenging assays. Reducing power of extracts was measured by the FRAP assay. In vitro
anti-diabetic (alpha-amylase inhibition) and neuroprotective (acetylcholine esterase
inhibition) assays of the extracts were also performed. Methanolic extracts showed higher
antioxidant, anti-diabetic and neuroprotective properties for all four species. Meanwhile, A.
pycnantha extracts showed the highest activity. Our results indicate that the leaves of
Australian Acacia are rich in biophenols with numerous pharmacological activities that
warrant further investigation for potential applications in nutraceutical and food industries.
RACI NPG One Day Symposium 2014
9
 Poster
 Oral Presentation
(please tick)
Discovery of the FFAs from the mushroom, Fulaga Dive (Amanitaceae) used
traditionally in Papua New Guinea
Edwin Castillo Martinez1*, Stewart W. Wossa1,2, Oromo Kevo3 & Russell A Barrow1,2
1
Research School of Chemistry, Australian National University, Canberra, ACT, 0200
2
University of Goroka, Goroka, Eastern Highlands, 441, Papua New Guinea
3
Kiovi Tribe, Eastern Highlands, Papua New Guinea
*
Corresponding author email address: [email protected]
As a part of an ongoing study examining the traditional use of fungi by communities’ in the
Highlands of Papua New Guinea our attention was focussed on the extract of the mushroom,
Fulaga Dive, used as a food source by the Kiovi tribe, in the Eastern Highlands Province.
The mushroom demonstrated antibacterial activity against the Gram (+) S. epidermidis (IC50
305 µg/mL) and represented a new species within the genus and subgenus Amanita as
determined by DNA analysis. Bioassay guided isolation and chemical structure elucidation
revealed the presence of novel furan fatty acids (FFAs) in abundant quantities. While FFAs
are rarely detected in nature they have recently gained special attention due to their
usefulness as active components of functional foods and their potential as antiinflammatories and radical scavengers [1,2]. The aim of the present work was to explore the
pathway from ethnomycology through isolation, structural elucidation and synthesis of these
compounds.
----------------------Acknowledgements: ECM acknowledges the ANU for an Australian Postgraduate Award. RAB
acknowledges funding from NHMRC (1028092).
References:
1. Vetter, W.; Wendlinger, C. (2013) Lipid Technology, 25 (1), 7-10.
2. Wakimoto, T.; Kondo, H.; Nii, H.; Kimura, K.; Egami, Y.; Oka, Y.; Yoshida, M.; Kida, E.; Ye, Y.;
Akahoshi, S.; Asakawa, T.; Matsumura, K.; Ishida, H.; Nukaya, H.; Tsuji, K.; Kan, T.; Abe, I. (2011)
Proceedings of the National Academy of Sciences, 108 (42), 17533-17537.
RACI NPG One Day Symposium 2014
10
 Poster
 Oral Presentation
(please tick)
Olive Biophenols: A Natural Weapon Against Alzheimer’s Disease
Syed H. Omar1,2,*, Christopher J. Scott1,2, Adam Hamlin1, Hassan K. Obied1,2
1
School of Biomedical Sciences and 2 Graham Centre for Agricultural Innovation,
Charles Sturt University, Wagga Wagga, Australia
*
Corresponding author email address: [email protected]
The most common form of dementia is Alzheimer’s disease (AD) which affects a high
percentage of people over the age 65 years. Globally, there are more than 36 million people
with dementia and it is predicted that numbers will reach 115 million by 2050. Currently, there
is no cure or treatment for AD. The FDA approved medicines have only shown a palliative
effect. Due to the severity of adverse effects and the narrow therapeutic index, researchers
suggested that some dietary and herbal constituents may offer certain cognitive benefits
alone or as adjunct to approved medication. Amongst various phytochemicals, biophenols
have attracted a great deal of attention. In addition to their well known antioxidant and radical
scavenging activities, biophenols are multi-potent chemical entities with a wide array of
pharmacological activities. Thus, they may be able to counteract or halt the progression of
neuronal damage in AD.
Olive (Olea europaea) fruit, leaf and extra-virgin oil are rich sources of biophenols. We
investigated the effect of these biophenols on the activity of key enzymes implicated in AD
disease: acetylcholine esterase, butyrylcholinesterase, β-secretase, tyrosinase and histone
deacetylase enzymes. Olive biophenols showed potent cholinesterase (quercetin: IC50= 9.42
μM), tyrosinase (quercetin: IC50= 10.73 μM) and histone deacetylase (Quercetin: IC50 =
105.1μM) inhibitory activities. In transgenic mice (APPswe), our results showed significant
improvement in memory and obvious reduction in amyloid plaques deposition in the brains of
olive extract treated mice. In conclusion, olive biophenols can present a powerful and safe
nutraceutical weapon to fight AD.
RACI NPG One Day Symposium 2014
11
 Poster
 Oral Presentation
(please tick)
Lead Compounds Targeting Hedgehog Signaling Pathway
from Natural Products
Yusnita Rifai1*, Midori A. Arai2, Samir K. Sadhu3, Masami Ishibashi2
1
2
Hasanuddin University,
Grad. Sch. Pharm. Sci., Chiba Univ.,
3
Khulna Univ.
*
Corresponding author email address: [email protected]
Hedgehog (Hh) signaling regulates numerous events in embryonic development and adult
tissue maintenance. The signaling is constitutively activated in several types of human
tumors such as basal cell carcinoma and medullablastoma due to mutations in Ptch or
Smoothened (Smo). To screen natural product libraries, a previous constructed cell screen of
expressed exogenous GLI1 in HaCaT under tetracycline control (T-REx system) was used.
To identify the cell viability of compounds, a fluorometric microculture cytotoxicity (FMCA)
method was performed. A screening program has identified Acacia pennata, Excoecaria
agallocha, Vallaris glabra, Ocimum gratissimum and Piper chaba as hit plants. Of 40 isolated
compounds, 1, 5, 6, 13, 14, 31, and 40 (Figure 1) exhibited strong inhibition of Hh/GLImediated transcription. Compounds were cytotoxic against PANC1, DU145 but less affect a
normal cell line (Figure 2). Western blotting results confirmed that compounds reduced the
expression of PTCH and BCL-2 proteins in a concentration-dependent manner. To further
understand the molecular mechanism underlying Hh signaling inhibitory effect, we examined
the expression of a GLI-related gene (Ptch) using real-time quantitative RT-PCR. Treatment
with 1 and 6 led to a significant decrease in mRNA expression of Ptch in PANC1, suggesting
that both compounds had an inhibitory effect on the transcription of Hh/GLI. In addition,
deleting the Smo function in PANC1 treated with led to downregulation of the mRNA
expression of Ptch (Figure 3).
RACI NPG One Day Symposium 2014
12
Figure 1
Figure 2
RACI NPG One Day Symposium 2014
13
Figure 3. Expression of Ptch mRNA (upper
panel) and Smo protein (bottom panel) in PANC1
treated with 6 after siRNA-mediated silencing
of Smo.
Figure 4. Effect of compound 6 on GLI1 and/or GLI-related
protein (PTCH and BCL2) levels in full, cytosolic and
nuclear PANC1 cells.
----------------------References
1. Rifai, Y.; Arai, M. A.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. J. Nat.Prod. 2010, 35, 995-997.
2. Rifai, Y.; Arai, M. A.; Sadhu, S. K.; Ahmed, F.; Ishibashi, M. Bioorg. Med.Chem. Lett. 2011, 21, 718722.
3. Rifai, Y.; Arai, M. A.; Koyano, T.; Kowithayakorn, T.; Ishibashi, M. J. Nat.Med. 2011, 65, 629-632.
RACI NPG One Day Symposium 2014
14
 Poster
 Oral Presentation
(please tick)
Genetic and Environmental Effects on Canola Oil Quality Traits,
Tocopherols and Carotenoids
Clare L. Flakelar1,2, David J. Luckett2,3, Julia A. Howitt1,4, Gregory Doran1,2,
Paul D. Prenzler1,2
1
School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga,
NSW 2650, Australia
2
Graham Centre for Agricultural Innovation (an alliance between Charles
Sturt University and NSW Department of Primary Industries), Pugsley Place,
Wagga Wagga, NSW 2795, Australia
3
NSW Department of Primary Industries, Agricultural Institute, Pine Gully
Road, Wagga Wagga 2650, NSW, Australia
4
Institute for Land, Water and Society, Charles Sturt University, Wagga
Wagga, NSW 2650, Australia.
Corresponding author email address: [email protected].edu.au
Canola oil has become an extremely important oil seed crop, both in Australia and overseas,
contributing millions of dollars to the food and agricultural industries. With the continuing rise
in canola production, new directions are being considered to further expand its marketability.
Recently, there has been an increased interest in enhancing any potentially health-beneficial
bioactive components present in the oil. Tocopherols and carotenoids are two classes of
bioactive components present in crude canola oil worthy of attention for their associated
health-benefits and antioxidant functionality. Few studies have been undertaken to analyse
these compounds in canola oil. Furthermore correlations between these components and
standard quality traits for oilseeds, as defined by AOF guidelines, have not been investigated
in depth. This study investigated 156 canola oil samples comprising 28 commercial varieties
collected from 14 different locations in southern NSW. Standard quality parameters were
assessed along with quantification of α-, γ-, and -tocopherol, β-carotene and lutein. A
subset of these samples (n=52) were analysed for FAP and oxidative stability, and correlated
against the traits above. Significant correlations were found amongst many traits, namely oil
content and: β-carotene (r = -0.33), lutein (r = -0.23), α-tocopherol (r = -0.49), γ-tocopherol (r
= 0.22), and δ-tocopherol (r = -0.30). Polyunsaturated fatty acid composition was significantly
(p < 0.01) positively correlated with carotenoid and tocopherol concentrations. Additionally, a
REML analysis of G  E effects illustrated significant varietal effects on levels of tocopherols
and carotenoids, and may provide useful information for breeding programs targeting
genotypes with enhanced levels of these compounds.
RACI NPG One Day Symposium 2014
15
 Poster
 Oral Presentation
(please tick)
Effects of canola proteins and hydrolysates on adipogenic differentiation of
C3H10T/2 mesenchymal stem cells
Adeola M. Alashi1, 2*, Christopher L. Blanchard1, 3, Rodney J. Mailer1, Samson O.
Agboola1, 2, A. John Mawson1, 2, Rotimi E. Aluko4 and Padraig Strappe1, 3
1
2
Graham Centre for Agricultural Innovation,
School of Agricultural and Wine Sciences, Charles Sturt University and
3
School of Biomedical Sciences, Charles Sturt University, Locked Bag
588, Wagga Wagga, NSW 2678, Australia
4
Department of Human Nutritional Sciences and the Richardson Centre
for Functional Foods and Nutraceuticals, University of Manitoba, Winnipeg
Canada, R3T 2N2
*
Corresponding author email address: [email protected]
Obesity is a health issue that is closely associated with the onset of other pathological
disorders including type II diabetes, hypertension, cancer and atherosclerosis. Inhibition of
adipocyte differentiation results in reduced fat accumulation in cells, which may have
application in treatment of obesity. This study investigates the ability of canola protein isolate
(CPI) and enzymatic hydrolysates (CPHs) to inhibit in vitro adipogenic differentiation of
C3H10T1/2 murine mesenchymal stem cells. CPH samples were obtained by individually
treating CPI with one of five digestive enzymes (Alcalase, trypsin, chymotrypsin, pepsin and
pancreatin). Viability of the C3H10T1/2 cells was maintained for all samples at a
concentration of 60 µg/ml. Samples showed anti-adipogenic differentiation activity with the
Alcalase hydrolysate demonstrating a higher reduction in differentiation through quantitation
by oil red O staining. Quantitative polymerase chain reaction (qPCR) analysis showed that
CPI and CPHs significantly inhibited expression of the peroxisome proliferator-activated
receptor gamma (PPARγ) gene, a key transcription factor involved in controlling adipocyte
differentiation. This was evident in a ~60-80 % fold reduction of PPARγ mRNA in CPI- and
CPH-treated cells. Immunofluorescence staining for PPARγ protein also showed a reduced
expression in cells treated with CPI and chymotrypsin, trypsin, and Alcalase hydrolysates
when compared to differentiated control cells. These results demonstrate that CPI and CPHs
contain bioactive components which can modulate in vitro adipocyte differentiation which
may have potential applications in a diet-based antiobesity intervention.
RACI NPG One Day Symposium 2014
16
 Poster
 Oral Presentation
(please tick)
Understanding oxidation and antioxidant activity in linoleic acid (LA) emulsion
through lipid-based antioxidant assay
M. A. Ghani1,2*, C. Barril2, D. R. Bedgood2, P. D. Prenzler1,2
1
Graham Centre for Agricultural Innovation, Charles Sturt University, Wagga Wagga,
NSW, Australia
2
School of Agricultural and Wine Sciences, Charles Sturt University, Wagga Wagga,
NSW, Australia
*Corresponding author email address: [email protected]
Polyunsaturated fatty acids such as linoleic acid (LA) are very susceptible to oxidation due to
reaction with oxygen and reactive oxygen species (ROS)1. LA is commonly used as a lipid
substrate in lipid-based antioxidant assay. In this assay, LA is oxidised for a certain period of
time and oxidative stability is studied using antioxidants from synthetic and natural sources2.
Our previous study showed that oxidation and antioxidant activity of Trolox in bulk LA in two
phase system might be problematic. In this perspective, an LA emulsion has been
investigated as alternative to study oxidation and antioxidant activity. In addition, temperature
effect of different antioxidants in this system has been taken into consideration. Consistent
oxidation and oxidation inhibition in a system are pre-requisite for a robust method to
measure antioxidant activity. Experimental design has been applied to find the stage of the
method where reproducibility was compromised by optimising oxidation time and different
concentrations of antioxidant. At the initial stage, different concentrations of synthetic
antioxidant Trolox were used to observe antioxidant activity. Emulsification was performed
giving average particle size of LA droplet ranging from 0.1 to 0.25 µm in diameter. Oxidation
and antioxidant activity were measured by a method adaptation on the basis of lipid-based
ferric thiocyanate (FTC) assay using absorbance at 500 nm. Results will be presented with a
particular view of using LA emulsion to more reliably assess antioxidant assay. The
understanding of this study will be helpful to develop a lipid-based method to measure
antioxidant activity of natural antioxidants.
1
-----------------------References (5 max)
1. Yin, H., & Porter, N. A. (2005). New insights regarding the autoxidation of polyunsaturated fatty
acids. Antioxidants & redox signaling, 7(1-2), 170-184.
2. McDonald, S., Prenzler, P. D., Antolovich, M., & Robards, K. (2001). Phenolic content and
antioxidant activity of olive extracts. Food Chemistry, 73(1), 73-84.
RACI NPG One Day Symposium 2014
17
 Poster
 Oral Presentation
(please tick)
Bioactive compounds in canola meal
Saira Hussain1,2.3, Ata-Ur-Rehman2,3, David J. Luckett 1, Padraig Strappe1,2,3 and
Christopher L. Blanchard 1,2,3*
1
2
3
Graham Centre for Agricultural Innovation, Charles Sturt University and NSW
Department of Primary Industries, Wagga Wagga NSW
School of Biomedical Sciences, Charles Sturt University, Wagga Wagga, NSW
ARC Industrial Transformation Training Centre for Functional Grains, Charles Sturt
University, Wagga Wagga NSW.
*
Corresponding author email address: [email protected]
Canola is produced for the production of oil; the meal which remains after oil extraction is of
relatively low value and used mainly for animal feed. This meal may have additional value in
the pharmaceutical industry if bioactive compounds can be identified which may have
potential health benefitssuch as, reducing the risk of disease, or contributing to optimal
nutrition. Meal extracts produced using different solvents were assessed in different
bioassays that measure potential anticancer, antidiabetic, antiobesity, antioxidant, and blood
pressure-lowering abilities. Several protease inhibitors have been implicated in the treatment
of different ailments including HIV and diabetes. Protease inhibitors (PIs) were extracted
from canola meal and purified to homogeneity from two different genotypes. Both extracts
exhibited different molecular weight and IEF properties. Extracts showed both the
topoisomerase (enzyme) poisoning and inhibition activities which are indicators of anticancer
properties. Inhibition of adipocyte differentiation without cell toxicity was observed with
acetone, butanol, and hexane extracts. The highest antidiabetic inhibition was observed in
butanol, acetone and water extracts. ACE inhibition (antihypertension) was also greatest in
water and methanol extracts, whereas the acetone and methanol extracts showed
antioxidant activity. These potential bioactive and health-functional properties of canola meal
extracts may increase the profitability for farmers, processors, food manufacturers, and the
pharmaceutical industry.
RACI NPG One Day Symposium 2014
18
 Poster
 Oral Presentation
(please tick)
Novel Grass Cell Wall Phenolic Acids
Wade J. Mace1*, Marty Faville1, Xuezhao Sun1, Casey Flay1, and Michelle Turner1
1
AgResearch Ltd, Grasslands Research Centre, Palmerston North, New Zealand
*
Corresponding author email address: [email protected]
The energy potential of forage is related to the nutritive value of the forage, and the voluntary
intake of the grazing animal. For a given nutritive value, a limiting factor becomes the
amount of forage an animal will consume, which is dependent on the rumen clearance rate,
which in turn is affected by the rate at which the insoluble forage (fibre component) degrades
in the rumen once consumed.
As evaluating forage for fibre degradation rate under rumen conditions involves an intensive
process requiring large quantities of material and resources, an alternate evaluation
procedure was sort. The relationship between the quantity of hydroxy-cinnamic acid (HCA)
monomers and dimers (major components of grass cell walls) released under mild base
conditions and degradation rate in the rumen was investigated. Initial studies have shown
such a correlation exists, therefore allowing chemical analysis of released HCA’s to be used
as a selection tool for predicting fibre degradation rate in a plant breeding context. The
chemical analysis for released HCA’s can be undertaken on a single plant basis, and is a
relatively quick and inexpensive method compared to direct measurement of rumen
degradation kinetics.
During the investigation of the HCA’s released and their correlations with degradation rate, it
was observed that novel dimeric HCA’s were released under the same mild base conditions.
This presentation explores the methods used to isolate and identify the novel HCA’s
released.
RACI NPG One Day Symposium 2014
19
 Poster
 Oral Presentation
(please tick)
Identification and localization of isohexenylnaphthazarins in mature roots and
seedlings of Paterson’s curse (Echium plantagineum)
Xiaocheng Zhu1, Brigette Ryan1, Dmitry V. Sokolov1, Geoff M. Gurr2 and
Leslie A. Weston*1
1
Charles Sturt University, Graham Centre for Agricultural Innovation, Life Sciences
Building, Wagga Wagga NSW 2678 Australia
2
Charles Sturt University, School of Agriculture and Wine Science, Orange NSW
2800 Australia
*Corresponding author email address: [email protected]
Paterson’s curse (Echium plantagineum L.) is a member of the Boraginaceae family. It is
native to the Iberian Peninsula and is currently listed as a noxious weed in Australia. It
currently infests over 30 M ha and losses due to infestation are over $125 M annually for the
meat and wool industries. We have previously annotated at least 25
isohexenylnaphthazarins, also known as naphthoquinones (NQs), in the root periderm of
this species. These compounds are lipophilic red pigments derived from shikonin/alkannin.
They have been identified in the out layers of the root of numerous species of
Boraginaceae. NQs have been widely studied for their antimicrobial, anti-inflammatory,
antitumor and wound healing properties. Previous bioassays also highlighted the strong
activity of NQs as plant growth inhibitors. Several studies have been reported on
biosynthesis and localization of these compounds in selected species of Boraginaceae.
Using dissecting, fluorescent and confocal microscopy, for the first time, we identified and
localized NQs in the living root tissues of E. plantagineum. These compounds are localized
primarily in the outer layer of cells in the root periderm. The ultra-structure of periderm cells
shows that large amounts of NQs are stored in the intercellular spaces between cells, while
NQs in the cell are deposited in numerous small cytoplasmic vesicles. NQs were also found
in E. plantagineum seedlings 48h after imbibition. Interestingly, root hairs actively exude red
NQs just 72h after imbibition. This is the first reported study of NQ localization in living plant
tissues of E. plantagineum. This study provides important information on biosynthesis of
these compounds and aids in our understanding of the invasive potential of E. plantagineum
across Australia.
Keywords: naphthoquinones, Boraginaceae, shikonin, light and confocal microscopy,
spectral imaging, absorbance, fluorescence
Tel: +61 (2)69334689; fax: +61 (2)69332429
RACI NPG One Day Symposium 2014
20
 Poster
 Oral Presentation
(please tick)
Multiple roles of flavonoids in plant-rhizobia symbioses
Samira Hassan1*, Anton Wasson2 and Ulrike Mathesius1
1
Department of Plant Sciences, Research School of Biology, The Australian National
University, Canberra, ACT
2
CSIRO Plant Industry - Black Mountain Laboratories, Black Mountain, ACT
*Corresponding author email address: [email protected]
Flavonoids are secreted by plant roots to activate and attract nitrogen fixing bacteria known
as rhizobia (Hassan and Mathesius, 2012). Rhizobia perceive flavonoids and reciprocate
with secretion of nod factors required to initiate nodule organogenesis. We explored the roles
flavonoids play in the roots during early nodulation of the model legume Medicago truncatula.
We used constitutive nod factor-expressing rhizobia to inoculate flavonoid silenced (CHSi)
roots and studied the transcriptomic changes 6 hours and 24 hours post inoculation. The
CHSi roots showed significant reduction in the nodule numbers compared to empty vector
control hairy roots (Wasson et al., 2006) that could be rescued with addition of flavonoids.
These roots also failed to accumulate auxin at the site of inoculation, a requisite to induce the
initial cell divisions for nodule formation. Using radiolabeled auxin, we found that the
flavonols and isoflavonoids are likely to be involved in regulating the transport of this
hormone in response to rhizobia inoculation. Inoculated CHSi roots over-accumulated many
defence related genes compared to controls, but failed to express structural enzymes that
could be involved in infection thread formation. GFP-labelled rhizobia were shown to form
infection threads in control but not CHSi roots, suggesting that flavonoids are involved in
infection of rhizobia. CHSi roots also did not trigger reactive oxygen species accumulation
required for cross-linking of glycoproteins in the matrix of infection threads. Together, our
findings suggest that flavonoids are versatile molecules that have likely been adapted to
perform various functions during the root-rhizobia interactions, including nod factor induction,
accumulation of auxin for nodule initiation and regulation of defence responses for successful
infection.
----------------------References
1. HASSAN, S. & MATHESIUS, U. 2012. The role of flavonoids in root-rhizosphere signalling:
opportunities and challenges for improving plant-microbe interactions. Journal of Experimental Botany,
63, 3429-44.
2. WASSON, A. P., PELLERONE, F. I. & MATHESIUS, U. 2006. Silencing the flavonoid pathway in
Medicago truncatula inhibits root nodule formation and prevents auxin transport regulation by rhizobia.
Plant Cell, 18, 1617-1629.
RACI NPG One Day Symposium 2014
21
 Poster
 Oral Presentation
(please tick)
Sample Handling and Plant Phenols: Too Important to Ignore
Hassan K. Obied*
Graham Centre for Agricultural Innovation, Wagga Wagga NSW and School of
Biomedical Sciences Charles Sturt University, Wagga Wagga NSW 2650
*
Corresponding author email address: [email protected]
Scientific research on plant phenolic compounds, biophenols, is exponentially growing.
Biophenols, the largest family of plant secondary metabolites, encompass close to 9000
different phenolic metabolites with varying structural complexities. The reactivity of the
phenolic hydroxyl group creates enormous potential for biochemical interactions and endless
possibilities for formation of new chemical derivatives. Furthermore, the biological activities of
biophenols in plants, humans and other organisms are currently areas of intense scientific
investigation driven by tantalising commercial opportunities in food, nutraceutical and
cosmetic industries. However, amidst all of these zealous and diligent endeavours to
understand biophenols chemistry and biological properties, there is a tendency to overlook or
underestimate the importance of sample handling and preparation prior to chemical or
biological analyses. In this review, light will be shed on the impact of sample handling on the
biophenolic composition of plant samples and subsequent repercussions on bioassay
outcomes.
RACI NPG One Day Symposium 2014
22
 Poster
 Oral Presentation
(please tick)
Backbone-fluorinated amino acids: synthesis and applications
Luke Hunter1*
1
School of Chemistry, UNSW Australia
* Corresponding author email address: [email protected]
One of the outcomes of incorporating fluorine atoms into an organic molecule is that the
conformation can be affected. The highly polarised C–F bond participates in a variety of
stereoelectronic interactions with adjacent functional groups, and these interactions can
favour certain molecular conformations over others. Thus, it is possible to rationally
“program” molecules to adopt desired conformations by decorating them with carefullydesigned patterns of fluorine substituents.1
This presentation will describe the synthesis of fluorinated backbone-homologated amino
acids (see Figure). These molecules have been characterised by NMR, X-ray crystallography
and molecular modelling, and it emerges that the different stereoisomers have very different
preferred conformations.2,3 Current work is focused on exploiting these shape-controlled
molecules in a variety of biological contexts, for example as GABA receptor ligands4 and as
components of anti-microbial and anti-angiogenic peptides.5
----------------------References
1. L. Hunter, Beilstein J. Org. Chem. 2010, 6, doi:10.3762/bjoc.6.38.
2. L. Hunter, K. A. Jolliffe, M. J. T. Jordan, P. Jensen, R. B. Macquart, Chem. Eur. J. 2011, 17, 2340.
3. R. Cheerlavancha, A. Lawer, M. Cagnes, M. Bhadbhade, L. Hunter, Org. Lett. 2013, 15, 5562.
4. I. Yamamoto, M. J. T. Jordan, N. Gavande, M. R. Doddareddy, M. Chebib, L. Hunter, Chem.
Commun. 2012, 48, 829.
5. X.-G. Hu, D. Thomas, R. Griffith, L. Hunter, Angew. Chem. Int. Ed. 2014, in press.
RACI NPG One Day Symposium 2014
23
 Poster
 Oral Presentation
(please tick)
To conduct biological screening, isolate and structurally elucidate bioactive
compounds from medicinal plants traditionally used by Indigenous people
Kaisarun Akter1, Joanne Jamie2, Subramanyam Vemulpad3, David Harrington4
Corresponding author email address: [email protected]
Traditional medicinal knowledge possessed by Indigenous people is a significant resource,
as exemplified by traditional medicine being the primary healthcare for ~80% of people in
developing countries1. The study of traditional medicine is also a successful avenue for drug
discovery. The aim of this study is to conduct biological screening, isolate and structurally
elucidate bioactive compounds from medicinal plants traditionally used by Indigenous people
of Nagaland India and NSW, Australia. Erythrina stricta (bark), which is used by Nagaland
people for skin infections, was selected for investigation following a literature review of 135
medicinal plants. The plant materials were sequentially extracted and the crude extracts
were screened against Staphylococcus aureus, Pseudomonas aeruginosa and Escherichia
coli using the disc diffusion and microdilution (MTT) assay. TLC bioautography was used for
the bioassay guided isolation. The dichloromethane extract of E. stricta showed activity
against S. aureus at 7.81 µg/ml and the ethyl acetate extract showed activity against S.
aureus at 125 µg/ml. Bioassay guided isolation of the dichloromethane extract lead to the
isolation of three compounds; alpinum isoflavone (1), wighteone (2) and luteone (3). Though
Alpinium isoflavone has been isolated from E. stricta2 before, this is the first report of isolation
of wighteone from this species and of luteone from the Erythrina family. Isolation of bioactive
compounds from the plant supports the traditional use of the plant by the Indigenous people
for the treatment of skin diseases.
----------------------References
1. Cragg GM, Newman DJ (1999). Discovery and development of antineoplastic agents from
natural sources. Cancer investigation 17: 153-163.
2. Hussain MM, Dastagir MG, Billah A, Ismail M, Hussain MM, Dastagir MG, et al. (2011).
Alpinum isoflavone from Erythrina stricta Roxb. Bol. Latinoam. Caribe Plantas M. 10: 88-90.
RACI NPG One Day Symposium 2014
24
 Poster
 Oral Presentation
(please tick)
Amaranthus toxicity in production livestock
Emily Birckhead1, Cecile Bouveret2, Dominik Skoneczny2,3, Allan E. Kessell4,
Leslie A. Weston2 and Jane C. Quinn2,4
1
2
3
School of Animal and Veterinary Sciences Charles Sturt University,
Wagga Wagga NSW
Graham Centre for Agricultural Innovation, (Charles Sturt University and NSW
Department of Primary Industries), Wagga Wagga NSW
School of Agriculture and Wine Sciences Charles Sturt University, Wagga Wagga
NSW
4
Gribbles Veterinary Pathology, Adelaide, SA
Corresponding author email address: [email protected]
Amaranthu spp. are known to cause toxicity and a renal syndrome in livestock, however
the toxic principal remains unknown. A case of Amaranthus hybridus poisoning in sheep
resulting in mortality and lowered pregnancy rate was recently documented in Australia
(in press). This research aims to identify the toxin(s) in A. hybridus (slim amaranth) and
describe its distribution in the plant. Mature A. hybridus plants were collected from
agricultural land in Wagga Wagga, NSW and methanolic extracts of leaf, seed/flower,
stem and roots were analyzed using solid phase extraction and HPLC coupled to
ESI/MS QToF. Preliminary results indicate a diverse number of potentially toxic
secondary metabolites in A. hybridus with a large number of nitrogen containing
constituents. These potential toxins were annotated in all plant extracts and include nonprotein amino acids, alkaloids and other N containing compounds that reportedly act as
neuro- and reproductive toxins in mammalian systems. Further research will be
conducted to putatively identify these metabolites, determine their relative abundance
and test for bioactivity in mammalian cell culture.
RACI NPG One Day Symposium 2014
25
 Poster
 Oral Presentation
(please tick)
Understanding photosensitization in grazing animals caused by ingestion of
the pasture legume Biserrula pelecinus
Cecile Bouveret1, Xiaocheng Zhu1, Jane C. Quinn1,2, and Leslie.A. Weston1*
1
2
Graham Centre for Agricultural Innovation, Charles Sturt University and NSW
Department of Primary Industries, Wagga Wagga NSW
School of Animal and Veterinary Sciences Charles Sturt University, Wagga Wagga
NSW
Corresponding author email address: [email protected]
Biserrula pelecinus was introduced to Australia for its exceptional performance as a pasture
legume in extreme climates and its ability to tolerate drought. However, sporadic and
occasionally severe outbreaks of photosensitization have been reported in sheep grazing
biserrula pastures. Photosensitivity results in severe dermatitis induced by heightened
reactivity of skin cells exposed to UV light after ingestion or contact with bioactive plant
pigments or secondary compounds. The resulting photosensitization can either be primary,
whereby plant secondary products (PSPs) cause direct damage to skin tissues; or
secondary, where underlying liver damage results in recirculation of PSPs causing
photosensitivity. Preliminary studies of crude extracts from biserrula, as well clinical data
collected from affected animals, have determined that photosensitisation resulting from
ingestion is caused by primary mechanism(s). To attempt identification of the PSPs involved
in biserrula photosensitization, experiments were designed to examine the effect of extracts
of different cultivars and life stages. We utilised both in vivo and in vitro bioassays to
examine the mechanism of photosensitization as well as cytotoxic and allelopathic activity
associated with bioactive biserrula extracts. A standard cell assay (NIH3T3 fibroblasts in 96well microplates) was designed to assess photosensitivity in extracts while cytotoxic and
allelopathic effects were evaluated using a cell and seed germination assay. Impact of
cultivar, life stage and location will be evaluated using all assay systems. In order to
determine which PSPs are associated with photosensitization and cytotoxicity, metabolic
profiling of bioactive purified extracts will be performed, using LC ESI/MS QToF. A bioassaydirected separation using thin layer and liquid column chromatography is also underway to
further isolate constituents associated with photosensitization and cytotoxicity.
RACI NPG One Day Symposium 2014
26
 Poster
 Oral Presentation
(please tick)
Acid Catalyzed Dimerization of Chromene Type Natural Products
Kenneth Kam-Chung Hong*, David StC Black, Graham Ball, Naresh Kumar
School of Chemistry, The University of New South Wales, UNSW, Sydney, NSW
2052, Australia
*
Corresponding author email address: [email protected]
Dependensin1 is a dimeric flavonoid isolated from the root bark of Uvaria dependens, whose
crude extract shows potent anti-malarial activity. The heterocyclic ring system present in
dependensin is quite unique and contains a dense array of functionality and stereochemistry,
which includes two fused benzopyran ring systems, four stereocentres and one trans double
bond. Various dependensin analgues with fluorine substituents were synthesized and their
anti-malarial activities were explored.
Kamalachalcone A is also a dimeric flavonoid, isolated from the fruit of Malloutus
philippensis2. It possesses a completely different but unique heterocyclic ring system
compared with dependensin. This ring system, according to our knowledge, is only found in
kamalachalcone series and morusynnansins B. This unique ring system is formed via an
acid-catalyzed dimerization of 5’-hydroxychromene forming three new stereocentres. The
biological application of kamalachalcones is not well studied but with our efficient synthesis,
the biological activities of kamalachalcone A can be explored in depth.
----------------------References (5 max)
1. Deodhar, M.; Black, D. S. C.; Kumar, N., Tetrahedron 2007, 63 (24), 5227-5235.
2. Tanaka, T., et al., Phytochemistry, 1998, 48(8): p. 1423-1427.
RACI NPG One Day Symposium 2014
27
 Poster
 Oral Presentation
(please tick)
The genoprotecitve effects of faba beans (Vicia faba)
Emma Kalle1,2,3, Wouter Kalle2,3, Hassan Obeid 1,2,3, Christopher L. Blanchard 1,2,3
1
2
3
Graham Centre for Agricultural Innovation, Charles Sturt University and NSW
Department of Primary Industries, Wagga Wagga NSW
School of Biomedical Sciences, Charles Sturt University, Wagga Wagga NSW
ARC Industrial Transformation Training Centre for Functional Grains, Charles Sturt
University, Wagga Wagga NSW.
Corresponding author email address: [email protected]
Pulses are an important food source in Australia and are known to contain high levels of
antioxidants. Numerous reagent-based assays have demonstrated potential antioxidant
activity, however, there have been only a limited number of in depth studies that have
assessed the functionality of pulse extracts at a cellular level. In this study compounds from
two faba bean varieties, rossa and nura, were extracted using 80% methanol and boiling
water. These extracts were tested for their genoprotective activity using the comet assay, a
biological test that shows the ability of compounds to protect cells from DNA damage. The
results showed that the methanol extracts are more effective in protecting against oxidative
DNA damage than boiling water extracts, and the rossa variety exhibits more pronounced
genoprotective activity than the nura variety. These preliminary results showed that faba
beans are a good potential source of antioxidants, and that methanol is a more effective
organic solvent for use in extraction of biologically active compounds.
RACI NPG One Day Symposium 2014
28
 Poster
 Oral Presentation
(please tick)
The Development of Isatin-based Prosthetic Groups for Peptide
Radiopharmaceuticals
S. K. Lim1*, N. Kumar1, A. Katsifis2
1
School of Chemistry, The University of New South Wales, UNSW, Sydney, NSW
2052, Australia
2
The Royal Prince Alfred Hospital, Camperdown, NSW 2050, Australia
*
Corresponding author email address: [email protected]
Isatin (1H-indole-2,3-dione) is an indole derivative obtained by Erdmann and Laurent
in 1841 from the oxidation of indigo using nitric acid and chromic acids. In nature,
isatin is found in plants such as Isatis tinctoria, Calanthe discolour and in Couroupita
guianensis. Isatin is a versatile molecule and has many applications such as colour
dyes and pharmaceutical intermediates. Our group has shown that N-acylisatins
undergo rapid ring-opening reaction with amines and amino acid esters to yield the
corresponding glyoxylamides in high yields. We are interested in developing isatins
as a prosthetic group for Positron Emission Tomography (PET). PET is a noninvasive nuclear imaging technique that utilises a radioactive substance known as a
tracer to detect diseases in the body by showing how organs or tissues are
functioning through measuring cellular-level metabolic changes. There is a current
need for better prosthetic groups which can incorporate the radionuclide into the
peptide. Therefore, in my work, radiolabelled isatins will be developed as prosthetic
groups using [18F]fluorine as the radionuclide.
RACI NPG One Day Symposium 2014
29
 Poster
 Oral Presentation
(please tick)
Pharmacokinetic Study of a Standardised Herbal Extract of Diospyros kaki
Leaves in Rats
Mitchell N. Low1, Dennis Chang1*, Cheang Khoo1, Chun G. Li1, Kelvin Chan1,
Manilar Nang1 and Alan Bensoussan1
1
National Institute of Complementary Medicine, University of Western Sydney,
Locked Bag 1797, Penrith, NSW 2751, Australia
*
Corresponding author email address: [email protected]
Introduction
Naoxinqing Pian (NXQ), a patented herbal medicine comprises the standardised extract of
dry leaves of Diospyros kaki. The extract has been reported to possess a wide range of
pharmacological activities including radical scavenging, thrombosis inhibition, blood pressure
lowing, and neuroprotective effects. The bioactive flavonoids including quercetin and
kaempferol have been suggested to play a major role in the pharmacological effects of NXQ.
Methods
This study was designed to establish the pharmacokinetic characteristics of quercetin and
kaempferol in rats. UPLC-MS/MS methods for quercetin and kaempferol were established
and validated. 32 rats were allocated to two active treatment groups (n=16 in each group)
receiving a single oral administration of a low (0.05 g/kg) and high dose (2.5 g/kg) of NXQ
respectively. Blood samples were taken regularly over 24 h and were later analysed to
determine the plasma concentrations of quercetin and kaempferol. Cmax, Tmax, T1/2, AUC and
MRT were calculated using PK Solutions 2.0.
Results
The UPLC-MS/MS methods were successfully applied to the analyses of quercetin and
kaempferol in rat plasma. Quercetin and kaempferol were both detected within the first 20
min after the oral administration. The maximum plasma concentrations of quercetin (low
dose: 0.42 ± 0.20 µg/ml; high dose: 1.62 ± 0.55 µg/ml in the high dose) and kaempferol (low
dose: 0.20 ± 0.10 µg/ml; high dose: 1.00 ± 0.44 µg/ml) were reached at 4 h. In both groups,
the plasma levels of quercetin were significantly higher than that of kaempferol; however,
kaempferol appeared to have a slower elimination rate evidenced by a longer elimination
half-life and mean residence time.
Conclusions
This study has established the pharmacokinetic profiles of quercetin and kaempferol in rats
treated with NXQ. Future work should be extended to the evaluation of bioavailability,
tissue/organ distribution and elimination profiles of the key bioactive components of NXQ.
RACI NPG One Day Symposium 2014
30
 Poster
 Oral Presentation
(please tick)
Changes in the composition of wine during ethanol reduction processing
L. Manera1, H. Ghantous1 B. Saha1,2, A. Deloire1, L. Schmidtke1, & P. Torley1
1
National Wine and Grape Industry Centre, School of Agricultural and Wine Science,
Charles Sturt University, Locked Bag 588, Wagga Wagga 2678 NSW, Australia
2
School of Chemical Engineering, University of New South Wales, Australia
Corresponding author email address: [email protected]
Rising alcohol levels is an issue for wine producers in many wine making countries1. People
are also being encouraged to reduce alcohol consumption due to health and social benefits2.
Several techniques have been developed to reduce ethanol content in wine before, during or
after the fermentation3. A common approach used in Australia is a membrane processing
technology that combines reverse osmosis and evaporative perstraction4.
In the present study membrane processing has been used to reduce the alcohol content of
wine made from different varieties (Chardonnay, Sauvignon Blanc, Shiraz) to 8% and 5%
and at different levels of grape maturity at harvest (Mature and Post-mature Shiraz) to 10%
and 8%.
The effect of membrane processing on wine composition (ethanol, pH, titratable acidity,
SO2), colour (CIELab) and headspace (HS-SPME/GC-MS) and liquid concentration of aroma
compounds were measured.
Ethanol and SO2 concentrations decreased with processing time, while pH, titratable acidity
and colour showed no significant change. Results indicated that the headspace
concentrations of most of the aroma compounds were significantly reduced in the 8% and
5% wines compared to the unmodified wines.
----------------------References
1. Alston, JM, Fuller, KB, Lapsley, JT, et al. Splendide mendax: False label claims about high and
rising alcohol content of wine. Journal of Wine Economics, (2011) 6: 135-159
2. Saha, B, Torley, P, Blackman, JW & Schmidtke, LM. 1st Oenoviti International symposium “Alcohol
level reduction in wine”. 2013. ISVV, Villenave d’Ornon, France
3. Pickering, G. J. (2000). Low- and Reduced-Alcohol Wine: A Review. Journal of Wine Research,
11(2), 129-144. Doi:10.1080/09571260020001575
4. Memstar. Wine membrane technology. http://www.memstar.com.au/modules/mastop_publish/
RACI NPG One Day Symposium 2014
31
 Poster
 Oral Presentation
(please tick)
Synthesis and Biological Activity of Novel Bis-indole Inhibitors of Bacterial
Transcription Initiation Complex Formation
M. Mielczarek1*, D. StC. Black1, R. Griffith2, P. J. Lewis3, N. Kumar1
1
School of Chemistry, 2 School of Medical Sciences, Department of Pharmacology,
UNSW Australia, Sydney, NSW 2052, Australia
3
School of Environmental and Life Sciences, University of Newcastle, Callaghan,
NSW 2308, Australia
* Corresponding author email address: [email protected]
A variety of biologically active bis-indoles has been derived from natural products, especially
marine organisms like sponges and tunicates. Topsentin-A (deoxytopsentin) 1a, topsentin-B1
(topsentin) 1b and topsentin-B2 (bromotopsentin) 1c, alkaloids found in a Mediterranean
sponge Topsentia genitrix, showed antibacterial, antiviral and antitumour activity.1,2 Hamacanthin A 2, trans-4,5-dihydrohamacanthin A 3 and hamacanthin B 4, alkaloids isolated from
a Korean sponge Spongosorites sp., have been found to inhibit sortase A (SrtA), a key
enzyme responsible for the adhesion of Staphylococcus aureus to specific organ tissues by
binding to cell-matrix proteins, such as fibronectin and fibrinogen.3
We have targeted the essential interaction between bacterial RNA polymerase and σ70/σA2.2
for the development of lead molecules exhibiting a novel mechanism of antibacterial activity. 4
Several classes of structurally related bis-indole inhibitors of bacterial transcription initiation
complex formation 5, 6 and 7 were synthesized and their antimicrobial activities were evaluated. The resulting compounds were found to efficiently inhibit the β′-CH-σ70/σA2.2 interaction
in ELISA assay and exhibited moderate antibacterial activity.5
---------------------References:
1. Bartik, K.; Braekman, J.-C.; Daloze, D.; Stoller, C.; Huysecom, J.; Vandevyver, G.; Ottinger, R.
Can. J. Chem. 1987, 65, 2118.
RACI NPG One Day Symposium 2014
32
2. Tsujii, S.; Rinehart, K. L.; Gunasekera, S. P.; Kashman, Y.; Cross, S. S.; Lui, M. S.; Pomponi, S.
A.; Diaz, M. C. J. Org. Chem. 1988, 53, 5446.
3. Oh, K.-B.; Mar, W.; Kim, S.; Kim, J.-Y.; Oh, M.-N.; Kim, J.-G.; Shin, D.; Sim, C. J.; Shin, J. Bioorg.
Med. Chem. Lett. 2005, 15, 4927.
4. Ma, C.; Yang, X.; Kandemir, H.; Mielczarek, M.; Johnston, E. B.; Griffith, R.; Kumar, N.; Lewis, P.
J. ACS Chem. Biol. 2013, 8, 1972.
5. Mielczarek, M.; Devakaram, R. V.; Ma, C.; Yang, X.; Kandemir, H.; Purwono, B.; Black, D. StC.;
Griffith, R.; Lewis, P. J.; Kumar, N. Org. Biomol. Chem. 2014, 12, 2882.
RACI NPG One Day Symposium 2014
33
 Poster
 Oral Presentation
(please tick)
Alkaloids in Australia: analysis of selected fungally produced alkaloids in
fifteen geographically distinct annual ryegrass ecotypes
Joseph R. Moore1, James E. Pratley1, Leslie A. Weston1 and Wade J. Mace2
1
2
Graham Centre for Agricultural Innovation, Charles Sturt University and NSW
Department of Primary Industries, Wagga Wagga NSW
Grasslands Research Centre, AgResearch, Palmerston North 4442, New Zealand
Corresponding author email address: [email protected]
Annual ryegrass (Lolium rigidum) is a persistent weed species of southern Australian farming
systems, infesting millions of hectares of land and costing hundreds of millions of dollars
each year to control. Fungal endophytes asymptomatically infect Lolium spp. and are known
to increase their host’s survival through production of bioactive alkaloids such as the antiinsect compound peramine. Ecological fitness benefits derived from these compounds differ
with each grass-fungal association and their importance is determined by the environmental
selection pressure encountered. Strictly vertically transmitted Epichloë endophytes, are
widely studied and commonly are found to be beneficial to their hosts. However, this has not
been established for the endophyte of annual ryegrass, Epichloë occultans, which has not
been properly surveyed for known alkaloids. This project provided an important opportunity
to advance the understanding of the types and quantities of beneficial alkaloids E. occultans
infected annual ryegrass produces. Plants of each ecotype were grown under controlled
glasshouse conditions and inspected for living endophyte presence before processing and
extraction. Loline alkaloids were assessed in individual plant tillers using GC-FID, including
predominant natural variants of lolines; N-acetylloline (NAL), N-formylloline (NFL) and Nacetylnorloline (NANL). In addition, bulked infected plant samples were assessed for each
ecotype, ergovaline, peramine, lolitrem B and 11, 12 epoxy-janthitrem were analysed using
LC-MS. Lolines were found in every infected ecotype in varying concentrations (0 - 1200
ppm). There was considerable variation in Loline concentrations within and between
ecotypes. Peramine, the only other alkaloid found, was detected in nine ecotypes (0.2 3.1ppm,). The widespread production of bioactive alkaloids implies an ecological purpose for
the endophyte, which may contribute to the success of annual ryegrass from decreased
herbivory.
RACI NPG One Day Symposium 2014
34
 Poster
 Oral Presentation
(please tick)
Phytochemical characterisation and analysis of Curcuma xanthorrhiza extracts
Jarryd Pearson1*, Cheang Khoo1, Alan Bensoussan1
1
The National Institute of Complementary Medicine, School of Health and Science,
University of Western Sydney, Campbelltown, NSW, 2560, Australia.
Introduction: Curcuma xanthorrhiza is traditionally used to treat stomach, liver and skin
inflammation1,2,3. Curcuma xanthorrhiza contains curcuminoids and sesquiterpenoids4. In
industry, the quality control of herbal extracts currently involves only safety testing, typically
for pesticides, heavy metals and microbial contamination. Currently only one or two analytes
are used to quantify the quality of a herb. The aim of this study was to develop and validate a
method to characterise the quality of Curcuma xanthorrhiza extracts using nine analytes to
provide a more accurate description of herbal quality.
Materials and Methods: The ground dried raw herb material was extracted in ethanol (EtOH),
70% aq. EtOH, 50% aq. EtOH, 35% aq. EtOH and water using sonication for 20 min. The
developed method was tested for accuracy and precision by replicate (n=7) analyte recovery
studies at three spiking levels (50, 100 and 150%).
Results: Ethanol extraction gave the best analyte recoveries and all recoveries were above
94% (range 94.04% to 99.85%) with an average precision recovery of 96.64% (range
94.15% to 99.34%). Calibration curves for all nine analytes gave a coefficient of
determination (R2) of >0.999.
Conclusion: Overall a method was developed and validated to quantify nine analytes in
curcuma xanthorrhiza extracts, which will help industry monitor the quality control aspect of
the manufacturing herbal extracts.
-----------------------References:
1. Yasni. et al. (1993) Food Chem Toxicol. 31: 3:213-218.
2. Ozaki. (1990) Chem Pharm Bull. : 1045 - 1048.
3. Park. et al. (2008) Phytother Res 2008: 695-698.
4. Uehara. et al. (1992) Yakugaku Zasshi. 112:817-823.
RACI NPG One Day Symposium 2014
35
 Poster
 Oral Presentation
(please tick)
Optimisation of small molecules targeting childhood cancers
Hoang An Phan1*, Naresh Kumar1 and Belamy-Bin Cheung2
1
2
School of Chemistry, The University of New South Wales, UNSW, Sydney, NSW
2052, Australia
Children’s Cancer Institute Australia for Medical Research, Lowy Cancer Research
Centre, University of NSW, Randwick, NSW 2052, Australia
*Corresponding author email address: [email protected]
Acute lymphoblastic leukaemia (ALL), neuroblastoma, and medulloblastoma, are cancers in
which children are at especially high risk of mortality and morbidity, particularly in later stages
of disease progression. Conventional therapeutic approaches have varied success, however,
there still remains a need for the development of new approaches that are effective against
cases of severe cancer growth and metastasis, with reduced long-term side effects. Over
expression of the MycN gene is a shared characteristic of these cancer cells, as is their
ability to grow in the absence of the growth factor, interleukin-7. With this knowledge of the
cancer cells’ ability to grow in these specific conditions, in concert with the use of organic
synthesis techniques for lead modification, this study aims to investigate the structure activity
relationships to develop novel molecules as targeted cancer treatments.
RACI NPG One Day Symposium 2014
36
 Poster
 Oral Presentation
(please tick)
Pomegranates as a possible source of toxic illness in cattle
Elizabeth Read1,2*, Myrna A. Deseo1*, Mark Hawes1 and Simone Rochfort1,2
1
Centre for AgriBio Science, Biosciences Research Division, Department of
Environment and Primary Industries, Bundoora, VIC
2
La Trobe University, Bundoora, VIC
Corresponding author email address: [email protected]
Pomegranates (Punica granatum) are a well-known and established source of antioxidants
for humans. Pomegranate juice is commonly added into juice blends or sold enriched for the
most abundant antioxidant present: punicalagin. Punicalagin is thought to have neuroprotective, hepato-protective and even anti-carcinogenic properties for humans. However,
this same compound (punicalagin) has also been isolated from the leaves of particular trees:
Terminalia catappa (Tropical almond) and Terminalia oblongata (Yellow-wood). In
Queensland, Australia, sheep and cattle that ate significant amounts of Yellow-wood leaves
often became sick: sheep would undergo sudden convulsive fits after a loud noise or
excitement and then recover, while cattle would often have severe liver injury and sometimes
kidney injury resulting in death. It was discovered that the punicalagin present in the leaves
was the cause of the toxicosis in both sheep and cattle using mouse models and feeding
experiments with sheep.
During times of significantly reduced feed availability farmers have been reported to
purchase pomegranate husks as a cheaper feed alternative. Cattle that have access to
pomegranate trees may also consume them either out of curiosity or hunger. After
consumption of the pomegranate husks, some cattle in the herd become ill and a number
can succumb to the toxicosis over a period of 1 week.
Pomegranate pieces collected from a cattle farm that recently experienced toxicity have been
analysed using HPLC-QTOF-MS techniques and the presence of punicalagin confirmed.
Punicalagin will be isolated and its toxicity assessed using cytotoxicity assays.
RACI NPG One Day Symposium 2014
37
 Poster
 Oral Presentation
(please tick)
Development of high throughput screening assays for determining endophyte
associated toxins
Priyanka Reddy1,2,3*, Myrna A. Deseo1, John Forster1,2,3 and Simone Rochfort1,2,3
1
Centre for AgriBio Science, Biosciences Research Division, Department of
Environment and Primary Industries, Bundoora, VIC
2
Dairy Futures Co-operative Research Centre, Australia
3
La Trobe University, Bundoora, VIC
*Corresponding author email address: pri[email protected]
Perennial ryegrass Lolium perenne L., is by far the most common perennial grass used for
temperate pasture in southern Australia. The established area is estimated at more than six
million hectares and such pasture forms the feed base for the wool, meat and dairy industries
of southern Australia. Grasses frequently form symbioses with endophytic fungi belonging to
the family Clavicipitaceae. Symbiotic interactions of grasses with fungal endophytes,
Epichloe species and their asexual relatives Neotyphodium, often provide the grass hosts
with major fitness enhancements. However, grasses infected by clavicipitaceous fungi can
be associated with a number of diseases occurring in grazing animals, including rye grass
staggers, fescue foot, summer syndrome and ergotism. Standard toxic endophyte strains can
create significant animal health issues, which if severe enough, can substantially reduce the
economic profitability of these same systems. Despite the negative impacts some
endophytes possess, the benefits outweigh these effects in an agricultural setting.
Current work for forage grass improvement involves the selection of novel endophytes that
produce less or none of the known toxins. The major toxins of concern are the lolitrem
metabolites and the ergot alkaloids. Endophytes are selected for progression into a breeding
program based on their toxin profiles but the associations are generally not tested in animals
until very late in the breeding program. The concern is that endophytes that do not produce
these toxins can still produce intermediates in the biosynthetic pathway. Some of these
intermediates are known toxins while the biological activity of many of these intermediates
remains unknown.
Large scale extraction and isolation will allow the toxicity of key intermediates to be
determined. This information will allow the construction of a ‘toxicosis map’ that can be used
to assess current and new endophyte ryegrass associations. A proposed scheme for the
isolation and purification of the target toxins is described. Preliminary optimization of high
throughput methodologies using UPLC-ESI-QqQ-MS and UPLC-ESI-QTOF-MS assays for
monitoring the levels of these metabolites will be discussed. The quantitative, rapid LC/MS
screening methods combined with the toxicosis map will allow better characterization of
novel associations reducing the risk of toxicosis issues arising later in the breeding cycle.
RACI NPG One Day Symposium 2014
38
 Poster
 Oral Presentation
(please tick)
The role of leaf surface chemistry and morphology in plant defense of two
related invasive weeds Echium vulgare and Echium plantagineum
Brigette Ryan1, Dominik Skoneczny1, Xiaocheng Zhu2, Paul A. Weston1,
Jane C. Quinn2, 3and Leslie A. Weston2
1
School of Agriculture and Wine Sciences Charles Sturt University, Wagga Wagga
NSW.
2
3
Graham Centre for Agricultural Innovation, (Charles Sturt University and NSW
Department of Primary Industries), Wagga Wagga NSW.
School of Animal and Veterinary Sciences Charles Sturt University, Wagga Wagga
NSW.
Corresponding author email address: [email protected]
Echium plantagineum and Echium vulgare are invasive Boraginaceous weeds introduced to
Australia in the mid-1800s. They are highly toxic to grazing livestock containing alkaloid
toxins which cause significant liver damage. Both plants have exceedingly pubescent leaves,
but little information is available regarding their trichome morphology or function. To address
this question, we performed studies on leaf and trichome morphology as well as leaf surface
chemistry in order to gain a deeper understanding of the role of the leaf surface in plant
defense against herbivory. We utilized SEM, light and confocal microscopy to perform
intensive examination of leaf morphology and related trichome structures of both Echium
species. Three morphologically different trichome types were identified in both Echium
species and these included previously undescribed sets of glandular and non-glandular
trichomes that are evidently characteristic to this genus of the Boraginaceae. In E.
plantagineum, large non-glandular and small glandular trichome height averaged 1.1mm and
0.05 mm, while E. vulgare trichomes averaged 1.6mm and 0.03mm, respectively. Metabolic
profiling of the leaf surface was performed by 1) briefly (1-3 sec) dipping leaves of each
species in ethanol, or 2) extracting leaf peels in ethanol and subjecting extracts to UPLC ESI
LC QToF (Agilent 6410). Leaf peel extracts contained ten pyrrolizidine alkaloids with
echimidine-N-Oxide, echiuplatine-N-oxide and 3’- O-acetylechiumine-N-oxide most
abundant. Leaf dips also revealed these compounds were present in high abundance on the
leaf surface of E. plantagineum. The metabolic profiles of the two species revealed
considerable qualitative and quantitative differences in the abundance of pyrrolizidine
alkaloids present on the leaf surface, with greater abundance of PAs associated with leaf
surface of Echium plantagineum compared to Echium vulgare. Morphological studies of the
leaf trichomes also revealed differences among the two species, providing critical information
for taxonomic separation based on leaf morphology. Our studies on leaf morphology and
surface chemistry provide insights into physical and chemical mechanisms associated with
plant defense in Echium spp. and our understanding of why E. plantagineum is a highly
successful and noxious weed invader in comparison to its less common congener, E.
vulgare.
RACI NPG One Day Symposium 2014
39
 Poster
 Oral Presentation
(please tick)
Impact of Paterson’s curse (Echium plantagineum L.) establishment on species
richness in invaded and native range
Dominik Skoneczny1, Xiaocheng Zhu2, Ragan M. Callaway3 and Leslie A. Weston2*
1
2
3
Charles Sturt University, Locked Bag 588, Wagga Wagga NSW 2678
Graham Centre for Agricultural Innovation, Locked Bag 588, Wagga Wagga NSW
2678
Division of Biological Science University of Montana, 32 Campus Dr, Missoula , Mt
59812, US.
*
Corresponding author email address: [email protected]
Geographically distinct random populations of Echium plantagineum L. (Paterson’s curse)
were selected across NSW Australia and Spain to examine the impacts of Paterson’s curse
interference upon establishment and biodiversity of common competitors, including both
native and non-native herbaceous species. At 16 study sites in NSW, 1 m2 quadrats in
undisturbed pastures, roadsides, croplands and natural areas were used to evaluate stand
counts of Paterson’s curse and any other co-established vegetation. Data collected included
stand counts of all species present and relative growth parameters related to selected
Paterson’s curse plants in each quadrat. At the same location, quadrats without Paterson’s
curse were evaluated for species richness by collecting stand counts and identifying all
established species. In the area surrounding Cooma NSW, comparative counts were also
taken in sites infested with another related member of the Boraginaceae, E. vulgare or
Viper’s bugloss. At the same time, ecological surveys of Paterson’s curse were performed in
Spain to investigate biodiversity and plant establishment in its native range. Six sites in Spain
were evaluated, using multiple 1 m2 quadrats at each site. We observed the negative
correlation of Echium spp. density with species richness in NSW, which is not observed in
Spain. These studies will provide insight into the mechanisms used by plant species for
successful invasion in non-native regions and the impacts of Paterson’s curse on species
biodiversity. Our results suggest that decrease in species richness in Australia at Paterson’s
curse infested sites might be related to plant interaction(s) on chemical level. We will discuss
our findings from year one surveys performed across Spain and Australia.
RACI NPG One Day Symposium 2014
40
 Poster
 Oral Presentation
(please tick)
Determination of nitric oxide inhibitory activities of selected raw and processed
Chinese materia medica
John Truong1*, Xian Zhou1, Valentina Razmovski-Naumovski1,2, Cheang Khoo1,
Kelvin Chan1,2
1
The National Institute for Complementary Medicine, School of Science & Health;
University of Western Sydney
2
Faculty of Pharmacy, The University of Sydney
*Corresponding author email address: [email protected]
Nitric oxide (NO) serves as an important biomarker in acute and chronic inflammatory
responses. Chinese materia medica (CMM) and its processed derivatives have been used
for centuries to promote good health and to treat ailments including inflammatory-related
conditions. Many raw CMM such as Zingiberis Rhizoma, Crataegi Fructus and Scutellariae
Radix have been screened for inhibitory activities towards NO generation, however, the
effect of herb processing has not been investigated.
To investigate the inhibitory effect of raw and processed Zingiberis Rhizoma, Crataegi
Fructus and Scutellariae Radix on lipopolysaccharide (LPS)-stimulated NO production in
RAW264.7 cells.
The samples were extracted in water using heat reflux for 1 h. RAW264.7 cells (1 × 106/well)
were seeded and incubated for 48 h and the extracts of three CMM in DMSO were added 2 h
prior to LPS (50 ng/mL) stimulation. NO release was determined by the Griess reaction with
absorbance measured at 540 nm. Cell viability was assessed using MTT assay.
Raw and processed products of Zingiberis Rhizoma (stir-baked) and Scutellariae Radix
(fried, wine-fried) dose-dependently suppressed NO release with IC50 values ranging from
8.13-69.0 µg/mL. The fried preparation of Scutellariae Radix (IC50 = 11.71 µg/mL)
demonstrated a more potent inhibitory effect than its raw CMM (IC50 = 24.9 µg/mL). Crataegi
Fructus CMM and its processed forms exhibited slight inhibitory effects. The observed
inhibitory effects were not due to their cytotoxicity.
Raw and prepared CMM of Zingiberis Rhizoma and Scutellariae Radix revealed promising
anti-inflammatory properties and warrants further studies using animal models.
RACI NPG One Day Symposium 2014
41
 Poster
 Oral Presentation
(please tick)
Flavonoids as potential scaffold for hybrid drug molecules
Yee M. H. E1, Kumar N.1
School of Chemistry, The University of New South Wales, Sydney, NSW 2052,
Australia
Corresponding author email address: [email protected]
Natural products provide numerous solutions to previously unmet medical needs, and have
influenced the direction of synthesizing many biologically active molecules1. One of the
commonly occurring classes of natural products is the isoflavonoids, which are present in a
variety of foods such as legumes, soy and tea. The most prominent isoflavonoids present in
these foods are genistin and daidzin, which are metabolized in the body to genistein and
daidzein. These have been shown to promote healthy function of various biological
processes. Genistein has shown promising biological activity against cancer cells, which
has led to the development of its derivative phenoxodiol, itself a natural product, for clinical
trials. This makes phenoxodiol a potential candidate for hybrid drug development. The
poster will present our recent work on the synthesis of phenoxodiol-propranolol hybrids and
their biological activities against cancer cells2.
--------------------References:
1. Newman, D. J. J. Med. Chem. 2008, 51, 2589.
2. Yee, E. M.; Pasquier, E.; Iskander, G.; Wood, K.; Black, D. S.; Kumar, N. Bioorganic & medicinal
chemistry 2013, 21, 1652.
RACI NPG One Day Symposium 2014
42
 Poster
 Oral Presentation
(please tick)
Aqueous extracts of Danshen-Sanqi herb-pair inhibit lipopolysaccharideinduced nitric oxide release in RAW264.7 macrophage cells through the PI3K
pathway
Xian Zhou1#*, Antony Kam1,2# , Valentina Razmovski-Naumovski1,2 , Kelvin Chan1,2
1
The National Institute for Complementary Medicine, School of Science & Health;
University of Western Sydney
2
Faculty of Pharmacy, The University of Sydney
#
Joint first authors
*
Corresponding author email address: [email protected]
Salvia Miltiorrhizae Radix & Rhizoma (Danshen) is used in combination with Notoginseng
Radix (Sanqi) in traditional Chinese medicine (TCM) formulations for managing
cardiovascular complications. As individual herbs, their anti-inflammatory activities have
been extensively evaluated, particularly nitric oxide (NO) release. However, the combined
effect of this herb-pair on NO release and its associated mechanistic pathway has not been
well-defined. This study aims to examine the inhibitory effects of Danshen-Sanqi
combinations on lipopolysaccharide (LPS)-induced NO release through the PI3K pathway.
Single and combinations (different ratios) of Danshen and Sanqi crude herbs were extracted
in water by heat reflux. The aqueous extracts were analysed for their effects on LPS-induced
NO release in RAW264.7 cells. To investigate the involvement of the PI3K pathway in
mediating the inhibitory effects, wortmannin (a specific PI3K inhibitor) was co-incubated with
the extracts.
Danshen-Sanqi combinational extracts had more potent inhibitory effects than Danshen and
Sanqi (IC50=1.21±0.3 mg/mL vs. 2.19 and 2.06 mg/mL, respectively). In particular, the
Danshen-Sanqi combination of 4:1 (IC50=0.54 mg/mL) was the most effective among the
tested ratios. Wortmannin treatment (2 μM) alone increased LPS-induced NO release by
30%, whilst the co-incubation of wortmannin and the 4:1 extract (1 mg/mL) significantly
reversed the inhibitory effects of the extract on NO release.
This study demonstrated that the Danshen-Sanqi combination inhibited LPS-induced NO
release in RAW264.7 cells more effectively than the single herbs and supports the TCM
practice of these herb-pair. The mechanism of the combination can be associated with the
activation of the PI3K pathway.
RACI NPG One Day Symposium 2014
43
NOTES
RACI NPG One Day Symposium 2014
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RACI NPG One Day Symposium 2014
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