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i
Bio-analytical Screening and Characterization of Antioxidant Compounds using Online Liquid
Chromatography Techniques and Mass Spectrometry
A thesis submitted in fulfillment of the requirements for the degree of Doctor of Philosophy
By Rashida Bashir
Department of Chemistry & Biotechnology, School of Science,
Faculty of Science, Engineering and Technology
Swinburne University of Technology, Australia
September 2017
ii
Abstract This thesis is positioned in the fields of natural product and bioanalytical
chemistry, and covers three main integrated studies along with some additional
explorations. Oxidative stress plays a leading role in the deterioration of human
health and to counter oxidative stress, the body relies on compounds called
antioxidants that are usually obtained from dietary sources. Pharmaceutical and
nutraceutical industries are interested in the identification and characterization of
new antioxidant compounds from natural products, which is a challenging task. In
the recent years conventional online post column antioxidant assays coupled with
chromatographic techniques have been used. The main drawback associated with
these conventional techniques is an extra post column dead volume which causes
peak broadening and loss of separation power. An alternative technique, namely
Reaction Flow Chromatography is an application of active flow management,
whereby, the derivatising reagent can be added directly into one of the inlet ports of
a parallel segmented flow column. This avoids reaction coils and utilizes the mixing
potential of a frit which results in high levels of sensitivity and resolution.
In an initial study, the aim was to develop Reaction Flow Chromatography
for a rapid post column ferric reducing antioxidant potential (FRAP) assay. The
reaction flow chromatography with multiplexed detection allows for an absolute
assignment of antioxidants with component identity.
An application of this analysis for the profiling of antioxidants for a range of
Australian native food plants has provided identification of many bioactive
compounds known to exhibit antioxidant, anticancer and anti-inflammatory
iii
activities. Native species in this study exhibited superior antioxidant capacity and
comprise predominantly of Flavonols, Anthocyanin, Phenolic acids and their
hydrolysable tannins. All of the evaluated Australian native plants were found to
contain antioxidants with known therapeutic potential in cardiovascular,
neurodegenerative and other chronic diseases that play a major role in the
prevention/delay of oxidative stress mediated diseases.
A further application explored the antioxidant potential of edible mushrooms
with detailed investigation revealing that Phenolic acids, Vitamin B3, Vitamin B5,
Ergosterol, L-Ergothioneine, Ergosterol peroxide, Vitamin D and its derivatives are
bioactive compounds, which make edible mushrooms a suitable source of
antioxidants. Untargeted metabolic profiling has not only indicated the presence of
antioxidants but has also demonstrated that edible mushrooms are a source of
anticancer and anti-inflammatory bioactive compounds. This suggests commercially
available mushrooms in Australia may provide comprehensive protection from
oxidative stress and other possible pronounced health benefits.
iv
Acknowledgements
I would like to thank my supervisors Dr. Peter Mahon and Prof. Enzo
Palombo for their help, support, guidance and patience throughout my candidature;
in particular a thank you to Dr. Peter Mahon as my primary supervisor for the expert
guidance, assistance and advice, who also always encouraged me to do my best. I
would like acknowledge Prof. Andrew Shalliker in Western Sydney University for
providing me the opportunity and assistance to work on this collaborative project. I
would like to acknowledge Dr. Sercan Pravadali for her initial contribution to the
project and Andrew Jones for helping me throughout this research. Thank you to LC-
MS Application Specialist Alex Chan from Thermofisher Scientific for his expertise
in Mass Spectrometry. Special thanks to my colleagues and friends at Swinburne
University for their help, support and most of all their friendship for the duration of
my PhD. Finally, as an expression of gratitude, thank you to my family and husband,
who have always supported and encouraged me throughout my candidature. I am
indebted to them for their love and care.
v
Declaration I, Rashida Bashir, declare that this thesis is my original work and contains no
material that has been accepted for the award of Doctor of Philosophy, or any other
degree or diploma, except where due reference is made.
I declare that to the best of my knowledge this thesis contains no material previously
published or written by any other person except where due reference is made.
Wherever contributions of others were involved, every effort has been made to
acknowledge the contributions of the respective workers or authors.
Rashida Bashir
April 2018
vi
Publications arising of this work
The following publications resulted and expected from the work carried out for this
thesis.
Published Journal Paper A. Jones, S. Pravadali-Cekic, G. R. Dennis, R. Bashir, P. J. Mahon and R. A.
Shalliker, Analytica Chimica Acta 2017, 967, 93-101.
Expected Journal Papers
Rashida Bashir, Andrew Jones, Alex Chan, Prof Andrew Shalliker, Prof
Enzo Palombo & Dr Peter Mahon. Bioactive profiling of Australian
Commercial Mushrooms with High Resolution Q-Exactive Mass
Spectrometry.
Rashida Bashir, Andrew Jones, Alex Chan, Prof Andrew Shalliker, Prof
Enzo Palombo & Dr Peter Mahon. Exploring Ethnopharmacological
Potential of Australian Native Plants with High Resolution Q-Exactive Mass
Spectrometry.
Conference Oral Presentations
Rashida Bashir, Prof Enzo Palombo & Dr Peter Mahon. Bioactives profiling
of Australian native Cordycep gunnii at the 5th international conference and
Exhibition on Pharmacognosy, Phytochemistry and Natural products
Toxicology held during July 24-25, 2017 in Melbourne, Australia
vii
Rashida Bashir, Andrew Jones, Alex Chan, Prof Andrew Shalliker, Prof
Enzo Palombo & Dr Peter Mahon. Untargeted metabolic profiling of
antioxidants within Australian native Quandong and Desert lime with RF-
PCD FRAP and LC-HR-MS at the 5th international conference and Exhibition
on Pharmacognosy, Phytochemistry and Natural products Toxicology held
during July 24-25, 2017 in Melbourne, Australia.
Rashida Bashir, Andrew Jones, Alex Chan, Prof Andrew Shalliker, Prof
Enzo Palombo & Dr Peter Mahon. Untargeted Metabolic Profiling of
Bioactive Compounds in Tasmanian Pepper with Reaction Flow
Chromatography and High Resolution Orbitrap Q-Exactive Mass
Spectrometry at the RACI-FNAC Student Symposium Competition held on
31st August 2017. 2nd Position Cash Prize
Rashida Bashir, Andrew Jones, Alex Chan, Prof Andrew Shalliker, Prof
Enzo Palombo & Dr Peter Mahon. Bioanalytical screening of Australian
white cup and Shimeji mushrooms using Online HPLC Assay and
characterisation with mass spectrometry. (Abstract accepted) at Analytical
separation meeting Japan
Conferences Poster Presentations
Rashida Bashir, Andrew Jones, Alex Chan, Prof Andrew Shalliker, Prof
Enzo Palombo & Dr Peter Mahon. Exploring the Ethnopharmacological
potential of Australian native Oldman saltbush and wattle seeds with
untargeted metabolic profiling and structural elucidation with Orbitrap mass
spectrometry” at the 8th World congress on Pharmacology and Toxicology
held during July 24-25, 2017 in Melbourne, Australia (Best Poster Prize)
Rashida Bashir, Prof Enzo Palombo & Dr Peter Mahon. Mechanistic study
of Ferric Reducing FRAP Assay with Cyclic Voltammetry and mass
spectrometry. (Abstract accepted) at Analytical Separation Meeting Japan.
viii
Abbreviations AF Active flow
AFT Active flow technology
AFM Active flow management
RF Reaction flow
PCD Post column derivatisation
HR High resolution
ESI Electrospray ionization
LC Liquid chromatography
MS Mass spectrometry
MS/MS Tandem mass spectrometry
PDA Photodiode Array
V/V Volume/Volume
W/V Weight/Volume
HPLC High performance liquid chromatography
i.d. Internal diameter
IEX Ion exchange chromatography
K Retention factor
K0 Specific permeability
L Column length
mAU Milliabsorbance units
mg Milligrams
MeOH Methanol
min Minute
mV Millivolts
mL Millilitres
N Theoretical plate count
n Number of peaks
NMR Nuclear magnetic resonance
NP Normal phase
ix
np Peak capacity
Pd Particle size
PDA Photodiode array
PFP Pentafluro-phenyl
PSF Parallel segmented flow
Q Quadrant
R Resolution
RI Refractive index
RP Reversed phase
RSD Relative standard deviation
S Sensitivity
SD Standard deviation
t0 Column dead time
TIC Total ion count
tR Retention time
uF Flow rate
UHPLC Ultra high performance liquid chromatography
x
List of Figures
Figure 1.1: Typical Post Column Derivatisation setup coupled with HPLC ............. 7
Figure 1.2: The AFT- Parallel Segmented flow column with multiport end fitting
............................................................................................................................ ..13
Figure 1.3: AFT-PSF column outlet used for Reaction Flow Chromatography ..... 15
Figure 1.4: Multiplex detection of natural complex mixtures with an AFT-PSF
Column .................................................................................................................. 16
Figure 1.5: Australian native foods used in the bioanalytical characterisation of
antioxidants… ........................................................................................................ 24
Figure 1.6: Conventional approach of untargeted metabolic profiling for Drug
Discovery from Natural Products… ....................................................................... 27
Figure 1.7: Untargeted metabolic profiling -A tool for Ethno pharmacological
studies.................................................................................................................... 28
Figure 2.1: Schematic representation of the process involved in the sample
preparation for the mushroom samples ................................................................... 34
Figure 2.2: Schematic representation of the process involved in the sample
preparation for the Australian Native Foods… ....................................................... 34
Figure 2.3: Schematic diagram of the Post Column Derivatisation setup coupled
with HPLC ............................................................................................................. 37
Figure 2.4: Active flow column attached with Frit-Thermo fisher Scientific (UK)
.............................................................................................................................. 38
Figure 2.5: Principle of Orbitrap mass spectrometry .............................................. 39
Figure 3.1: The reaction mechanism for the FRAP Assay. .................................... 46
Figure 3.2: Reaction flow chromatography - RF column in multiplexed mode. .... 50
Figure 3.3: Chromatograms of a solution containing 1000 mg/L p-coumaric acid,
hesperidin, Naragingin and hesperitin and 10 mg/L Trolox derivatized using the
FRAP reagent and analysed using (a) Conventional PCD and (b) RF-PCD at 593 nm,
corresponding to the FRAP analysis wavelength .................................................... 52
Figure 3.4: Chromatograms of Decaffeinato Intenso espresso coffee analysed using
HPLC-PCD analysis with the FRAP reagent. The data corresponds to (a)
conventional PCD with a 500-µL reaction loop and (b) RF-PCD ........................... 56
xi
Figure 4.1: Chromatograms showing the response for the Portobello mushroom
extract (a) UV-Vis, (B) PCD-DPPH at 520 nm and (C) RF-PCD-FRAP at 593 nm
............................................................................................................................. 70
Figure 4.2: Chromatograms showing the response for the Dried Porcini mushroom
extract (a) UV-Vis, (B) PCD-DPPH at 520 nm and (C) RF-PCD-FRAP at 593 nm
.............................................................................................................................. 70
Figure 4.3: RF-PCD FRAP response of Vitamin D mushroom at 593 nm .............. 74
Figure 4.4: RF-PCD FRAP response of Shimeji mushroom at 593 nm .................. 74
Figure 4.5: RF-PCD FRAP response of Brown Cup mushroom at 593nm ............. 75
Figure 4.6: RF-PCD FRAP response of White Button mushroom at 593 nm ......... 76
Figure 4.7: Summary of antioxidants identified within edible mushrooms ............. 81
Figure.4.8. Showing HR-MS analysis of mushroom Dried Porcini. ...................... 85
Figure 5.1: RF-PCD-FRAP profile of Old man saltbush prepared with (A) direct
extraction and (B) sonication ................................................................................. 92
Figure 5.2: RF-PCD-FRAP assay showing the antioxidant capacity of Australian
native Old man saltbush ........................................................................................ 93
Figure 5.3: RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Gumbi Gumbi ...................................................................................................... 100
Figure 5.4: RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Quandong ............................................................................................................ 108
Figure 5.5: RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Lemon grass ........................................................................................................ 117
Figure 5.6: Compounds from Lemon grass that has a potential role in the treatment
of headaches and migraines .................................................................................. 123
Figure 5.7: RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Desert lime ............................................................................................. 124 Figure 5.8: RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Tasmannia lanceolata .............................................................................. 130
Figure. 5.9: Showing high resolution mass spectrometry analysis of Tasmanian
pepper .................................................................................................. 134
Figure 5.10: RF-PCD-FRAP analysis of Australian native Wattle seed.............. 136
xii
List of Tables
Table 3.1: Quantitative performance of antioxidant standards with conventional
PCD ....................................................................................................................... 54
Table 3.2: Quantitative performance of antioxidant standards with RF-PCD ......... 54
Table 4.1: Bioactive compounds within Dried Porcini ........................................... 71
Table 4.2: Bioactive compounds within Portobello mushroom .............................. 72
Table 4.3: Bioactive compounds within Vitamin D mushroom .............................. 73
Table 4.4: Bioactive compounds within Shimeji mushroom .................................. 74
Table 4.5: Bioactive compounds within Brown cup mushroom ............................ 75
Table 4.7: Bioactive/antioxidant compounds within White Button mushroom ....... 76
Table 5.2: Structural elucidation of antioxidants within Australian native Old man
saltbush .................................................................................................................. 94
Table 5.3: Structural elucidation of antioxidants within Australian native Gumbi
gumbi. ................................................................................................................ 102
Table 5.4: Structural elucidation of antioxidants within Australian native Quandong
............................................................................................................................ 109
Table 5.4: Structural elucidation of antioxidants within Australian native Lemon
grass .................................................................................................................... 118
Table 5.5: Structural elucidation of antioxidants within Australian native Desert. 126
Table 5.6: Structural elucidation of antioxidants within Australian native Tasmannia
lanceolata ............................................................................................................ 131
Table 5.7: Structural elucidation of antioxidants with Australian native Wattle seeds
............................................................................................................................ 137
xiii
Table of Contents
Abstract ............................................................................................................................ ii
Acknowledgements ........................................................................................................ iv
Declaration ...................................................................................................................... v
Publications arising for this work ............................................................................... vi
Abbreviations ................................................................................................................ viii
List of figures .................................................................................................................. x
List of Tables ................................................................................................................. xii
Table of Contents ........................................................................................................ xiii
Chapter 1 ..................................................................................................................... 1
Literature Review .................................................................................................. 1
1.1.Liquid Chromatography ........................................................................................ 2
1.2. Post column derivatisation Coupled with HPLC ................................................ 4
1.2.1. Basic configuration for Post Column derivatisation..................................... 6
1.2.2. Limitations of Conventional Post Column Derivatisation ........................... 9
1.3. Reaction Flow Chromatography ........................................................................ 10
1.3.1. Background of Active Flow Column Technology ..................................... 10
1.3.2. Active Flow Technology .............................................................................. 12
1.3.3. Reaction Flow Chromatography .................................................................. 14
1.3.3.1. Multiplexing AFT-RF Column ............................................................... 16
1.3.4. Mass Spectrometry ....................................................................................... 18
1.3.4.1.High Resolution Orbitrap Mass Spectrometry ...................................... 20
1.4. Natural Products as a Source of Antioxidants ................................................... 21
1.4.1. Oxidative stress and Antioxidants ............................................................... 21
1.4.2. Australian Native Plants as a source of Antioxidants ................................ 23
1.4.3. Australian Mushrooms as a source of Antioxidants ................................... 25
1.5. Untargeted Metabolomics- A Potential Tool of Drug Discovery .................... 26
Chapter 2 .................................................................................................................... 29
Materials and Methods .......................................................................................... 29
xiv
2.1. Materials ............................................................................................................... 30
2.1.1. Standards and Samples ................................................................................. 30
2.1.1.1. Mushrooms ........................................................................................... 30
2.1.1.2. Australian Native Plants ...................................................................... 30
2.1.1.3. Coffee Samples ..................................................................................... 30
2.1.1.4. Standard Solutions ................................................................................ 30
2.1.2. Chemical reagents ....................................................................................... 31
2.1.2.1. Mobile Phases ....................................................................................... 31
2.1.2.1.1. Milli Q water .................................................................................. 31
2.1.2.1.2. Sodium acetate buffer ..................................................................... 31
2.1.2.1.3. Methanol ......................................................................................... 31
2.1.2.2. Post Column Derivatisation Reagents .................................................. 32
2.1.2.2.1. DPPH Reagent ................................................................................. 32
2.1.2.2.2. FRAP Reagent ................................................................................ 32
2.1.3. Extraction Methods ................................................................................. 33
2.1.3.1. Solvent Extraction with Shaking ...................................................... 33
2.1.3.2. Sonication Method ............................................................................ 33
2.2. Instrumentation ................................................................................................... 35
2.2.1. Post Column Derivatisation Coupled with HPLC-Conventional Method 35
2.2.2. Post Column derivatisation using Reaction Flow Chromatography ........ 37
2.2.3.LC-HRMS-Q-ExactiveTM Hybrid Quadrupole Orbitrap Mass Spectrometer
......................................................................................................................................... 39
2.3. Optimization of Chromatography and MS Method ........................................... 40
2.3.1. Optimization of segmentation ratios within RF Chromatography ............. 40
2.3.2. Optimization of Flow rate within RF Chromatography .............................. 41
2.3.3. Optimization of Gradient conditions ............................................................ 42
2.3.4. Optimization of MS conditions ..................................................................... 43
2.4. Data Interpretation and Analysis......................................................................... 44
Chapter 3 ................................................................................................................ 45
Ferric Reducing Potential(FRAP) of Antioxidants Using Reaction
Flow Chromatography ........................................................................................... 45
xv
3.1. Introduction ......................................................................................................... 46
3.2. Material and Methods ......................................................................................... 48 3.2.1. Chemicals ....................................................................................................... 48
3.2.2. Columns ........................................................................................................ 48
3.2.3. Reagent, Sample and Standard Preparations................................................ 48
3.2.4. Instrumentation .............................................................................................. 49
3.2.5. Chromatographic Conditions ........................................................................ 50
3.2.6. Quantitative Performance Measures ............................................................. 51
3.3.Results and Discussion ......................................................................................... 51
3.3.1.Conventional PCD .......................................................................................... 51
3.3.2.RF-PCD ........................................................................................................... 53
3.3.3.Comparision of Conventional PCD and RF-PCD ........................................ 53
3.3.4.FRAP Analysis of Decaffinated Coffee ........................................................ 55
3.3.5 Antioxidant and FRAP Assay Kinetics Using Reaction Flow
Chromatography ............................................................................................................. 57
3.4. Conclusions .......................................................................................................... 59
Chapter 4 .................................................................................................................... 60
A Rapid Antioxidant Capacity Analysis (FRAP) Of Australian
Mushrooms Using Reaction Flow Chromatography and Structural
Elucidation with Mass Spectrometry ................................................................. 60
4.1. Introduction ......................................................................................................... 61
4.2. Materials and Reagents ........................................................................................ 63
4.2.1. Chemicals and Reagents ................................................................................ 63
4.2.2. Mobile Phases ............................................................................................... 63
4.2.3. Derivatisation Reagent .................................................................................. 63
4.2.4. Sample Preparation ....................................................................................... 63
4.3. Instrumentation and Chromatographic Conditions ............................................ 64
4.3.1. Column ........................................................................................................... 64
4.3.2. Instrumentation Setup .................................................................................... 64
4.3.2.1. Conventional PCD-DPPH Assay Detection Setup ................................ 64
4.3.2.2. RF PCD-FRAP Assay Detection Setup ................................................. 64
xvi
4.3.2.3. High Resolution Mass Spectrometry...................................................... 65
4.3.3. Chromatographic Analysis ............................................................................ 65
4.3.3.1.Conventional PCD-DPPH and RF-PCD FRAP Assay Detection Setup
......................................................................................................................................... 65
4.3.3.2. High Resolution Mass Spectrometry...................................................... 66
4.3.4. Data Analysis ................................................................................................. 66
4.4. Results and Discussion ........................................................................................ 67
4.4.1.General Observations ..................................................................................... 67
4.4.1.1. Comparison of PCD Techniques .......................................................... 67
4.4.1.2. LC-HR-MS Detection ............................................................................ 68
4.4.2. Mushroom Sample Analysis .......................................................................... 69
4.4.2.1. Dried Porcini ............................................................................................ 71
4.4.2.2. Portobello .................................................................................................. 72
4.4.2.3. Vitamin D ................................................................................................. 72
4.4.2.4. Shimeji ..................................................................................................... 73
4.4.2.5. Brown Cup ............................................................................................... 74
4.4.2.6. White Button ............................................................................................ 75
4.4.3. Structural Characterization of Antioxidants within Australian Mushrooms
......................................................................................................................................... 76
4.4.3.1. Phenolic Acids ......................................................................................... 76
4.4.3.2. Water-Soluble Vitamins ........................................................................... 77
4.4.3.3. Ergothioneine ........................................................................................... 77
4.4.3.4. Ergosterol and Derivatives ...................................................................... 77
4.4.3.5. Vitamin D2 and Analogues ...................................................................... 80
4.4.4. Confluence of RF-PCD and LC-HR-MS ...................................................... 82
4.5. Conclusion ........................................................................................................... 85
Chapter 5 .................................................................................................................... 87
A FRAP-based Rapid Antioxidant Capacity Analysis of Australian
Native Plants Using Reaction Flow Chromatography and
Characterization with Mass Spectrometry ..................................................... 87
5.1. Introduction ....................................................................................................... 88
xvii
5.2. Materials and Reagents ...................................................................................... 89
5.2.1. Chemicals and Reagents.............................................................................. 89
5.2.2. Mobile Phases ............................................................................................. 89
5.2.3. Sample Preparation ..................................................................................... 89
5.3. Instrumentation and Chromatographic Conditions .......................................... 90
5.3.1. Column ....................................................................................................... 90
5.3.2. Instrumentation Setup ............................................................................... 90
5.3.3. Chromatographic Analysis ....................................................................... 91
5.3.4. Mass Spectrometry .................................................................................. 91
5.3.5. Untargeted Antioxidant Identification with LC-HR-MS ........................ 91
5.4. Results and Discussion ......................................................................................... 92
5.4.1.Comparison of Extraction Methods ................................................................ 92
5.4.2.Antioxidant Analysis and Structural Elucidation ......................................... 92
5.4.2.1.Oldman SaltBush ....................................................................................... 92
5.4.2.1.1.Rhamnetin and Isorhamnetin .............................................................. 94
5.4.2.1.2.Kynurenic acid..................................................................................... 95
5.4.2.1.3.Glycitein ............................................................................................... 95
5.4.2.1.4.Polyphenol and Phenolic acids ........................................................... 96
5.4.2.1.5.Aminoacids and Nucleosides.............................................................. 97
5.4.2.1.6.Alkaloids .............................................................................................. 98
5.4.2.1.7.Vitamins and Fatty acids ..................................................................... 99
5.4.2.2.Gumbi Gumbi .......................................................................................... 100
5.4.2.2.1.Pheophorbide and its Methyl Esters ................................................. 100
5.4.2.2.2.Chlorogenic acids .............................................................................. 104
5.4.2.2.3.Esculetin ............................................................................................. 105
5.4.2.2.4.Luteolin 7-Sulfate .............................................................................. 105
5.4.2.2.5.Phenolic and Organic acids .............................................................. 106
5.4.2.2.6.Flavonoids and Flavonoids Glycosides ........................................... 106
5.4.2.2.7.Miscellaneous Compounds ............................................................... 107
5.4.2.3.Quandong ................................................................................................. 107
5.4.2.3.1.Chlorogenic Acids ............................................................................. 108
5.4.2.3.2.Phenolic and Organic acids .............................................................. 111
5.4.2.3.3.Flavanoids and Glycosides ............................................................... 112
xviii
5.4.2.3.4.Fatty acid and Fatty acids esters ....................................................... 115
5.4.2.4.Australian Native Lemon grass .............................................................. 116
5.4.2.4.1.Flavonoids and Anthocyanin ............................................................ 117
5.4.2.4.2.Phenolic and Organic acids ............................................................. 121
5.4.2.4.3.Vitexin and Iso-Vitexin..................................................................... 121
5.4.2.5.Australian native Desert lime ................................................................. 123
5.4.2.1.1.Catechin and Procyanidins ............................................................... 124
5.4.2.1.2.Flavanoids and Flavanoid Glycosides.............................................. 129
5.4.2.6.Australian Native Tasmanian Pepper ..................................................... 130
5.4.2.6.1.Polygodial and Sesquiterpenes ......................................................... 131
5.4.2.6.2.Flavanoids and Glycosides ............................................................... 133
5.4.2.6.3.Miscellaneous Compounds ............................................................... 135
5.4.2.7.Australian native Wattle seeds ................................................................ 135
5.4.2.7.1.Phenolic Acids ................................................................................... 135
5.4.2.7.2.Bioactive Lipids ................................................................................ 136
5.4.2.7.3.Miscellaneous Compounds ............................................................... 136
5.5. Conclusion ......................................................................................................... 138
Chapter 6 ................................................................................................................. 139
Conclusion and Future Directions ................................................................. 139 6.1. Conclusions ...................................................................................................... 140
6.1.1. Scope of the RF-PCD FRAP assay for Natural Products Screening ..... 140
6.1.2. Ethnopharmacological potential of Australian Mushrooms .................. 141
6.1.3. Ethnopharmacological potential of Australian Native Plants ................ 141
6.2. Future directions ............................................................................................... 143
1
Chapter 1
Literature Review
2
1.1. Liquid Chromatography
The analysis of samples of biological origin is problematic due to the
complexity and diversity of the chemical components. Careful sample preparation
can reduce the diversity but further separation is required if the individual substances
are to be characterized. Russian biologist Mikhail Tsvet invented column
chromatography to separate plant pigments about a century ago [1]. Pressurization of
the liquid phase with controlled flow rate [2], along with the introduction of reduced
particle sizes, resulted in the development of High Performance Liquid
Chromatography (HPLC) [3, 4]. HPLC is an analytical chromatographic technique
for the separation and analysis of compounds that has found wide applications in
various fields such as environmental sciences, pharmaceutical analysis and clinical
sciences [5, 6].
In HPLC, the compounds within a mixture are separated on a stationary
phase and several modes of HPLC, such as reversed-phase (RP), normal-phase (NP),
size exclusion chromatography (SEC), ion exchange chromatography (IEX), chiral
chromatography and more are currently used in analytical laboratories [7]. The
amount of time that an analyte takes to elute from the column is known as the
retention time and is dependent on the selective interactions between the solute with
the stationary and mobile phases [8-10]. Resolution is a quantitative term used to
define the separation of one component from another within a chromatographic
separation [8, 9]. HPLC elution can be either isocratic or gradient [8] with the
relationship between isocratic and gradient elution well documented in the literature
and the “linear solvent strength theory” model has been used to explain elution
strength within RPLC [11-15].
3
Resolution is based primarily on the selection of stationary and mobile
phases, temperature, flow rate and solvent pH [9]. Efficiency can be further
optimized with appropriate selection of solvent and stationary phases [10]. The
choice of stationary phases is vast and column architecture with fully porous
particles are preferred over core shell particles because they can be utilized at higher
backpressures [94]. Otherwise, the peak capacity is exceeded with limited
information derived from such chromatographic separations [1-3].
RP-HPLC is the most common separation mode used for analytical purposes.
It employs a non-polar stationary phase with a polar mobile phase, typically
aqueous/organic mixtures [12]. Nowadays, HPLC columns with small, micron-sized
particles are packed under high pressure and this advancement has changed the field
of separation science. Until recently, a conventional HPLC system with column
dimensions of 250 mm length and an internal diameter of 4.6 mm packed with 5 μm
particles was used as an appropriate setup for analysis [11]. The majority of
analytical studies are performed with C18 stationary phases with a particle size of 3
μm or more [13]. The particle size of HPLC columns has become smaller with the
development of ultra-high performance liquid chromatography (UHPLC) technology
[14, 15]. The performance of these analytical columns has significantly improved
[16] but newer column technology might be incompatible with existing HPLC
equipment due to increased backpressures [17]. Column lengths of 250 mm were
commonplace but recently column lengths of 50 mm and 150 mm, which allow rapid
analysis and efficient separation, have become more popular [18].
The particle size of the column packing influences the backpressure of HPLC
systems. An efficient separation can be achieved through using long columns packed
4
with small particles as it provides more theoretical plates. Column length and
particle size both contribute to system pressure [19, 20].
Some limitations of HPLC have been discussed in detail by renowned
researcher Georges Guiochon [21]. The pressure limitation of the instrument and
column restricts the availability of sufficient theoretical plates for good separation.
Due to the heterogeneity of the packed column bed and viscous friction, non-
isothermal conditions result with solutes in warmer regions migrating faster than
solutes in cooler regions of the column bed [21].
1.2. Post-Column Derivatisation Coupled with
HPLC
Identification and characterization of bioactive compounds from complex
mixture is a challenging task [22]. For conventional methods, bioassay guided
fractionation followed by biological screening is expensive and time consuming
[23]. However, in the last decade or so, hyphenated techniques have been developed
that couple high performance liquid chromatography with online post-column
assays. Different parallel detection methods like diode-array detection, mass
spectrometry and nuclear magnetic resonance have been used to identify and
quantify bioactive compounds [24]. These methods are not only limited to online
post column assays but also useful for assays based on enzymes and receptors. These
advanced strategies have proved to be useful for rapid analysis of complex mixtures
for natural drug based discovery [25].
Derivatization coupled with HPLC is a powerful analytical technique [26-29]
with the choice of derivatization modes dependent on a range of considerations such
5
as hardware availability, desired separation characteristics and physicochemical
properties of analytes [30]. Different system analytical parameters are usually
optimized and these parameters include column dimension, stationary phase, particle
size and geometry of the reaction loop [11].
In post-column derivatization (PCD), the derivatization reagent(s) are added
to the eluent stream in between the column and the detector [31, 32]. PCD has
several advantages:
Post-column detection coupled with HPLC is used to detect compounds
which are usually not detectable with available detectors [26, 33, 34]. For
example, compounds which are not fluorescent and do not absorb UV-
Visible light such as amino acids and biogenic amines [30, 33].
In the most analytical techniques, such as capillary electrophoresis,
derivatization improves sensitivity [4].
Chemical entities having low molecular mass such as thiols are stabilized
with derivatization [5].
Analytes which are incompatible with selected analytical techniques, such as
reversed phase chromatography and gas chromatography, are also derivatised
to make them compatible [35].
Complexity of the sample matrix is a critical variable in the selection of a
derivatization method. In PCD, separation of compounds takes place prior to
reagent addition and mixing which reduces side reactions [3] and matrix
effects can be avoided [36].
Derivatisation is also useful to increase the signals of analytes of interest with lower
limits of quantification and detection [33]. Post column derivatization can also be
6
used to avoid matrix and interference effects [37]. Furthermore, enhanced detection
of analytes can also be achieved by changing the nature of the mobile phase [38].
Pre-column derivatization reactions, where the reagent is added before the
column, has several advantages [30] and disadvantages [27]. Compared to PCD, the
considerations include:
Pre-column derivatization reaction requires sample handling or automated
input. While in the case of PCD, once the derivatization reagent is pumped
into the system, no further handling is required.
In PCD methods, the derivatizing reagent does not enter the column whereas
in pre-column derivatization, the derivatizing reagent can form large peaks.
Method development for post-column derivatization is comparatively easy.
Product formation in pre-column detection occurs prior to detection and the
product is stable throughout chromatographic run. In PCD techniques, the
product forms just before detection.
In PCD techniques, multiple detectors can be used to detect derivatized and
underivatized products by managing the flow directions. In pre-column
derivatization, only the derivatized product will be detected.
1.2.1.Basic configuration for Post-Column Derivatization
There are two main aspects of the post-column system. One is the pumps and
reactors while the other is the less common components, such as static mixers and
flow regulators. Recent improvements in the instrumentation of analytical chemistry
has seen the development of highly sensitive detectors [30] but sample preparation
and pre-treatment is the main challenging task in terms of throughput [39].
7
Pumps are the main instrumental components of the post-column setup and
they are required to deliver reagent with low or high-pressure at a constant flow rate
[31]. Three types of pumps, including peristaltic, piston and syringe-based, are
commercially available and are typically used in the PCD setup as shown in Figure
1.1. All of these pumps have advantages and disadvantages [40]. Piston-based
pumps are accurate in terms of flow rate and corrosion resistance due to the use of
PEEK tubing with low pulse flow rate [14] and backpressure [15] achieved through
the use of a pulse dampener. Alternatively, syringe pumps have advantages in terms
of reproducibility and flow accuracy. They are designed to supply a fixed volume
and need periodic refilling after each analysis. Flow rates at the low microlitre per
minute level can be achieved so they are used in capillary LC-PCD [30]. PEEK
tubing is used to deliver reagent from the vessel to the post-column reactor and the
chemical compatibility of this flexible tubing with the derivatization reagent is a
critical analytical parameter [41].
Figure 1.1. Typical Post Column Derivatization setup coupled with HPLC.
8
Connectors, reactors and mixers are an important component of the PCD
setup. They influence the yield of reaction, flow and efficiency of the
chromatographic separation [30, 42]. Reactors are used to facilitate and enhance the
mixing of the reagents in a PCD setup and are also useful for delaying the analytes
so as to achieve the required products formation [43]. Reactors such as rotating flow
mixing devices are also used to facilitate mixing [17, 32]. Comparison of direct UV
and post-column detection of captopril has shown that the post-column extra volume
increases peak width and decreases theoretical plates [44].
An important consideration for the selection of the design and dimensions of
flow reactors is the kinetics of the post-column reaction [42]. Air or liquid
segmented reactors are used for reactions with slow kinetics (reaction time > 5 min)
and tubular or less packed reactors are used for reactions with fast or intermediate
kinetics [45, 46]. PCD reactions with slow kinetics are facilitated with elevated
temperature and the Ninhydrin online derivatization at about 130 °C is a classic
example of slow reaction kinetics [27]. In conventional setups, water baths and
column heaters are used to facilitate appropriate temperature regulation. However,
increasing the temperature of the LC mobile phase and PCD reagents usually causes
an increase in the noise of the baseline. Also, back pressure regulators should be
used for the minimization of air bubbles [30].
Coiled tubing is usually used for PCD derivatization because it reduces band
broadening [46, 47] with narrow reaction coils (< 0.5 mm i.d.) preferably used [27].
Connectors such as Tee, Cross and 5-port are commercially available and resistant to
temperature and pressure. Commercially available Nano Y connectors with low dead
9
volume are used for micro column liquid chromatography [48]. In capillary
chromatography extremely low dead volumes micro-Tee connectors are used [49].
An extra post-column HPLC pump is connected to a detector in the
conventional setup. If the HPLC pump is directly connected to the detector, the
baseline from the detector will show periodic noise with the pulse time period related
to the pump stroke. Commercial pulse dampeners are usually added between the
post-column pump and the detector to improve the baseline as the noise is less
pronounced as the dampener absorbs most of the pulses from the pump [41].
Pickering has described several requirements which should be optimized for
the implementation of a PCD system [28, 50]. The derivatization reagent solution
should be stable for the complete series of chromatographic runs as quantitation is
only possible if results are reproducible. Reagents with high detector response could
be a source of noise and improper mixing can cause poor detector responses. On the
other hand, the underivatized reagent should produce minimal signal in the detector.
Components of the solution should be soluble so that flow lines and the detector are
not blocked [28].
1.2.2. Limitations of Conventional Post-Column
Derivatization
PCD methods include reaction loops that are available in different volumes in
comparison to rest of the system. The volume varies usually from 100 μL to 2000 μL
[18, 26, 33, 51-76] and one method with a volume of about 12000 μL has been
reported in the literature [77]. Any extra volume of the reaction loop leads to peak
broadening and the choice for the reaction loop is usually a compromise between
10
being large enough to allow adequate response and not too long that peak broadening
interferes with the chromatographic separation power [78, 79]. Long reaction coils
are usually used to enable increased reaction times and proper mixing. Peak
broadening also results in wider peaks with higher limits of quantitation and
detection. Post-column dead volume also causes peak deterioration and affects the
detectability of a derivatized molecule [80]. Our research collaborator, Andrew Jones
has reviewed about 87 PCD methods published from 2009 to 2014 [11] and his
critical review has found that reaction loops varied in size with most having a
volume greater than 500 μL. Recent experiments have revealed that resolution and
loss of separation efficiency occurs when the size of a reaction coil is increased
beyond 100 μL [11].
1.3. Reaction Flow Chromatography
1.3.1. Background of Active Flow Column Technology
Columns packed with 5 and 10 μm fully porous particles have dominated the
field for 30 years [81]. However, column technology has rapidly evolved in the last
decade with Merck Pharmaceutical commercializing the first 100 mm × 4.6 mm
monolithic silica column [82]. Nakanishi and Tanaka developed a method for the
preparation of silica based columns [83] encapsulated in PEEK tubing [84].
Instrument manufacturers and pharmaceutical companies began to produce and
commercialize columns with fully porous materials, ranging in size from 1.5 to 5 μm
[81]. Recent technologies have significantly improved analysis time and plate
heights as fast separations and improved resolution can be achieved with these
columns [85].
11
Chromatographers have argued for a long time about the “wall effect” as no
clear visual scientific evidence was available to illustrate this phenomenon.
Chromatographic columns do not have uniform cross sectional flow and this results
in radial heterogeneity and a temperature differential between the column centre and
the peripheral walls [86, 87]. Electrochemical analysis of three analytical columns,
namely 100 mm × 4.6 mm C18, 150 mm × 4.6 mm C18 column packed with 2.7 μm
particles and 150 mm × 4.6 mm C18 column packed with 3 μm fully porous
particles, has demonstrated that all these columns are not radially homogenous.
Polarographic detection of chromatographic band profiles has shown that the wall
effect phenomenon is present and increases with an increase in mobile phase flow
velocity along the walls [88]. There is a loss of separation efficiency along the wall
region of the column compared to the central region [89] with radial heterogeneity
being a long standing issue in chromatographic separations [90].
Radial heterogeneity arises from the difference between packing density
along the wall and the central region of the column bed. Moreover with the uneven
distribution of the sample band, the resultant effect is a decrease in separation
efficiency due to the formation of a parabolic like sample band [91]. The solute plug
moves as a parabolic band with a higher flow rate at the centre of column compared
to along the wall and more theoretical plates are required for the separation of
parabolic bands rather than flat profiles [92]. Further studies conducted to find the
correlation between the radial heterogeneity of a column with the peak shape of the
elution profile have demonstrated that peak tailing and fronting phenomenon are also
affected due to radial heterogeneity and increase with an increasing amplitude of the
radial distributions and efficiency of the column [93, 94].
12
1.3.2. Active Flow Technology
Active flow technology (AFT) is based on a novel end-fitting for
chromatography columns that are designed to reduce solute band broadening and
peak volume by eliminating the wall effect due to bed heterogeneity. This novel end-
fitting separates the peripheral flow from the central flow [95]. In active flow
chromatography, the AFT fitting design has a frit with ports, which allows
segmentation of the flow. The eluent from the column bed enters the AFT fitting
with part of the eluent exiting through the central port and the remaining eluent
exiting through the three outer peripheral ports [95]. The parallel segmentation ratio
is adjusted using differential outlet pressure by simply altering the length of the
connected tubing [96, 97].
Active flow management has a few different configurations [98]. A Curtain
flow column has multiport end-fittings at both the inlet and outlet of the column. The
analytical performance of curtain flow chromatography columns compared to
conventional chromatographic conditions revealed that the limit of detection and
quantitation was lowered by a factor of three [99, 100].
Alternatively, Parallel segmented flow describes conditions where only the
outlet of column has the novel end-fitting (Figure 1.2). This outlet fitting separates
central flow from peripheral flow and as a result of this flow segmentation, column
efficiency was improved by 20% with gains in sensitivity by as much as 22% and a
decrease in peak volume by up to 85% [101, 102]. Further analysis has shown that
there is no difference in the retention times between the peripheral ports with the
standard deviation in the retention time of the three peripheral ports observed to be
less than 0.6%. It was also demonstrated that the peak maxima obtained at the
13
peripheral ports lags behind the central port and the degree of difference increases as
the retention time increases [103, 104].
Figure 1.2. The AFT - Parallel segmented flow column with multiport end fitting [95].
Active Flow - Parallel Segmented Flow (AFT-PSF) columns have several
advanatges:
The most appropriate measure of separation performance is the evaluation of
the chromatographic profiles as a function of peak volume [101]. Active flow
management has reduced the peak volume for a sample as compared to
normal mode and has showed improved transport of solvent from one
dimension to the second dimension in 2D-HPLC [95]. Reduction in peak
volume results in efficient analysis in MS mode and the necessity to remove
solvent is greatly reduced [105].
Active flow management improves analytical sensitivity compared to the
conventional mode of operation because the analyte to mobile phase ratio is
14
increased; only a portion of the flow that elutes from the column passes
through the detectors [106, 107].
Separation efficiency on the analytical scale is increased by 25% with an
increase in sensitivity by as much as 52% compared to conventional
columns. The significant improvement in separation efficiency is due to
reduced plate height and, consequently, an increase in theoretical plates [107,
108].
Viscous fingering [109] and thermodynamic differences for spatially
heterogeneous solvent environments are limiting conditions in
chromatography [110]. Active flow columns reduce the solvent load and
minimize these effects [19].
AFT has been used as a platform for multiplexed detection, with sample
analysis being undertaken from each of four ports of parallel segmented flow
columns [103].
Reaction flow chromatography is a new application of parallel segmented
flow column. Rapid post-column derivatisation can be achieved without a
reaction coil with an elimination of band broadening of chromatographic
peaks [80, 111].
1.3.3. Reaction Flow Chromatography
Reaction Flow (RF) chromatography is an application of active flow
management, whereby the derivatization reagent is added directly into one of the
outlet ports of a parallel-segmented flow column. RF-PCD is based on removing the
reaction coils and utilizing the mixing potential of the frit, resulting in overall higher
efficiency and improved resolution than conventional methods [80, 111] as shown in
15
Figure 1.3. RF chromatography has shown an increased sensitivity, theoretical plate
counts and resolution. This significant increase in performance is due to less peak
broadening because of a reduction in the post column dead volume [80]. RF
chromatography of amino acids using fluorescamine reagent has shown an
improvement in the response signal of analyte while minimizing the baseline noise
compared to conventional PCD methods [111]. The RF-PCD method provided a
greater linear range with a high correlation coefficient value, and the reduction of
baseline noise which further allows lower LODs and LOQ [80].
Figure 1.3. AFT-PSF column outlet used for Reaction Flow Chromatography [80].
RF-PCD using DPPH (2,2-diphenyl-1-picryl-hydrazyl-hydrate) is an
application of an antiradical scavenging assay for the screening of antioxidants from
natural complex mixtures. The mixing between the derivatizing reagent and solute is
very efficient and removes the need to employ a reaction coil. Comparative studies
between conventional and reaction flow setups have shown that resolution,
sensitivity and separation efficiency of analytical compounds has improved with RF-
16
PCD Chromatography [80]. Another application of RF-PCD involved the use of two
derivatization reagents, 4-aminoantipyrene and potassium ferricyanide, for the
detection of phenols where peak broadening and resolution of peaks were improved
[112]. RF chromatography with fluorescamine reagent and ultraviolet-visible (UV-
Vis) detection was used for the analysis of amino acids with the limits of detection
and quantification being reduced by 50% compared to a conventional column setup
[111].
1.3.3.1 Multiplexing AFT-RF Columns
AFT enables multiple detectors to be used on the outlet ports where the
segmentation ratio is set in such a way that each port carries an exact flow rate [113]
as shown in Figure 1.4.
Figure 1.4. Multiplex detection of natural complex mixtures with an AFT-PSF Column [114].
AFT-PSF columns enable multiplex detection without compromising
separation performance, unlike conventional multi-detection setups such as split-
flow or serial detector setups [113-115]. In RF-multiplexed detection, derivatizing
17
reagents are introduced, using a reagent pump, into one or two of the outer peripheral
ports against the direction of the mobile phase flow. The column eluent, after mixing
with the reagent, is passed to the detector via a free outer port and this flow setup can
be used for more than one reagent. The central flow can be used for the detection of
underivatized analytes [103]. Multiplexing by simultaneously connecting up to four
detectors is also possible using destructive and non-destructive detectors without
additional dead volume tubing [103].
Several types of detectors have been employed in the multiplexed AFT-RF
setup. The UV-Vis detector is one of the most important and widely used detectors
in HPLC. It is primarily used for those compounds which absorb radiation in the
wavelength range of 160 – 800 nm and has several applications in the field of natural
product chemistry [116]. It has some limitations as it is only applicable to those
chromophores that absorp within the UV-Vis region and provides very little spectral
information about unknown compounds [117]. Fluorescence detectors (FLD) are not
considered as a general detector and are known to be selective for only those
compounds which fluorescence when excited by UV-Vis radiation. It is more
sensitive than UV-Vis detection but limited to those compounds which show
excitation. The difference in the basic aspects of detectors may be an important tool
to design a detection strategy in multiplexed detection [116]. The refractive index
detector (RID) is the most commonly used detector for sugars but it responds to
gradient changes of mobile phase, which limits its applications in the field of natural
product chemistry. Other detectors such as evaporation light scattering detectors,
corona discharge detectors are not detectors of choice in natural product chemistry
[118].
18
Mass spectrometry (MS) is an important tool in natural product chemistry
[119] and a specially designed fitting provides segmentation between central and
peripheral flow which reduces the solvent load to the MS [120]. Under high
throughput conditions, fast separations within six seconds using mobile phase flow
rates in the order of 5–6 mL/min have been recorded [120, 121].
The multiplexed AFT-RF setup enables PCD techniques with reaction flow
setups. A significant advantage of multiplexing AFT-RF is the rapid characterization
of bioactive compounds as it allows for easy peak matching and detection [103, 113,
122].
1.3.4. Mass Spectrometry
Mass spectrometry (MS) is an integral technique in the characterization of
biomolecules and it is the only technique which provides so much information with
so little sample [119]. It is a standard technique for analytical investigations of
molecules, in determining elemental composition and structural insights of natural
products [123, 124]. It has applications in the field of biotechnology, and in clinical,
forensic, food and polymer analysis [125, 126] and is becoming an important tool to
revolutionize the medical field [127-129]. MS continues to play a significant role in
pharmaceutical analysis [130, 131] and the selection of drug candidates [132].
Structural elucidation of small molecules using MS is current practice in
modern sciences [126, 133, 134]. It has become an efficient and high thorough-put
screening method [135] used for characterization [136]. It has applications in several
stages of the drug discovery process, such as selection, screening, de-replication and
identification of unknown bioactive compounds [137] and has proven to be the
technique of choice [138]. MS is not only a preferred technique for discovery
19
purposes but also a tool for drug development purposes. Electrophilic bio-activation
of drugs and their binding with tissues and macromolecules, such as lipids and fatty
acids, is a current area of research as it gives information about the structural
fragments of electrophilic by-products used for therapeutic purposes [139-141].
Initial drug discovery procedures involve biochemical screening and MS-
based assays are widely used [137]. Hyphenated MS systems are particularly useful
in pharmaceutical and drug discovery projects [142]. Multidimensional LC-MS
approaches are powerful tools for pharmaceutical analysis and are an emerging area
of drug discovery and development [136].
LC-MS systems include an auto sampler, the HPLC, the ionization source
(which interfaces the LC to the MS) and the mass spectrometer [143-145]. LC is
very efficient for separating compounds within mixture but requires a suitable
detector to efficiently identify components within a mixture [146, 147]. MS is based
on the production of ions according to their mass-to-charge (m/z) ratio and mass
spectra present the ion intensity as a function of the m/z [148].
Several types of instrument are currently available for analysis, including ion
trap (IT), triple quadrupole (QQQ), time-of-flight (TOF), Fourier transform-ion
cyclotron resonance (FT-ICR), and the newest mass analyzer, the FT-Orbitrap.
Different mass analyzers have specific merits and limitations [149] in terms of
speed, sensitivity, ease of use and robustness, mass accuracy, mass resolution, and
their cost to own and operate [150].
The analysis of bioactive compounds is dependent on the type of ion source,
mass analyzers and the inlet system [151]. Typically, MS has two ionization modes -
electrospray ionization (ESI) and atmospheric pressure chemical ionization (APCI).
20
ESI is based on the formation of concentrated charged droplets via the evaporation
of solvent molecules from the droplet surface and APCI is achieved through gas
phase chemical ionization [152]. Chemical ionization MS is a relatively new
technique for analyzing compounds formed by ion molecule reactions [153].
Tandem MS (or MS/MS) uses multi-staged mass selection and separation processes
to obtain structural information of molecules [154]. Target identification and
development is achieved [155] due to an increased sensitivity, range and selectivity
with several modes of detection [156]. MS has evolved to that point where it can be
used throughout natural product discovery and development [130].
1.3.4.1. High Resolution Orbitrap Mass Spectrometry
The Orbitrap mass analyzer was developed in the early 2000s and is a mass
analyzer of the Fourier transform family that operates based on harmonic ion
oscillations in electrostatic fields [157]. For high resolution MS, measurement ions
are accumulated in the linear ion trap and passed on to the orbitrap analyzer. The
concept of orbital trapping can be traced back to 1923 [158], where it was observed
that ions with high tangential velocity orbit around a wire. Experimental work for
half a century, reviewed in 2008 [159], has improved the efficiency of electrostatic
trapping but researchers were still not able to figure out how to use this concept as a
mass analyzer. This critical problem was successfully solved by Makarov [160],
followed by significant innovation in technology to make the C-trap, which brings
the electrostatic mass analyzer to practice [161]. This storage device has enabled
coupling of the orbitrap to any instrument, which has mass fragmentation
capabilities. The C-trap linear orbitrap becomes more efficient and versatile with the
addition of higher collision energy dissociation [162]. Implementation of electron
transfer dissociation in the linear orbitrap [163] has enabled the study of post-
21
transformational changes within proteomics [164]. A dual pressure linear ion trap
configuration has increased the sensitivity and speed. These technological
improvements have enabled the acquisition of up to ten fragmentation spectra per
second [165]. In the Orbitrap Elite instrument, the resolving power of the mass
spectrometer has been increased to 24000 at m/z 400 with 20 CID scans all within a
2.7 s cycle time.
Fourier transform ion cyclotron resonance mass spectrometry, FT-ICR, also
has an ability to accumulate longer transients [166]. In comparison to orbitrap, FT-
ICR is able to achieve hyper resolution due to the transient lasting several minutes
[167]. In an orbitrap, this long transient phenomenon is achieved by adding the high
field analyzer and Fourier transform algorithm [166]. In the Orbitrap Exactive
instrument, the benchtop mass spectrometer ion source is directly linked to the C-
trap [168] which enables the detection of metabolites with high selectivity, dynamic
range and scan speed [169]. Rapid screening and identification of target constituents
using hybrid LTQ-Orbitrap mass spectrometer is a current area of research [170].
The high-resolution q-Exactive orbitrap mass spectrometer is capable of separating
mass fragments at the fourth and the fifth decimal place (exact mass) [171, 172]
which provides data about molecular fragmentation and isotopic abundance for
solutes after chromatographic separation [173]. Conventional instrumentation is
limited to single-digit mass units or integer mass.
1.4. Natural Products as a Source of Antioxidants 1.4.1. Oxidative stress and Antioxidants
Oxygen is as essential component for living organisms but oxygen is also
poisonous and aerobic organisms only survive because they contain antioxidant
22
defense systems [174]. Singlet oxygen is an unpaired electronically excited state and
is important in biological chemistry. It was first identified by Carl Wilhelm Scheele
and Joseph Priestly in the 18th century [175]. Understanding the chemistry of singlet
oxygen is important due to its destructive impact on organisms [176] and photo
physics effects within our lives [177, 178]. Molecular oxygen absorbs energy in the
presence of photons and this leads to significant degradation as well [179].
Oxidative stress is usually defined on the basis of reactive oxygen species
and other free radicals [129]. It is important to understand the mechanism of the
formation of free radicals and their detrimental effects [180]. The theory of free
radicals originated in 1950 with the essential role of free radicals and their
metabolites being demonstrated as important in the ageing process [181, 182].
Oxidative stress due to reactive oxygen species results in DNA damage and is
responsible for several diseases in humans [183-187]. It is also a leading cause of
cardiovascular diseases such as pathogenesis of atherosclerosis, lung inflammation
[188-190], vascular inflammation, hypertension and endothelial dysfunction [191-
195]. Oxidative and glycol-oxidative stress increases in hyperglycemia diabetes and
metabolic disorders [196-199]. Neurodegenerative diseases usually result from free
radical attacks on neuronal cells [200, 201], and pathological conditions such as
Parkinsonism [202, 203] and Alzheimer disease [204-206] are common examples of
neurodegenerative diseases due to oxidative stress. Oxidative stress contributes
towards HIV pathogenesis, including viral replication, inflammation, decreased
immune cell proliferation and apoptosis [207-209]. Oxidative stress with hepatitis C
virus contributes towards cirrhosis and hepatocellular carcinoma [210-212].
Antioxidants are substances which are present in small quantity compared to
an oxidizable substrate and they inhibit or delay oxidation of that substrate [213,
23
214]. Antioxidants are classified depending on their mode and duration of action
with antioxidant enzymes, chain breaking antioxidants and transition metal binding
proteins being the three main classifications of antioxidant systems [214].
1.4.2. Australian Native Plants as a source of Antioxidants
Indigenous Australian were the first to harvest, process and prepare
Australian native foods which have medicinal, nutraceutical, cosmetic and
pharmaceutical properties [215, 216]. In recent years, Australian native plants have
been recognized for many groundbreaking antimicrobial, cyto-protective, pro-
apoptotic and antioxidant benefits [217, 218]. In this study, several Australian native
plants are used to investigate bioactive phytochemical profiles.
Quandong, Santalum acuminatum, is an Australian native shrub or tree
belonging to the family Santalaceae with their habitat in Southern Australia. It is a
semi-parasite attached to the roots of a host plant for nutrients. Ripe fruit are 15–25
mm in size with a shiny, yellow to red skin and are also known as sweet quandong,
wild peach, desert peach and native peach [219]. Aboriginal communities ate the
fresh fruit and used kernel extracts for various medicinal purposes [220]. Quandong
was also a favorite fruit among early European explorers and the Commonwealth
Scientific and Industrial Research Organisation (CSIRO) of Australia has conducted
research on its propagation since 1973 [219]. Quandong was one the first Australian
native bush plants to be considered for scientific research, with a paper on
propagation and cultivation by Grant and Buttrose appearing in 1978 [221].
Desert lime (Citrus glauca) grows in the Queensland and New South Wales
[222] and is known as a medicinal plant for indigenous people. It has a good
potential as a commercial crop and its fruiting capacity is highly variable. Desert
24
lime trees grow easily and can be stored for supply throughout the year [221, 223].
Desert lime is known to have health enhancing components such as folates, vitamin
C and antioxidants [224].
Tasmannia lanceolata, commonly referred to as Tasmanian Native Pepper, is
a native shrub of Tasmania and southeast Australia that can reach heights of up to 5
m and produce black, berry like fruits [225]. It is one of the more popular native food
ingredients to have appeared on the culinary horizon in the last 20 years [181].
Figure 1.5. Australian native foods used in the bioanalytical characterisation of antioxidants.
Wattle seed belongs to the subfamily Mimosoideae of the family Fabaceae.
Indigenous people used it in bakery products and it has found applications in the
cosmetic and pharmaceutical industries. Acacia victoriae (Bentham), also known as
prickly wattle, is one of the most common of the approximately 960 species of wattle
plant found in Australia [226].
25
Gumbi Gumbi (Pittosporum angustifolium) is a small fruiting tree and
Indigenous people consider it as God’s medicine. It was collected in June 2008 in
Central Queensland [227]. Phylogenetic analysis has shown that it belongs to genus
Pittosporum and about 20 species of Pittosporum angustifolium have been found in
Australia [228].
Old man saltbush (Atriplex nummularia) is one of the best regarded
saltbushes in the world [229]. It was very common is the Murray-Darling Basin in
the 19th Century and the potential of saltbush has been recognized in the farming
industry [230].
1.4.3. Australian Mushrooms as a source of Antioxidants
Medicinal mushrooms can be defined as macroscopic fungi, mostly higher
Basidiomycetes, which are used for prevention, nutritional and healing purposes
[221]. The global market of antioxidants is expected to be double from $103.6
million in 2011 to reach $246.1 million in 2018 [231, 232]. They are considered as
functional foods and consumed by the people due to their nutritive and medicinal
properties [233]. Edible mushrooms are known for their high nutritional value and
therapeutic properties as they are a promising source of antioxidants and reduce the
level of oxidative stress [234-250].
Agaricus, Lentinula, Flammulina, Pleurotus, Ganoderma lucidum, Agaricus
blazei are the most commonly cultivated mushrooms [251, 252]. Mushrooms are a
source of bioactive compounds, which help them survive in their natural
environment. Many of the bioactive compounds within mushrooms have anti-
inflammatory and antioxidants properties [253-256]. They not only possess
antioxidant and antimicrobial properties but are also one of the richest sources of
26
anticancer and immunomodulating agents [257, 258]. Medicinal mushrooms have
therapeutic applications such as cardioprotection [259, 260] hepatoprotective [261]
and antihypertensive effects [262]. Scientists have shown great interest in research of
medicinal mushrooms. It is suprising that in 2010, many people across the world
were still unaware of the therapeutic value of medicinal mushrooms, which have
broad spectrum of pharmacological activities [263, 264].
1.5. Untargeted Metabolomics - A Potential Tool of
Drug Discovery
Searching for new bioactive compounds in natural products is still an
emerging area and untargeted methods are new to the rapidly developing field of MS
[265-267]. Natural extracts comprise thousands of molecules which have potential to
be bioactive and searching for novel compounds is a laborious task. Targeted
metabolomics is the measurement of the defined groups of metabolites [268]. By
contrast, untargeted metabolomics measure all detectible metabolites in a sample,
including chemical unknowns [269]. Natural product scientists are using MS for
untargeted identification and structural elucidation of molecules. Over the next
decade, the availability of efficient ionization sources and sensitive detectors will
enable in-depth analysis of natural products. One can envision that these advanced
technologies will make MS an essential tool for bioactive profiling [270]. The
untargeted metabolic driven approach has also been applied to screen bioactive and
their metabolites in in-vivo systems [271].
Natural remedies have become more popular during the last decade and
simultaneous determination of compounds within natural products is a current area
of interest. In particular, LC-MS can be used to make qualitative and quantitative
27
analysis of metabolites in complex mixtures, with significant advantages over
techniques such as nuclear magnetic resonance (NMR) spectroscopy [272].
Figure 1.6. Conventional approach of untargeted metabolic profiling for Drug Discovery from Natural Products.
The high selectivity, sensitivity and versatility of natural products make them
ideal for study [273, 274]. Novel techniques in MS with ultrahigh resolution to data
independent MS/MS have advanced technology to such an extent that hypothesis
driven validation can be achieved [170]. Sample preparation is a critical step in
natural products metabolomics due to matrix effects and its implications in
separation performance of chromatographic columns due to increased back pressure
[275]. In order to maintain wide coverage of metabolites, sample pre-treatments are
usually avoided. An ideal sample preparation for metabolomics should be non-
selective, reproducible, simple and fast [276].
28
Figure 1.7. Untargeted metabolic profiling -A tool for Ethnopharmacological studies.
High-resolution MS-based metabolomics, taking advantage of technological
advances, modern analytical techniques, as well as the power of data interpretation
tools, comprises a new approach. It promises a holistic view of the phytochemical
profile of every natural product, aiming for a more comprehensive and multifaceted
view in phytochemistry and evidence-based natural products. According to the scope
of this study, bioactive profiling was applied in a targeted or non-targeted manner to
explore the ethno-pharmacological potential of native plants. It is, indeed, worth
mentioning that the applications of high-resolution Orbitrap-based metabolomics and
drug discovery has become elevated in the area of natural products.
29
Chapter 2
30
2.1. Materials
2.1.1. Standards and Samples
2.1.1.1. Mushrooms
Fruiting bodies of different commercially available mushrooms were selected
from local grocery stores in Victoria, Australia and were frozen slowly, first at -20
˚C and then at -80 ˚C. Freeze drying using a CRYODOS-50 (Dynavac, Melbourne,
Australia) operating at -50 ˚C and 1 mbar was used to remove moisture. The
processing time for drying varied from 48-60 hrs, depending on the amount of
material and moisture content within the samples.
2.1.1.2. Australian Native Plants
Australia native plants were harvested and collected by wholesaler Lyle
Dudley in the Southern Flinders Ranges, South Australia. Samples were transported
to the laboratory at Swinburne University of Technology via courier under ambient
conditions and were stored in Ziploc® bags in a cold room at 4 ˚C for two months.
2.1.1.3. Coffee Samples
Coffee (Nestlé Nespresso – Ristretto and Decaffeinated, Sydney, NSW,
Australia) was purchased from a local store in Western Sydney and coffee samples
were prepared using an Expresso coffee machine.
2.1.1.4. Standard Solutions
Standard antioxidants (±)-6-hydroxy-2,5,7,8-tetramethylchromane-2-
carboxylic acid (Trolox), rosmarinic acid, chlorogenic acid, caffeic acid, rutin
hydrate, quercetin, morin hydrate, epicatechin, hesperitin, hesperidin, p-coumaric
acid and naringin were purchased from Sigma-Aldrich (Castle Hill, NSW,
Australia). All materials were used as received.
31
For optimization, a 100 mg/L standard solution containing Trolox and
rosmarinic acid was prepared by dissolving 10 mg of each in 100 mL of methanol. A
standard containing 10 mg/L Trolox and 1000 mg/L hesperitin, hesperidin, p-
coumaric acid and narigingin was prepared as follows: Trolox (50 mg) was dissolved
in 50mL of methanol to produce a 1000 mg/L solution; Hesperitin, p-coumaric acid
and narigingin (each 100 mg) were dissolved in methanol and 1 milliliter of the 1000
mg/L Trolox solution was added to the mixture. The resultant solution was
subsequently diluted to 100 mL with methanol.
2.1.2. Chemical reagents
2.1.2.1. Mobile Phases
2.1.2.1.1. Milli Q water
Ultrapure Milli-Q water (18.2 MΩ) was obtained from a Milli-Q® Plus
purification system (Millipore, Germany) and filtered through a 0.2 μm filter.
2.1.2.1.2. Sodium acetate buffer
Sodium acetate buffer (300 mM, pH 3.6) was prepared by dissolving 40.8 g
of sodium acetate trihydrate in 500 mL of Milli-Q water with the aid of ultrasonic
agitation. The pH of the solution was then adjusted to 3.6 (±0.1) with glacial acetic
acid and diluted to 1 L with Milli-Q water. HCl (40 mM) was prepared by diluting
3.3 mL of concentrated hydrochloric acid to 1 L with Milli-Q water.
2.1.2.1.3. Methanol
Methanol was used as solvent B in all reversed phase HPLC and LC-HR-MS
analysis. Methanol was purchased from VWR (Tingalpa, Queensland, Australia) and
was filtered through a 0.2 μm membrane using a 1000 mL vacuum suction filtering
32
apparatus. The automated degasser system on the Shimadzu HPLC provided further
degassing of mobile phases.
2.1.2.2. Post Column Derivatisation Reagents
Reagent reservoirs were washed with laboratory detergent and hot water. The
inlet tubes were wiped with methanol to minimise contamination. All reservoir
containers were properly labelled following GLP guidelines.
2.1.2.2.1. DPPH Reagent
2, 2-Diphenyl-1-picrylhydrazyl free radical (DPPH•) was purchased from
Merck. For conventional HPLC, the DPPH reagent (0.25 mM) was prepared by
adding 98.8 mg of DPPH to 1000 mL of methanol, sonicating for 10 mins to ensure
complete dissolution and stored in a container covered with aluminium foil to
prevent exposure to light.
2.1.2.2.2. FRAP Reagent
The FRAP reagent was prepared according to the method outlined by Benzie
and Strain [277]. TPTZ (10 mM) was prepared by dissolving 62.5 mg of TPTZ in 20
mL of 40 mM HCl with the aid of ultrasonic agitation. Ferric chloride (20 mM) was
prepared by dissolving 108.1 mg of ferric chloride hexahydrate in 20 mL of Milli-Q
water with the aid of ultrasonic agitation. The final FRAP reagent was prepared by
combining 500 mL of 300 mM sodium acetate buffer pH 3.6, 20 mL of 10 mM
TPTZ and 20 mL of 20 mM ferric chloride.
33
2.1.3. Extraction Methods
2.1.3.1. Solvent Extraction with Shaking
Powdered Australian mushrooms and native foods (5 g) were placed in a
conical flask and extracted with methanol solvent (50 mL) in a shaker at 150 rpm for
24 hrs as shown in Figures 2.1 & 2.2. Extraction mixtures were then filtered through
a Whatman No.1 filter paper.
2.1.3.2. Sonication Method
Native foods were homogenized in an electric grinder with methanol as
extracting solvents. The flask containing methanol and the powdered sample was
then placed in an ultrasonic bath that was maintained at room temperature for 30
min. In order to avoid the loss of solvent, the flask was covered with aluminum foil.
The obtained extracts were filtered through a Whatman No. 1 filter paper and the
filtrate was concentrated in a rotary evaporator under controlled vacuum at 37 ˚C.
The concentrated extract was redissolved in 2 mL of methanol to prepare the final
sample.
34
Figure 2.1. Schematic representation of the process involved in the sample preparation for the mushroom samples.
Figure 2.2. Schematic representation of the process involved in the sample preparation for the Australian Native Foods.
35
2.2. Instrumentation
2.2.1. Post Column Derivatization coupled with
Conventional HPLC
In this project, studies were conducted with an analytical Shimadzu HPLC
that consisted of a controller (SCL-10AVP), a Low-Pressure Gradient Valve (FCL-
10ALVP), a Pump (LC-20AD), an Injector (SIL-10ADVP) and a PDA detector
(SPD-M10ADVP).
1. Solvent Reservoirs: Mobile phase solvents were contained in glass
reservoirs. The mobile phase was a mixture of Methanol and Milli-Q water
whose respective concentrations were varied depending on the gradient
conditions.
2. Pump: A pump (LC-20AD) aspirates the mobile phases from the solvent
reservoir and forces it through the column PES C18 and detector. Depending
on a number of analytical factors, an operating pressure of up to 42000 kPa
(about 6000 psi) could be generated.
3. Sample Injector: An automated system injector (SIL-10ADVP) was used
for batch runs. The (SIL-10ADVP) draws precise amounts of the sample with
high reproducibility and is able to inject under high pressure (up to 4000 psi).
4. Column: For conventional and RF-PCD methods, a PES C18 (150 × 4.6
mm, DP = 5 µm) column sourced from Thermo Fisher Scientific (Runcorn,
Cheshire, United Kingdom) was used. The column and mobile phase
temperatures were kept constant throughout an analysis.
36
5. Detector: A UV-Vis detector (SPD-M10ADVP) operating at a single
selectable wavelength detected the analytes as they eluted from the
chromatographic column.
6. Data Collection: Chromatographic data were processed using LC-
Solution software that produced integrated peak responses.
7. Degassing: A Phenomenex Degassex DG-4400 4-Channel On-line
Degasser was used to improve flow rate stability and reduce detector noise
level.
8. Reaction coils: PEEK tubing reaction coils of varying lengths that
included 20 µl, 50 µl, 100 µl and 500 µl were used for the post column
derivatisation reactions.
Reagent solutions, DPPH and FRAP, were pumped by a Shimadzu LC-20AD
pump fitted with an external pulse dampener and added to the eluent exiting the
column via a zero-dead volume T-piece. One end of reaction coil was attached to the
outlet of the T-piece and other end was attached to the detector. The flowing stream
passed through one of a 20 µL, 50 µL, 100 µL, or 500 µL reaction coil prior to
entering the detector. Detection wavelengths of 520 nm and 573 nm were selected
for DPPH and FRAP reaction systems, respectively. The PCD instrument setup is
represented in Figure 2.3.
37
Figure 2.3. Schematic diagram of the Post Column Derivatisation setup coupled with
HPLC.
2.2.2. Post Column Derivatization using Reaction Flow
Chromatography
Most of the analytical parameters used in the conventional PCD method were
applicable in RF-PCD. Reaction Flow chromatography is based on the principle of
parallel segmentation ratios and the ratio of flows between the central and peripheral
ports of an active flow column. This parallel segmentation was achieved with tubing
of different lengths because length and internal diameter of the tubing contribute to
the backpressure and flow rate of the system. In this study, flow stability and
pressure of the reagent pump was considered because an unstable flow affects the
limit of detection and quantitation [278].
In reaction flow chromatography, the reaction coil is replaced with a frit
enclosed within the AFT column. The derivatization reagent(s) are pumped against
38
the flow of mobile phase into either one or two of the outer ports of the column
where they mix with the column effluent inside a frit housed within the column end
fitting. This technique allows for more efficient mixing of the column effluent and
the derivatization reagent(s) meaning that the volume of the reaction loops can be
minimized or even eliminated altogether. It was found that RF-PCD methods
performed better than conventional PCD methods in terms of observed separation
efficiency and signal to noise ratio.
Figure 2.4.Active flow column attached with Frit-Thermo fisher Scientific (UK)
The HPLC system described in Section 2.2.1. was used with the conventional
column replaced by a multiport Active Flow Technology (AFT) column. With a flow
rate of 1 mL/min, the inlet of the column was connected to the HPLC instrument and
tubing of 15 cm length and 0.13 mm i.d. was connected to the outlet central port of
the column for the underivatized effluent to be monitored. The post column reagent
pump line was connected to a peripheral port on the outlet of the column and one
other outlet peripheral port was connected to the UV-Vis detector using a 15 cm
length of 0.13 mm i.d. tubing. The unused peripheral port on the outlet of the column
was blocked using a column stopper. For the FRAP reaction flow studies, a PES C18
39
(150 × 4.6 mm, DP = 5 µm) column with a RF 2:1 frit and 4-port outlet head-fitting
was used, sourced from Thermo Fisher Scientific (Runcorn, Cheshire, United
Kingdom) as shown in Figure 2.4.
2.2.3. LC-HRMS-Q-Exactive TM Hybrid Quadrupole
Orbitrap Mass Spectrometer
Liquid chromatography-high resolution mass spectrometry, LC-HRMS, was
used for the identification of antioxidants compounds. The mass spectrometer used
in this study is a Quadrupole Orbitrap, in which an electrostatic field is established
that possesses sufficiently high tangential velocity where the ions orbit the rods,
rather than directly colliding with them [158]. The Q Exactive HF-X Mass
Spectrometer uses a high-capacity transfer tube for maximum ion loading, an
electrodynamic ion funnel that accommodates and transmits ions over a broader
mass range [171, 279, 280].
Figure 2.5. Principle of Orbitrap mass spectrometry
After ionization, ions with different mass to charge ratios (m/z) are
transported to the quadrupole, which is performed via a radio frequency (RF) lens.
40
The quadrupole can operate as a mass filter and in the storage quadrupole, the ion
velocity can be reduced by collision with an inert gas and by the application of
electric fields. The orbitrap analyser consists of an inner and outer electrode, which
creates an electric field that causes the initiation of trajectory around the inner
electrode. All injected ions show an equal amplitude during administration of a
specific electrical field and the frequency of this axial oscillation is directly related
with the m/z ratio of the ions. The axial frequency in Orbitrap MS is not affected by
the chemical characteristics of the ions. In this advanced ion trap system, the C-Trap
has improved the space charge capacity. A high-energy collision dissociation (HCD)
cell can be applied to obtain improved mass spectra and collision induced
dissociation (CID) has also become possible in the C-trap device.
2.3. Optimization of Chromatography and MS
Methods
2.3.1. Optimization of segmentation ratios within RF
Chromatography
The ratio of flow exiting through the column central port relative to the
peripheral ports is known as the segmentation ratio and can be tuned to optimize
chromatographic performance.
Two clean and dry vessels were weighed. One was labelled as central (C) and
other as peripheral (P).
Mobile phase existing from the central port was collected in the central vessel
(C) for 1.0 min and vessel (C) was re-weighed.
41
The effluent exiting from the detector that is attached to the peripheral port of
the RF column was collected and the weight after 1.0 min of flow was
determined.
The percentage of flow coming from the central and peripheral ports is as
follows:
% Central Port = Weight of Central Port (g) / (Weight of Central Port (g) +
Weight of Peripheral Port (g)) x 100
% Peripheral Port = Weight of Peripheral Port (g) / (Weight of Central Port
(g) + Weight of Peripheral Port (g)) x 100
The segmentation ratio between central and peripheral flow was adjusted as
required in experimental protocols.
Adjustment of segmentation ratios, pre-or post-detector, depends on the type
of detectors. If a post column reaction based detector is used, then the flow ratio is
measured post detector without addition of the reagent. If two or more destructive
detectors are used, then the flow ratio is measured pre-detector. System pressure of
destructive detector such as mass spectrometry is an important consideration while
adjusting flow percentage pre-detector [281].
A shutdown procedure was employed once all the samples were injected and
the LC run was finished. The derivatization reagent pump flow was stopped and the
reagent pump line was removed from the peripheral port with the port then closed
with a stopper. The column was equilibrated with the mobile phase in which it is to
be stored by allowing the mobile phase to pass through the column at 1 mL/min for
10 min. Flow of the mobile phase pump on the HPLC system was stopped and the
reagent was replaced with methanol and the additional pump was purged then the
HPLC system was turned off.
42
2.3.2. Optimization of flow rate within RF Chromatography
The flow rate of post column reagent is an important analytical parameter and
its optimization is a requirement for reliable and reproducible online-PCD [282]. The
flow rate of the DPPH reagent has a significant impact on the observed detector
response. A solution containing 100 mg/L of both Trolox and rosmarinic acid was
used to optimize the flow rate of the reagent. In this study, the optimized flow rates
of 0.8 mL/min and 0.5 mL/min for DPPH and FRAP reagents, respectively.
For analysis using the AFT column, first optimal segmentation flow ratio
between the central and peripheral ports was found to be 50:50 central:peripheral,
which was also used for all subsequent analyses using the reaction flow column. An
optimisation was performed using a reaction flow column that was operated with a
flow ratio of 50:50 central:peripheral. The optimal flow rate for the FRAP reagent
was found to be 0.5 mL/min, which was used for all subsequent analyses in the
conventional post column derivatisation method. Whilst keeping all other conditions
the same, the solution containing Trolox and Rosmarinic acid was injected. On
subsequent injections, the FRAP reagent was pumped into the system at various flow
rates ranging from 0.1 mL/min to 1.2 mL/min. The signal to noise ratio of the each
of the peaks was measured.
2.3.3. Optimization of gradient conditions
Gradient elution is a method used to improve resolution and detection of
compounds. It is able to separate complex compounds in a short analysis time.
Gradient separation is required for complex samples because it is usually not
possible to elute components between retention factors of 1 to 10 under isocratic
conditions.
43
For an optimization of the post column DPPH assay experiments, an isocratic
elution with 40 % Milli-Q and 60 % methanol was used as the mobile phase. Once
the system was optimized, all subsequent analyses were performed using a gradient
elution where mobile phase A was Milli-Q water and mobile phase B was methanol.
A linear gradient was used starting at 100% A and changing to 100 % B over 20
minutes, which was then held for a further 5 minutes. The flow was then returned to
100 % A over 1 minute and allowed to equilibrate for 5 minutes prior to the next
injection.
For the optimization of reaction flow chromatography experiments, an
isocratic elution with 40 % 30 mM sodium acetate pH 3.6 and 60 % methanol was
used as the mobile phase. Once the system was optimized, subsequent analyses were
performed using a gradient elution where mobile phase A was 30 mM sodium
acetate pH 3.6 and mobile phase B was methanol. A linear gradient was used starting
at 100% A and changing to 100 % B over 20 minutes, which was then held for a
further 5 minutes. The flow was then returned to 100 % A over 1 minute and allowed
to equilibrate for 5 minutes prior to the next injection. For all experiments, the flow
rate of the mobile phase was 1.0 mL/min and an injection volume of 20 µL was
used. The detector was set to an analysis wavelength of 593 nm (2 nm bypass) and
520nm for FRAP and DPPH, respectively unless otherwise specified.
2.3.4. Optimization of MS Method
The LC-HR-MS system was composed of a ThermoFisher ULTIMATE 3000
system equipped with an analytical column ThermoFisher C18 (2.1 mm × 100 mm,
2.2 μm particle size), coupled to a single-stage Exactive Orbitrap MS system,
ThermoFisher Scientific (Australia).
44
All LC-MS spectra were acquired using a Thermo Scientific Q Exactive
hybrid quadrupole-orbitrap mass spectrometer. Sheath gas, auxiliary gas flow rate,
sweep flow, ion source temperature, capillary voltage, capillary temperature, probe
temperature, spray voltage, sheath gas pressure, auxiliary air pressure were set to the
default values. An analysis cycle that performed a full MS scan (AGC target 1e6,
resolution 70,000 within scan range of 120 to 750 m/z) in which major ions were
selected, fragmented and detected in the linear ion trap (AGC target 1e4). The
collision-induced dissociation energy was 30% NCE and the activation time was 30
ms. Ions were added to a dynamic exclusion list after being fragmented twice.
MS/MS parameters include mass resolution of 17,500, AGS target of 1e5 having
maximum injection time of 200 ms within isolation window of 2.0 m/z.
2.4. Data Interpretation and Analysis
Acquisition parameters were specified and raw data files were stored after
analysis. The raw data files were processed with a Compound Discoverer™ software
(ThermoScientific) which is a flexible solution that offers a full suite of tools to
address MS-based small molecule analysis, identification, and mapping. It can be
used for complete bioactive molecule identification and characterization.
Identification of compounds of interest in complex natural mixtures is one of the
greatest challenges in many research applications. This software includes the most
confident elemental composition prediction for unknowns compounds utilizing fine
isotopic structures from high resolution Orbitrap full MS, as well as MS/MS
information to improve predictions. Compound Discoverer™ is combined with
parallel library searching such as the mzCloud™ online fragmentation library
45
(mzCloud.org) and molecular weight or formula-based searches of Chemspider™
and other custom databases.
46
Chapter 3
47
3.1. Introduction
High performance liquid chromatography coupled with post column
derivatization may be used to measure antioxidant capacity of individual compounds
within a complex mixture. An antioxidant capacity assay based on single chemical
reaction seems unrealistic because of the numerous methods that have been used in
the literature to evaluate antioxidant capacity [283]. A simple bench assay, Ferric
reducing ability of plasma (FRAP) assay, has been developed for assessing the
antioxidant properties of human plasma [277]. It is based on direct electron transfer,
with ferric reduced to ferrous at low pH [283]. The Ferric-tripyridyltriazine salt,
Fe(III)(TPTZ)2Cl3, is used as the oxidant and provides an indicator of the reaction
due to the absorbance of the reaction product Fe(II)(TPTZ)22+
at 593 nm [277]
(Figure 3.1).
Figure 3.1. The reaction mechanism for the FRAP Assay.
Although the FRAP assay is easy to use and effective in measuring
nonspecific antioxidants within a sample, it is not able to differentiate between the
48
different antioxidant components within a sample. One drawback associated with the
conventional bio-assay guided identification and characterisation of antioxidants is
their stability. To obtain information about the individual compounds, the
development of online coupling of antioxidant assays with HPLC is gaining ground
[284] and a possible option is the direct coupling of the FRAP assay. A post column
FRAP assay coupled with HPLC was developed by Raudonis and his colleagues, for
the online evaluation, separation and characterisation of antioxidants within natural
products [285, 286]. In an initial experimental design, the FRAP reagent entered a
reaction coil through a T-piece where the reaction coil was used to increase the
reaction time for the reagent and the separated solutes before UV detection.
The most commonly used reaction coils employed in PCD setups are
typically 500 µl or greater [287-290]. The use of these large reaction coils is no
longer compatible with the development of modern HPLC columns. The current
trend is towards short and/or narrow columns packed with sub-3 micron particles
that typically yield around 180,000 to 250,000 plates/metre. In hyphenated online
HPLC systems, both destructive and non-destructive detectors are employed to
identify and characterize the antioxidants within the sample. If an additional
destructive detector, such as a mass spectrometer, is attached then a post column
splitter must be used. However, addition of a splitter increases the extra-dead volume
of the system and splitting the flow between two detectors results in a loss of
sensitivity. The use of large mixing coils almost entirely nulifies the benefit of these
columns, since the mixing coil volume may be larger than the peak volume [114]. In
order to achieve the best results, any extra column volume should be avoided with
the shortest possible length of tubing recommended as a part of method and
instrument optimization [291].
49
The aim of this Chapter is to introduce an application of Reaction Flow
Chromatography based on the rapid post column FRAP assay. In particular, we
investigate the ability of RF-PCD to minimise the reaction loop volume providing
greater efficiency and selectivity with observed gains in sensitivity compared to the
conventional PCD techniques.
3.2. Material and Methods
3.2.1. Chemicals
Ultrapure Milli-Q water (18.2 MΩ) was prepared in-house and filtered
through a 0.2 µm filter. Sodium acetate trihydrate, glacial acetic acid, hydrochloric
acid (12M), TPTZ, ferric chloride hexahydrate, (±)-6-hydroxy-2,5,7,8-
tetramethylchroman-2-carboxylic acid (trolox), rosmarinic acid, chlorogenic acid,
caffeic acid, rutin hydrate, quercetin, morin hydrate, epicatechin, hesperitin,
hesperidin, p-coumaric acid and narigingin were used as received.
3.2.2. Columns
For the conventional PCD FRAP method, a PES C18 (150 × 4.6 mm, DP = 5
µm) column was used (Thermo Fisher Scientific, Runcorn, Cheshire, United
Kingdom). For the FRAP RF-PCD studies, a PES C18 (150 × 4.6 mm, DP = 5 µm)
column with a RF 2:1 frit and 4-port outlet head-fitting was used (Thermo Fisher
Scientific, Runcorn, Cheshire, United Kingdom).
3.2.3. Reagent, Sample and Standard Preparations
The FRAP reagent was prepared according to the method outlined by Benzie
and Strain [13]. The final FRAP reagent was prepared by combining 500 mL of 300
50
mM sodium acetate buffer pH 3.6, 20 mL of 10 mM TPTZ and 20 mL of 20 mM
ferric chloride. For optimization, a 0.1 mg/mL standard solution containing trolox
and rosmarinic acid was prepared. A 1 mg/mL standard solution containing trolox,
rosmarinic acid, chlorogenic acid, caffeic acid, rutin hydrate and quercetin was
prepared. A second standard containing 1 mg/mL trolox, morin hydrate and
epicatechin was prepared and serial dilutions were performed. A third standard
containing 10 mg/L trolox and 1000 mg/L hesperitin, hesperidin, p-coumaric acid
and narigingin was prepared.
A fresh sample of espresso coffee (Decaffeinato intenso) was prepared prior
to analysis by extraction using an espresso coffee machine into a 30 mL shot. The
solution was diluted four-fold with of Milli-Q water and then filtered through a 0.45
µm nylon filter.
3.2.4 Instrumentation
All sample analyses were performed on the Shimadzu HPLC System as
described in Section 2.4 with an additional Shimadzu LC-10AT VP pump used for
the addition of the post column FRAP reagent.
51
Figure 3.2. Reaction flow chromatography - RF column in multiplexed mode.
3.2.5. Chromatographic Conditions
A linear gradient was used starting at 100% mobile phase A (30 mM sodium
acetate pH 3.6) changing to 100 % mobile phase B (Methanol) over 20 minutes,
which was the held for a further 5 minutes. The flow was then returned to 100 % A
over 1 minute and allowed to equilibrate for 5 minutes prior to the next injection. For
all experiments, the flow rate of the mobile phase was 1.0 mL/min and an injection
volume of 20 µL was used. The optimal flow rate for the FRAP reagent was found to
be 0.5 mL/min. The detector was set to scan from 190 to 800 nm with an analysis
wavelength of 593 nm used for all measurements unless otherwise specified.
3.2.6. Quantitative Performance Measures
A series of standards were used to verify the performance of the RF-PCD
FRAP analysis. All of the standards contained trolox as a constant for direct
52
comparison. Standards containing between 0.5 mg/L and 1000 mg/L were prepared
and analysed using the RF-PCD FRAP analysis technique. A total of 13 standards
were prepared across this range. It was found that a number of antioxidants did not
show a linear response up to 1000 mg/L and where this occurred, the standards with
concentrations greater than the linear range were not used to calculate the
performance data. Loss of linearity was due to the complete consumption of the
FRAP reagent rather than detector saturation. The use of a more concentrated FRAP
reagent was investigated but this caused problems with solution stability and
precipitation.
The limits of detection and quantitation were defined as the estimated
concentration where a signal to noise ratio of 10 and 3 were obtained, respectively,
based on the signal to noise ratio of the standards used. The r2 value and the relative
response to trolox were calculated based on the line of best fit through the data.
Instrument precision was defined as the %RSD of the areas of ten consecutive
injections of the 100 mg/L standard.
3.3. Results and Discussion
3.3.1. Conventional PCD
Characterisation of the conventional PCD method with a 500 µL reaction
loop as employed by Raudonis et al. [67] was performed. An example chromatogram
for 1000 mg/L p-coumaric acid, hesperidin, naragingin and hesperitin and 10 mg/L
trolox is shown in Figure 3.3(a) and Table 3.1 summarizes the performance measures
for conventional PCD.
53
(a) (b)
(a)
54
Figure 3.3. Chromatograms of a solution containing 1000 mg/L p-coumaric acid,
hesperidin, naragingin and hesperitin and 10 mg/L trolox derivatized using the FRAP
reagent and analysed using (a) Conventional PCD at 593 nm, corresponding to the
FRAP analysis wavelength (b) RF-PCD at 593 nm and (c) RF-PCD at 338 nm.
(c)
55
Table.3.1. Quantitative performance of antioxidant standards with conventional
PCD.
Antioxidant Precision
(% RSD)
Linear Range
(mg/L)
Correlation
Coefficient (r2)
LOQ
(mg/L)
LOD (mg/L) Response
Relative to
Trolox
Trolox 0.682 3 – 75 0.9976 3 1 1
Chlorogenic Acid 1.248 10 - 1000 0.9937 10 3 0.3029
Caffeic Acid 1.167 5 – 100 0.9913 5 1.5 0.7857
Rosmarinic Acid 0.975 8 – 250 0.9946 8 3 0.5254
Rutin 1.671 60 – 1000 0.991 60 20 0.0547
Quercetin 0.837 3 – 100 0.9974 3 1 0.8784
Epicatechin 1.879 40 – 1000 0.9968 40 10 0.2501
Morin 1.278 5 – 1000 0.9912 5 2 0.3801
3.3.2. RF-PCD
The chromatogram obtained under reaction flow conditions is shown in
Figure 3.3(b) and the performance measures are presented in Table 3.2.
Table.3.2. Quantitative performance of antioxidant standards with RF-PCD.
Antioxidant Precision
(% RSD)
Linear Range
(mg/L)
Correlation
Coefficient (r2)
LOQ
(mg/L)
LOD (mg/L) Response
Relative to
Trolox
Trolox 0.485 1.5 – 100 0.9967 1.5 0.5 1
Chlorogenic Acid 0.841 7.5 – 1000 0.9996 7.5 2.5 0.1239
Caffeic Acid 0.764 3 – 250 0.9985 3 1 0.2895
Rosmarinic Acid 0.743 4 – 500 0.9975 4 1.2 0.2920
Rutin 1.124 40 – 1000 0.9990 40 12 0.0394
Quercetin 0.524 2 – 250 0.9990 2 0.5 0.7434
Epicatechin 1.324 25 – 1000 0.9999 25 7.5 0.2075
Morin 0.943 3 – 1000 0.9999 3 0.9 0.2868
3.3.3. Comparison of Convention PCD and RF-PCD
In general, the working linear ranges for the antioxidants were found to cover
at least two orders of magnitude for each of the analytes investigated. Limits of
detection and quantitation were found to be in the low ppm region, which
56
corresponds to less than 1 µg of analyte. The antioxidants tested showed varying
responses to the FRAP reagent. Of all of the antioxidants tested, trolox showed the
highest response, while the antioxidant with the lowest response was found to be
rutin whose response was around 5 % of that of the trolox peak. Due to the varying
response factors, the FRAP PCD method is not suitable for the general assay of
antioxidant mixtures as specific standards are required for each analyte. Although the
FRAP assay is simple and reproducible, insufficient information is available in the
scientific literature about the behaviour of different antioxidants that respond in the
FRAP assay.
For all molecules, the linear range of the method was smaller in the
conventional PCD method compared to the RF-PCD method. The relative amount of
antioxidant that is availbale to be derivatized in the conventional method is greater
compared to the RF method because only part of the RF flow is mixed with the
FRAP reagent. This has the effect of saturating the reagent at a lower concentration
and, therefore, lowering the maximum concentration for the linear range.
Additionally, for all analytes, the conventional method showed worse limits
of detection and quantitation, higher %RSD measurements and lower correlation
coefficients when linearity was measured compared to the RF-PCD method. This is
due to the increased baseline noise when using the conventional method compared to
the RF-PCD method (Figure 3.3). Despite an increase in peak height, the increased
baseline noise leads to a lower signal to noise ratio. However, the % RSD results
obtained for all antioxidants across both methods were less than 1.5 %, indicating
that acceptable levels of precision were obtained using both methods. The linearity
was similarly acceptable for all antioxidants across both methods with correlation
coefficients of greater than 0.99 obtained.
57
Finally, there were distinct differences in the relative response factors
obtained in both of the methods. Trolox was the antioxidant with the highest
response in both methods but the responses relative to trolox were greater for the
conventional PCD method compared to the RF-PCD method. This likely indicates
that for the antioxidants considered in this study, trolox is the antioxidant that reacts
fastest with the FRAP reagent. It also follows that the post column residence time
between the mixing of the eluent and the FRAP reagent is less for the RF-PCD
system.
3.3.4. FRAP Analysis of Decaffinated Coffee
The chromatograms from the analysis of the coffee sample using the
conventional PCD and RF-PCD are presented in Figure 3.4. The loss of separation
efficiency for the conventional PCD compared to RF-PCD can be cleary seen due to
the poorly resolved peaks. As an example, the single peak with a retention time of
4.5 minutes in (a) is shown to be two peaks in chromatogram (b).
Similar to the analysis of the standards in Figure 3.3., the signal intensity and
the baseline noise is much greater for (a) compared to (b). Therefore, it would be
expected that the quantitative performance measures that were produced for the
standards would also apply here.
58
Figure.3.4. Chromatograms of Decaffeinato Intenso espresso coffee analysed using HPLC-PCD analysis with the FRAP reagent. The data corresponds to (a) conventional PCD with a 500 µL reaction loop and (b) RF-PCD.
0 5 10 15-60000
-40000
-20000
0
20000
40000
60000
80000
100000
120000
140000
160000
Abso
rbanc
e
Time (min)
0 5 10 15-60000
-40000
-20000
0
20000
40000
60000
80000
100000
120000
140000
160000
Abso
rban
ce
Tinme (min)
(a)
(b)
59
3.3.5. Antioxidant and FRAP Assay Kinetics Using Reaction
Flow Chromatography
A detailed literature survey revealed that for most of the studies based on the
FRAP assay are not very reliable because the extent of the direct electron transfer
reaction is very sensitive to environmental factors. There are thermodynamic and
kinetic influences on antioxidant measurements, with high selectivity and very fast
reaction kinetics suggesting it should be a promising candidate for an HPLC post
column system [292]. However, the difficulty with the FRAP assay is that the
reaction is based on the ability of water-soluble antioxidants to reduce the ferric ion.
Hesperitin, hesperidin, p-coumaric acid and nariginin did not show any response in
the RF-PCD FRAP assay and this unexpected inactivity requires detailed
investigation that can only be understood from kinetics studies of these antioxidants
[293].
The FRAP reagent was prepared in 300 µM acetate buffer at pH 3.6 which,
after mixing with the mobile phase, reacts with the antioxidants within the reaction
flow frit and studies have demonstrated that the solvent composition has an effect on
the antioxidant potential of flavonoid and polyphenols [294]. Previous research has
revealed that different antioxidants such as nariginin, hesperidin, hesperitin and p-
coumaric acid are affected by solvent composition [295]. These antioxidants are
soluble in water-alcohol mixtures and also soluble in fat and oil [296] and the
kinetics of these antioxidants change with different solvent environments [297, 298].
Studies conducted with naringin, hesperidin and hesperitin have also demonstrated
that all compounds possess antioxidant activity in a hydrophilic environment. On the
contrary, reduction in the antioxidant capacity of hesperitin, hesperidin was reported
in lipophilic environments.
60
The intense antioxidant activity of flavonoids is due to the chemical
configuration of molecules within the hydrophilic environment. In a polar
environment, molecules are present as ions that are acidic and stable due to more
resonance forms. Ortho and para substitution on molecules show more antioxidant
activity than meta-radical substitution [299].
Another important aspect that can be considered is the relationship between
the electron transfer rate and the limit of detection using the RF-PCD FRAP assay.
The reaction kinetics of these antioxidants can be discussed with reference to their
electron transfer behaviour. Minimum detectable amounts of the fast reacting trolox
and quercetin was observed to be 0.001µg. Slow reacting rutin and epicatechin were
detected at the limits of 0.012µg and 0.075 µg, respectively. The mechanism for the
oxidation of slow reacting rutin is a complex process and proceeds in a cascade
manner [300]. Previously studied voltammetric behaviour for trolox showed that the
electron transfer rate is fast and environment dependent. However, the potential
difference was large enough to cause a redox reaction between trolox and the radical
[301].
Electrochemical characteristics of flavonoids plays a crucial role in their
antioxidant activity [302]. Electrochemical studies of these antioxidants have
demonstrated that the lower the oxidative peak potential then the higher the electron
donor ability will be, whilst the higher the reductive peak potential then the greater
will be the rate and/or number of electrons transferred [302-304]. Experimental
conditions and chemical properties of antioxidants have an interdependent role on
electrochemical reactions [304]. Cyclic voltammetric analysis of p-coumaric acid has
shown one irreversible peak due to the oxidation of the hydroxyl group on the
aromatic ring of the molecule. Oxidation of p-coumaric acid with a pH below the
61
pKa occurs with the transfer of one electron and one proton [305, 306]. The electron
transfer behaviour of nariginin was studied with differential pulsed voltammetry but
it has shown no activity under RF-PCD FRAP conditions [307]. A novel
voltammetric method was used to study the redox behaviour of hesperitin which
showed a significant voltammetric response with the transfer of two electrons but no
response within RF-FRAP is possibly due to slow reaction kinetics [308].
Electrochemical techniques have potential applications in the direct determination of
antioxidant activity or capacity [309] and multiplexing them with reaction flow
chromatography can be a useful approach in the critical study of reaction kinetics for
antioxidants.
3.4. Conclusions
Three important outcomes can be derived from this study of a rapid Post
Column Derivatization assay using reaction flow chromatography.
(1) The mixing between the FRAP reagent and the mixture inside the peripheral
region of the outlet frit on the reaction flow chromatography column is very
efficient.
(2) The direct feed of FRAP reagent into the outlet fitting of the RF column
eliminates the need for mixing T-pieces, and reduces the post-column extra-dead
volume to no more than required in chromatography using conventional modes of
detection.
(3) The reaction flow mode of operation with dual multiplexed detection allows for
an absolute assignment of antioxidants within complex mixture.
62
Chapter 4
A Rapid Antioxidant Capacity Analysis (FRAP) Of Australian Mushrooms Using Reaction Flow
Chromatography and Structural Elucidation with Mass Spectrometry
63
4.1. Introduction
Antioxidants are important in the regulation of oxidative homeostasis and are
extensively promoted for their medicinal benefits in health care and pharmaceutical
industries. Market driven demand of natural antioxidants is booming and is expected
to more than double, from $103.6 million in 2011 to reach $246.1 million in 2018
[310-312]. In the last decade, restrictions on the use of synthetic antioxidants has
increased the interest in natural antioxidants [313] and natural sources of
antioxidants are being investigated by various research groups [314].
Edible mushrooms are widely used as functional foods and have a tendency
to protect against oxidative stress [313, 315, 316]. Different types of mushroom
antioxidants are associated with pharmacological action with different mechanisms
[263, 313, 317, 318]. Fruiting bodies are the main source of antioxidants within
mushrooms and several types of antioxidants, such as phenolic acids,
polysaccharides, tocopherols, Ergothioneine and ascorbic acid, are the main
compounds that have been previously identified in mushrooms [317-324].
The phytochemical profile of antioxidants in mushrooms is complex and can
be unstable. Constituents are in low concentration, sensitive to heat/light and the
separation of these antioxidants is a challenging task. Advanced chromatographic
and electrophoretic techniques are routinely employed to separate complex mixtures
and despite the advances in chromatography, some mixtures cannot be fully
separated [325]. Ultraviolet-visible-photodiode array along with mass spectrometry
techniques are usually used to detect individual compounds [326].
In this chapter, one of the most popular antioxidant assays for the
identification of antioxidant compounds involving the discoloration of DPPH is used
64
to measure the scavenging activity of antioxidants. DPPH is moderately stable
between pH 5.0 to 6.5 [327] with a nitrogen atom reduced by receiving a hydrogen
atom from an antioxidant to form the corresponding hydrazine [328], which becomes
a more stable diamagnetic molecule [329]. This assay measures the hydrogen
donating activity of antioxidant molecules and is known by the terms free radical
scavenging activity or antioxidant capacity [328, 330, 331]. Antioxidants reacting
with DPPH have revealed that there are different kinetic orders dependent on the
structure of molecules [332] with the reaction time and stoichiometry varying
amongst antioxidant classes [333].
Post-column derivatization (PCD) is the most widely used method for online
analysis [334] and, in this study, both conventional and rapid reaction flow methods
are employed to study the antioxidant profile for edible mushrooms. In the
conventional PCD-DPPH analysis, the antioxidant reaction with the DPPH reagent
results in an absorbance change at 515 nm and is recorded as a negative peak [334].
In Chapter 3, the RF-PCD FRAP analysis demonstrated significant advantages as
compared to the conventional technique in terms of greater signal to noise ratio,
linear range and separation efficiency.
The quest to find a comprehensive characterization method for the analysis of
complex samples is an important area of natural product research. In this study,
conventional PCD-DPPH, RF-PCD-FRAP and high resolution mass spectrometry
are employed to screen and elucidate the chemical structure of the
antioxidant/bioactive compounds within edible mushrooms. This chapter is aimed
towards the further comparison of the conventional and reaction flow methodologies,
differences in antioxidant screening and detailed chemical profiling of edible
mushrooms.
65
4.2. Materials and Reagents
4.2.1. Chemicals and Reagents
Chemicals and reagents were prepared as described in section 2.1.2.
4.2.2. Mobile Phases
Mobile phases were prepared as described in Section 2.1.2.1.
4.2.3. Derivatizing Reagent
The DPPH and FRAP reagents were prepared as described in Section 2.1.2.2.
4.2.4. Sample Preparation
Fresh samples were frozen slowly, first at -20 °C and then at -80 °C. Frozen
samples stored at -80 °C were freeze-dried (CRYODOS-50 (Dynavac, Melbourne,
Australia), conditions: -50 °C and 1 mbar) where the processing time varied from
48-60 hrs, depending on the moisture content. The freeze-dried mushroom samples
were ground to homogeneity using a domestic electric blender and a 5 g sample was
weighed out and transferred into a 50 mL flask. Further extraction was carried out
with methanol in an orbital shaker for 24 hr. Approximately 20 mL of each filtrate
was added into FalconTM 50 mL conical centrifuge tubes and the methanol was
evaporated using a rotary evaporator (EZZI vision Vacuum Technology system,
Savant, NY, USA) at 35 °C. The final sample was prepared by redissolving the dry
66
mass in 2 mL of methanol. About 1 mL of this sample was filtered with a 0.45-μm
membrane and 100 μL was then transferred into LC sample vials. For RF-PCD
FRAP and LC-ESI-MS analysis, the samples were injected in triplicate and
duplicate, respectively.
4.3. Instrumentation and Chromatographic
Conditions
4.3.1. Column
For RF-PCD methods, a PES C18 (150 × 4.6 mm, dp = 5 µm) column with
RF frit, 2:1 frit and 4-port outlet head-fitting was sourced from Thermofischer
Scientific (Runcorn, Cheshire, United Kingdom). The end fitting has four outlet
ports, three that channel flow from the wall region and a central port that captures
flow from radial center of the column. Flow between peripheral and central ports
was optimized by adjusting the segmentation ratio.
4.3.2. Instrumentation Setup
4.3.2.1. Conventional PCD-DPPH Assay Detection Setup
The conventional PCD-DPPH experiments were conducted using a Shimadzu
LC-20AD pump fitted with an external pulse dampener and added to the eluent
exiting the column via a zero dead volume T-piece. The flow stream passed through
a 20-µL reaction coil maintained at room temperature prior to entering the UV-Vis
detector (PDA) that was set to a wavelength of 520 nm.
67
4.3.2.2. RF-PCD-FRAP Assay Detection Setup
This was as described in Section 2.2.2.
4.3.2.3. High Resolution Mass Spectrometry
Mass spectrometry detection was carried out using a Q Exactive™ Plus
Hybrid Quadrupole-Orbitrap™ Mass Spectrometer, equipped with heated
electrospray ion (HESI) source (Thermo Scientific, Bremen, Germany). Samples
were analysed under both positive and negative mode conditions within a run time of
15 min. A Full MS-Data Dependent MS/MS Mode was applied with a mass
resolution was 70,000 in Full Scan mode and the AGC target value set to 1e6. The
maximum injection time was 10 ms within a scan range of 120 to 750 m/z. MS/MS
was conducted on the HCD cell with a normalized collision energy of 30% and other
parameters included mass resolution of 17,500, AGS target of 1e5 having maximum
injection time of 200 ms within an isolation window of 2.0 m/z.
4.3.3. Chromatographic Analysis
4.3.3.1. Conventional PCD-DPPH and RF-PCD-FRAP Assay
Detection Setup
All analyses were performed using gradient elution where mobile phase A
was 30 mM sodium acetate pH 3.6 and mobile phase B was methanol. A linear
gradient was used starting at 100% mobile phase A and changing to 100 % mobile
phase B over 20 minutes, which was held for a further 5 minutes. The flow was then
returned to 100 % A over 1 minute and allowed to equilibrate for 5 minutes prior to
the next injection. For all experiments, the flow rate of the mobile phase was 1.0
68
mL/min and an injection volume of 20 µL was used. The UV-Vis detector was set to
an analysis wavelength of 593 nm for all measurements unless otherwise specified.
The optimal flow rate for FRAP reagent was found to be 0.5 mL/min. For analyses
using the reaction flow column, the optimal flow ratio between the central and
peripheral ports was found to be 50:50 central: peripheral.
4.3.3.2. High Resolution Mass Spectrometry
For LC-HR-MS analysis, a linear gradient was used starting at 98% mobile
phase A and changing to 100 % mobile phase B over 10 minutes, which was held for
a further 2.5 minutes. The flow was then returned to 98 % A over 0.1 minute and
allowed to equilibrate for 2.4 minutes prior to the next injection.
4.3.4. Data Analysis
Data analysis was undertaken with Microsoft Excel. The MS instrument was
controlled using Xcalibur™ software and the untargetted data analysis was
performed using Compound Discoverer™ (ThermoFisher Scientific, San Jose USA).
4.4. Results and Discussion
As a consequence of the multiple interrelated aspects investigated in this
chapter, the technique specific observations will be initially described and then the
mushroom sample analysis results will be presented. A final section discusses the
veracity of the MS identification of the various classes of compound that have been
discovered.
69
4.4.1. General Observations
4.4.1.1 Comparison of PCD Techniques
An essential requirement of the conventional PCD-DPPH assay is efficient
mixing between the DPPH reagent and the mobile phase. Previously, it had been
demonstrated that system performance decreases markedly when reaction loops
greater than 50 µL are used [80]. In this derivatization assay, a reaction coil of 20
µL was connected to the T-piece at the outlet of the column and the use of this small
reaction loop maximized chromatographic resolution resulting in sharp peaks.
Another factor is the relative DPPH concentration and the analyses were conducted
with a flow rate of 1.5 mL/min for the DPPH solution and 1.0 mL/min for the mobile
phase. The higher flow rate for DPPH has a negative effect on the S/N ratio and our
collaborators have previously demonstrated that complex chromatographic
separations can be improved by the application of power functions where noise
added due to the post column reaction can be minimized [335].
Active flow columns can provide sharp, intense and properly resolved peaks
because the frit design allows the introduction of samples into the centre of the
column thereby avoiding the wall effects as described by the principle of the
“infinite diameter column” [336]. Furthermore, sensitivity should be improved to be
twice that of conventional systems because of the reduction in the post-extra column
dead volume [337]. It was apparent that RF chromatography has enabled improved
separation performance and sensitivity of antioxidant compounds within edible
mushrooms.
70
4.4.1.2. LC-HR-MS Detection
Fully validated and rigorously optimized mass spectrometry analysis was
also used to characterize the bioactive compounds. A systematic and methodical
approach, without the use of standards, was adopted for identification and
characterisation of antioxidants within the edible mushrooms. Considerations were
given to the elution order, selective response at 593 nm, high mass accuracy m/z
values as well as previously published literature.
The elution series of bioactives (from shortest to longest retention time) was
predicted based on the substitution pattern for each molecule in the series.
For example, compounds with more hydroxyl groups will have the greatest
degree of polarity, thus eluting first, while compounds with the most
methoxy groups give a more hydrophobic character which increases the
retention time on a reversed phase column. Identification of compounds was
further ensured by the reproducibility of retention times among the different
mushroom samples.
The proximity of accurate mass (experimentally determined) to exact mass
(calculated mass of ion whose elemental formula, isotopic composition and
charge states are known) was observed. The experimentally measured values
were compared with theoretical calculations of the most abundant isotope for
the chemical formula at high resolution with MS Spec Plotter [262].
Calculated error measurements were less than 2 ppm for all identified
compounds.
71
Literature investigation confirmed compound identification, based on the
accurate masses and fragmentation patterns obtained from MS-MS data.
As a note of caution, it is important to remember that the elution order can vary as
the chromatographic parameters and setups were different. High mass accuracy
measurements, with generally less than 1 ppm error, has enabled the differentiation
of two species without the use of standards. Identity confirmation was achieved by
observing the presence of the correct m/z ions and the daughter ions of compounds
using MS-MS with the results summarized in respective tables.
4.4.2. Mushroom Sample Analysis
The mushroom samples were analysed using HPLC with UV-Vis, DPPH and
FRAP detectors. Few of the samples have shown any response to the DPPH reagent
with Portobello and Dried Porcini having the strongest antioxidant responses. A
comparison of the chromatograms for these two mushrooms with each detector are
shown in Figure 4.1 and Figure 4.2.
72
Figure 4.1. Chromatograms showing the response for the Portobello mushroom extract (a) UV-Vis, (b) PCD-DPPH at 520 nm and (c) RF-PCD-FRAP at 593 nm.
a
b
c
73
Figure 4.2. Chromatograms showing the response for the Dried Porcini mushroom extract (a) UV-Vis, (B) PCD-DPPH at 520 nm and (C) RF-PCD-FRAP at 593 nm.
Most of the mushroom samples demonstrated significant antioxidant
responses against FRAP reagent except Shimeji and Enoki where the activity was
considerably low. As shown in Figure 4.1, the Portobello mushroom extract has
unresolved peaks and in order to improve the separation, further enhancement in the
separation space is required. Aside from a varation in the signal intensities between
the different samples, there is a very similar retention profiles across all samples.
74
The high resolution of eluting peaks avoids the possibility of mistakenly assigning
antioxidant responses to the co-eleuting peaks.
4.4.2.1. Dried Porcini
As seen in Figure 4.1, Dried Porcini showed strong antioxidant responses
against DPPH and FRAP reagents. Conventional online PCD-DPPH assay displayed
one intense antioxidant peak while the RF-PCD-FRAP assay indicated the presence
of three fully resolved peaks. HR-MS structural characterization indicated the
presence of Ergothioneine, phenolic acids, vitamins and Oleamide.
Table.4.1. Bioactive/antioxidant compounds within Dried Porcini.
4.4.2.2. Portobello
For Portobello, RF-PCD-FRAP analysis indicated the presence of eight
antioxidant peaks while the online PCD-DPPH assay showed the presence of only
one negative antioxidant peak response. Poorly resolved and early eluting
LC-HR-MS
RT(min)
Tentative Assignments Molecular
Formula
Exact mass Measured mass Error(ppm)
0.534 L-Ergothioneine C9H15N3O2S 229.08851 229.08864 0.6
0.647 Niacin C6H5NO2 123.03204 123.03214 0.8
0.654 L(-) Methionine C5H11NO2S 149.05106 149.05089 -1.1
0.733 L-Tyrosine C9H11NO3 181.07391 181.07409 1.0
1.204 Adenosine C10H13N5O4 267.09678 267.09682 0.1
2.581 Vitamin B5-Pantothenic acid C9H17NO5 219.11070 219.11064 -0.3
2.972 Unidentified compound C7H8O3 140.04736 140.04740 0.3
2.825 Tryptophan C11H12N2O2 204.08989 204.08997 0.4
3.033 Unidentified compound C9H16N4 180.13750 180.13764 0.8
4.711 Coumaric acid C9H8O3 164.04736 164.04658 -0.5
4.820 N-Acetyl-L-Leucine C8H15NO3 173.10521 173.10532 0.1
5.407 Suberic acid C8H14O4 174.08923 174.08858 0.1
5.452 Indole 3- Acetic acid C10H9NO2 175.06334 175.06351 1.0
6.363 Azelaic acid C9H16O4 188.10488 188.10428 -3.3
11.415 Oleamide C18H35NO 281.27186 281.27183 0.02
11.778 25-Hydroxyvitamin D2 C28H44O2 412.33414 412.33461 1.1
13.303 Ergosta-4,6,8(14),22-tetraen-3-one C28H40O 392.30792 392.30754 -1.0
75
antioxidant peaks imply the presence of polar compounds, which is further
confirmed with the presence of phenolic acid as characterised by HR-MS.
Table 4.2. Bioactive/antioxidant compounds within Portobello mushroom.
4.4.2.3. Vitamin D
The Vitamin D mushroom extract showed an antioxidant peak for RF-PCD -
FRAP assay (Figure 4.3) but post column DPPH assay did not show any antiradical
activity. Structural characterization with HR-MS indicated the presence of water-
soluble vitamins, phenolic acids and vitamin D. Ergosterol peroxide, a strong
anticancer compound, is present in the sample.
LC-HR-MS
RT(min)
Tentative Assignments Molecular
Formula
Exact
mass
Measured
mass
Error(ppm)
0.427 L-Histidine C6H9N3O2 155.06949 155.06954 0.3
0.693 L-Tyrosine C9H11NO3 181.07402 181.07401 -0.05
0.853 Nicotinamide C6H6N2O 122.04802 122.04812 0.8
0.642 Niacin –Vitamin B3 C6H5NO2 123.03204 123.03221 0.8
1.142 Propionylcarnitine C10H19NO4 217.13143 217.13121 -0.8
1.476 L-Phenylalanine C9H11NO2 165.07899 165.07941 2.5
1.48 Adenosine C10H13N5O4 267.09678 267.09673 -0.2
1.563 Cinnamic acid C9H8O2 148.05244 148.05242 -0.1
4.699 p-Coumaric acid C9H8O3 164.04736 164.0465 -0.5
11.414 Oleamide C18H35NO 281.27186 281.27188 0.07
11.788 Pro-vitamin D2 C28H44O 396.33922 396.33974 1.3
76
Figure 4.3. RF-PCD FRAP response of Vitamin D mushroom at 593 nm.
Table.4.4. Bioactive/antioxidant compounds within the Vitamin D mushroom.
4.4.2.4. Shimeji
Shimeji mushroom did not show a significant antioxidant response with RF-
PCD-FRAP assay and anti-radical activity was not observed for this sample. LC-
LC-HR-MS
RT(min)
Tentative Assignments Molecular
Formula
Exact mass Measured
mass
Error(ppm)
0.420 Gluconic acid C6H12O7 196.05834 196.05760 -3.8
0.421 L-Ornithine C5H12N2O2 132.08989 132.0899 0.07
0.644 Niacin –Vitamin B3 C6H5NO2 123.03204 123.03221 1.4
0.839 Nicotinamide C6H6N2O 122.04802 122.04813 0.9
1.230 Adenosine C10H13N5O4 267.09678 267.09664 -0.5
1.453 phenylalanine C9H11NO2 165.07899 165.07918 1.2
2.884 Tryptophan C11H12N2O2 204.08989 204.08995 0.3
2.518 Vitamin B5-Pantothenic acid C9H17NO5 219.1107 219.11055 -0.7
6.365 Azelaic acid C9H16O4 188.10488 188.10411 -4.1
11.772 25-Hydroxy vitamin D2 C28H44O2 412.33414 412.33443 0.7
12.028 Ergosterol peroxide C28H44O3 428.32906 428.32912 0.1
12.276 Vitamin D2-Ergocalciferol C28H44O 396.33922 396.33974 1.3
77
HR-MS indicated the presence of water-soluble amino acids, vitamins, phenolic
acids and vitamin D. Ergosterol peroxide is also present in this sample.
Figure 4.4. RF-PCD FRAP response of Shimeji mushroom at 593 nm.
Table.4.5. Bioactive compounds within Shimeji mushroom.
4.4.2.5. Brown Cup
The Brown cup mushroom demonstrated strong antioxidant responses for the
RF-PCD -FRAP assay as shown in Figure 4.5 and DPPH anti-radical activity was
not observed for this sample. High-resolution mass spectrometry analysis indicated
LC-HR-MS
RT(min)
Tentative Assignments Molecular
Formula
Eaxct mass Measured
mass
Error(ppm)
0.654 L(-) Methionine C5H11NO2S 149.05106 149.05089 -1.1
0.709 L-Tyrosine C9H11NO3 181.07391 181.07402 0.6
1.487 L-Phenylalanine C9H11NO2 165.07899 165.07938 2.4
1.603 D-Phenylalanine C9H11NO2 165.07899 165.07938 2.4
2.571 Vitamin B5-Pantothenic acid C9H17NO5 219.1107 219.11064 0.3
2.884 L-Tryptophan C11H12N2O2 204.08989 204.08995 0.3
3.979 5’-S-Methyl-5’-thioadenosine C11H15N5O3S 297.08958 297.08965 0.2
6.185 Azelaic acid C9H16O4 188.10488 188.10428 -3.2
11.416 Oleamide C18H35NO 281.27186 281.27185 -0.07
11.782 Pro Vitamin-D2 Ergocalciferol C28H44O 396.33922 396.33939 0.4
11.935 25-Hydroxy vitamin D2 C28H44O2 412.33414 412.33428 0.3
11.977 1-Stearoylglycerol C21H42O4 358.30833 358.30840 0.2
12.03 Ergosterol peroxide C28H44O3 428.32906 428.32912 0.1
12.253 Vitamin D2-Ergocalciferol C28H44O 396.33922 396.33974 1.3
78
the presence of water-soluble amino acids, vitamins, phenolic acids and vitamin D.
Ergosterol peroxide is present in this sample.
Figure 4.5. RF-PCD FRAP response of Brown Cup mushroom at 593nm.
Table.4.6. Bioactive compounds within Brown cup mushroom.
4.4.2.6. White Button
The White Button mushroom demonstrated strong antioxidant responses with
LC-HR-MS
RT(min)
Tentative Assignments Molecular
Formula
Exact mass Measured
mass
Error(ppm)
0.427 L-Histidine C6H9N3O2 155.06949 155.06954 0.322
0.644 Niacin –Vitamin B3 C6H5NO2 123.03204 123.03221 1.4
0.817 Nicotinamide C6H6N2O 122.04802 122.04813 0.9
1.428 Adenosine C10H13N5O4 267.09678 267.09673 -0.8
1.476 L-Phenylalanine C9H11NO2 165.07899 165.07941 2.5
2.518 Vitamin B5-Pantothenic acid C9H17NO5 219.1107 219.11055 -0.7
2.868 DL-Tryptophan C11H12N2O2 204.08989 204.08970 -0.9
4.705 p-Coumaric acid C9H8O3 164.04736 164.0465 -5.2
6.363 Azelaic acid C9H16O4 188.10488 188.10428 -3.2
8.893 Unidentified monoterpenes C15H22O2 234.16199 234.16214 0.6
11.214 16-Hydroxyhexadecanoic acid C16H32O3 272.23516 272.23546 1.1
11.285 Hexadecanamide C16H33NO 255.25621 255.2562 0.04
11.414 Oleamide C18H35NO 281.27186 281.27188 0.07
11.537 25-Hydroxy vitamin D2 C28H44O2 412.33414 412.33428 0.3
11.786 Pro-Vitamin D2 C28H44O 396.33922 396.34014 2.3
12.031 Ergosterol Peroxide C28H44O3 428.32906 428.32912 0.1
13.978 Spermidine C7H19N3 145.1579 145.15792 0.1
79
RF-PCD -FRAP assay as seen in Figure 4.6 and no anti-radical activity was
observed for this sample. High-resolution mass spectrometry analysis indicated the
presence of water-soluble amino acids, vitamins, phenolic acids and vitamin D.
Ergosterol peroxide is present in this sample.
Figure 4.6. RF-PCD FRAP response of White Button mushroom at 593 nm.
Table.4.7. Bioactive compounds within the White Button mushroom.
LC-HR-MS
RT(min)
Tentative Assignments Molecular
Formula
Exact mass Measured
mass
Error(ppm)
0.618 L-Tyrosine C9H11NO3 181.07391 181.07408 0.9
0.638 Niacin –Vitamin B3 C6H5NO2 123.03204 123.03221 1.4
0.817 Nicotinamide C6H6N2O 122..04802 122.04813 0.9
1.220 Adenosine C10H13N5O4 267.09678 267.09673 -0.2
2.571 Vitamin B5-Pantothenic acid C9H17NO5 219.1107 219.11064 -0.3
11.414 Oleamide C18H35NO 281.27186 281.27188 0.07
11.55 25-Hydroxy vitamin D2 C28H44O2 412.33414 412.33428 0.3
12.031 Ergosterol Peroxide C28H44O3 428.32906 428.32912 0.1
13.978 Spermidine C7H19N3 145.1579 145.15792 0.1
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4.4.3. Structural Characterization of Antioxidants within
Australian Mushrooms
4.4.3.1. Phenolic Acids
Mass spectrometry analysis of the edible mushrooms identified the presence
of phenolic compounds such as Azelaic acid and Cinnamic acid but other phenolic
acids such as p-hydroxybenzoic and Protocatechuic acid were not found in these
samples. The absence of these commonly found phenolic acids might be attributed to
losses during sample pre-treatment and other matrix effects. In addition, possible
losses due to the storage conditions should be acknowledged. In LC-ESI-MS
analysis, Cinnamic acid exhibited a positive molecular ion at m/z 166.0865
[M+NH4]+, Coumaric acid indicated a mass at m/z 163.03921 [M-H]-, Azelaic acid
had an m/z of 188.10440 [M-H]- and vanillin exhibited m/z at 151.06023. Phenolic
acids are considered the most widely occurring groups within mushrooms and are of
pharmacological importance within the human body as they have strong antioxidant
activity.
4.4.3.2. Water-soluble Vitamins
Four vitamins were identified in the mushroom samples and the
fragmentation patterns are in good agreement with previous studies [338, 339]. The
water-soluble vitamins – niacin (nicotinic acid), nicotinamide (vitamin B3) and
pantothenic acids (vitamin B5) – were detected in the samples. Interestingly, the
response of samples containing these vitamins varies; this might be due to the
interference of matrix, pH of sample and quantity of analytes within different
samples. For pantothenic acid and nicotinamide, the [M+H]+ ions were detected at
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m/z 220.11806 and m/z 123.05552, respectively, and niacin exhibited an [M-H]- ion
at m/z 124.03961, which are similar to the results observed by other research groups
[338, 339].
4.4.3.3. Ergothioneine
Ergothioneine is a 2 thiol-L-histidine-betaine and is known for its
antioxidant, anti-inflammatory and Cyto-protective properties [340-343]. Mass
spectra of Ergothioneine indicated that the most intense peaks are the protonated
molecular ion at m/z 229.08864 and major fragment ions at m/z 186.10597 and
127.3258, which are in good agreement with the previous research conducted to
study antidepressant effects of Ergothioneine with LC-MS/MS [344]. Dried Porcini
and Enoki mushroom samples were identified as a source of Ergothioneine.
4.4.3.4. Ergosterol and Derivatives
Analysis revealed that edible mushrooms are a promising source of bioactive
sterols with the presence of Ergosterol and its peroxide derivative observed.
Provitamin D2, also known as Ergosterol, eluted at RT11.788 min and transitions
were monitored at m/z 397.34668 to m/z 379.33630, as observed in previous studies
[345] [346]. The Ergosterol derivative 22, 23-dehydroergosterol has an extremely
similar structure to ergosterol and previous studies were used as a reference to assign
the identity. One such study was conducted to investigate sterol microemulsions and
demonstrated that ergosterol eluted before 22, 23-dihydroergosterol on a C18
column [347]. In ESI positive ion mode, 22, 23-de-hydroergosterol exhibited
precursor ions m/z 383.29140 and 368.38828.
82
In the MS/MS spectrum, ions of m/z 446.3629 [M+NH4]+ with fragment ion
377.32001 [M+NH4-[O2+CH3+H2O]+ suggests the presence of the C28-sterol,
ergosterol peroxide (EP; 5α, 8α-epidioxy-22E-ergosta-6, 22-dien-3β-ol) within the
mushroom extracts. MS identities confirmed the presence of ergosterol peroxide in
all edible samples except Dried Porcini and Enoki mushroom extracts. Mushroom
derived ergosterol peroxide is pharmacologically known for anti-inflammatory,
antioxidant and anticancer activities [348] and is a promising candidate for drug
development [349]. Ergosterol was identified in the both positive and negative
modes, implying that ergosterol peroxide is a peroxidated derivative of ergosterol.
Both identified compounds, ergosterol and its peroxide, have lipophilic character and
it is uncommon to identify these compounds within methanolic extracts, which is the
solvent of choice for polyphenolic compounds with hydrophilic character. This
unexpected observation might be attributed to co-dependent enhanced solubility of
non-polar compounds within natural products and the high sensitivity of the mass
spectrometer detector.
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4.4.3.5. Vitamin D2 and Analogues
The presence of ergosterol indicates the potential presence of vitamin D2 and
related compounds in the mushroom samples with the production of vitamin D2 from
mushrooms upon sunlight exposure well-established [350]. Indeed, vitamin D and its
related compounds were detected within the mushroom samples. It is generally
assumed that mushrooms only contain vitamin D2 but the results have demonstrated
that pro-vitamin D2 (Ergosterol) and 25-hydroxy vitamin D2, are also abundantly
present in these edible mushrooms. Mass spectra showing transitions from m/z
397.3300 to m/z 379.3300 was identified as Vitamin D2, as previously reported
[351]. 25-hydroxy vitamin D2 was identified with the observation of the protonated
molecules at m/z 413.34116 with the most abundant fragment ion m/z 395.3300, as
previously described [352]. Ergosterol and Ergocalciferol have the same molecular
formula and nominal masses but it has been reported that pro-vitamin D2 elutes
before vitamin D2 on a C18 column [353] and identities were assigned according to
this published work. Pro-vitamin D2 with experimental m/z 396.33974 and vitamin
D2 with experimental m/z 396.33956 are identified in both Portobello and Vitamin D
mushrooms.
These results demonstrated that the LC-HRMS is sufficiently sensitive and
selective to differentiate between slight mass differences for similar chemical
species, without the use of standards. Careful consideration is required during data
interpretation and assigning identities as some bioactives with the same chemical
formulae will have the same precursor ions but there will be differences in the
fragmentation patterns detected by MS/MS. These compounds were confirmed by
different identification steps mentioned above and were in good agreement with the
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previous scientific literature. The average mass accuracy of m/z values was less than
1 ppm error, except for Caffeic acid.
However, there are barriers for the accurate and reproducible assessment of
vitamin D2 with the research gap around the analysis of vitamin D briefly outlined
here. Previous photobiology studies have demonstrated that pre-vitamin D2, vitamin
D2, vitamin D3, provitamin D4, vitamin D4 are present in mushrooms [353] but there
are relatively few studies that demonstrate the chemical configuration of all the
available forms of vitamin D and its metabolites within natural products. There is
also currently little information about fragmentation patterns of these metabolites
with mass spectrometry. A major challenge will be to develop reliable and accurate
methods for the simultaneous detection of different forms of pre-vitamin D2, vitamin
D2, Tachysterol and lumisterol that have the same molecular formula, C28H44O,
within a complex matrix. Further exploration of vitamin D3, vitamin D4 within edible
mushrooms might be warranted as most of them are exposed to sunlight.
Figure 4.7. Summary of antioxidants identified within edible mushrooms.
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4.4.4. Confluence of RF-PCD-FRAP and LC-HRMS
LC-HRMS results suggested that the antioxidant activity might be from the
polar lipophilic compounds rather than the non-polar compounds. The methanolic
extracts of the different mushroom samples contained and a considerable number of
antioxidants and had similar antioxidant profiles. However, these results do not
coincide with the RF-PCD-FRAP chromatograms, which showed noticeably
different antioxidant peak intensities among mushroom samples with a remarkably
low response in the Enoki and Shimeji samples.
Figure.4.8. Showing LC-HRMS analysis of mushroom Dried Porcini.
Compared to RF-PCD-FRAP, LC-HRMS analysis of the mushroom extracts
showed very different profiles and MS chromatograms were dominated by peaks
which did not appear in the FRAP chromatograms as shown in Figure 4.11. These
experiments were conducted on separate instruments under different gradient
conditions but with the same C18 column chemistry. Clearly, the RF-PCD-FRAP
and LC-HRMS retention times do not correspond but methodical interpretation of
86
the data enabled some meaningful information to be obtained. The RF-PCD-FRAP
peaks eluting earlier on the reversed phase column could be assigned to
Ergothioneine and hydrophilic vitamins due to their polar natures, while intense
antioxidant peaks observed later might be due to phenolic acids which are fast
reacting with the FRAP reagent. Responses identified after 20 minutes in the FRAP
chromatograms are probably related to the ergosterol peroxide, vitamin D and its
analogues. It has been previously demonstrated that the FRAP reagent in RF-PCD
mode does not react with every antioxidant, indicating that certain antioxidants
identified with MS did not react fast enough with the FRAP without an extended
reaction loop. It was also observed that the response factor varied dramatically from
antioxidant to antioxidant, indicating that the kinetics of the reaction is dependent on
each individual antioxidant. It is important to understand that FRAP only measure
antioxidant activity based on single electron transfer, hence, other antioxidant
compounds present in mushrooms, which have different modes of action, will not be
observed. Finally, FRAP does not measure antioxidant capacity of those compounds
whose redox potential is below the FRAP detection threshold, such as antioxidants
with thiol group. The RF-PCD-FRAP assay can be used as a selective detection
technique for certain type of antioxidants within a complex mixture.
Similar studies conducted by our research collaborators, with simultaneous
multiplexed detection using active flow technology columns coupled with a mass
spectrometer, also revealed some unexpected results. Chromatograms of tobacco
leaves that had very intense peaks in MS were not detected in DPPH chromatograms
and some responses observed with UV-Vis and DPPH did not appear in the MS
scans, indicating that some species did not ionize in the MS conditions used in the
method [354]. In order to gain a greater understanding of the retention behaviour of
87
these compounds and the RF-PCD FRAP responses, analysis of the complex
mushroom samples should be conducted by coupling reaction flow chromatography
with high resolution mass spectrometry that were used separately in this study.
Nicotinamide appeared to elute in White Button and Portobello mushrooms
with retention times of 0.817 min and 0.853 min, respectively, and although
identification was based on matched m/z values, there was a slight variation in
retention times between the runs. The former depends on the mass accuracy and
resolution of the mass spectrometer while the latter largely depends on the
reproducibility of analytical method used. Impurity peaks from the sample
preparation were also detected which may complicate the analysis and has a direct
impact on untargeted metabolomics results. It is recommended that every peak
should be included from the raw LC/MS data files in the analysis of untargeted
metabolic profiles [355]. On the other hand, the inclusion of impurity peaks can
complicate the statistical analyses and may prevent interesting results from being
found [356]. Unexpected compounds were also observed and a library search of
metabolomics identified that spermidine eluted at 13.978 min but is rarely observed
in mushrooms, with the m/z value matching the molecular formula C7H19N3. The
experimental value recorded corresponded to less than 1 ppm error and had a
matching fragmentation pattern. This compound still seemed unusual for mushroom
samples because of its chemical nature but it appears plausible that it might be
present in trace quantities.
At present, the major bottleneck in metabolomics studies of natural products
is compound identification. Our work has demonstrated that the state-of-the-art tools
for library-assisted compound identification, including annotation of adducts and
88
fragments, with determination of the molecular formula has made characterization
easier. The results can be justified for the proposed identifications based on the
additional knowledge from previous scientific literature. High-resolution mass
spectrometry has proved to be of real value for identification but detailed structural
elucidation still requires 2-D NMR and X-ray crystallography.
4.5. Conclusions
As far as we know, this is the first report demonstrating the presence of
phenolic acids, vitamins, ergosterol, its derivatives, and ergosterol peroxidase within
edible mushrooms as shown in Figure 4.10. Bioanalytical screening of antioxidant
capacity of natural products with RF-PCD-FRAP assay has never been reported
before and there are limitations observed when characterizing exceedingly complex
samples. Post column derivatisation assays using reaction flow chromatography
display better resolution and sensitivity than the conventional online DPPH assay.
Until now, there has been no published study performed for rapid and
accurate characterization of antioxidant compounds in edible mushrooms by liquid
chromatography coupled to high-resolution mass spectrometry. Nevertheless, further
data mining has potential to explore more antioxidants within mushrooms.
Characterisation of vitamins and other antioxidant compounds is certainly not novel;
however, there are limited publications describing simultaneous determination of
phenolic acids, vitamins and their derivatives from natural products. The majority of
the previously published methods involved complicated procedures and
impractically long run times but untargeted metabolic profiling with LC-HRMS has
enabled the identification of individual compounds and has provided an additional
89
insight into the range of natural products . In conventional practice, single collision
energy cannot be used to generate good data for compounds with widely different
masses. The ion trap source used in this study worked on the principal of resonance
excitation to induce fragmentation. The recently developed normalized collision
energy approach to achieve fragmentation produces reproducible MSn spectra and
reproducible libraries can be now generated, as mass spectra produced on one
instrument will be the same as those from another.
Finally, edible mushrooms are a promising source of bioactive molecules that
predominantly have antioxidant properties. Of the extracts tested in this study, the
maximum antioxidants were detected in a sample of Dried Porcini. Detailed
investigation has revealed that phenolic acids, vitamin B3, vitamin B5, Ergosterol,
Ergothioneine, Ergosterol peroxide, vitamin D and its derivatives are bioactive
compounds, which make edible mushrooms as a suitable source of antioxidants.
More than fifteen antioxidants were identified and their chemical structures were
proposed based on high-resolution mass spectrometry. Untargeted metabolic
profiling has not only indicated the presence of antioxidants but has also
demonstrated edible mushrooms as a source of anticancer and anti-inflammatory
bioactive compounds. This suggests that commercially available mushrooms in
Australia may provide comprehensive protection from oxidative stress, and possibly
more pronounced health benefits.
90
Chapter 5
A FRAP-based Rapid Antioxidant Capacity Analysis of Australian Native Plants Using
Reaction Flow Chromatography and Characterization with Mass Spectrometry
91
5.1. Introduction
To the indigenous Aboriginal people of Australia, edible native fruits have
served as a source of food and medicine for thousands of years [357]. Over the last
decade, a number of commercially significant fruits and bush foods have become
popular in the search for new novel functional foods [221]. Australian native fruits,
such as Muntries, Tasmanian pepper berry, Davidson’s plum and raspberries, have
been identified as sources of antioxidants [358] with Australian grown herbs and
spices also containing very high levels [217]. Bioactivity studies of Australian native
plants and fruits have shown that polyphenols and major classes of compounds, like
flavonoids and carotenoids, are abundantly present [359]. At present in Australia,
there are 77 growers of native fruits and spices who offer 91 primary products [360].
These exotic native foods are a rich source of antioxidants and could have a potential
role in health promotion [5]. This study represents the first attempt to understand the
complete phytochemistry of Australian native plants and further research may lead
towards identification of other groups of compounds.
The untargeted metabolomics approach provides an alternative procedure for
natural products drug discovery that uses high content screening to reveal the
identities of individual bioactive compounds within complex natural products based
on ever-developing libraries. This approach could help to improve
ethnopharmacological identifications and be used as an additional strategy for the
discovery of the next generation of natural product inspired drug leads. Advanced
bioanalytical approaches should enable rational selection of traditional medicinal
plants as preventive medicines based on their biological and/or chemical novelty.
92
In this Chapter, the previously demonstrated RF-PCD FRAP assay was used
for the bioanalytical screening of antioxidants in Australian native plants. It further
demonstrates the significant advantages compared to the conventional technique, in
terms of greater signal-to-noise ratio, linear range and separation efficiency [361].
Plant extracts have complex chemical profiles and comprehensive characterization of
complex samples is a critical step when correlating the chemical composition with
any biological function. In this study, RF-PCD-FRAP and high resolution mass
spectrometry were employed separately to screen and elucidate the chemical
structure of the antioxidant/bioactive compounds contained in Australian native
foods.
5.2. Materials and Reagents
5.2.1. Chemicals and Reagents
As described in Section 2.1.2.
5.2.2. Mobile Phases
As described in Section 2.1.2.1.
5.2.3. Sample Preparation
Wattle seeds, the dried fruits of Desert lime, Tasmanian lanceolata and
Quandong, and the leaves of Australian native Lemon grass, Old man saltbush and
Gumbi gumbi were ground to homogeneity using a domestic electric blender. Two
methods of extraction were compared. The first was a direct extraction method
where 5 g of each plant sample was weighed out and transferred into a 50 mL glass
93
flask. Extraction was carried out with methanol in an orbital shaker for 24 hrs. Then
20 mL of each filtrate was placed in 50 mL FalconTM conical centrifuge tubes and the
methanol was evaporated in an EZZI vision Vacuum Technology instrument
(Savant, NY, USA) at 35 ˚C. The final sample was prepared by dissolving the dry
mass in 2 mL of methanol.
For the alternative sonication-based extraction method, the flask containing
50 mL of methanol and 5 g of powdered sample was placed in an ultrasonic bath that
was maintained at room temperature for an hour. In order to avoid the loss of
solvent, the flask was covered with aluminum foil. The obtained extracts were
filtered through a Whatman No. 1 filter paper and the filtrate was concentrated in a
rotary evaporator under controlled vacuum at 37 ˚C. The concentrated extract was
re-dissolved in 2 mL of methanol to produce the final sample. Approximately 1 mL
of the final sample was filtered through a 0.45 μm membrane and 100 μL was then
transferred into the LC vials. For the RF-PCD-FRAP and LC-HRMS analyses, the
samples were injected in duplicate.
5.3. Instrumentation and Chromatographic
Conditions
5.3.1. Column
As described in Section 4.3.1.
5.3.2. Instrumentation Setup
As described in Section 4.3.2.
94
5.3.3. Chromatographic Analysis
As described in Section 4.3.3.
5.3.4. Mass Spectrometry
As described in Section 4.3.3.2
5.3.5. Untargeted Antioxidant Identification with LC-HRMS
LC-HRMS offers the potential to identify several bioactive metabolites
contained within complex biological extracts of the Australian native plants in a
single analytical run. Furthermore, LC-HRMSMS has enabled an undoubted
structural elucidation for the metabolites and elemental composition of unknown
metabolites is one of the most challenging tasks in untargeted metabolomics studies.
In this study, compound identification started with the prediction of molecular
formulae by matching accurate masses against comprehensive databases using the
Compound DiscovererTM data analysis software. Many chemical structures can have
an identical molecular formula and the automated fragmentation capability of MSMS
mode, when combined with the Compound DiscovererTM software, enabled library
searching to predict structural identities.
5.4. Results and Discussion
5.4.1. Comparison of Extraction Methods
The RF-PCD-FRAP assay was used to measure the total antioxidant capacity
of redox active compounds within the Australian native plant extracts and a short
95
study for the selection of an appropriate sample preparation method was performed.
RF-PCD-FRAP profiles of the Australian native extracts obtained using the direct
extraction and the alternative sonication methods showed that the direct extraction
for all the native samples had a larger number and more intense antioxidants peaks.
An example in Figure 5.1 of the RF-PCD-FRAP profile for the extraction of Old
man saltbush shows that there are over 30 fully resolved peaks for the direct
extraction method while the sonicated sample has only five antioxidant peaks. It
follows that the direct extraction method provides the best results.
96
Figure 5.1. UV-Vis and RF-PCD-FRAP profile of Old man saltbush prepared with (a) direct extraction and (b) sonication method.
(a)
(b)
97
5.4.2. Antioxidant Analysis and Structural Elucidation
5.4.2.1. Old man saltbush
RF-PCD-FRAP analysis of Old man saltbush showed thirty-one resolved
peaks and has strong antioxidant responses between 24 mins to 30 mins (Figure 5.2).
Figure 5.2. RF-PCD-FRAP assay showing the antioxidant capacity of Australian native
Old man saltbush.
Table 5.1 contains the compounds that were identified along with their measured
monoisotopic molecular masses and calculated molecular formulae.
98
Table 5.1. Structural elucidation of antioxidants within Australian native Old man
saltbush.
5.5.2.1.1. Rhamnetin and Isorhamnetin
Compound DiscovererTM identified the presence of isorhamnetin (97.2%) and
rhamnetin (92.5%). The fragmentation pattern for the isomers of rhamnetin and
isorhamnetin were compared with MSMS reference spectra in the mzCloudTM library
and scientific literature [362, 363]. A compound with a precursor ion [M-H]- m/z
315.05130 presented the most abundant fragment ion m/z 300.02756 due to the
LC-HRMS (min)
Tentative Assignments Molecular Formula
Exact mass Measured mass Error(ppm)
0.542 Trigonelline C7H7NO2 137.04768 137.04773 -0.3
0.729 L-Tyrosine C9H11NO3 181.07391 181.07403 0.6
1.404 Adenosine C10H13N5O4 267.09678 267.09650 -0.1
1.588 L-Phenylalanine C9H11NO2 165.07899 165.07930 1.8
2.661 Pantothenic acid C9H17NO5 219.11070 219.11030 -1.0
2.669 Quinol glucronide C12H14O8 286.06891 286.06920 1.1
2.714 Pyrogallol C6H6O3 126.03171 126.03200 -0.2
2.876 Tryptophan C11H12N2O2 204.08989 204.09001 0.5
2.912 Salicylic acid C7H6O3 138.03171 138.03160 -0.7
2.949 Protocatechuic acid C7H6O4 154.02663 154.02660 -0.1
2.963 Trans-3-indolacrylic acid C11H9NO2 187.06334 187.06350 0.8
3.099 3-Hydroxysuberic acid C8H14O5 190.08415 190.08420 0.2
3.389 Kynurenic acid C10H7NO3 189.04260 189.04238 -1.1
3.758 4-Pyridoxic acid C8H9NO4 183.05318 183.05250 -0.3
3.839 Chlorogenic acid C16H18O9 354.09513 354.09540 0.7
3.908 Unidentified phenolic acid C9H8O4 180.04228 180.04160 -3.7
4.002 5-S-Methyl-5-thioadenosine C11H15N5O3S 297.08958 297.08965 0.2
4.085 Coumaroyl hexoside C15H18O8 326.10021 326.10040 0.5
4.269 3-O-Feruloylquinic acid C17H20O9 368.11078 368.11090 0.2
4.884 Coumarin C9H6O2 146.03679 146.03650 -1.9
5.081 Unidentified compound C10H10O4 194.05793 194.05730 -0.3
5.134 8-Hydroxyquinlone C9H7NO 145.05276 145.05290 -0.4
5.386 Suberic acid C8H14O4 174.08923 174.08850 -0.4
5.756 Rhamnetin C16H12O7 316.05834 316.05824 -0.3
6.351 Azelaic acid C9H16O4 188.10488 188.10425 -3.3
6.611 Vitamin D5 C29H48O9 540.32998 540.33050 0.9
6.710 Camphoric acid C10H16O4 200.10488 200.10430 -0.2
7.393 Rhamnetin C16H12O7 316.05834 316.05843 0.2
7.400 Iso-Rhamnetin C16H12O7 316.05834 316.05860 0.2
8.047 Asarone C12H16O3 208.10996 208.11010 0.6
8.452 Glycetein C16H12O5 284.06860 284.06856 -0.1
10.278 Unidentified compound C18H30O3 294.21951 294.21963 0.4
10.258 Unidentified compound C15H22O 218.16707 218.16175 -24.3
10.751 Unidentified compound C20H32O2 304.24040 304.24020 -0.6
11.410 Oleamide C18H35NO 281.27186 281.27182 -0.2
12.202 Pyropheophorbide A C33H34N4O3 534.26311 534.26354 0.8
12.820 Alpha-tocospiro A C29H50O4 462.37093 462.37107 0.3
13.535 Vitamin E quinone C29H50O3 446.37601 446.37620 0.4
13.948 Spermidine C7H19N3 145.15790 145.15790 0.0
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dissociation of aglycones, which was produced by the loss of a methyl group [364].
The fragmentation of rhamnetin is indistinguishable from that of isorhamnetin but
these two isomers are easily identified by their facile chromatographic resolution on
reversed phase packing based on previous literature reports [365, 366]. It was
demonstrated that isorhamnetin eluted before rhamnetin and differentiation is
possible between these two isomers without use of any commercial standards.
5.5.2.1.2. Kynurenic acid
The MS spectra of a compound with the protonated precursor ion [M+H]+
m/z 190.04993 was unambiguously assigned as kynurenic acid. It has a predicted
match of 99.5% based on the standard spectrum of kynurenic acid in the mzCloudTM
library as revealed by Compound DiscovererTM. The fragmentation pattern was
further confirmed based on previous literature reported for plants and herbs [367].
Despite the fact that the role of this bioactive is not yet fully known, it is indicated
that it has strong antioxidant [368], antiinflammatory [367], anticancer [369],
antidepressant and gastrointestinal tract protective activities. The controversial role
of Kynurenic acid in psychological disorders has been previously reported [370, 371]
and individuals using Old man saltbush as a herb should be monitored for any
possible psychological reactions.
5.5.2.1.3. Glycitein
The presence of the O-methylated isoflavone, Glycitein, was identified based
on the precursor ion [M+H]+ m/z 285.07584 and fragment ions m/z 270.1681, m/z
286.07892, m/z 252.16812, m/z 109.10152, m/z 107.08570 and m/z 95.08595. This is
in good agreement with the standard spectra in mzCloudTM (93.7%) and previous
100
literature [372, 373]. Glycitein has shown an inhibitory effect on hydrogen peroxide
induced cell damage in lung fibroblasts [374] and prevents atherosclerotic
cardiovascular diseases [375]. A potential therapeutic role of Old man saltbush in
respiratory and cardiovascular diseases could be an interesting aspect of future
research.
5.5.2.1.4. Polyphenol and Phenolic acids
Azelaic acid, C9H16O4, with a 95.5% mzCloudTM match was identified based
on a precursor ion of [M-H]- m/z 187.09694 and the most abundant fragment ion m/z
125.09614. Other fragments ions were also in agreement with standard mass spectra
from previous literature studies [376, 377] that have provided unambiguous
identification. The presence of Azelaic acid in an Old man saltbush extract has
revealed its promising scope in dermatological applications such as antioxidant,
antiinflammatory, hyperpigmentation disorders [378] and antiaging [379]. Other
acids such as suberic acid was also predicted by Compound DiscovererTM but the
MSMS spectra showed some variation of the fragmentation patterns from the
corresponding standard mass spectra in mzCloudTM library. Further validation
studies are required to ensure the reliability of the Compound DiscovererTM
identification.
The compound with molecular formula C17H20O9 that eluted at 4.269 min
with [M-H]- m/z 367.10361 was identified as the polyphenol, feruloylquinic acid
[380], which fragments to produce the ion m/z 193 [381]. Another phenolic acid
derivative, C15H18O8, eluting at 4.085 min with precursor ion [M-H]- m/z 325.09311
that fragmented to the most abundant product ion m/z 163, corresponding to the loss
of a coumaroyl radical [382], was tentatively assigned as coumaroyl hexoside.
101
The chlorogenic acid, C16H1809, caffeoyl quinic acid ester was also identified
at 3.839 min and the presence of m/z 191.05556 indicates the linkage of caffeoyl
group to quinic acid according to a previously documented fragmentation pattern
[383]. The linkage position of caffeoyl groups on quinic acid is important for
structural characterization and the cinnamic acid moiety, quinic acid moiety, H2O
and CO are the common chemical groups that are eliminated from [M−H]- ions of
chlorogenic acids to afford their respective diagnostic product ions [384, 385].
5.5.2.1.5. Amino acids and Nucleosides
The Old man saltbush chemical profile also contains the amino acids, L-
phenylalanine, L-tryptophan, L-tyrosine. In addition to these essential amino acids
there are also the pharmacologically important nucleosides, adenosine and
guanosine. Compound Discoverer has identified tryptophan with a 96% match at
2.876 min with the experimental molecular mass of 204.09001 [386]. Precursor ions
for both correspond to m/z 205.09727 with the most abundant fragment ion m/z
188.07065 [387, 388].
Phenylalanine, C9H11NO2 m/z 165.07930 with product ions C9H9O2 m/z
149.0710 and C7H7O m/z 107.04961, was identified using Compound DiscovererTM
and literature [389]. Phenylalanine eluted at 1.588 min and were identified
according to the previously reported scientific report [390].
Spermidine, C7H19N3, is a polyamine [391] and identified with a retention
time of 13.948 mins. It has a precursor ion [M+H]+ m/z 146.16521 that further
fragmented to m/z 130.05005 with the preferred product C7H14N+ m/z 112.11376
102
[392]. It has been reported that spermidine has anticancer [393], antiaging and
cardioprotective potential [394, 395].
5.5.2.1.6. Alkaloids
5-S-methyl-5-thioadenosine, C11H15N5O3S, with precursor ion [M+H]+ m/z
298.09692, was identified with a retention time of 4.002 min. The methylsulfanyl
group substitutes for the hydroxy group at C-5' substituted 5-S-Methyl-5-
thioadenosine and loss of this group produces m/z 136.06369, which is major
fragment ion of adenosine after removal of sugar moiety. This compound was also
previously identified within bioactive herbs [396] and has alkaloid chemistry [397].
Another bioactive alkaloid compound, C7H7NO2, with precursor ion peak [M+H]+
m/z 138.05492, was tentatively assigned to be trigonelline and has been previously
reported [398, 399]. Trigonelline acts as an antidiabetic [400], ameliorates oxidative
stress [401] and improves insulin signaling pathways [402].
5.5.2.1.7. Vitamins and Fatty acids
The bioactive lipid oleamide, C18H35NO, with [M+NH4]+ m/z 299.30563, was
observed at 11.410 min and fragmentation resulted in two daughter ions [M+H-
NH₃]+ m/z 265.2516 and [M+H-NH₃-H₂O]+ m/z 247.2405, as previously reported
[403, 404].
The compound that eluted at 13.535 min was tentatively assigned as α-
tocopherol quinone, with chemical formula C29H50O3. The protonated ion [M+H]+
m/z 447.38290 further fragmented to form the most abundant product ions m/z
165.09366. Other fragment ions observed are m/z 401.34844, m/z 247.17064, m/z
103
233.15323, m/z 205.12224, m/z 191.10670, m/z 177.09373 and m/z 163.11182. The
strong antioxidant response of this compound has been previously established [405].
The phytochemical profile of Old man saltbush includes alkaloids,
flavonoids, polyphenols, phenolic acids, amino acids, triterpenes, sterols and lipids.
High-resolution mass spectrometry analysis indicated the presence of the significant
bioactive molecules kynurenic acid, rhamnetin, isorhamnetin, tocopherol glycitein,
and azelaic acid. To the best of our knowledge, this is the first report of these
bioactive compounds in Old man saltbush extracts. Further, detailed data mining
demonstrated that Old man saltbush is also a rich source of bioactive lipids,
sterols and alkaloids but these identifications are not discussed in this section.
104
5.5.2.2. Gumbi gumbi
As seen in Figure 5.3, the RF-PCD-FRAP assay of the extracted sample
generated many intense peaks, which indicates that the Gumbi gumbi sample
contains relative high concentrations of readily oxidizable compounds with the
presence of 15 antioxidant peaks. Both the extracted and sonicated samples have
closely related profiles signifying their fundamentally similar chemical composition.
Figure 5.3. RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Gumbi gumbi.
A careful evaluation of the metabolic contents for Gumbi gumbi with high-
resolution mass spectrometry revealed a high degree of complexity and that Gumbi
gumbi is a rich source of bioactives. Table 5.3 provides a list of identified
compounds.
105
5.5.2.2.1. Pheophorbide and its Methyl Esters
The compound that eluted at 11.736 min, C35H36N4O5, [M+H]+ m/z
593.27606 was assigned as Pheophorbide a (chlorophyll catabolite) based on
published literature [406]. It is a breakdown product of chlorophyll and is used as a
photosensitizer due to its anticancer activity [407]. This compound was identified
with precursor ion [M+H]+ m/z 593.27539, which further fragmented to m/z
565.28333, m/z 533.25439, m/z 460.22528, m/z 285.11218, m/z 149.13246, m/z
135.1162, m/z 109.10140 and m/z 95.08572. The product ions C33H32N4O3 m/z 533
and C30H26N4O m/z 460 were previously observed [406].
106
Table 5.2 Structural elucidation of antioxidants within Australian native Gumbi
gumbi.
LC-HRMS (min)
Tentative Assignments Molecular Formula
Exact mass Measured mass Error (ppm)
0.438 L-Threonic acid C4H8O5 136.03720 136.03659 -4.5
0.446 D-(-)Quinic acid C7H12O6 192.06342 192.06280 -3.2
0.524 Citric acid C6H8O7 192.02704 192.02646 -3.0
0.625 Unidentified compound C11H22O10 314.12135 314.12160 0.8
0.667 Unidentified compound C4H9NO4 135.05318 135.05356 2.8
1.330 Adenosine C10H13N5O4 267.09678 267.09682 0.1
2.920 DL-Tryptophan C11H12N2O2 204.08989 204.08998 0.4
2.909 Salicylic acid C7H6O3 138.03171 138.03167 -0.3
3.230 Unidentified compound C14H16O9 328.07945 328.07964 0.6
3.355 Kynurenic acid C10H7NO3 189.04260 189.04265 0.3
3.366 Caffeic acid C9H8O4 180.04228 180.04140 -4.9
3.344 C-glycoside of 4-O-methyl gallic
acid
C14H16O9 328.07945 328.07964 0.6
3.530 Unidentified compound C15H18O9 342.09550 342.09552 0.1
3.584 Unidentified compound C33H32N4O14 708.19158 708.19111 -0.7
3.570 Unidentified compound C15H18O9 342.09550 342.09559 0.3
3.596 Unidentified compound C17H20O11 400.10062 400.10084 0.5
3.629 Caffeoyl Quinic acid C16H18O9 354.09513 354.09494 0.9
3.679 Unidentified compound C7H16O5S 212.07139 212.07141 0.1
3.762 Caffeoyl Quinic acid C16H18O9 354.09513 354.09545 0.9
3.840 Esculetin C9H6O4 178.02663 178.02655 -0.4
3.871 Caffeic acid-3-glucoside C15H18O9 342.09513 342.09497 -0.5
3.884 Unidentified compound C15H18O9 342.09513 342.09547 1.0
3.954 Unidentified compound C17H24O10 388.13700 388.13765 1.7
3.963 p-Coumaric acid glucoside C15H18O8 326.10021 326.10048 0.8
3.992 Caffeoyl alcohol C9H10O3 166.06301 166.06227 -4.5
4.093 Unidentified compound C18H22O12 430.11119 430.11165 1.1
4.365 Unidentified compound C16H18O8 338.10021 338.10063 1.2
4.378 Coumaroyl quinic acid C16H18O8 338.10019 338.10021 0.05
4.424 Unidentified compound C16H18O9 354.09513 354.09545 0.9
4.584 Unidentified compound C18H22O12 430.11119 430.11165 1.1
4.680 3-O-Feruloylquinic acid C17H20O9 368.11078 368.11122 1.2
4.936 Coumaroyl quinic acid C16H18O8 338.10021 338.10064 1.3
5.009 Unidentified compound C18H24O10 400.13700 400.13746 1.1
5.566 Quercetin-3β-glucoside C21H2OO12 464.09554 464.09544 -0.2
6.060 Luteolin 7-Sulfate C15H10O9S 366.00460 366.00490 0.8
6.086 Cynaroside C21H20O11 448.10139 448.10136 -0.1
6.322 Azelaic acid C9H16O4 188.10488 188.10515 1.4
6.831 Cyanidin C15H10O6 286.04777 286.04780 0.1
6.836 Luteolin C15H10O6 286.04777 286.04778 0.0
7.404 Chrysoeriol C16H12O6 300.06342 300.06328 -0.5
8.822 Unidentified compound C17H31NO5 329.22025 329.22022 -0.1
9.550 Unidentified compound C17H26O4 294.18313 294.18311 -0.1
9.623 Unidentified compound C17H24O3 276.17256 276.17260 0.1
9.654 Unidentified compound C15H18O3 246.12561 246.12560 0.0
9.716 Unidentified compound C15H20O3 248.14126 248.14125 0.0
9.729 Unidentified compound C20H34O3 322.25081 322.25066 -0.5
107
Table 5.2.(cont.) Structural elucidation of antioxidants within Australian native
Gumbi Gumbi.
This is the first time that this compound has been reported for Gumbi Gumbi and the
presence of Pheophorbide a within this extract could be a possible reason for its use
LC-HRMS (min)
Tentative Assignments Molecular Formula
Exact mass Measured mass Error (ppm)
9.801 Unidentified compound C18H28O 260.21402 260.21338 -2.5
9.910 Unidentified compound C10H16O 152.12012 152.12009 -0.2
9.912 Unidentified compound C11H18O 166.13577 166.13584 0.4
9.965 Unidentified compound C15H22O 218.16707 218.16709 0.1
9.970 Unidentified compound C18H28O3 292.20386 292.20381 -0.2
10.009 Unidentified compound C30H50O5 490.36585 490.36590 0.1
10.057 Unidentified compound C18H28O2 276.20894 276.20893 0.0
10.069 Unidentified compound C17H24O2 260.17764 260.17757 -0.3
10.116 Unidentified compound C22H30O2 326.22459 326.22429 -0.9
10.117 Unidentified compound C28H46O5 462.33455 462.33449 -0.1
10.157 Unidentified compound C15H24O 220.18272 220.18274 0.1
10.258 Nookatone C15H22O 218.16707 218.16715 0.4
10.057 Unidentified compound C18H28O2 276.20894 276.20893 0.0
10.069 Unidentified compound C18H30O3 294.21951 294.21991 1.4
10.157 Unidentified compound C15H24O 220.18272 220.18274 0.1
10.158 Unidentified compound C27H46O4 434.33963 434.33980 0.4
10.241 Unidentified compound C20H30O4 334.21443 334.21343 -3.0
10.254 Unidentified compound C15H22O 218.16707 218.16709 0.1
10.312 Unidentified compound C32H52O5 516.38150 516.38176 0.5
10.326 Unidentified compound C18H22O 254.16707 254.16700 -0.3
10.546 Unidentified compound C30H50O3 458.37601 458.37595 -0.1
10.568 Unidentified compound C9H14O 138.10447 138.10446 -0.1
10.964 Unidentified compound C18H28O 260.21402 260.21338 -2.5
10.972 Unidentified compound C21H36O4 352.26138 352.26131 -0.2
11.21 16-Hydroxyhexadecanoic acid C16H32O3 272.23516 272.23560 1.6
11.231 Alpha-linolenic acid C18H30O2 278.22459 278.22488 1.0
11.238 Hexadecanamide C16H33NO 255.25621 255.25610 -0.4
11.302 Unidentified compound C23H38O4 378.27703 378.27684 -0.5
11.326 Unidentified compound C22H36O3 348.26646 348.26625 -0.6
11.355 Unidentified compound C17H24 228.18780 228.18779 0.0
11.402 Unidentified compound C27H48O3 420.36036 420.36028 -0.2
11.424 Oleamide C18H35NO 281.27186 281.27180 -0.2
11.623 Unidentified compounds C30H50O 426.38617 426.38643 0.6
11.736 Pheophorbide a C35H36N4O5 592.26860 592.26877 0.3
12.094 Methyl Pheophorbide a C36H38N4O5 606.28425 606.28429 0.1
13.353 Lanosterol C30H50O 426.38617 426.38600 -0.4
13.704 Phytonadine (Vit K1) C31H46O2 450.34979 450.34953 -0.6
13.797 phosphatidylethanolamine C39H74NO8P 715.51524 715.51463 -0.9
13.844 Unidentified compound C37H69NO6 623.51252 623.51286 0.5
108
by Australian indigenous people for the treatment of various kinds of cancers.
Previous reports suggest that it has a role in endothelial cell dysfunction modulation
[408], with antitumor [409] and anticancer [410] activity. Gumbi gumbi is a
promising candidate for photodynamic therapy and tumor treatment, which can be
applied to treat various diseases such as psoriasis, rickets, vitiligo and skin cancer
[410]. Formulations that include Gumbi gumbi for skin cancer prevention and
treatment could be a future direction of this research.
The compound C36H38N4O5, with experimental m/z 606.28429, eluted at
12.094 min and was identified as the methyl ester of pheophorbide a. The protonated
ion [M+H]+ m/z 607.29150 fragmented to produce ions m/z 547.27008, m/z
548.27338, m/z 487.25598, m/z 459.22629 and m/z 461.24054 and this fragmentation
pattern was in agreement with previous reports [411]. Pheophorbide a methyl ester
has anticancer activity [412] and is a known candidate for photodynamic therapy to
directly kill tumors by shutting down tumor vasculature [413]. Drug development
has recently modified this molecule by reacting it with amines to produce chlorin e6
13-caboxamide, a strong photosensitizer used in photodynamic therapy [414].
5.5.2.2.2. Chlorogenic acids
Four types of chlorogenic acids were identified in Australian native Gumbi
gumbi and all are reported for the first time from this source. A review of the
literature revealed that the bioactive compound with retention time 4.378 min
[M+H]+ m/z 339.10733 that further fragmented to m/z 147.04611 is a cinnamate
ester, coumaroyl quinic acid. [415-417]. Another bioactive metabolite with the same
chemical formula C16H18O8, eluting at 4.936 min, was also identified as coumaryl
quinic acid as previously reported with linear Orbitrap mass spectrometry studies
109
[418, 419]. Caffeoyl quinic acid, C16H18O9, eluted at 3.629 and 3.783 min,
respectively, and were identified with characteristic precursor peak [M+H]+ m/z
355.10217 with diagnostic product ion m/z 163.03900 [420].
5.5.2.2.3. Esculetin
The compound C9H6O4, eluting at 3.840 min, was identified as esculetin (6,7
dihydroxycoumarin) with a 96% match from mzCloudTM. The most abundant ion
observed was m/z 179.03389, corresponding to the protonated ion. Further MSMS
fragmentation showed diagnostic product ions with m/z 151.03902 due to the loss of
CO and [M+H-2CO]+ m/z 123.0443, the ion [M+H-H2O-CO]+ m/z 133.03026 was
also observed. The traditional use of Gumbi gumbi to alleviate fever and pain might
be attributed to the presence of esculetin [421], which has been shown to inhibit
lipoxygenase activity [422], platelet aggregation [423] and oxidative stress [423]. In
addition, the traditional anticancer role of this native plant could also be attributed to
the presence of esculetin [424-427].
5.5.2.2.4. Luteolin 7-Sulfate
Luteolin 7-sulfate, C15H10O9S, was identified as eluting at 6.060 min with the
deprotonated precursor ion peak [M-H]- m/z 364.99759 fragmented to form the most
abundant product ion m/z 285.04077 due to the removal of the sulphate group.
Luteolin 7 sulfate has anti-melanogenic effects [428]. Sulfate conjugates of
flavonoids are easily hydrolysed and required careful handling during sample
preparation [429] and luteolin is also observed at 6.836 mins, which is known to
have antiinflammatory and anticancer [430-432] activity. These results indicate that
Gumbi gumbi is a potentially attractive candidate for the development of topical
lotions with dermatological applications
110
5.5.2.2.5. Phenolic and Organic acids
L-threonic acid, D-quinic acid, citric acid, kynurenic acid, azelaic acid and
salicylic acid are present as predicted by Compound DiscovererTM and confirmed by
the literature [433, 434]. Quinic acid has been identified at various retention times
within phytochemical profiles of the Australian native extracts used in study and this
is in agreement with the previous studies as six isomers of quinic acid occur within
natural products [435, 436].
5.5.2.2.6. Flavonoids and Flavonoids Glycosides
High-resolution mass spectrometry provided detailed metabolomic
fingerprinting of flavonoids and their conjugated glycosides. However, most of the
constituents with potential flavonoids and glycosides were not assigned
identifications due to limited reference mass spectra to differentiate compounds of
the same class with similar empirical formula. The fragmentation pattern of
flavonoids in natural extracts varies with different conditions [437-440] and
instrumental setups [441-443].
Chrysoeriol, C16H12O6, the 3'-methoxy derivative of luteolin was identified at
7.404 mins with [M+H]+ m/z 301.07068 and product ions m/z 286.04691 and m/z
265.18240 [444]. Replacement of the hydroxyl group with a methoxy group or a 7-
O-β-D-glucopyranoside side chain in luteolin results in Chrysoeriol and Cynaroside,
respectively [444]. The compound, C21H20O11, eluted at 6.086 min with observed ion
[M-H]- m/z 447.09409 and was tentatively identified as Cynaroside. The
fragmentation pathway of the O-glycosylated flavonoids starts with the cleavage of
the glycosidic bonds and elimination of the sugar moieties with charge retention on
111
aglycones. Removal of the glucopyranoside side chain resulted in the most abundant
diagnostic fragment ion for luteolin with m/z 285.04080.
5.5.2.2.7. Miscellaneous Compounds
An anthocyanin with protonated molecular ion [M+H]+ m/z 287.05505 eluted
at 6.831 min and was identified as cyanidin. The phytochemical profile contains
about 1600 compounds and includes the presence of bioactive lipids, alkaloids and
phytosterols. Further data interpretation is required to explore the known and
unknown compounds, which would reveal the complete ethnopharmacological
potential of Gumbi gumbi.
In summary, Gumbi gumbi is a well-known bush medicine that has been
shown to be effective against a range of afflictions from cold to cancer, and our
study has demonstrated this anticancer and antitumor activity might be attributed to
the presence of abundant chlorophyll catabolites.
5.5.2.3. Quandong
Quandong is a well-known indigenous food and has been used in traditional
medicine. However, due to the intrinsic complexity of the chemical constituents,
minimal information about the overall chemical profile of this native food is
available. Quandong fruit has revealed a specific chemical profile mostly
characterized by endogenous components that are mainly connected to nutritional
and pharmaceutical importance. The RF-PCD-FRAP assay showed an interesting
profile and the methanol-extracted sample had eight antioxidant peaks with the most
intense antioxidant response at 9.644 mins. This is proposed to be due to chlorogenic
112
acids as identified from the most abundant antioxidant-type compound found in the
sample based on the LC-HRMS analysis (Table 5.3).
Figure 5.4. RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Quandong.
5.5.2.3.1. Chlorogenic acids
The major chlorogenic acid and its sequential products exhibited [M-H]- ions
and [M+H]+ ions with abundant structural information in the respective negative and
positive modes. Six types of chlorogenic acids were identified in Australian native
Quandong and all are reported here for the first time from this source. Compound
DiscovererTM did not predict their presence due to unavailability of reference spectra
in the mzCloudTM library but literature investigations revealed that the major
bioactive compound, C16H18O8 which eluted at 3.416 min, is a cinnamate ester, 3-O-
p-coumarylquinic acid [415]. Another bioactive metabolite with same chemical
formula eluted at 4.441 mins and was identified as 4-O-p-coumarylquinic acid, as
previously reported[361].
113
In previous reports, the diagnostic product ions were identified based on the
high-resolution and low-resolution MS data [15, 19]. For example, the fragment ions
[quinic acid-H]- m/z 191, [caffeic acid-H]- m/z 179 and [quinic acid-H-H2O]- m/z 173
were observed [445]. 4-O-p-coumarylquinic acid and
Table 5.3. Structural elucidation of antioxidants within Australian native
Quandong.
LC-HRMS (min)
Tentative Assignments Molecular Formula
Exact mass Measured mass Error(ppm)
0.416 L-Hydroxyproline C5H9NO3 131.05830 131.05826 0.3
0.488 D-(-)Quinic acid C7H12O6 192.06251 192.06342 -4.7
0.525 Pipecolic acid C6H11NO2 129.07895 129.07899 -0.3
1.398 Adenosine C10H13N5O4 267.09636 267.09678 -1.6
1.59 5-Hydroxymethyl-2-
furaldehyde
C6H6O3 126.03182 126.03171 0.9
1.699 Hydroxyquinol C6H6O3 126.03182 126.03171 0.9
2.347 Unidentified C12H16O8 288.08437 288.08456 -0.7
2.576 3-O-Caffeoylquinic acid C16H18O9 354.09490 354.09513 -0.6
2.720 Hippuric acid C9H9NO3 179.05829 179.05826 0.2
2.735 4-O-Caffeoylquinic acid C16H18O9 354.09490 354.09513 -0.6
3.046 Gallic acid C7H6O5 170.02160 170.02155 0.3
3.416 3-O-p-Coumarylquinic acid C16H18O8 338.10000 338.10021 -0.6
3.434 4-Coumaric acid C9H8O3 164.04737 164.04736 0.1
3.451 Coumarin C9H6O2 146.03662 146.03679 -1.2
3.498 Coumaric acid C9H8O2 146.03642 146.03679 -2.5
3.594 Unidentified compound C7H7NO3 153.04250 153.04260 -0.7
3.925 Unidentified compound C9H10O6 214.04760 214.04777 -0.8
3.985 4-Hydroxycoumarin C9H6O3 162.03154 162.03171 -1.0
3.989 5-O-Caffeoylquinic acid C16H18O9 354.09494 354.09513 -0.5
4.177 Quinolin-8-olate C9H9NO 147.06838 147.06842 -0.3
4.441 4-O-p-Coumarylquinic acid C16H18O8 338.10000 338.10021 -0.6
4.451 Caffeic aldehyde C9H8O3 164.04737 164.04736 0.1
4.522 Phlorizin C21H24O10 436.13680 436.13700 -0.5
4.689 Coumaric acid C9H6O2 146.03661 146.03679 -1.2
4.512 Unidentified compound C15H14O5 274.08330 274.08415 -3.1
4.785 Unidentified C12H14O6 254.07890 254.07907 -0.7
4.785 Unidentified C15H12O6 288.06310 288.06342 -1.1
4.845 Unidentified C12H16O5 240.09976 240.09980 -0.2
5.046 (+) Ouratea-Catechin C16H16O7 320.08938 320.08964 -0.8
5.285 Naringenin-7-O-glucoside C21H22O10 434.12114 434.12135 -0.5
5.286 Naringenin C15H12O5 272.06819 272.06850 -1.1
5.337 Phloretin C15H14O5 274.08333 274.08415 -3.0
5.362 Unidentified compound C12H16O4 224.10468 224.10488 -0.9
5.523 4-Coumaroyl Shikimate C16H16O7 320.08940 320.08964 -0.7
114
Table 5.3.(cont.) Structural elucidation of antioxidants within Australian native
Quandong.
LC-HRMS (min)
Tentative assignment Molecular Formula
Exact mass Measured mass Error ppm
5.870 Cynidine-3-glucoside C21H22O11 450.11623 450.11627 -0.1
6.071 Saponarin C27H30O15 594.15856 594.15855 0.1
6.345 6-hydroxy coumarin C9H6O3 162.03174 162.03171 0.2
6.635 (+) Abscisic acid C15H20O4 264.13608 264.13618 -0.4
6.730 Glycyroside C27H30O13 564.18468 564.18436 0.6
7.138 Flavanol-3- rutinoside C27H32O12 548.18956 548.18944 0.2
7.712 Unidentified compounds C12H14O4 222.08922 222.08923 0.0
7.829 Unidentified compounds C15H30O5 290.20910 290.20935 -0.9
8.045 Unidentified compounds C12H16O3 208.10997 208.10966 1.5
8.832 Β-Asarone C12H16O3 208.11000 208.10997 0.1
8.845 Polygodial C15H22O2 234.16194 234.16199 -0.2
8.858 Unidentified C15H22O3 250.15681 250.15691 -0.4
9.353 Unidentified C15H22O2 234.16197 234.16199 -0.1
9.460 Unidentified C15H20O 216.15142 218.16699
9.729 Unidentified C15H20O3 248.14121 248.14126
9.827 Unidentified compounds C16H22O4 278.15180 278.15183 -0.1
10.266 Alpha tocospiro A C29H50O4 462.37080 462.37093 -0.3
10.969 Glycerophospholipid(18:2) C26H50NO7P 519.33269 519.33252 0.3
11.212 Alpha tocotrienol C29H48O2 428.36560 428.36544 0.4
11.274 Hexadecanamide acid C16H33O 255.25610 255.25621 -0.4
11.398 Unidentified compound C20H30O 286.22955 286.22967 -0.4
11.277 Glycerophospholipid(18:1) C26H52NO7P 521.34829 521.34817 0.2
11.418 Oleamide C18H35NO 281.27180 281.27186 -0.2
11.974 1-Stearoylglycerol C21H42O4 358.30830 358.30833 -0.1
13.409 Alpha-Tocopherol C28H46O3 430.34480 430.34471 0.2
13.543 Vitamin E Quinone C29H50O3 446.37160 446.37061 2.2
3-O-p-coumarylquinic acid not only possess potent antioxidant and antimicrobial
activity [446], they also have antinflammatory [447], cardioprotective [448, 449],
hepatoprotective, renoprotective, neuroprotective, antidiabetic [450] and
antilipidemic activities. Chlorogenic acids also inhibits human platelet activation and
thrombus formation [451]. Fatty diet induced hepatic steatosis and insulin resistance
can be improved with chlorogenic acid-rich Quandong fruit [452]. Furthermore,
extracts can be used to ameliorate brain damage by inhibiting cerebral ischemia
115
[453] and might have potential application to alleviate hyperalgesia in neuropathic
pain [454].
Caffeoyl quinic acids esters of chlorogenic acid [383], C16H1809, were also
identified with the isomers, 3-O-caffeoyl quinic acid, 4-O-caffeoyl quinic acid, and
5-O- caffeoyl quinic acid observed at retention times 2.576 min, 2.735 min and
3.989 min, respectively. For their structural identification, the linkage position of
caffeoyl groups on quinic acid could be determined according to the relative
intensities of the ESI-MSMS base peak ion and dominant product ions [20]. The
fragmentation patterns should be similar with those of the chlorogenic acids. Thus,
the cinnamic acid moiety, quinic acid moiety, H2O, and CO should be common
chemical groups that are easily eliminated from the [M-H]- ions of chlorogenic acids
to afford their respective product ions.
When the caffeoyl group was linked to quinic acid at 3-OH or 5-OH, m/z 191
was the base peak ion and m/z 179 was much more significant for 3-OH. When m/z
173 was the prominent peak, the caffeoyl group was linked at 4-OH. Additionally,
chromatographic peaks were identified as 3- and 4- according to the documented
elution patterns described from previous studies [455]. Both of the coumaroyl quinic
acid isomers were differentiated based on the relative intensities of their product ions
[445, 456, 457]. Caffeoyl quinic acid esters possess potent antioxidant [458],
antibacterial [459, 460], antiinfluenza and neuroprotective [461] properties.
5.5.2.3.2. Phenolic and Organic acids
Commonly occurring phenolic acids, coumaric acid, pipecolic acid, gallic
acid, quinic acid and abscisic acid, were abundantly present within Australian native
Quandong fruit. The identification of phenolic and organic acids is in agreement
116
with the previous reported studies [462, 463]. However, some compounds
resembling phenolic and organic acids were not identified due to the absence of
reference mass spectra in mzCloudTM and the scientific literature.
5.5.2.3.3. Flavonoids and Glycosides
Major types of flavonoids, namely isoflavones, isoflavans, flavones and
dihydroisoflavones, were differentiated by characteristic fragment ions with accurate
mass measurements. Naringenin, β-asarone and polygodial, adenosine, pipecolic
acid, 4-coumaric acid, chlorogenic acid (3.416min) and 5-hydroxymethyl-2-
furaldehyde were unambiguously identified by Compound DiscovererTM and were
confirmed by comparing their mass spectra with those of the reference spectra within
mzCloudTM and scientific literature [464].
β-asarone, C12H16O3, was identified at 8.832 min and had a 91% similarity
match with the mzCloudTM library mass spectra. The protonated precursor ion
[M+H]+ m/z 209.11728 of β-asarone further fragmented to the most abundant
fragment ion m/z 168.07811 and other fragmented product ions observed were m/z
178.09877, m/z 181.08589, m/z 177.09097, m/z 151.07520, m/z 149.09616, m/z
121.06493, m/z 99.04414 and m/z 87.04421. Another compound with the same
molecular formula eluted at 8.045 min and although the spectra has a similar
protonated ion m/z 209.11705, the most abundant product ion m/z 168.07809 was
observed. Other fragmentation products found in this spectrum were m/z 178.09872,
m/z 181.08569, m/z 169.08159, m/z 145.04941 and m/z 67.84345. The compound
assignment of β-asarone was based on a previous study where the elution pattern for
α and β isomers showed that α-asarone eluted before β-asarone [465]. Further
investigation is required to identify these compounds unambiguously.
117
Naringenin was identified at 5.286 min based on the characteristic protonated
ion [M+H]+ m/z 273.07550 and fragmented product ion, m/z 153.1808,
corresponding to retro Diels-Alder fragmentation in the C-ring involving 1,3-
scission [466]. Another related compound eluting at 5.285 min was tentatively
assigned as naringenin-7-glucoside m/z 435.12842 with an MSMS ion corresponding
to the loss of hexose moiety [M+H-hexose] m/z 273.07574 [467]. Compounds
identified at 5.870 min and 6.071 min were assigned as cyanidine-3-glucoside and
kaempferol 3-O-rutinoside, respectively, based on their mass spectra and literature
references [468, 469].
Phloretin, C15H14O5, was tentatively identified at a retention time of 4.512
min due to the precursor ion [M+H]+ m/z 275.09039 and the most abundant fragment
ion m/z 139.04077. The glycosylated chalcone, phlorizin, C21H24O10, with a retention
time of 4.522 min, is the O-linked glucoside of phloretin and was tentatively
identified in the studied extract as [M+NH4]+ m/z 454.17059 which fragmented to
products ions m/z 275.09212 and m/z 139.03893 [470].
Vitamin E and its analogues were also identified in the Quandong extract
with tocopherol and tocotrienol being the main forms found in the sample. Previous
studies have demonstrated that tocopherol and tocotrienol have a common basic
structure with a chromanol ring that has an alkyl side chain at position 2 on the ring.
The structural difference between tocotrienol and tocotrienol is due to the side chain
where tocopherol has a saturated side chain and tocotrienol is unsaturated. An
identical fragmentation pathway is observed in MS due to the common structure and
diagnostic fragment ions are produced in MSMS due to the different side chains
[471]. The compound, C29H50O4, eluted at 10.266 min and was tentatively assigned
as tocospiro A based on the protonated precursor ion m/z 463.37811 that fragmented
118
to form the most abundant ion m/z 165.09103 with the other ions m/z 388.29672, m/z
z 299.06158, m/z 223.06342, m/z 166.09686 and m/z 147.04599 observed. No
reference data was available in the mzCloudTM library and a detailed literature search
did not revealed any relevant studies. Tocospiro is an antioxidant [472] and is an
antituberculosis bioactive compound [473] that has been found in the several fruits
and plants [472, 474].
The vitamin E analogue, α-tocopherol, was identified at 13.409 min based on
the precursor ion m/z 431.35211 with a diagnostic fragment ion m/z 165.09363,
which is in agreement with previous studies [475, 476]. α-tocopherol is a clinically
proven antioxidant [477] that alleviates oxidative stress and a recent study in rat
striatal culture has demonstrated that α-tocopherol can exert antiapoptotic,
neuroprotective action independently of its antioxidant property [478]. These
findings suggest that the Quandong extract could be a promising source of
neuroprotection independent of its antioxidant properties.
The compound eluting at 11.212 min was identified as α-tocotrienol from the
positive molecular ion m/z 429.37286 with the most abundant fragment ions m/z 165.
09363 and m/z 205.12576, as previously documented in related research on plant
extracts [472, 479]. A compound eluting at 13.543 min was tentatively assigned as
α-tocopherol quinone, with chemical formula C29H50O3 and experimental m/z
446.37160. The protonated ion [M+H]+ m/z 447.38290 further fragmented to the
diagnostic most abundant product ion m/z 165.09366. Other fragment ions observed
are m/z 401.34824, m/z 247.17014, m/z 233.15321, m/z 205.12224, m/z 191.10670,
m/z 177, 09373 and m/z 163, 11182. Detailed investigation of the literature revealed
no authentic reference for vitamin E-quinone identification with mass spectrometry
and mzCloudTM library has no reference spectrum. Identification of this compound
119
requires confirmation with the commercially available standard. Vitamin E quinone
has shown greater potency in suppressing platelet function compared to vitamin E
suggesting that is a better antithrombotic agent and is considered to be responsible
for in-vivo effects previously attributed to vitamin E [480-482]. Homologues of
vitamin E can be simultaneously identified based on the reversed phase C18 elution
pattern as previously documented [483-485]. This suggests that the Quandong
extract is a potential candidate for antithrombotic and cardioprotective
pharmaceutical applications.
5.5.2.3.4. Fatty acid and Fatty acids esters
The antioxidant lipids oleamide, 1-stearoylglycerol and hexadecanamide acid
were predicted from Compound DiscovererTM with high probabilites and spectra
were confirmed by manual inspection of mzCloudTM and literature [486, 487]. The
compounds with chemical formulae C26H52NO7P and C26H50NO7P eluting at 10.968
min and 11.277 min, respectively, were abundantly present in Quandong fruit
extracts. A review of the literature revealed that these compounds are
glycerophospholipids with similar mass spectra [488]. LC-HRMS detected the
presence of large numbers of compounds with lipid structural configurations but they
are not discussed in detail in this Chapter. Glycerophospholipid from plants and
fruits have therapeutic roles in cancer, neurodegeneration and metabolic syndrome
[489]. The presence of dietary bioactive lipids indicated that the Quandong lipidomic
profile should be investigated in detail to establish any related therapeutic potential.
Fatty acids esters of coumaryl acids were also observed and these esters are usually
localized in the fruit peel. Ripened Quandong fruit, where the peel had a red color,
was used in this study and it has been found that the amount of unsaturated esters
120
and compounds containing Z-p-coumaryl alcohol are greater compared to unripened
fruit [490].
Research conducted by Food Science Australia has previously demonstrated
the potential physiological activities of Quandong fruit in cell-based assays [491,
492]. However, there has been no research on the comprehensive chemical profile
of Quandong, and the active ingredients are still unclear, which is hampering its
broader application.
In this work, an untargeted approach based on LC-HRMS has discovered
that the major bioactive metabolites from the phytochemical profile are
chlorogenic acids. Chlorogenic acids along with naringenin, phloretin, phlorizin,
vitamin E, glycyroside, cynidine-3-glucoside, naringenin-7-O-glucoside and other
related antioxidants might be responsible for previously documented antidiabetic
and antiobesity activities of Quandong in cell-based assays. The untargeted profile
of the whole extract showed 1123 constituents and Compound DiscovererTM along
with relevant scientific literature has enabled the identification of 56 bioactive
compounds with significant antioxidant and other pharmacological activities.
Detailed investigation has also determined that Quandong is a rich source of
bioactive lipids, sterols and alkaloids but these were not discussed in this Chapter.
5.5.2.4 Australian native Lemon grass
The chromatogram obtained with the RF-PCD-FRAP assay (Figure 5.5)
shows that Australian native Lemon grass has a substantial number of antioxidant
compounds. The chromatogram has an intense and sharp antioxidant peak at 13.8
121
min and a number of additional peaks were observed, though they appear with lower
intensity. Table 5.4 contains a listing of the results from LC-HRMS analysis.
Figure 5.5. RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Lemon grass.
5.5.2.4.1 Flavonoids and Anthocyanin
Positive ionization generally results in more extensive fragmentation than
negative ionization [437, 439, 440] and remains popular due to the amount of
structural information that may be obtained using this mode [438, 439]. Fortunately,
the general ionization and fragmentation behaviour of flavonoids is largely
independent of the ionization source and instrument used [441-443] although relative
ion intensities may vary [493, 494]. One of the main bioactive compounds identified
within native Lemon grass is apigenin with a 99.9% mzCloudTM match which was
further confirmed with literature. The precursor ion [M+H]+ m/z 271.06000 further
fragmented to form the major product ion m/z 243.06635 as well as m/z 219.17430,
122
m/z 217.15845 and m/z 159.11903. In negative ionization conditions, m/z 269.04578
further fragmented to m/z 149.02341, m/z 225.14949, m/z 117.0346 and m/z
151.00258 [470]. The compound C15H10O6 eluted at 6.850 min and was identified by
Compound DiscovererTM to be cyanidin with a 98% match. The parent peak m/z
287.05493 further fragmented to m/z 231.0653, m/z 213.185, m/z 147.11919, m/z
118.08612 and m/z 109.10280. The compound C10H16O eluted at 6.326 min with
[M+H]+ m/z 153.12710 and was identified as Pulegone. Pulegone is the main
monoterpene present in the extract [495] .
123
Table 5.4. Structural elucidation of antioxidants within Australian native Lemon
grass. LC-HRMS
(min) Tentative Assignments Molecular
Formula Exact mass Measured mass Error(ppm)
0.446 D-(-)Quinic acid C7H12O6 192.06342 192.0628 3.2
0.453 Unidentified compound C4H6N6O4 202.04507 202.04526 -0.9
0.506 Unidentified compound C7H15NO3 161.10521 161.10526 -0.3
0.511 Unidentified compound C7H7NO2 137.04768 137.04766 0.1
0.513 Unidentified compound C8H19NO7 241.11619 241.11609 0.4
0.519 Unidentified compound C7H15NO2 145.11029 145.11025 0.3
0.537 Dihydroascorbic acid C6H6O6 174.01647 174.01578 4.0
0.542 Unidentified compound C6H6O3 126.03171 126.03189 -1.4
0.556 Unidentified compound C5H15NO4 153.10013 153.10007 0.4
0.598 Unidentified compound C6H11NO2 129.07899 129.07913 -1.1
0.632 Unidentified compound C6H7NO2 125.04768 125.04788 -1.6
0.649 Unidentified compound C8H13NO7 235.06924 235.06917 0.3
0.661 Unidentified compound C5H11N3O 129.09022 129.09036 -1.1
0.672 Unidentified compound C5H7NO3 129.04260 129.04277 -1.3
0.682 Unidentified compound C11H20N2O3 228.14741 228.14739 0.1
0.649 Unidentified compound C6H11NO4 161.06883 161.06886 -0.2
0.745 Unidentified compound C7H13NO2 143.09464 143.09465 -0.1
0.813 Nicotinamide C6H6N2O 122.04802 122.04829 -2.2
0.868 Isoleucine C6H13NO2 131.09464 131.09475 -0.8
0.917 Unidentified compound C7H15N2O 145.11845 145.11025 56.5
1.020 Unidentified compound C12H23NO7 293.14749 293.14723 0.9
1.200 Unidentified compound C10H23NO7 269.14749 269.14726 0.9
1.518 L-Phenylalanine C9H11NO2 165.07899 165.07904 -0.3
1.699 Unidentified compound C9H15NO7 249.08489 249.08495 -0.2
1.845 Unidentified compound C9H17NO8 267.09546 267.09562 -0.6
1.891 Unidentified compound C6H9NO2 127.06334 127.06352 -1.4
2.668 Unidentified compound C9H14O 138.10447 138.10448 -0.1
2.786 D-Pantothenic acid C9H17NO5 219.11070 219.10300 35.1
2.920 DL-Tryptophan C11H12N2O2 204.09890 204.09730 7.8
3.273 Unidentified compound C9H10O2 150.06809 150.06810 -0.1
3.355 Kynurenic acid C10H7NO3 189.04260 189.04265 -0.3
3.605 Unidentified compound C12H16O5 240.09980 240.09976 0.2
3.730 Unidentified compound C9H10O3 166.06301 166.06316 -0.9
3.825 Unidentified compound C12H16O5 240.09980 240.09976 0.2
3.840 Esculetin C9H6O4 178.02663 178.02655 0.4
3.871 Caffeic acid-3-glucoside C15H18O9 342.09513 342.09497 0.5
3.906 Unidentified compound C12H16O5 240.09980 240.09976 0.2
3.917 Hydroxy Cinnamic acid C9H8O4 180.04170 180.04140 1.7
4.308 Cinnamaldehyde C9H8O 132.05752 132.05765 -1.0
4.365 Unidentified compound C9H14O3 170.09431 170.09439 -0.5
4.372 Unidentified compound C9H6O3 162.03171 162.03172 -0.1
4.397 Chlorogenic acid C16H18O9 354.09513 354.09543 -0.8
4.425 Vanillin C8H8O3 152.04736 152.04738 -0.1
4.495 Methyl Sinapate C12H14O5 238.08415 238.08415 0.0
4.535 Unidentified compound C9H14O3 170.09431 170.09445 -0.8
4.589 Unidentified compound C9H18O4 190.12053 190.12059 -0.3
124
Table 5.4.(cont.) Structural elucidation of antioxidants within Australian native Lemon grass.
LC-HRMS (min)
Tentative Assignments Molecular Formula
Exact mass Measured mass
Error(ppm)
4.915 Unidentified compound C12H14O5 238.08415 238.08415 0.0 4.933 Coumarin C9H6O2 146.03679 146.03683 -0.3 5.063 Unidentified compound C12H16O4 224.10488 224.10492 -0.2
5.067 Trans-cinnamic acid C21H2OO12 464.09554 464.09544 0.2
5.088 Unidentified compound C10H10O4 194.05793 194.05739 2.8
5.134 Unidentified compound C9H7NO 145.05276 145.05278 -0.1
5.159 Unidentified compound C13H18O3 222.12561 222.12559 0.1
5.214 Unidentified compound C9H12O4 184.07358 184.07359 -0.1
5.295 Unidentified compound C9H14O3 170.09431 170.09447 -0.9
5.329 Iso Vitexin C21H20O10 432.10567 432.10570 -0.1
5.333 Unidentified compound C9H10O3 166.06301 166.06316 -0.9
5.402 Suberic acid C8H14O4 174.08923 174.08856 3.8
5.539 Apigetrin C21H20O10 432.10670 432.10570 2.3
5.557 Unidentified compound C12H16O5 240.09980 240.09978 0.1
5.559 Unidentified compound C12H19NO 193.14667 193.14687 -1.0
5.581 Kaempferol 7-O-glucoside C21H20O11 448.10062 448.10060 0.0
5.600 Unidentified compound C11H14O5 226.08415 226.08414 0.0
5.606 Unidentified compound C9H14O2 154.09939 154.09934 0.3
5.679 Unidentified compound C11H10O4 206.05793 206.05783 0.5
5.668 Unidentified compound C10H20O3 188.14126 188.14134 -0.4
5.703 Unidentified compound C10H12O3 180.07866 180.07871 -0.3
5.716 Unidentified compound C28H32O15 608.17420 608.17449 -0.5
5.785 Unidentified compound C12H16O4 224.10488 224.10485 0.1
5.825 Unidentified compound C12H16O5 240.09980 240.09976 0.2
5.948 Unidentified compound C14H21N 203.16740 203.16747 -0.3
5.983 Unidentified compound C10H14O 150.10447 150.10454 -0.5
5.997 Unidentified compound C22H26O9 434.15773 434.15776 -0.1
6.013 Vitexin C21H20O10 432.10570 432.10567 0.1
6.018 Unidentified compound C9H16O3 172.10996 172.11004 -0.5
6.022 Unidentified compound C13H16O4 236.10488 236.10478 0.4
6.032 Vanillic acid C12H16O4 224.10488 224.10490 -0.1
6.145 Unidentified compound C20H22O6 358.14167 358.14159 0.2
6.157 Unidentified compound C14H18O5 266.11545 266.11534 0.4
6.188 Unidentified compound C12H20O5 244.13110 244.13119 -0.4
6.282 Sinensetin C20H20O7 372.12094 372.12105 -0.3
6.311 Unidentified compound C9H16O2 156.11504 156.11507 -0.2
6.326 Pulegone C10H16O 152.12012 152.12020 -0.5
6.332 Unidentified compound C9H6O3 162.03171 162.03178 -0.4
6.344 Azelaic acid C9H16O4 188.10488 188.10515 -1.4
6.441 Unidentified compound C19H32O7 372.21484 372.21481 0.1
6.467 Elimicin C12H16O3 208.10996 208.11003 -0.3
6.545 Dodecanedioic acid C12H22O4 230.15183 230.15161 1.0
6.627 Unidentified compound C15H20O4 264.13618 264.13627 -0.3
6.631 Abscisic acid C15H20O4 264.13618 264.13652 -1.3
6.828 Unidentified compound C9H14 122.10955 122.10980 -2.0
6.850 Cyanidin C15H10O6 286.04777 286.04766 0.4
6.857 Luteolin C15H10O6 286.04777 286.04778 0.0
7.252 Unidentified compound C13H18O4 238.12053 238.12036 0.7
7.340 Apigenin C15H10O5 286.04777 286.04778 0.0
7.347 Unidentified compound C10H10O5 210.05285 210.05321 -1.7
125
Table 5.4.(cont.) Structural elucidation of antioxidants within Australian native Lemon grass.
LC-HRMS (min)
Tentative Assignments Molecular Formula
Exact mass Measured mass Error(ppm)
7.375 Iso Coumarin C9H6O2 146.03679 146.03649 2.1
7.380 Aflatoxin G2 C17H14O7 330.07399 330.07399 0.0
7.858 Unidentified compound C13H16O4 236.10488 236.10487 0.0
8.007 Peregrinine C24H35NO6 433.24646 433.24650 -0.1
8.208 Unidentified compound C18H32O5 328.22500 328.22516 -0.5
8.731 Unidentified compound C13H18O 190.13577 190.13591 -0.7
9.181 Unidentified compound C15H20O3 248.14126 248.14118 0.3
9.224 Unidentified compound C10H20O 156.15142 156.15167 -1.6
9.606 Unidentified compound C15H22O2 234.16199 234.16203 -0.2
9.895 Unidentified compound C12H26O4S 266.15520 266.15532 -0.5
9.971 Unidentified compound C15H20 200.15650 200.15663 -0.6
9.874 Unidentified compound C10H18O4 202.12053 202.12005 2.4
10.083 Unidentified compound C19H24O4 316.16748 316.16759 -0.3
10.148 Unidentified compound C15H24O 220.18272 220.18271 0.0
10.424 Unidentified compound C9H8O3 164.04736 164.04752 -1.0
10.469 Unidentified compound C15H24 204.1878 204.18796 -0.8
10.575 Unidentified compound C15H20 200.1565 200.15663 -0.6
10.589 Unidentified compound C9H16O4 188.10488 188.10501 -0.7
10.618 Unidentified compound C20H28O4 332.19878 332.19883 -0.2
10.620 Unidentified compound C12H14O3 206.09431 206.09426 0.2
10.626 Unidentified compound C19H26O3 302.18821 302.18830 -0.3
10.753 Unidentified compound C25H42O9 486.28293 486.28340 -1.0
11.095 Unidentified compound C15H24 204.18780 204.18796 -0.8
11.216 16-Hydroxyhexadecanoic acid C16H32O3 272.23516 272.23560 -1.6
11.238 Hexadecanamide C16H33NO 255.25621 255.25610 0.4
11.424 Oleamide C18H35NO 281.27186 281.27180 0.2
11.824 Unidentified compound C14H24O4 256.16748 256.16744 0.2
11.989 Unidentified compound C12H14O3 206.09431 206.09429 0.1
12.042 Unidentified compound C46H72O4 688.54308 688.54326 -0.3
12.047 Unidentified compound C19H28O2 288.20894 288.20903 -0.3
12.065 Unidentified compound C15H22O 218.16707 218.16710 -0.1
12.783 Unidentified compound C9H16O4 188.10488 188.10490 -0.1
12.835 Unidentified compound C25H42O4 406.30833 406.30824 0.2
12.847 Unidentified compound C21H34O4 350.24573 350.24578 -0.1
12.889 Unidentified compound C18H32O3 296.23516 296.23516 0.0
12.913 Unidentified compound C28H50O3 434.37601 434.37691 -2.1
12.923 Unidentified compound C29H48O2 428.36544 428.36556 -0.3
12.981 Unidentified compound C29H48O 412.37052 412.37035 0.4
12.985 Unidentified compound C19H32O 276.24532 276.24538 -0.2
13.016 Unidentified compound C26H46O2 390.34979 390.34984 -0.1
13.026 Unidentified compound C28H52O3 436.39166 436.39209 -1.0
13.103 Unidentified compound C19H34O4 326.24573 326.24564 0.3
13.123 Unidentified compound C23H44O3 368.32906 368.32906 0.0
13.146 Unidentified compound C30H53NO4 491.39748 491.39742 0.1
13.151 Unidentified compound C26H48O2 392.36544 392.36538 0.2
13.418 Unidentified compound C25H46O 362.35487 362.35494 -0.2
13.459 Unidentified compound C29H52O4 464.38658 464.38689 -0.7
identified as pulegone [495]. Pulegone is the main monoterpene present in the
extract and reportedly is an analgesic and psychoactive drug [496].
126
5.5.2.4.2 Phenolic and Organic acids
Trans-cinnamic acid, suberic acid, vanillic acid, iso-coumarin,
dehydroascorbic acid and several unidentified phenolic and organic acids were
present within Australian native Lemon grass.
5.5.2.4.3 Vitexin and Iso-Vitexin
Iso-vitexin, C21H20O10, eluted at 5.329 min and was identified based on the
protonated precursor ion [M+H]+ m/z 433.11295 [497] with the characteristic
fragment m/z 313.07050 resulting from the loss of 120 Da due to cross-ring sugar
cleavages [498]. Vitexin is an isomer of iso-vitexin and eluted at 5.539 min with the
protonated ion [M+H]+ m/z 433.11310. Both isomers fragmented to the most
abundant product ion m/z 283.06000 and Compound DiscovererTM predicted the
identity match at 96%. They are differentiated on the basis of a previously
documented elution pattern as iso-vitexin eluted before vitexin on a reversed phase
HPLC column [499]. Another unidentified compound with same predicted formula
C21H20O10 eluted at 6.013 min with the protonated precursor ion [M+H]+ m/z
433.11298 and the most abundant fragment ion m/z 271.06003.
The mass spectra of deprotonated vitexin has not shown any diagnostic ions
to differentiate it from iso-vitexin and previous research has demonstrated that even
during the second stage of ionization, there was an ion m/z 311 resulting exclusively
from the loss of CO for both isomers. The difficulties with using MS fragmentation
differentiation for these isomers is a good example of why the HPLC separation
pattern is important and other ionization modes should be explored that might create
different types of precursor ions with more characteristic fragmentation properties.
127
Infusions and decoctions of Cymbopogon ambiguus have been used
traditionally in Australia for the treatment of headache, chest infections and muscle
cramps [500]. Previous evidence-based research conducted to explore the
mechanism of native Lemon grass has demonstrated that eugenol, elimicin and trans
iso-elimicin are the main bioactive compounds which established the basis of the
therapeutic use for headaches via antiplatelet activity [500]. Clinical, biochemical,
and pathological findings reported in migraine have demonstrated that platelets taken
from patients have significantly higher spontaneous platelet aggregation and
adhesion than platelets from controls. Oxidative stress is a plausible unifying
principle behind the headaches and also a triggering cause of migraine attack.
Vitexin has a therapeutic role to alleviate endothelial injury [501] and protect cardiac
hypertrophy [502], treat oral cancer [503] and a regulatory effect on stress-mediated
autophagy [504]. Our study supports Griffith University researchers’ findings that
native Lemon grass is as potent as aspirin in relieving headaches and migraines [505]
not only due to the presence of antiplatelet molecules but also due to the presence of
bioactive vitexin and iso-vitexin which inhibit inflammatory pain [506] and have
previously demonstrated antinociceptives effects in mouse models with post-
operative pain [507]. Figure 5.6 summarizes the compounds identified in Lemon
grass that have been demonstrated to have a potential role in the treatment of
headaches and migranes.
MS profiling also revealed the presence of aflatoxin within the methanolic
extract and identification was predicted with high probability by Compound
DiscovererTM and confirmed by literature [508, 509]. The presence of aflatoxin G2,
eluting at 7.380 mins, demonstrates the potential health threat due to contamination
by toxigenic fungi within medicinal herbs and spices in Australia. This study
128
provides a basis for assessing the degree of potential mycotoxin contamination in
Australian native traditional medicinal plants as previously reported in natural
products [510-513].
Untargeted metabolic profiling has revealed about 2030 compounds within
Australian native Lemon grass. Unidentified terpenes, alcohols, ketones and
flavonoids constitute the major portion of the metabolic profile and contribute to the
previously demonstrated pharmacological and therapeutic potential [514].
Figure 5.6. Compounds from Lemon grass that have a potential role in the treatment of headaches and
migraines.
5.5.2.5. Australian native Desert lime
Historically, Australian native Desert lime fruit was used by indigenous
Australians as a cordial and infused drink. Several studies have previously been
conducted to explore the bioactive potential of Desert lime. As far as we know, this
is the first scientific study to reveal the extensive phytochemical profile and
129
ethnopharmacological potential of Australian native Desert lime. The analysis of
antioxidants from a complex sample of Australian native Desert lime leaves was
obtained from the RF-PCD-FRAP assay with the detection of 21 compounds (Figure
5.7) where the most intense response was observed at 11.07 min. LC-HRMS was
again employed to provided a more detailed profile with the results listed in Table
5.5. The LC-HRMS analysis found that sugars were abundantly present and the
superior total reducing capacity effect observed in the RF-PCD-FRAP assay could
be due to the large quantity of sugars in the methanolic extract.
Figure 5.7. RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Desert lime.
5.5.2.5.1 Catechin and Procyanidins
The main bioactive compounds within Desert lime are catechins and
procyanidins. As previously reported in research on reversed phase C18 column with
mixed standards, the catechins were observed to elute in the following order - gallic
130
acid, gallocatechin, epigallocatechin, catechin, epicatechin, epigallocatechin gallate,
gallocatechin gallate and epicatechin gallate [515, 516].
The compound C15H14O7 eluted at 1.751 min and was identified as
gallocatechin. The protonated precursor ion m/z 307.08115 fragmented to form the
131
Table. 5.5. Structural elucidation of antioxidants within Australian native Desert lime. LC-HRMS
(min) Tentative Assignments Molecular Formula Exact mass Measured mass Error (ppm)
0.508 Quinic acid C7H12O6 192.06342 192.06265 4.0
0.533 Pyrogallol C6H6O3 126.03171 126.03184 -1.0
0.551 Shikimic acid C7H10O5 174.05285 174.05221 3.7
1.101 Pantothenic acid C9H17NO5 219.11070 219.11061 0.4
1.130 Coumaric acid C6H8O3 128.04736 128.04750 -1.1
1.519 2-Isopropylmalic acid C7H12O5 176.06850 176.06775 4.3
1.731 Protocatechuic acid C7H6O4 154.02663 154.02677 -0.9
1.751 Gallocatechin C15H14O7 306.07399 306.07384 0.5
1.761 (-) Epigallocatechin C15H14O7 306.07399 306.07384 0.5
2.908 Salicylic acid C7H6O3 138.03171 138.03160 0.8
3.034 Unidentified Compound C9H16O5 204.09980 204.09933 2.3
3.497 (-) Epigallocatechin C15H14O7 306.07399 306.07405 -0.2
3.579 Catechin C15H14O6 290.07907 290.07881 0.9
3.849 Esculetin C9H6O4 178.02663 178.02655 0.4
3.981 Procyanidin B1 C30H26O12 578.14249 578.14275 -0.4
3.992 Brevifolincarboxylic Acid C13H8O8 292.02196 292.02211 -0.5
4.028 Aucubin C15H22O9 346.12643 346.12639 0.1
4.081 Gallic acid C7H6O5 170.02155 170.02165 -0.6
4.237 Caffeic acid C9H8O4 180.04228 180.04160 3.8
4.307 Epicatechin C15H14O6 290.07907 290.07896 0.4
4.386 Unidentified Compound C10H20O3 188.14126 188.14127 -0.1
4.389 Unidentified Compound C10H16O 152.12012 152.12016 -0.3
4.681 Ferulic acid C10H10O4 194.05793 194.05799 -0.3
4.759 Ionone glycoside C19H32O8 388.20976 388.20963 0.3
4.772 Eugenol C10H12O2 164.08374 164.08377 -0.2
4.889 Ionone glycoside C19H32O8 388.20976 388.20964 0.3
4.905 Catechin gallate C22H18O10 442.09005 442.08988 0.4
4.931 Taxifolin C15H14O7 306.07399 306.07397 0.1
5.019 Unidentified Compound C12H22O4 230.15183 230.15173 0.4
5.040 Ascorbic acid C6H8O6 176.03212 176.03129 4.7
5.169 Picrocrocin C16H26O7 330.16789 330.16765 0.7
5.370 Combretol-3,7,3',4',5'-O-methylation of myricetin
C21H20O12 464.09554 464.09578 -0.5
5.364 Suberic acid C8H14O4 174.08923 174.08840 4.8
5.370 Hyperoside C21H20O12 464.09554 464.09578 -0.5
5.523 Unidentified Compound C17H28O10 392.16830 392.16853 -0.6
5.862 Myricetin C15H10O8 318.03748 318.03743 -0.1
5.926 Unidentified Compound C16H28O7 332.18354 332.18355 0.0
5.968 Quercitrin C21H20O12 464.09554 464.09557 -0.1
5.992 Kaempferol-7-O-Glucoside C21H20O11 448.10062 448.10066 -0.1
6.006 Unidentified Compound C10H16O2 168.11504 168.11512 -0.5
6.053 Rhamnetin 3 glucoside C22H22O12 478.11119 478.11116 0.1
6.241 Flavonoids C15H10O7 302.04271 302.04262 0.3
6.280 Quercetin 3-(6’-O-Caffeoyl)-beta-D-glucopyranoside
C30H26O15 626.12725 626.12733 -0.1
6.312 Procyanidin B2 C30H26O12 578.14249 578.14273 -0.4
6.312 Azelaic acid C9H16O4 188.10488 188.10417 3.8
6.324 7-Hydroxy Coumarin C9H6O3 162.03171 162.03174 -0.2
6.354 Linarionoside A C19H34O7 374.23049 374.23033 0.4
6.413 Picrionoside B C19H32O7 372.21484 372.21486 -0.1
6.518 Apigenin 7-O-glucoside C21H20O10 432.10570 432.10564 0.1
6.587 Flavonoid C22H22O11 462.11627 462.11608 0.4
6.624 Unidentified Compound C15H20 200.15650 200.15640 0.5
6.634 Unidentified Compound C20H36O9 420.23598 420.23625 -0.6
6.612 Abscisic acid C15H20O4 264.13618 264.13620 -0.1
6.663 Quercetin C15H10O7 302.04269 302.04270 0.0
6.843 Cyanidin C15H10O6 286.04777 286.04760 0.6
132
Table. 5.5.(cont.) Structural elucidation of antioxidants within Australian native Desert lime. LC-HRMS
(min) Tentative Assignments Molecular Formula Exact mass Measured mass Error
(ppm)
6.844 Scutellarein C15H12O6 288.06342 288.06346 -0.1
6.845 Luteolin C15H10O6 286.04777 286.04783 -0.2
6.930 Unidentified Compound C10H18O2 170.13069 170.13073 -0.2
6.794 Unidentified Compound C11H20O3 200.14126 200.14129 -0.1
6.938 Unidentified Compound C10H12O2 164.08374 164.08388 -0.9
7.143 Unidentified Compound C11H20O2 184.14634 184.14639 -0.3
7.165 Sebacic acid C10H18O4 202.12053 202.11987 3.3
7.202 Unidentified Compound C17H22O4 290.15183 290.15172 0.4
7.347 Unidentified Compound C11H20O3 200.14126 200.14127 0.0
7.527 Unidentified Flavonoid C15H10O6 286.04777 286.04780 -0.1
7.464 Isoeugenol C10H12O2 164.08374 164.08388 -0.9
7.812 Unidentified Compound C11H20O2 184.14634 184.14634 0.0
7.886 Methoxylated Flavonol C19H18O8 374.10021 374.10011 0.3
8.036 Unidentified Compound C15H20 200.15650 200.15654 -0.2
8.181 Limonene C10H14 134.10955 134.10958 -0.2
8.129 Terpenoid C10H14O 150.10447 150.10453 -0.4
8.267 Hyperinone C19H20O8 376.11586 376.11565 0.6
8.340 Unidentified Compound C11H20O2 184.14634 184.14639 -0.3
8.353 Unidentified Flavonoid C15H14O6 290.07405 290.07907 -17.3
8.353 Unidentified Compound C19H20O7 360.12112 360.12094 0.5
8.367 Unidentified Compound C10H14 134.10955 134.10962 -0.5
8.459 Methoxylated Flavonol C19H18O8 374.10011 374.10021 -0.3
8.769 Methylated Flavonol C19H18O7 358.10515 358.10529 -0.4
9.111 Unidentified Compound C16H22O2 246.16196 246.16199 -0.1
9.398 Phytosphingosine C18H39NO3 317.29289 317.29301 -0.4
9.432 Unidentified Compound C16H20O 228.15140 228.15142 -0.1
9.350 Unidentified Compound C25H28O6 424.18892 424.18862 0.7
9.954 Bilobol-Alkyl Resorcinol C21H34O2 318.25582 318.25589 -0.2
9.649 Unidentified Compound C17H24O3 276.17260 276.17256 0.1
9.767 Unidentified Compound C20H32O4 336.22987 336.23008 -0.6
9.833 Unidentified Compound C15H20 200.15654 200.15650 0.2
10.017 Unidentified Compound C21H32O4 348.22997 348.23008 -0.3
10.046 Unidentified Compound C19H30O5 338.20903 338.20935 -0.9
10.141 Unidentified Compound C11H20O3 200.14124 200.14126 -0.1
10.142 Unidentified Compound C21H32O 300.24526 300.24532 -0.2
10.430 Unidentified Compound C15H22O 218.16710 218.16707 0.1
10.436 Unidentified Compound C16H24O 232.18270 232.18272 -0.1
11.049 Bilobol –Alkyl resorcinol C21H34O2 318.25578 318.25589 -0.3
11.141 Unidentified Compound C16H22O 230.16710 230.16707 0.1
11.211 Unidentified Compound C20H30O 286.22972 286.22967 0.2
11.235 Cadalene C15H18 198.14093 198.14085 0.4
11.242 Unidentified Compound C15H22O 218.16710 218.16707 0.1
11.360 Unidentified Compound C29H50O4 462.37100 462.37093 0.2
11.527 Unidentified Compound C20H30O 286.22972 286.22967 0.2
11.530 Unidentified Compound C15H22O 218.16710 218.16707 0.1
11.976 1-Stearoylglycerol C21H42O4 358.30840 358.30833 0.2
12.258 Stigmasterol C29H48O 412.37053 412.37052 0.0
12.296 Unidentified Compound C27H36O 376.27675 376.27662 0.3
12.817 Unidentified Compound C29H50O4 462.37120 462.37093 0.6
12.586 Elasterol C29H48O 412.37053 412.37052 0.0
12.953 Unidentified Compound C29H50O4 462.37120 462.37093 0.6
13.086 Triterpene C29H48O3 444.36060 444.36036 0.5
13.153 Citroneyllyl oleate C28H52O2 420.39711 420.39674 0.9
133
most abundant diagnostic product ion m/z 139.01080. Mass fragment ions m/z
261.07693, m/z 219.06580, m/z 139.03923 and m/z 125.02337 were observed and
correspond to the loss of CO2, C4H6O2, C8H6O4 and C9H8O4, respectively. In
accordance with previous characterization, two adjacent ions with the same mass
were identified as (−)-gallocatechin and (−)-epigallocatechin based on the
chromatographic elution order for reversed phase HPLC [515, 516].
Procyanidins, formed due to linkage of catechin molecules, were also
detected. A compound eluted at 3.981 min, with the deprotonated precursor ion [M-
H]- m/z 577.13544 which further fragmented to product ions m/z 125.02336, m/z
289.07193, m/z 407.07781, m/z 425.08752 and m/z 451.10425. This fragmentation
pattern is consistent with the previously studied procyanidin B [517-522]. Likewise,
the mass spectra from the peak eluting at 5.129 min had a similar molecular ion and
fragmentation pattern [523, 524], which led us to believe that this compound is also
a B-type compound [470, 518-520, 525, 526]. Fragmentation pathways of
procyanidin m/z 577 include the quinone methide cleavage of the interflavanoid
bond leading to m/z 289.07193 [527], heterocyclic ring fission resulting in m/z
451.10425 [528] and retro Diels-Alder fission with m/z 425.08752 of the
heterocyclic ring system subunits which are distinctive of proanthocyanidins [518,
527, 529]. In these compounds the cleavage of the upper interflavanoid bond is
observed with mass fragment ions m/z 289 [521].
The difference between A-type and B-type dimers is due to an additional C-
O-C linkage within Procyanidin B compounds. In B-type procyanidins, quinone
methide cleavage produces fragment ions m/z 289 and m/z 287 while in case of A-
type procyanidins fragment ions m/z 289 and m/z 285 are produced and this
difference of 4 Da is due to an additional C-O-C linkage within procyanidin B
134
compounds [530]. These compounds have already been reported from this source
[531].
5.5.2.5.2 Flavonoid and Flavonoid Glycosides
Quercetin, [M-H]- m/z 302.04262, was identified at 6.663 min and further
fragmented to the diagnostic fragment ion m/z 178.99794, due to the removal of
C8H3O5, as well as other characteristic fragments [M-H-C6H6-CO2-CO]- m/z
151.00256, [M-H-C6H6-2CO2-CO]- m/z 107.01373, m/z 112.98438 and m/z 89.02314
were also observed [532, 533].
Hyperoside, quercetin-3-O-galactoside, was identified with the characteristic
deprotonated molecule [M-H]- m/z 463.08856 and the ion corresponding to the
deprotonated aglycone [A-H]- m/z 301.03491 due to neutral loss of 162 Da (loss of
tetrahydroxylated hexose) [534] which is formed by the loss of the sugar moiety
from the glycoside [535]. The observed fragment ions at m/z 178.99760 and m/z
151.00260 correspond to the quercetin fragmentation pattern [536].
Myricetin was identified at 5.862min with a 94% percent match by
Compound DiscovererTM and was confirmed using literature [537]. Esculetin eluted
at 3.849 mins and was identified with precursor ion [M-H]- m/z 175.01859 and the
most abundant fragment ion [M-H-CO2]- m/z 133.0852 [538], m/z 130.08633, m/z
101.02313, m/z 105.03313 and m/z 92.99463 [539]. Esculetin was also identified in
Gumbi Gumbi and Lemon with similar retention times.
Cyanidin, with protonated precursor ion [M+H]+ m/z 271.06140, was
predicted with a 97% match using Compound DiscovererTM. Luteolin was also
identified with a 95% match and confirmed using a literature source. Taxifolin,
kaempferol-7-O-glucoside, rhamnetin 3 glucoside, apigenin 7-O-glucoside,
135
quercitrin, quercetin 3-(6’-O-caffeoyl)-beta-D-glucopyranoside, orientin, apocynin
and p-coumaric glucoside were tentatively assigned as previously reported [540-
543]. Most of these compounds were identified for the first time in Australian native
Desert lime.
5.5.2.6. Australian native Tasmannia lanceolata
The exceptionally high antioxidant content of Tasmannia lanceolata has been
previously studied and is considered to be four-fold higher than blueberries [224,
544]. The antioxidant capacity of T. lanceolata was evaluated using the RF-PCD-
FRAP method and the chromatogram (Figure 5.8) showed intense antioxidant peaks
at 9.4 mins and 11.0 mins. Owing to the complex nature of the sample, it is clear that
there are numerous unresolved peaks and significant further separation will be
required. For instance, plants extracts presented here have provided significant
chemical information but multidimensional separation can be employed to achieve
comprehensive analysis [545, 546]. Although LC-HR-MS spectra has provided an
improved and intense response as shown in Figure 5.7. but multidimensional
separation techniques are often used to increase resolution and separation of peaks,
which outweigh the desire for fast separations [547]. Further analysis using LC-
HRMS is reported in Table 5.6.
136
Figure 5.8. RF-PCD-FRAP Assay showing antioxidant capacity of Australian native
Tasmannia lanceolata
Figure. 5.9. High resolution mass spectrometry analysis of Australian native
Tasmannia lanceolata
5.5.2.6.1 Polygodial and Sesquiterpenes
137
The molecular formula, C15H22O2, was determined for a compound eluting at
9.106 min and was identified as polygodial [548].
Table 5.6. Structural elucidation of Antioxidants within Australian native Tasmannia lanceolata. LC-HRMS
(min) Tentative Assignments Molecular
Formula Exact mass Measured mass Error (ppm)
0.426 Ethyl Gallate C6H15NO6 197.09010 197.08997 0.7
0.433 Unidentified compound C8H14O8 238.06833 238.06891 -2.4
0.511 Quinic acid C7H12O6 192.06261 192.06342 -4.2
0.518 Unidentified compound C9H18O10 286.09004 286.09005 0.0
0.526 Malic acid C4H6O5 134.02152 134.02155 -0.2
0.574 Shikimic acid C7H10O5 174.05221 174.05265 -2.5
0.619 Unidentified compound C8H16O8 240.08412 240.08456 -1.8
0.664 Unidentified compound C6H6O6 174.01564 174.01647 -4.8
0.700 Unidentified compound C13H27NO11 373.15840 373.15847 -0.2
0.719 Unidentified compound C6H6O5 158.02147 158.02155 -0.5
0.740 Unidentified compound C6H12O5 164.06857 164.06850 0.4
0.778 Unidentified compound C9H10O6 214.04767 214.04777 -0.5
0.813 Unidentified compound C7H14O6 194.07909 194.07907 0.1
0.827 Nicotinamide C6H6N2O 122.04824 122.04802 1.8
0.906 Alpha-hydroxy glutaric acid C5H8O5 148.03724 148.03720 0.3
0.971 Unidentified compound C12H16O8 288.08440 288.08456 -0.6
1.005 Unidentified compound C6H13NO3 147.08951 147.08956 -0.3
1.110 Unidentified compound C14H21NO9 347.12160 347.12168 -0.2
1.112 Unidentified compound C6H10O3 130.06303 130.06301 0.2
1.142 Unidentified compound C7H17NO5 195.11081 195.11070 0.6
1.189 Unidentified compound C10H20O7 252.12089 252.12094 -0.2
1.349 Unidentified compound C10H20O7 252.12089 252.12094 -0.2
1.434 Unidentified compound C13H19NO9 333.10576 333.10603 -0.8
1.523 Unidentified compound C5H11NO5 165.06395 165.06375 1.2
1.728 Dihydroxybenzoic acid C7H6O4 154.02665 154.02663 0.1
2.429 Vanillin C8H8O3 152.04740 152.04736 0.3
2.667 D-Pantothenic acid C9H17NO5 219.11020 219.11070 -2.3
2.708 Pyrogallol C6H6O3 126.03188 126.03171 1.3
2.915 Unidentified compound C7H6O3 138.03160 138.03171 -0.8
3.603 Catechin C15H14O6 290.07894 290.07907 -0.4
3.801 Camphoric acid C10H16O4 200.10490 200.10488 0.1
3.830 Chlorogenic acid C16H18O9 354.09512 354.09513 0.0
3.924 Unidentified compound C9H8O4 180.04243 180.04228 0.8
4.051 Unidentified compound C9H8O5 196.03659 196.03720 -3.1
4.124 Caffeic acid-glucuronide C15H16O10 356.07438 356.07439 -0.1
4.289 Unidentified compound C15H22O7S 346.10860 346.10866 -0.2
4.461 Unidentified compound C9H8O4 180.04243 180.04228 0.8
4.478 Kaempferol 3-O-rutinoside C27H30O15 594.15886 594.15855 0.5
4.518 Genipin C11H14O5 226.08408 226.08415 -0.3
4.618 Europine N-Oxide C16H27NO7 345.17885 345.17879 0.2
4.631 Hyperoside C21H22O12 466.11121 466.11119 0.0
138
Table 5.6.(cont.) Structural elucidation of Antioxidants within Australian native Tasmanian Lanceolata. LC-HRMS
(min) Tentative Assignments Molecular Formula Exact mass Measured
mass Error (ppm)
4.685 Unidentified compound C16H24O7 328.15228 328.15224 0.1 4.914 Unidentified compound C15H12O8 320.05320 320.05326 -0.2 5.069 Unidentified compound C9H8O2 148.05247 148.05244 0.2 5.128 8-Hydroxy Quinoline C9H7NO 145.05283 145.05276 0.5 5.174 Coumarin derivative C9H6O4 178.02591 178.02663 -4.0 5.423 Unidentified compound C9H8O6 212.03158 212.03212 -2.5 5.600 Unidentified compound C28H26N4O12 610.15317 610.15479 -2.7
5.615 Quercetin -3-β-D glucoside C27H30O15 594.15857 594.15855 0.0
5.828 Apigenin-8-C glucoside C21H20O10 432.10561 432.10570 -0.2
6.072 Quercitrin C21H20O11 448.10051 448.10062 -0.2
6.123 Unidentified compound C29H40N4O12 636.26326 636.26434 -1.7
6.342 Azelaic acid C9H16O4 188.10422 188.10488 -3.5
6.426 Kaempferol 3-glucoside C21H20O11 448.10051 448.10062 -0.2
6.645 Quercetin C15H10O7 302.04289 302.04269 0.7
6.871 Unidentified compound C15H22O5 282.14674 282.14675 0.0
6.849 Luteolin C15H10O5 286.04775 286.04765 0.3
7.093 Abscisic acid C15H20O4 264.13624 264.13618 0.2
7.228 Unidentified compound C22H34O11 474.21037 474.21017 0.4
7.346 Apigenin C15H10O5 270.05296 270.05285 0.4
7.770 Unidentified compound C30H47NO8 549.33031 549.33021 0.2
7.949 Unidentified compound C15H20O5 280.13117 280.13110 0.2
8.035 Unidentified compound C12H16O3 208.10998 208.10996 0.1
8.143 Unidentified compound C15H22O3 250.15694 250.15691 0.1
8.177 Flavonoid C16H12O6 300.06342 300.06339 0.1
8.273 Unidentified compound C15H22O4 266.15173 266.15183 -0.4
8.209 Matricin C17 H22 O5 306.14667 306.14675 -0.3
8.302 Unidentified compound C14H20O4 252.13618 252.13628 -0.4
8.360 Unidentified compound C16H24O4 280.16734 280.16748 -0.5
8.500 Unidentified compound C14H23NO3 253.16778 253.16781 -0.1
8.693 Unidentified Isoflavone C16H12O5 284.06865 284.06850 0.5
8.709 Unidentified compound C15H25NO3 267.18356 267.18346 0.4
8.714 Unidentified compound C30H47NO6 517.34068 517.34037 0.6
8.731 Unidentified compound C17H29NO3 295.21470 295.21476 -0.2
8.776 Unidentified compound C19H30O9 402.18922 402.18903 0.5
8.806 Unidentified compound C15H22O3 250.15689 250.15691 -0.1
8.823 Unidentified compound C30H44O5 484.31881 484.31890 -0.2
8.902 Unidentified compound C14H22O3 238.15673 238.15691 -0.8
8.963 Unidentified compound C36H58O11 666.39845 666.39797 0.7
9.022 Unidentified compound C14H22O3 238.15673 238.15691 -0.8
9.067 Unidentified compound C15H22O4 266.15193 266.15183 0.4
9.106 Polygodial C15H22O2 234.16203 234.16199 0.2
9.268 Unidentified compound C16H18O9 354.09512 354.09513 0.0
9.317 Unidentified compound C15H22O2 234.16202 234.16199 0.1
9.337 Unidentified compound C15H25NO2 251.18866 251.18854 0.5
9.426 Unidentified compound C18H24O4 304.16725 304.16748 -0.8
9.495 Unidentified compound C16H26O7 330.16801 330.16789 0.4
9.453 Rotundone C15H22O 218.16711 218.16714 0.5
9.710 Baicelien C15H10O5 270.05302 270.05285 0.6
9.771 Unidentified compound C16H24O3 264.17255 264.17256 0.0
9..826 Unidentified compound C14H22O2 222.16173 222.16199 -1.2
10.164 Unidentified compound C15H20O 216.15151 216.15142 0.4
10.370 Unidentified compound C15H20O 216.15151 216.15142 0.4
10.477 Unidentified compound C18H32O4 312.23030 312.23008 0.7
11.200 Unidentified compound C18H41N8O4P 464.29900 464.29886 0.3
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Table 5.6 (cont.) Structural elucidation of Antioxidants within Australian native Mountain pepper.
LC-HRMS (min)
Tentative Assignments Molecular Formula
Exact mass Measured mass Error (ppm)
11.347 Unidentified compound C18H34O3 298.25080 298.25081 0.0
11.465 Unidentified compound C25H41N8O2P 516.30930 516.30902 0.5
11.48 Unidentified compound C24H34O3 370.25080 370.25081 0.0
11.519 Unidentified compound C19H34O4 326.24580 326.24573 0.2
11.749 Unidentified compound C24H48O3 384.36050 384.36036 0.4
11.892 Citral C10H14O 150.10450 150.10447 0.2
12.151 Gamma-tocotrienol C28H42O2 410.31860 410.31849 0.3
13.230 Unidentified compound C27H46N4 426.37140 426.37225 -2.0
13.288 Alpha-tocotrienol C29H44O2 424.33425 424.33414 0.3
13.423 Unidentified compound C27H54O4 442.40250 442.40223 0.6
13.621 Unidentified compound C31H59N8O3P 622.44526 622.44479 0.8
13.78 Unidentified compound C23H46O3 370.34480 370.34471 0.2
Another compound with predicted formula C15H22O eluted at 9.453 min and
was tentatively identified as rotundone, a commonly found sesquiterpene in black
pepper [549]. Although these sesquiterpenes are volatile and are commonly analyzed
with gas chromatography, there are some traces that can be identified by high
resoluttion mass spectrometry [550]. Black peppers are considered as a major source
of sesquiterpenes [551, 552].
5.5.2.6.2 Flavonoids and Glycosides
Apigenin, C15H10O5, was identified based on the deprotonated precursor ion
[M-H]- m/z 269.04568 with the fragment ion [M-H-CO2]- m/z 225.14917 [185, 186]
and other characteristic fragment ions m/z 207.13849 and m/z 177.12778 [553].
Another compound with similar molecular formula eluted at 9.710 min and was
tentatively assigned as the flavanoid, baicalein.
Quercetin, C15H10O7, was identified with a deprotonated precursor ion [M-
H]- m/z 301.03537 eluting at 6.645 min that fragmented further to m/z 151.00270,
m/z 178.99783 and m/z 273.04077 as predicted by Compound DiscovererTM with a
140
93% match and against a previous study [554]. This compound was also found in
Desert lime with a retention time of 6.663 min. Another compound with the same
molecular formula eluted at 6.051 mins with the most abundant fragment ion m/z
165.01839 but could not be identified.
Kaempferol 3-O-rutinoside, C27H30O15, a bitter tasting flavanols glycoside
eluting at 4.478 mins was identified with precursor ion peak [M+H]+ m/z 595.16614
and characteristic fragment ion m/z 287.05484 as previously reported in similar
studies [555].
The flavonoids, luteolin 7-O-glucoside (Orientin) eluting at 6.426 min and
luteolin 4-O-glucoside at 6.072 min have a similar predicted formula C21H20O11 and
precursor ion [M+H]+ m/z 449.10770. The most abundant diagnostic fragment ion
observed for both luteolin glycosides was C15H9O6 m/z 287.05496. The
identifications of these isomers were assigned based on the reversed phase C18
elution pattern as previously reported [436, 555].
The compound eluting at 5.828 min was identified as apigenin-8-C-glucoside
due to the observed precursor ion [M+H]+ m/z 433.11310 and characteristic fragment
ion m/z 313.07050 with the loss of 120 Da due to cross-ring sugar cleavage [498].
Quercetin-3β-D-glucoside eluted at 5.615 min and was identified from the
protonated precursor ion [M+H-H2O]+ m/z 449.10785 that further dissociated to the
most abundant fragment ion m/z 303.04987. Another compound at 4.631 min, with
molecular formula C21H22O12, was tentatively assigned as quercetin-3-O-galactoside
(hyperoside) [540, 556].
141
5.5.2.6.3 Miscellaneous Compounds
The phenolic acids azelaic acid, camphoric acid, chlorogenic acids, quinic
acid, malic acid and shikimic acid were identified with good probability by
Compound DiscovererTM and previous literature [557]. Bioactive lipids and alkaloids
were also abundantly present within phytochemical profile of the T. lanceolata.
Monoterpenes and sesquiterpenes were the main constituents obtained from the
untargeted metabolic profile and will need further data mining and interpretation.
This study has revealed that T. lanceolata has anticancer, immunopharmacological,
cardioprotective, dermatological and neuroprotective potential based on the
identified compounds.
5.5.2.7. Australian native Wattle seeds
RF-PCD-FRAP analysis of the Wattle seed extract (Figure 5.9) showed that
this sample is not a very promising source of antioxidant activity. The main peaks
are very close to the void time and therefore are likely to be weakly-retained polar
antioxidant compounds. Previous studies conducted with Wattle seed has shown the
presence of phenolic acids and flavonoids with the LC-HRMS results from this study
listed in Table 5.7.
5.5.2.7.1 Phenolic Acids
Untargeted metabolic profiling of wattle seeds with LC-HRMS indicated the
presence of gallic acid, ferulic acid, malic acid and citric acid. Ferulic acid,
C10H10O4, eluted at 4.681 min and was identified from the precursor ion [M-H]- m/z
193.04997 and the diagnostic fragment ion m/z 149.05977. Another compound with
142
the same predicted formula and fragmentation pattern eluted at 4.894 min and was
identified as iso-ferulic acid based on literature elution data [558-560].
Figure 5.10. RF-PCD-FRAP assay of Australian native Wattle seed.
5.5.2.7.2 Bioactive Lipids
Fatty acids and their bioactive derivatives are the major constituent of the
phytochemical profile of Wattle seeds. Hexadecanedioic acid, 3-hydroxy myristic
acid, oleamide, linoleic acid and 2-hydroxy docosanoic acid were identified by
Compound DiscovererTM and confirmed with reference literature [561]. The
phospholipid and lipidomic profile of Wattle seed are still unidentified but it is
certain that Wattle seeds are a promising source of therapeutic oil.
5.5.2.7.3 Miscellaneous Compounds
The bioactive nucleosides, tannins and antharnilic-type compounds, were
also identified within the phytochemical profile. These compounds might be
143
responsible for the previously demonstrated antifungal, antiviral and
antiinflammatory activities of Wattle seeds [562-564].
Table.5.7. Structural elucidation of antioxidants with Australian native Wattle
seeds.
LC-HRMS (min)
Tentative Assignments Molecular Formula
Exact mass Measured mass Error (ppm)
0.491 Antharnilic acid C7H7NO2 137.04768 137.04761 0.5
0.511 Quinic acid C7H12O6 192.06342 192.06287 2.9
0.516 Malic acid C4H6O5 134.02155 134.02154 0.1
0.518 Citric acid C6H8O7 192.02704 192.02632 3.7
0.530 Alpha hydroxy glutaric acid C5H8O5 148.03720 148.03650 4.7
0.631 Adenine C5H5N5 135.05443 135.05450 -0.5
0.906 Unidentified compound C7H6O3 138.03171 138.03176 -0.4
0.929 Unidentified compound C8H9NO2 151.06314 151.06334 -1.3
1.387 Adenosine C10H13N5O4 267.09678 267.09648 1.1
2.937 p-Hydroxy benzoic acid C7H6O5 170.02158 170.02155 0.2
3.203 L-Threo-3-phenyl serine C9H11NO3 181.07391 181.07362 1.6
3.468 D-Pantothenic acid 4”-0-beta
glucose
C15H27NO10 381.16355 381.16371 -0.4
4.037 3-Hydroxyanthranilic acid C7H7NO3 153.04260 153.04268 -0.5
4.472 4-p-Coumaroylquinic acid C16H18O8 338.10021 338.10051 -0.9
4.681 Ferulic acid C10H10O4 194.05793 194.05760 1.7
4.686 Unidentified compound C10H10O3 178.06301 178.06232 3.9
4.789 Unidentified compound C10H10O3 178.06301 178.06330 -1.6
4.894 Iso Ferulic acid C10H10O4 194.05793 194.05758 1.8
4.943 Unidentified compound C11H10O5 222.05285 222.05241 2.0
5.053 Sinapinic acid C11H12O5 224.06850 224.06817 1.5
6.681 Unidentified compound C10H12O4 196.07358 196.07313 2.3
7.142 Apocynin C10H13NO2 179.09464 179.09402 3.5
8.332 Rosamanol C20H26O5 346.17805 346.17821 -0.5
10.192 Hexadecanedioic acid C16H30O4 286.21443 286.21467 -0.8
10.46 3- Hydroxy myristic acid C14H28O3 244.20386 244.20382 0.2
11.413 Oleamide C18H35NO 281.27186 281.27169 0.6
11.042 Unidentified compound C18H32O3 296.23516 296.23530 -0.5
11.520 Linoleic acid C18H32O2 280.24024 280.24043 -0.7
13.205 2-Hydroxydocosanoic acid C22H44O3 356.32906 356.32915 -0.3
The identified compounds are ubiquitous primary metabolites, which are
important for the basic function of the living cells, and secondary metabolites, which
might be sample-specific. The proximity of accurate mass (experimentally
determined) to exact mass (calculated mass of ion whose elemental formula, isotopic
composition and charge state are known) was observed for all bioactive compounds.
The experimentally measured values were scrutinized using theoretical calculations
of the most abundant isotope of the chemical formula at high resolution and have
144
revealed that the error of measurement was less than 3 ppm for most of the
compounds.
5.6 Conclusion
The properties of Australian native medicines have been established over
many generations of trial and error. Furthermore, these native plants have remained a
popular resource for indigenous Australians. However, a major drawback for their
use, until now, has been a lack of evidence of the bioactives chemicals that they
contain and potentially mediate pharmacological effects for therapeutic purposes.
RF-PCD FRAP assay and untargeted metabolomics have revealed a phytochemical
picture of these products and identified many potentially active compounds in these
extracts.
The phytochemical chemical profile of native species composed
predominantly of flavonoids, flavonols, anthocyanin, alkaloids, phenolic acids and
their hydrolysable tannins. Vitamins and their analogues are also present in these
sources. Cholorogenic acid and their esters are abundantly present in Quandong.
Gumbi Gumbi has provided scientific evidence for their traditional use for cancer.
Lemon grass metabolic profile containing flavonoids and phenolic acids has
indicated its potential value in cancer and pain disorders. Pyrogallol and polygodial,
previously known strong antimicrobial, has provided scientific reason for Tasmanian
pepper traditional use in rheumatoid arthritis due to microbes. Procyanidins and
catechins have constituted the major portion of the bioactive profile of desert lime. In
general, all samples have antioxidants profiles with known therapeutic potential in
cardiovascular, neurodegenerative and other chronic diseases.
145
These advanced approaches can be further employed for the evaluation of
pre-clinical and clinical studies. These results will thus provide a promising
contribution toward efforts to accelerate the future development of phyto-
pharmaceuticals.
146
.
Chapter 6
Conclusion and Future Directions
147
6.1. Conclusions
6.1.1. Scope of the RF-PCD-FRAP assay for Natural
Products Screening
The quest to find a fast and comprehensive characterization method for the
analysis of complex natural extracts is an important area of science and is driven by
numerous factors, such as the search for new medical drugs, nutraceuticals and
forensic analysis. The research in this thesis studied aspects of antioxidant
characterization, principally aimed at exploiting new analytical approaches to yield
rapid analysis with characterization by high-resolution mass spectrometry.
Reaction flow chromatography has the ability to overcome inefficiencies due
to high volume reaction coils that are typically used in conventional post-column
reaction systems by using more efficient mixing with resultant improvement in the
signal-to-noise response while maintaining separation efficiency. Assessment of two
methods of HPLC-PCD antioxidant analysis based on the FRAP assay, in both
conventional and reaction flow PCD modes, found that the reaction flow technique
demonstrated significant advantages over the conventional technique in terms of
signal-to-noise, linear range, precision and observed separation efficiency. The RF-
PCD-FRAP assay improved the separation and resolution but the individual response
factors for the analysis still needs further investigation; this would require a critical
study of the reaction kinetics for each antioxidant.
The present study investigated the application of the RF-PCD FRAP assay as
a tool for the screening of antioxidants within Australian mushrooms and native
148
plants. The present work is one of the first studies on the extensive antioxidant and
bioactive profile of Australian native plants. These results have enhanced the validity
of traditional medicines, highlighting the Australian environment as a global
biodiversity hotspot.
6.1.2. Ethnopharmacological potential of Australian
mushrooms
The RF-PCD-FRAP assay enabled rapid antioxidant profiling of Australian
mushrooms and the presence of phenolic acids, vitamins, Ergosterol, its derivatives
and Ergosterol peroxide within edible mushrooms was determined using LC-HRMS.
An untargeted metabolic profiling approach not only indicated the presence of
antioxidants but also demonstrated edible mushrooms as a source of anticancer and
anti-inflammatory bioactive compounds. This suggests that commercially available
mushrooms in Australia might provide comprehensive protection from oxidative
stress and possibly have more pronounced health benefits. Further data mining has
the potential to explore unknown bioactive within mushrooms.
6.1.3. Ethnopharmacological potential of Australian Native
Plants
Bench level bio-guided and cell-based assays involve metabolite–cell
interactions with the extract rather than individual compounds. Untargeted
metabolomics is a promising approach for the discovery and characterization of
bioactive metabolites from plant extracts. In the present study, phytochemical
screening of methanol extracts showed the presence of alkaloids, steroids,
149
flavonoids, terpeniods, nonterpenoids, glycosides and some other non-classified
chemicals. Mass spectrometry was used for the identification of the bioactive
compounds but nuclear magnetic resonance is required for the structural elucidation
of the compounds isolated from various Australian native plants.
The phytochemical profile of the Australian native Lemon Grass,
traditionally used for the treatment of headaches and migraines, was explored and
the constituents responsible for this activity may have been identified.
Psychopharmacological studies should be conducted to explore clinical uses of
Australian native Lemon Grass. The identification of compounds possessing
anticancer potential from Australian native Lemon Grass is reported for the first time
in this thesis. Likewise, compounds in Tasmanian Pepper were identified that could
be promising candidates for immunotherapy. Bioactive compound profiling of
Desert Lime and Quandong provided valuable information about potential
applications in dermatology. Gumbi is a promising candidate for photodynamic
therapy and tumor treatment and can be applied to treat various diseases such as
psoriasis, rickets and vitiligo, and skin cancer. Saltbush extract has revealed its
promising scope in diabetes and dermatological applications such as antioxidant,
anti-inflammatory and hyperpigmentation disorders. An evidence-based approach
provided scientific validation for the use of these plants by Aboriginal people.
Molecular pharmacology and pharmacogenomics of these traditional medicinal
extracts need to be further explored to reveal their mode of action and mechanism.
150
6.2. Future directions
Reaction flow chromatography incorporated into a 2D HPLC-AFT system
could be a future direction of this research. Multidimensional separation of
compounds will be possible with high sample volumes in the AFT-MS setup. In this
thesis, detailed investigations have revealed that the direct coupling of reaction flow
chromatography with high-resolution mass spectrometry would be an ideal approach
for sample characterization. An untargeted metabolomic approach provided
significant results and enabled a much more efficient and far more accurate route to
unknown compound identification. Compound mapping of extracts exposes the
ethno pharmacological potential of plants by directly revealing the identities and
biological functions of individual bioactive compounds. The detailed structural and
functional information offered by this tool may help to improve the integration of
natural products with modern high content, high-throughput screening and provide
an additional strategy for the discovery of the next generation of natural product-
inspired drug leads and chemical probes within neutraceutical industry.
Greater understanding of the reaction kinetics of individual bioactives will
inform the apparently incoherent body of measurements that surrounds the various
assays developed to measure the antioxidant properties of samples. Multiplexing
electrochemical techniques with reaction flow chromatography should be a useful
approach in the critical investigation of reaction kinetics for antioxidants.
Furthermore, this study has confirmed the value of Australian traditional
medicinal plants as novel sources of bioactive compounds for medicinal and drug
development applications. It should encourage further investigations to discover
additional bioactive compounds using different sources of Australian native
151
traditional medicinal plants. Indigenous people have used these traditional medicinal
native plants for thousands of the years and phytochemical profiling of these natural
remedies should reveal that these native plants not only act prophylactically but also
help to alleviate the symptoms of many diseases. An important future scope of this
research is to develop these traditional medicines as commercial products and
establish their role as preventive medicines.
A critical element of these unexplored traditional medicines is
pharmacokinetics studies. Personalized Australian traditional medicines, based on
therapeutic monitoring and reverse pharmacology, represents one of the most
important challenges in natural product therapy. Chinmedomics, an integrative
method of serum pharmacochemistry with metabolomics from traditional Chinese
plants, can be adapted to investigate the effectiveness and safety aspects of
Australian native medicinal plants. The psychopharmacology of Australian native
Lemon Grass should be explored based on the phytochemical profile described in
this thesis to demonstrate the already established therapeutic value in the treatment
of headaches and migraines. Additionally, phytochemical profiling indicated the
presence of alkaloids within Australian native extracts. Alkaloids can have
pharmacological actions such as hallucinogenic and anticholinergic. Therefore,
further effort is required to explore the possible side effects of these extracts within
the human body.
This thesis has increased the awareness and the importance of Australian
native plants and mushrooms as a source of medicines. Many of the plant extracts
that have been investigated in this research project contain compounds that are
known to have anti-inflammatory, anticancer, antioxidant and cardio-protective
152
potential. The presence of these anticancer and antiangiogenic compounds could be
suitable to explore the use of these extracts in symptom management for cancer
palliative care. Moreover, the immuno-pharmacological potential of Tasmanian
Pepper and the photodynamic therapeutic value of Gumbi Gumbi provide scientific
argument to investigate their possible metronomics, the next generation of targeted
chemotherapy, to cure more cancers.
Advanced nanotechnology is an attractive tool to ensure the higher efficacy
and stability of these Australian native natural products necessary for efficacy
against various diseases. Nano-medicine-based natural products can be a successful
approach in addressing the solubility problems associated with these natural
bioactives. It is feasible that nanotechnology combined with multifunctional
Australian native natural products has the potential to treat diseases in the near
future.
Industrial and commercial significance of this technology can be a rapid and
an efficient high throughput analysis of biologically complex clinical and non-
clinical samples. Reaction flow chromatography coupled with high resolution
Orbitrap mass spectrometry can be a potential commercial product. However, this
thesis has also explored ethnopharmacological profiles of Australian native plants in
detail and provided an evidence based approach to carry out large scale introduction
and cultivation of Australian native herbal resources to develop pytopharmaceutical
with preventive and treatment therapies.
153
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