Bio-analytical screening and characterization of ......the recent years conventional online post...

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.


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


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.


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


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


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


Quandong ............................................................................................................ 108


Lemon grass ........................................................................................................ 117

Figure 5.6: Compounds from Lemon grass that has a potential role in the treatment

of headaches and migraines .................................................................................. 123


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


5.4.2.7.Australian native Wattle seeds ................................................................ 135

5.4.2.7.1.Phenolic Acids ................................................................................... 135

5.4.2.7.2.Bioactive Lipids ................................................................................ 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

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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).


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)


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)


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



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


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)


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


1.603 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


11.977 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)


Formula

Exact mass Measured

mass

Error(ppm)

0.427 L-Histidine C6H9N3O2 155.06949 155.06954 0.322



1.428 Adenosine C10H13N5O4 267.09678 267.09673 -0.8



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


11.786 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)


Formula

Exact mass Measured

mass

Error(ppm)

0.618 L-Tyrosine C9H11NO3 181.07391 181.07408 0.9


0.817 Nicotinamide C6H6N2O 122..04802 122.04813 0.9

1.220 Adenosine C10H13N5O4 267.09678 267.09673 -0.2


11.414 Oleamide C18H35NO 281.27186 281.27188 0.07


12.031 Ergosterol Peroxide C28H44O3 428.32906 428.32912 0.1

13.978 Spermidine C7H19N3 145.1579 145.15792 0.1

80

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.

83

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

84

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.

85

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


5.3.2. Instrumentation Setup


94

5.3.3. Chromatographic Analysis


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.


5.4.1. 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


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


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


10.258 Unidentified compound C15H22O 218.16707 218.16175 -24.3


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

99

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)


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


0.667 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


3.355 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


3.584 Unidentified compound C33H32N4O14 708.19158 708.19111 -0.7



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



3.963 p-Coumaric acid glucoside C15H18O8 326.10021 326.10048 0.8

3.992 Caffeoyl alcohol C9H10O3 166.06301 166.06227 -4.5



4.378 Coumaroyl quinic acid C16H18O8 338.10019 338.10021 0.05



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


5.566 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






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)





9.912 Unidentified compound C11H18O 166.13577 166.13584 0.4









10.258 Nookatone C15H22O 218.16707 218.16715 0.4













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



11.355 Unidentified compound C17H24 228.18780 228.18779 0.0


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


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).


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)



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


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




3.985 4-Hydroxycoumarin C9H6O3 162.03154 162.03171 -1.0


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






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


5.523 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



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


11.277 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.


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






0.537 Dihydroascorbic acid C6H6O6 174.01647 174.01578 4.0






0.661 Unidentified compound C5H11N3O 129.09022 129.09036 -1.1


0.682 Unidentified compound C11H20N2O3 228.14741 228.14739 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



1.518 L-Phenylalanine C9H11NO2 165.07899 165.07904 -0.3





2.786 D-Pantothenic acid C9H17NO5 219.11070 219.10300 35.1

2.920 DL-Tryptophan C11H12N2O2 204.09890 204.09730 7.8


3.355 Kynurenic acid C10H7NO3 189.04260 189.04265 -0.3




3.840 Esculetin C9H6O4 178.02663 178.02655 0.4

3.871 Caffeic acid-3-glucoside C15H18O9 342.09513 342.09497 0.5


3.917 Hydroxy Cinnamic acid C9H8O4 180.04170 180.04140 1.7

4.308 Cinnamaldehyde C9H8O 132.05752 132.05765 -1.0



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



124

Table 5.4.(cont.) Structural elucidation of antioxidants within Australian native Lemon grass.

LC-HRMS (min)


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


5.134 Unidentified compound C9H7NO 145.05276 145.05278 -0.1




5.329 Iso Vitexin C21H20O10 432.10567 432.10570 -0.1


5.402 Suberic acid C8H14O4 174.08923 174.08856 3.8

5.539 Apigetrin C21H20O10 432.10670 432.10570 2.3


5.559 Unidentified compound C12H19NO 193.14667 193.14687 -1.0

5.581 Kaempferol 7-O-glucoside C21H20O11 448.10062 448.10060 0.0









5.948 Unidentified compound C14H21N 203.16740 203.16747 -0.3



6.013 Vitexin C21H20O10 432.10570 432.10567 0.1



6.032 Vanillic acid C12H16O4 224.10488 224.10490 -0.1




6.282 Sinensetin C20H20O7 372.12094 372.12105 -0.3


6.326 Pulegone C10H16O 152.12012 152.12020 -0.5


6.344 Azelaic acid C9H16O4 188.10488 188.10515 -1.4


6.467 Elimicin C12H16O3 208.10996 208.11003 -0.3

6.545 Dodecanedioic acid C12H22O4 230.15183 230.15161 1.0


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


7.340 Apigenin C15H10O5 286.04777 286.04778 0.0


125

Table 5.4.(cont.) Structural elucidation of antioxidants within Australian native Lemon grass.

LC-HRMS (min)



7.375 Iso Coumarin C9H6O2 146.03679 146.03649 2.1

7.380 Aflatoxin G2 C17H14O7 330.07399 330.07399 0.0


8.007 Peregrinine C24H35NO6 433.24646 433.24650 -0.1






9.895 Unidentified compound C12H26O4S 266.15520 266.15532 -0.5














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






















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.


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


1.519 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


5.040 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


5.862 Myricetin C15H10O8 318.03748 318.03743 -0.1


5.968 Quercitrin C21H20O12 464.09554 464.09557 -0.1

5.992 Kaempferol-7-O-Glucoside C21H20O11 448.10062 448.10066 -0.1


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


6.612 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





7.165 Sebacic acid C10H18O4 202.12053 202.11987 3.3



7.527 Unidentified Flavonoid C15H10O6 286.04777 286.04780 -0.1

7.464 Isoeugenol C10H12O2 164.08374 164.08388 -0.9


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


8.353 Unidentified Flavonoid C15H14O6 290.07405 290.07907 -17.3


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


9.398 Phytosphingosine C18H39NO3 317.29289 317.29301 -0.4



9.954 Bilobol-Alkyl Resorcinol C21H34O2 318.25582 318.25589 -0.2



9.833 Unidentified Compound C15H20 200.15654 200.15650 0.2





10.430 Unidentified Compound C15H22O 218.16710 218.16707 0.1


11.049 Bilobol –Alkyl resorcinol C21H34O2 318.25578 318.25589 -0.3



11.235 Cadalene C15H18 198.14093 198.14085 0.4





11.976 1-Stearoylglycerol C21H42O4 358.30840 358.30833 0.2

12.258 Stigmasterol C29H48O 412.37053 412.37052 0.0



12.586 Elasterol C29H48O 412.37053 412.37052 0.0


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


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


0.511 Quinic acid C7H12O6 192.06261 192.06342 -4.2


0.526 Malic acid C4H6O5 134.02152 134.02155 -0.2

0.574 Shikimic acid C7H10O5 174.05221 174.05265 -2.5









0.906 Alpha-hydroxy glutaric acid C5H8O5 148.03724 148.03720 0.3










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


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



4.124 Caffeic acid-glucuronide C15H16O10 356.07438 356.07439 -0.1

4.289 Unidentified compound C15H22O7S 346.10860 346.10866 -0.2


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


6.342 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


6.849 Luteolin C15H10O5 286.04775 286.04765 0.3

7.093 Abscisic acid C15H20O4 264.13624 264.13618 0.2


7.346 Apigenin C15H10O5 270.05296 270.05285 0.4





8.177 Flavonoid C16H12O6 300.06342 300.06339 0.1


8.209 Matricin C17 H22 O5 306.14667 306.14675 -0.3




8.693 Unidentified Isoflavone C16H12O5 284.06865 284.06850 0.5











9.106 Polygodial C15H22O2 234.16203 234.16199 0.2






9.453 Rotundone C15H22O 218.16711 218.16714 0.5

9.710 Baicelien C15H10O5 270.05302 270.05285 0.6


9..826 Unidentified compound C14H22O2 222.16173 222.16199 -1.2




11.200 Unidentified compound C18H41N8O4P 464.29900 464.29886 0.3

139

Table 5.6 (cont.) Structural elucidation of Antioxidants within Australian native Mountain pepper.

LC-HRMS (min)








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




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)



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



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



4.894 Iso Ferulic acid C10H10O4 194.05793 194.05758 1.8


5.053 Sinapinic acid C11H12O5 224.06850 224.06817 1.5


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


11.520 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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