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Analysis of Coffee-Herbal Beverages

For Potential Benefits Against

Dementia Diseases

A thesis submitted in fulfilment of the requirements for the degree of

Master of Science by Research

Tao Yu

Bachelor of Pharmaceutical Science

School of Science,

College of Science Engineering and Health

RMIT University

May 2017

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To my wife Mao YE

For her on-going support and sacrifices,

I dedicate my research to you

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DECLARATION

I certify that except where due acknowledgement has been made, the work is that of the candidate

alone; the work has not been submitted previously, in whole or in part, to qualify for any other

academic award; the content of the thesis is the result of work which has been carried out since the

official commencement date of the approved research program; any editorial work, paid or unpaid,

carried out by a third party is acknowledged and ethics procedures and guidelines have been

followed.

Tao Yu

1st May 2017

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ACKNOWLEDGEMENTS

This thesis represents not only my laboratory work, but also the contributions and efforts of those

who guided and helped me. I would like to take this opportunity to express my gratitude for all their

generous input to make this project a success.

First and foremost, I would like to thank my major supervisor Associate Professor Helmut Hugel, for

his expertise, understanding and patience, especially his vast knowledge on herbal protecting

dementia and for his guidance and assistance in writing report (i.e., milestones review, human study

application and this thesis). I have so enjoyed our conversations; and the door of his office was

always open whenever I had a problem or a question about my project or writing.

Secondly Dr Jeff Hughes my surrogate supervisor without whom I would not have achieved my goals.

I am forever grateful for your support, encouragement, and for providing the many opportunities to

attend various workshops to improve my knowledge and skills.

I am appreciative to Mr Paul Morison, for his technical assistance in supporting our SPME-GC/MS

and HPLC-DAD analysis, and Mr Frank Antolasic for making available your equipment and your

assistance in many ways. I am grateful for your friendship, your ongoing words of encouragement

and support during my whole candidature.

I gratefully acknowledge Professor Ewan Blanch and Dr Saeideh Ostovar for Thioflavin T (ThT)

fluorescence assay assessment. I would like to gratefully acknowledge Dr Neale Jackson, who was a

technical consultant on the human sensory evaluation for coffee herbal functional beverage.

To Ms Ruth Cepriano-Hall and Ms Zahra Homan: not only would I like to acknowledge your technical

skills that made it possible for me to perform laboratory work during my studies. I am appreciative

to Dr Oliver Jones, for your assistance that brought me to the chromatography workshop. To the

chromatography workshop supervisor, Dr Ste e P i é , I g ateful fo ha ing an analytical

technical workshop and thanks to your charming personality that daily brightened me up.

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I would like to acknowledge those people within School of Science at RMIT who assisted me with my

project in one way or another (in alphabetical order): Ms Dianne Mileo, Dr James Tardio, Dr Joel Van

Embden, Mr Karl Lang, Associate professor Kay Latham, Ms Nadio Zakhartchouk, Associate professor

Nichola Porter, and Mr Trevor Rook.

To Dr Lisa Dias and Dr Emma Goethals, I am grateful for your assistance at the every stage during my

studies, which allowed me to complete the milestones successfully.

To my Office (3.2.10) o s a d these f ie ds f o Chi a, I ha e so e jo ed ou offee ti e a d

laughs.

I am eternally blessed to have a loving family who has supported me every step of the way during

stud hi h I su e at ti es as a halle gi g task; pa ti ula l the last 100 meters where the

sprint to the finish line has been exhausting. They have brightened up my life with their smiles.

ABSTRACT

The research strategy is focused on the potential prevention and treatment of cognitive

impairment by recommending the consumption of herbal-coffee beverages as early as possible

in life. General methods applicable for the analysis and extraction that are suitable for the

preparation of a range of herbal-coffee beverages have been found. The herb-bioactive

compounds are effectively extracted simply by using a coffee machine or microwave heating.

These herbal-coffee beverages may provide significant neuro-protection against dementia and

Alzhei e s disease, i ludi g the de elop e t of the fi st e a ples o tai i g gi ge , liquorice

and ginseng in coffee brews. Caffeine, caffeic acid and Vitamin B1 combined with HEWL to

strongly inhibit ThT fluorescence. A small study of the perception and sensory responses to the

new beverages was undertaken.

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JOURNAL PUBLICATIONS ARISING FROM THIS THESIS

Hügel HM, Yu T and Jackson N, The Effects of Coffee Consumption on Cognition and Dementia

Diseases, Gerontology & Geriatric Research 2015(4): 1-6

PRESENTATIONS

1. Tao Yu, Frank Antolasic, Jeff Hughes, Helmut Hugel. TGA-IR-GC-MS Enabling the Analysis of

the Aroma of Roasted Coffee Beans. presented at the Annual Research Day School of

Applied Sciences RMIT, Melbourne, June 2014. (poster)

2. Tao Yu, Paul Mo iso , F a k A tolasi , Jeff Hughes, Hel ut Hugel. The a al sis of he al-offee ta get o pou ds p ote ti e agai st Alzhei e s disease p ese ted at the Annual

Research Day School of Applied Sciences RMIT, Melbourne, June 2015. (poster and oral)

3. Tao Yu, Paul Morison, Frank Antolasic, Neale Jackson, Jeff Hughes, Helmut Hugel. The analysis of the constituents of herbal-coffees for protection against dementia disease , presented at the completion seminar at RMIT University, June 2016. (Oral)

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TABLE OF CONTENTS

Chapter 1: Introduction……………………………………………………………………………………………………………….16

1.1 Project background………………………………………………………………………………………………………………

1.2 Natural compound extraction methods and quality control……..……………..………………………….

1.2.1 Decoction extraction………………………………………………………………………………………………

1.2.2 Percolation extraction…………………………………………………………………………………………….

1.2.3 Soxhlet Extraction…………………………………………………………………………………………………..

1.2.4 Microwave-assisted extraction……………………………………………………………………………….

1.2.5 SPME extraction……………………………………………………………………………………………………..

1.3 Natural compound quality control [QC] via chromatographic analysis………………………………… 1.3.1 High Performance Liquid Chromatography (HPLC)………………………………………………….

1.3.2 Gas chromatography (GC)………………………………………………………………………………………

1.3.3 Mass spectrometry (MS)…………………………………………………………………………………………

1.4 Thioflavin T (ThT) fluorescence assay…………………………………………………………………………………..

1.4.1 Fluorescence spectrometry measurement…………………………………………………………….

1.5 Coffee herbal Functional beverage………………………………………………………………………………………

1.5.1 Coffee aroma induces/invites functional beverage usage……………………………………….

1.5.2 Herbs are widely used ingredients/nutrients to improve health in food science......35

1.5.3 Functional beverage sensory measuring…………………………………………………………………

1.6 Project Hypothesis and Scope…………………………………………………………………………………………….

1.6.1 Herbal against dementia selection criteria as functional beverage i this p oje t.….

1.6.2 Key questions of the project……………………………………………………………………………………

1.7 Reference……………………………………………………………………………………………………………………………..

Chapter 2: Coffee Herbal Bioactive Compound’s Potential to Prevent Dementia ….……………….45

. Multiple ta gets agai st Alzhei e s disease by nature compounds……………………………………

. . Ta get : A loid β peptide is p odu ed p oteases……………………………………………

2.1.2 Target2: Neurodegeneration accelerated by inflammation and oxidant stress……….

2.1.3 Ta get : Choli este ase i hi itio agai st Alzhei e s disease………………………………

. . Ta get : Ade osi e e epto s agai st Alzhei e s disease…………………………………….

2.2 Coffee protects against Dementia………………………………………………………………………………………..

2.2.1 Caffeine and coffee Compounds exert protective agai st AD……..…………………………

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2.2.2 Coffee protection against dementia on hu a Studies…………………………………………

2.3 Liquorice protects against dementia…………………………………………………………………………………….

2.3.1 Compounds in liquorice that a affe t Alzhei e s disease……………………………….

2.3.2 Liquorice protection against dementia o hu a studies……………………………………..

2.4 Ginseng protection against dementia…………………………………………………………………………………

2.4.1 Compounds in ginseng that a affe t Alzhei e s disease…………………………………..

2.4.2 Ginseng protection against dementia i hu a studies………………………………………..

2.5 Ginger protects against dementia………………………………………………………………………………….. …..

2.5.1 Compounds in ginger that a affe t Alzhei e s disease……………………………………..

2.6 References……………………………………………………………………………………………………………………………

Chapter 3: The analysis of coffee herbal beverages…………………………………………………………………61

3.1 Chromatography method validation parameters………………………………………………………….. …..

3.1.1 Quantification can be achieved by external standards…………………………………………

3.1.2 Peak ide tifi atio ….…………………………………………………………………………………………..

3.1.3 A u a e o e ……………………………………………………………………………….……………

3.1.4 Precision………………………………………………………………………………………………………………

3.1.5 Linearity……………………………………………………………………………………………………………….

3.1.6 Limits of detection……………………………………………………………………………………………….

. . Ko áts i de ete tio i de ……………………………………………………………………………..

3.2 The introduction to the coffee and herbal compounds analysis method ….……………………….

3.3 Coffee herbal compounds extraction procedure…………………………………………………………… …..

3.3.1 Coffee, herbal and analysis Instruments and reagents Information………………………

3.3.2 Mode 1: Comparison beverage with an increasing ratio via HPLC and GC/MS………

3.3.3 Mode 2: Comparison of different extraction methods for coffee herbal via HPLC…

3.4 The analysis methods of coffee-herbal beverage……………………………………………………………….

3.4.1 HPLC protocol for coffee and Liquorice………………………………………………………………….

3.4.2 HPLC protocol for ginseng compounds………………………………………………………………….

3.4.3 GC/MS analysis of coffee-herbal beverage…………………………………………………………….

3.4.4 Analytical standards information…………………………………………………………………………..

3.5 Thioflavin T (ThT) spectroscopic assay materials and methods…………………………………………..

3.6 Coffee-herbal beverage analysis results…………………………………………………………………………….

3.6.1 Validation of the HPLC assay………………………………………………………………………………….

3.6.2 Validation of the GC/MS assay………………………………………………………………………………

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3.6.2.1 Method validation by Kováts index (KI)…………………………………………………..

. . . Ca phe e ali atio u es…………………………………………………………………

3.6.3 Mode 1: Compounds concentration in coffee-herbal beverage……………………………..

3.6.4 Herbal compounds of camphene concentration in coffee-herbal beverage…………..

3.6.5 Mode2: Coffee herbal beverage with various extraction methods…………………………

3.7 Thioflavin T (ThT) assay measurement……………………………………………………………………………….

. Refe e e…………………………………………………………………………………………………………………………

Chapter 4: Coffee herbal beverage sensory estimation……………………………………………. …….90

4.1 Functional beverage sensory measurement………………………………………………………………………

4.2 Coffee herbal beverage constituents on sensory ……………..………………………………………………

4.3 Sensory assessment (9-point hedonic scale) applications in food science…………………………

4.4 Participant recruitment ethod ………………………………………………………………………………………

4.5 Coffee-herbal beverage preparation ………………………………………………………………………………

4.6 Coffee herbal beverage sensory assessment procedure………………………………………………… …

4.7 Sensory measurement protocol………………………………………………………………………………………..

4.8 Hedonic scale statistical analysis……………………………………………………………………………………….

4.9 Hedonic scale record discussion………………………………………………………………………………………..

. Refe e es………………………………………………………………………………………………………………………..

Chapter 5: Conclusions and further work…………………………………………….…………………………………98

5.1 Co lusio ………………………………………………………………………………………………………………………….

5.2 suggestion for further work……………………………………………………………………………………………..

Appendix 1 Herbal Coffee Beverage Sensory Analysis Evaluation………………………………………………….

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LIST OF FIGURES

Chapter 1

1.1 a The Alzhei e s Disease de elop e t stage; Neu oi agi g f o a health pe so a d a patie t ith Alzhei e s disease……………………………………………………………………………….

1.2 (a)Percolation extraction; (b)Conventional Soxhlet Extraction;(c)Microwave-assisted

extraction…………………………………………………………………………………………………………………………….

1.3 (a)Manual Solid-phase microextraction (SPME) Holder; (b) SPME extraction process

of absorption and release……………………………………………………………………………………………….……

1.4 (a)The components separation process; (b)Individual retention time of the compound A

a d B elutio f o the olu …………………………………………………………………………………………….

1.5 (a) Schematic of HPLC separation procedure and data obtained displays; (b) HPLC instrument;

(c) HPLC-DAD(diode-array detection ) compo e ts.……………………………………………………………

1.6 Schematic of a typical simple GC-MS……………………………………………………………………………….....

1.7 Configuration of quadrupole mass filter…………………………………………………………………………..….

1.8 (a) The structure of Thioflavin T(ThT); (b)Magnified view of the ThT– i di g β a loid sheet deposits. (c) Essential component of a fluorescence spectrometer (d)Characteristic increase

in ThT fluorescence upo i di g to a loid fi ils…………………………………………………………….

1.9 Typical sample preparation and separation steps involved in GC/MS and HPLC…………………..

Chapter 2

2.1 Amyloid cascade hypothesis……………………………………………………………………………………………….

2.2 Enzymatic processing of transmembrane APP: the non-amyloidogenic(a) and

a loidoge i path a s ……………………………………………………………………………………………….

. Caffei e, t igo elli e a d affei a id………………………………………………………………………………….

. β-Glycyrrhizic acid converts to a stable inclusion complex with other

molecules (GA do ut i g ………………………………………………………………………………………………… 2.5 Glycyrrhizic acid (GA) and Glabridin…………………………………………………………………………………….

2.6 a Gi se osides R , Rg ; α-Pi e e, β-Pinene and Camphene ……………………………………

Chapter 3

3.1 HPLC chromatogram of standard solution of Tigonelline, Caffeine and Glycyrrhizic acid (GA)73

3.2 HPLC chromatogram of standard solution of Ginsenoside Rb1………………………………………… ….

3.3 Calibration curves of Tigonelline, Caffeine and Glycyrrhizic acid (GA) and Ginsenoside

Rb1 the regression equations of the four compounds………………………………………………………. ..74

3.4 GC chromatogram of standard of n-alkanes (C8-C20)……………………………………………………………

3.5 GC chromatogram of coffee-ginger beverage……………………………………………………………………….

3.6 Calibration curves of Camphene the regression equations in the both range……………………….

3.7 GC/MS chromatogram of standards solution of Camphene and Caffeine……………………………..

3.8 Coffee herbal beverage sample HPLC chromatogram of Trigonelline, caffeine and GA

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and dilution (1:10) with 25% methanol…………………………………………………………………………………

3.9 Coffee herbal beverage sample HPLC chromatogram of Rb1(AG) and dilution (1:10)

with 25% Acetonitrile……………………………………………………………………………………………………………

3.10 Coffee herbal beverage sample GC/MS chromatogram of camphene and caffeine………………

3.11 The coffee herbal beverage compounds concentration measurements via various

extraction methods………………………………………………………………………………………………………………

3.12 The amount of compounds extraction via microwave oven extraction…………………………………

3.13 Semi-quantitative curve of protein fibrillation in the presence or absence of different

Natu e o pou ds……………………………………………………………………………………………………………….

Chapter 4

4.1 Texture of Hedonic Scales values in coffee herbal beverage………………………………………………

4.2 Flavors of Hedonic Scales values in coffee herbal beverage………………………………………………. .

4.3 Tasty of Hedonic Scales values in coffee herbal beverage…………………………………………………….

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LIST OF TABLES

Chapter 1

1.1 A brief summary of the experimental conditions for various methods of extraction for

herbal material……………………………………………………………………………………………………………………

Chapter 3

3.1 Coffee and herbs products information list………………………………………………………………………….

3.2 Coffee a d he al i tu e i g edie ts a d atio list…………………………………………………………….

3.3 Standards information list……………………………………………………………………………………………………

3.4 Regression equations and detection limits of components determined for the presented

HPLC assay…………………………………………………………………………………………………………………………..

3.5 Intra-day precision and accuracy for the HPLC assay of four components……………………………

3.6 Comparison of three times n-alkanes (C8-C20) Kovats Indices and the percent of quality

ass at h…………………………………………………………………………………………………………………………

3.7 Comparison of three times monoterpenoid and caffeine Kovats Indices and the percent

of quality mass match………………………………………………………………………………………………………….

3.8 Regression equations and detection limits of components determined for the

presented GC/MS assay……………………………………………………………………………………………………….

3.9 Intra-day precision for the GC/MS assay of ginger component (n=4)……………………………………

3.10 Amounts peak area and the concentration of caffeine, trigonelline in coffee-herbal

beverage (n=4)…………………………………………………………………………………………………………………….

3.11 Comparison of peak area and the concentration of GA from liquorice powder (LP), GA

from liquorice liquid extract(LL) in coffee-herbal beverages (n=3)……………………………………….

3.12 Comparison of peak area and the concentration of Rb1 from American ginseng (AG),

Rb1 from unidentified ginseng source (UG) in coffee-herbal beverage (n=3)……………………….

3.13 Comparison of peak area and the concentration of camphene from fresh ginger (FG),

and from Ginger capsule(GC) in coffee-herbal beverages …………………………………………..

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LIST OF ABBREVIATIONS

Aβ A loid β peptides

ACh Acetylcholine

AChE Acetylcholine cholinesterase,

AD Alzhei e s disease

APP Amyloid precursor protein

BuChE Butyryl-cholinesterase

CI Chemical Ionisation

DAD Diode Array Detector

DHA Docosahexaenoic acid

GA Glycyrrhizic acid

GC Gas Chromatography

Rb1 Ginsenoside Rb1

Rg1 Ginsenoside Rg1

EI Electron ionization

FID Flame ionization deection

HPLC High Performance Liquid Chromatography

KI Kováts index

m/z mass to charge ratio

MAE Microwave-assisted extraction.

MCI Mild cognitive impairment

MQ water Milli-Q water

MS Mass spectrometry

NMDA Receptor N-methyl D-aspartate Receptor

NPD Nitrogen–phosphorus

PBS Phosphate buffer saline

PDMS Polydimethylsiloxane

PMAE Pressurized microwave-assisted extraction

QC Quality control

SFMAE Solvent-free microwave-assisted extraction

SPME Solid phase microextraction

RPC Reversed-phase chromatography

RSD% The percent of Relative standard deviation

SD Standard deviation

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S/N Signal to Nosie ratio

SIM selected ion monitoring

TIC total ion chromatogram

ThT Thioflavin T

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1

Introduction

The ore i porta t reaso is that the resear h itself provides a i porta t lo g-run perspective on the

issues that we face on a day-to-day asis.

Ben Bernanke

1.1 Project Background

By 2050 it is estimated that the number of people over the age of 80 will triple globally and cognitive

decline and dementia could become the greatest challenge of our times. According to the 2003

World Health Estimates Report, dementia inflicts 11.2% elderly people (>60 years) more than stroke,

cardiovascular disease and various cancers [1]. Dementia is a collection of different disorders that

influence brain function such as progressive memory loss, judgment decline and other symptoms;

Alzhei e s disease (AD) is the most common type of memory disorder and contributes to between

50% and 70% of all types of dementia [2]. From neuroimaging, AD advances in three stages: an early

and preclinical stage with no symptoms; a middle stage of mild cognitive impairment (MCI); and a

final stage of AD. [3] [Fig.1.1 (a)] The pathological process of AD can have a long development time

even to 40-50 years (including the early and MCI periods) [4].

MCI is defined as the clinical condition of memory between normal aging and AD; in other words,

patients with MCI incur more memory loss problems than healthy people of a similar age, however

they do not yet have the cognitive function issues and criteria for AD [5]. According to

epidemiological studies, elderly people have been classified as the highest risk of population to

suffer from AD [6]. For example, the many memory complaints elderly people have include, having

trouble remembering friends names they meet often, having a greater probability to misplace things

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yet have normal judgment and thinking skills. These people can suffer from MCI for extended

periods of time. They are at high risk (approximately 80%) of accelerating to a dementia disorder

such as AD (mostly in 6 years); however their dementia may be slowed even stabilised after

satisfactory therapeutic and lifestyle interventions [7]. Therefore, aging associated memory

impairment is a major risk factor for the general degradation of memory that can progress from MCI

[8,9] symptoms to AD [10]. Although it is difficult to dete i e hethe a pe so s e o loss

reflects MCI symptoms. Aging is the highest risk factor that necessitates medical treatment for AD

for which there is no known cure.

Fig.1.1 (a) The AD development stages (left). (b) Neuroimaging from a healthy person (the left of

picture b) a d a patie t ith Alzhei e s disease ight [Rep odu ed f o efe e es 3, 14]

MCI symptoms and dementia is most likely caused by amyloid fibrils neurodegeneration that affect

brain cells to communicate neural information, resulting in memory loss and behaviour and feeling

changes. Damage in the memory and judgement functional regions such as the temporal lobe and

corpus callosum [Fig.1.1 (b)] is the cause of memory loss and one of the earliest symptoms of AD.

Amyloid fibrils are aggregated by normally soluble amyloid protein and are found in a wide range of

misfolded β-sheet amyloid protein resistant to neuro-degradation [11]. Research studies indicate the

structure of fibrils are mainly composed of β-sheet structure in a characteristic cross-β o fo atio

[12]. Therefore, a loid β peptides Aβ a e the ai o po e ts of the a loid pla ues fou d i

the brains of Alzheimer patients where it is deposited abnormally in senile plaques containing both

http://en.wikipedia.org/wiki/Alzheimer%27s_disease

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Aβ a d Aβ . Aβ is produced from the amyloid precursor protein (APP) by the sequential cleavage

ia β- and γ-secretases [13]. Currently there are no medications that can remove or affect the

dissolution of toxic Aβ f o eu o s to i p o e og iti e a ilit .

Symptomatic treatments have been widely applied for AD since the mid-1990s. There are two types

of medications, cholinesterase inhibitors (Donepezil, Rivastigmine, Galantamine) and NMDA

receptor antagonist (Memantine), that are approved by the USA Food and Drug Administration to

relieve the cognitive symptoms (such as memory loss, confusion) of mild, moderate and severe AD

[14] These drugs are based on sustaining the acetylcholine (ACh) neurotransmitter levels

(galantamine and the physostigmine-derivative rivastigmine) [15] in AD patients with cognitive

degeneration have cholinergic neurons deficits in due to a decline in the levels of ACh in the

hippocampus, temporal and parietal neocortex. ACh is hydrolytically destroyed by two

cholinesterases, AChE and butyryl-cholinesterase (BuChE). As AD progresses, AChE activity can

decrease by up to 67% of normal levels in the temporal lobe and hippocampus [16]. Thus,

Cholinesterase inhibitors are commonly used for the treatment of symptoms of AD cognitive decline,

but does not stop/prevent neuronal damage. NMDA glutamate receptors are composed of abundant

glutamate-gated cation channels with high calcium permeability, which are distributed throughout

the central nervous system (CNS). They have important roles in the process that learning, memory,

and neuroplasticity. Some neurological disorders including cognitive disorders are caused by

receptor over activation (excitotoxicity) and subsequent neurodegeneration [17,18]. Many AD

patient s memory loss and cognitive loss is due to prolonged Ca2+ excess and results in loss of

synaptic function, followed by synaptotoxicity and ultimately cell death [19]. It is known that NMDA

receptor inhibition and overactivation, leads to impairment of neuronal plasticity (learning) or cell

death, and dysfunctional cognition [20]. Memantine is a low affinity, voltage-dependent and

noncompetitive NMDA receptor antagonist, which has a higher affinity for Mg2+ and blocks Ca2+

influx. It has fast off-rate kinetics in the receptor channel, and so is only of benefit for dysfunctional

synapses found in AD patients without affecting the physiological stimuli of the NMDA receptor [21].

Many AD patients have been recorded to have personality changes, behaviour and sleep difficulties

even at the early stage. For example, the major behaviour alteration was apathy (72%), agitation

(60%) and anxiety (48%), which significantly correlates to the cognitive deterioration caused by

neuron cell damage in the brain [22]. Additionally, some medications may worsen these symptoms.

Therefore, atypical antipsychotics such as risperidone, quetiapine, olanzapine, aripiprazole were

utilized to treat the symptoms of short-term severe aggression in AD. However, these drugs have

limited cognitive benefits, particularly risperidone that poses a significant risk for cerebrovascular

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events [23, 24]. Currently, all antidepressants such as citalopram, sertraline are not licenced for AD

treatment.

In summary, many researchers acknowledge that current Alzhei e s drugs cannot cure AD. These

drugs and non-drug clinical conditions may ameliorate cognitive and behavioural symptoms for

improving the quality of life for some patients with AD.

A better strategy is to prevent AD onset or delay aging through the reduction of dementia risk

factors; as the medications used to treat mild cognitive or dementia do not reverse the formation of

eu ofi illa ta gles . There are seven risk factors that have been associated with one in three

Alzheimer's disease cases. These include diabetes, midlife hypertension, midlife obesity, physical

inactivity, smoking, depression, or low educational attainment [25,26]. These risk factors could

promote AD with aging. The current lifestyle interventions preventing AD include better diets, brain

games (Sudoku puzzles) and regular exercise. For example, research suggests that daily intake of fish

with docosahexaenoic acid (DHA), and omega-3 fatty acid decreases the risk for AD [27]. Neurons

and synapses are composed of long chain unsaturated fatty acids similar to omega-3; also lowering

the dementia risk factors is related to the protection against cardiovascular disease [28].

Scientific research demonstrates that strategies such as exercise and diet, significantly delay or

prevent aging cognitive decline or memory loss, many of which are based on small group and short-

term studies. Obviously the dietary nutrition brain benefits are variable and it is common for people

to take different kinds of foods containing vitamins, antioxidants and their effects are difficult to

quantify. There is room for improvement, especially in generating personalised treatments, however

the greatest advantage of dietary modifications is that they generally do not raise human safety

issues.

The health claims of traditional herbal remedies, their effectiveness and safe use are growing

rapidly. Functional foods are also popular for consumers who are seeking specific health effects to

promote physical fitness. Recent research found that many herbal compounds can affect the mind,

mood, cognitive function or health. For example, ginseng has been reported to improve short-term

memory performance when consumed as a dietary supplement. S. lavandulaefolia Vahl. (Spanish

sage) essential oil composition i ludes α-pinene (4%- % , β-pinene (5%-12%), 1,8-cineole (15%-

30%) and camphor (20%-30%, and it has been reported to improve accuracy for cognitive mental

status examination after 6 week of use [29]. Certain herbs may be potentially important sources of

http://topics.sciencedirect.com/topics/page/Diabetes_mellitus

http://topics.sciencedirect.com/topics/page/Hypertension

http://topics.sciencedirect.com/topics/page/Major_depressive_disorder

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drug candidates against the onset of AD. For example, Zingiber officinale (ginger), Ginkgo biloba

(ginkgo), Cinnamum cassia (Chinese cinnamon) extracts effectively protect primary neuronal cells

agai st Aβ -42 insult [30]. Huperzine A, originally isolated from Huperzia serrata, a type of moss

used in traditional Indian and Chinese medicine and its derivative, ZT-1, is being developed as a new

anti-AD drug for AD treatment. Yokukansan is composed of 7 herbs and is a useful formulation for

the effective treatment of dementia symptoms in 106 patients diagnosed with AD that was safe to

use [31].

The production of a functional beverage using safety tractional herbs and to encourage its long term

consumption would provide a sound strategy for neuroprotection against dementia and be of

general benefits for society. Firstly, these herbal effective compounds can be extracted into water

via a convenient procedure; moreover, these ingredients can be analytically measured the amount

consumed can be quality controlled, allowing the opportunity to monitor/measure the benefits over

several years; the last but not the least factor is the functional beverage can be modified according

to personal sensory perceptions and blended into daily consumption similar to coffee or tea intake.

1.2 Natural compound extraction methods and quality control

In order to quantify the herbal benefits for human health, the first important step is herb

constituent extraction, separation, identification and quantitation/concentration of the herbal

ingredients, as the bioactive compound concentration are product quality-related and also to herbal

efficiency. These herbal bioactive substances can be classified into families of compounds including

essential oils, alkaloids, steroids, saponins. Various classes of compounds have known methods for

extraction for optimal pharmaceutical application [32]. For example, some bioactives found in plants

are water soluble whilst others are hydrophobic/insoluble. Modern and traditional extraction and

analysis techniques play an important role in food industry [33,34]. These techniques include

decoction, percolation, Soxhlet extraction, solid phase micro-extraction, and microwave-assisted

extraction, that are available in the lab or in domestic use. Chromatographic techniques have proven

to be the most powerful and effective technology available for herbal analysis [35].

1.2.1 Decoction extraction

The usage of green extraction technology (also traditional extraction techniques) is able to protect

the natural environment and minimizes harmful chemical affects on human and environmental

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health. Traditional Chinese herbal teas and Turkish coffee are commonly prepared in this way. For

decoction methods, the extractive yield is determined by time/temperature and the chemical nature

of the herb. Botanic herbs such as stem and root from licorice, ginseng and ginger, are finely ground

to enhance maximum herb extraction [36]. Water is biocompatible and an eco-friendly medium,

which can be used to extract many bioactive constituents including alkaloids, flavonoids, saponins,

vitamins and others [37]. The herbs to water ratio used ranges from 1:4 to 1:10. Depending on the

consistency of the parts to be extracted, the decoction times of herbs generally decreases in the

order: roots > stems > leaves and flowers, when boiled in water for 5 to 60 minutes [38,39]. During

this time the evaporated water must be replaced. Essential oils are isolated by distillation of the

volatile organic compounds.

1.2.2 Percolation extraction

Percolation is an extraction process whereby the slow passage of a solvent through fine substrate

particles extracts constituents that pass through the filter into the collector. A coffee percolator is a

good example of the percolation process, which was invented by Hanson Goodrich in 1889 [40].

Water is heated in the percolator chamber, passes through the vertical tube as steam-powered

vacuum and is sprayed over the coffee grounds (that is continuously recycled through the grounds

until complete) [Fig. 1.2(a)]. Extracted coffee was filtered into the bottom chamber. Percolation is a

short and efficient extraction extraction procedure that can be applied to the extraction of certain

herbs [41]. As required, the coffee or herbs should be ground into powder form with a coffee

blender or commercial grinder. Some procedures require a preparatory step, to allow crude herbal

particles to swell to their maximum size as dense dry fine particles may block the filter mesh after

swelling and resist solvent flow, giving rise to inefficient extraction [42].

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Fig.1.2. (a)Percolation extraction (left); (b)Conventional Soxhlet Extraction (middle); (c)Microwave-

assisted extraction (right) [Reproduced from references 50, 53]

1.2.3 Soxhlet Extraction

Soxhlet extraction was first described in 1879 by Franz von Soxhlet, who applied the apparatus to

quantify fat in milk. It was then widely used for lipid extraction in agricultural chemistry [43]. The

apparatus facilitates the separation of soluble compounds of interest from the insoluble solids with a

solvent. The solvent reticulation enables continuous sample extraction from a few hours to days.

Figure [Fig.1.2 (b)] illustrates the Soxhlet components of condenser, extractor and distillation flask.

When the distillation flask is heated, the solvent vapour travels via the side-arm, liquefies on the

condenser jacket and flow back through the thimble in the extraction chamber containing the

crushed sample; the extractor chamber refills with the warm solvent slowly and extracts/dissolves

the desired compounds from the crude material. The concentrated solvent returns to the distillation

flask as the full chamber is emptied through the siphon arm. The fill-empty cycle is repeated until

the extraction is deemed satisfactory [44]. The Soxhlet extractor has the advantage that it allows

fresh recycled solvent to continuously extract the solid sample. The soluble compounds constantly

dissolve in freshly condensed solvent and return to the extraction flask until all the soluble

constituents are exhaustively extracted. The procedure operates automatically and is a gentle

extraction process without agitation. The concentrated constituents are readily collected when

completed [45]. This method was used for extraction of organic compounds from medicinal plant

extracts including isoflavonoids from ginger [46], caffeine[47] from coffee and glycyrrhizin from

liquorice [48]. Soxhlet extraction has been less used, as microwave-assisted extractions (MAE),

supercritical fluid extraction (SFE), and pressurized solvent extraction (PSE) techniques are preferred.

However, modified Soxhlet extractor systems such as high-pressure Soxhlet extraction, ultrasound-

assisted Soxhlet extraction, microwave-assisted Soxhlet extraction have been developed [50]. These

new systems employ auxiliary apparatus to decrease leaching time and improve the extraction yield.

1.2.4 Microwave-assisted extraction

Microwave-assisted extraction (MAE) [Fig.2 (c)] is an efficient extraction technique, which applies

microwave energy to heat the solvent/sample matrix containing plant tissue or herbs, allowing these

natural compounds to be rapidly extracted from raw materials [51]. The technique was first used in

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the laboratory in 1975 by Abu-Samra, who conducted the trace analysis of metals in biological

samples and has become a very cost effective method [52]. The technique utilizes microwaves,

electromagnetic energy waves are formed from electric and magnetic fields, with the spectral

window between X-rays and infrared frequency from 0.3 to 300 GHz [53]. In general, the alternating

current of electromagnetic energy interacts/aligns with the dipole moment of polar samples and/or

polar solvents and this constant electric field-sample dipole rotation/realignment generates

molecular friction converted to heat energy [54]. Only dielectric materials (such as porcelain

(ceramic) glass and plastics) or solvents with permanent dipoles such as water are microwave active.

A ceramic cup containing coffee or herbal tea can be heated in a domestic microwave. These herbal

products contain minute traces of moisture that when heated by microwaves generate

energy/pressure to break the plant cell walls so that many secondary metabolite bioactive

compounds or aroma molecules can be dissolved in water [55]. MAE has many advantages, most

significantly it reduces the extraction time compared to traditional methods such as Soxhlet

extraction, also increases extraction efficiency and yields. There are several advanced MAE

instruments commercially available including pressurized microwave-assisted extraction (PMAE) and

solvent-free microwave-assisted extraction (SFMAE) technologies.

1.2.5 SPME extraction

'Headspace' refers to volatile extraction techniques of the gas above the sample of liquid or solid

phase in a sealed chromatography vial, that started to be utilized in the late of 1950s. Headspace gas

components are usually collected by a SPME fiber. Its advantage is simplicity of use, effectiveness

and is a very clean method, which delivers volatile analytes into a gas chromatograph for analysis.

Solid-phase microextraction (SPME) fibers were invented by Janusz Pawliszyn and patented at the

University of Waterloo in Ontario, Canada [56]. At present Supelco Analytical (a division of Sigma-

Aldrich Co.) maintains the distribution of the materials for SPME. Many disciplines are using this

technique, including the analysis of food, aroma and pharmaceutical samples. When volatile sample

constituents distribute into the gas phase and reach equilibrium between solid/liquid phase and gas

phase, the volatiles are adsorbed (or absorbed) in the headspace over liquid/solid samples, then are

dispensed to a GC column [49,57]. Thus, there are absorption and adsorption characteristic SPME

types fiber coating. The most widely applied coating, polydimethylsiloxane (PDMS), consists of

absorption via a high viscosity rubbery liquid. However, it appears as a solid and converts to liquid

under desorption temperature. The other adsorption materials PDMS–DVB (divinylbenzene),

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Carbowax–DVB and Carboxen are commercially available, that are mixed coatings via a porous

extracting phase [58]. In this project, different kinds of fiber were used according to the molecular

weight and polarity of the analytes to be studied. For instance 100 µM PDMS are used for the most

volatile compounds with molecular weights from 60 to 275 [59].

The SPME fiber is assembled in a modified barrel, the manual SPME holder [Fig. 1.3(a)]. In the

standby position, the fiber is situated in a protective septum-piercing needle. Prior to sampling, the

liquid or solid samples are settled in the sealed GC vials, leaving enough headspace (more than the

fiber exposed including the needle) over the sample; the fiber needle is forced into the vial and

pushes the plunger retaini g s e i the iddle of the Z slot. The SPME fiber, fused silica rod

t pi all lo g a d . μ i.d.) coated such as PDMS, is immersed the headspace contains the

volatile compounds from the sample. After a time-interval (2-15 min), these compounds are

subsequently absorbed and reach equilibrium. The fiber is withdrawn after the needle is taken out

of the sampling vial. The needle is then inserted into the GC injector, and the volatile analytes are

immediately thermally desorbed in 1-2 minutes and enter the GC column. The fiber still needs to be

kept in the needle when removed from the GC injector [60] [Fig. 1.3(b)]. The SPME fiber assembly is

disposable and may be used for approximately 100 injections.

Fig.1.3 (a) Manual Solid-phase micro-extraction (SPME) Holder (left) (b) SPME extraction process of

absorption and release (right) [Reproduced from reference 60].

Table 1 A brief summary of the experimental conditions for various methods of extraction

for herbal products

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Decoction

Extraction

Percolation

Extraction

Soxhlet

Extraction

Microwave-assisted

Extraction

SPME

Extraction

Common Solvents

used

Mainly

water

Mainly water Methanol,

ethanol,

or mixture of

alcohol and

water

Water, ethanol,

or mixture of

alcohol and water

Any solvent

Temperature (°C) 100 90-100 Depending on

solvent used

Depending on

solvent used

Low/high

Time required i i 3–18 hr i 2-30min

Volume of solvent

required (ml)

Depending

on herbal

weight

100-500 150–200 100-300 2-10

Types of herbs Stem, root,

seeds

Ground:

leaves, flower,

seed, roots,

stem

Stem, root,

seeds

Any parts of herbs Herbal oil

1.3 Natural compound quality control [QC] via chromatographic analysis

Chromatography technology was first developed by Mikhail Tswett in 1901, separates the

components in a mixture according to the different partitioning behaviours between a mobile phase

and a stationary phase. The most common techniques are HPLC (High Performance Liquid

Chromatography) and GC (Gas Chromatography). The stationary phase is packed into the column,

through which the mobile phase (solvent or gas) continually passes. The sample is injected into the

column. The individual components elute from the column as determined by their partition

coefficient in the mobile phase, and are collected and measured by the detector measuring UV

adsorption (diode array detector, DAD), molecular weight (Mass spectrometry detector, MS) [61].

[Fig.1.4(a, b)]

Compound B

Compound A

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Fig.1.4 (a) The components separation process. (left) (b)Individual retention time of the compound A

and B elution from column. (right) [Reproduced from Agilent Technologies, Inc.]

Resolution(R) is utilized to describe the extent of the separation of compounds A and B. The

resolution of two species (A and B), is defined as:

R=√𝑁4 𝛼−1𝛼 1+𝜅κ where N expresses the number of theoretical plates, by lengthening the column leads to an

increase in retention time, but in practice HPLC standard columns are fixed at about 15, 25 and

; α, the sele ti it fa to , is usually described by the ratio of the retention (capacity) factors (κ)

between the two compounds A and B peaks distances and determined by chemical characteristics;

thus it is a priority to adjust retention (capacity) factors κ to improve the separation. When the

amount of R achieved is 1.5, it is evaluated as good separation between the two compounds. The

most useful optimization method is changing the solvent strength, for example the use of

acetonitrile instead of methanol to increase the separation in reverse phase HPLC. Others include

mobile phase solvent pH and column temperature adjustment. The column length and stationary

phase composition can be changed when there are no improvements made by changing other

variables [62].

1.3.1 High Performance Liquid Chromatography (HPLC)

High Performance Liquid Chromatography (HPLC) is a liquid chromatography system with a pump,

sampler injector, column compartment (column and oven), detector (such as diode array detector,

fluorescence detector or MS detector) and computer control system, some of which have a vacuum

degasser. HPLC is an important technique for the qualitative and quantitative analysis for non-

volatile compounds, for instance natural product extracts, proteins, salts, polymers and

pharmaceuticals. HPLC separation procedure [Fig.1.5 (a) and (b)] involves injection of a liquid

sample (5ul-10ul) into the column packed with fine particles (3- μ i dia ete a d high p essu e

from a pump onto individual components of the sample through the column for separation. Finally,

when these constituents are eluted/separated from the column, the detector measures the eluted

amount (peak area) converted into an electrical signal output.

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(a)

(b) (c)

Fig.1.5 (a) Schematic of HPLC separation procedure and data obtained displays (b) HPLC instrument

(c) HPLC-DAD (diode-array detection) components [reproduced from Agilent Technologies, Inc.]

1966 was the start of HPLC, when Piel published a method describing slurry-packed finely ground

silica as a high liquid pressure (3500 psi) column to separate the samples [63]. Since the 1970s, the

development of HPLC in column material and instrumentation in the hardware—pumps, detectors

evolved. The octadecylsilane, [also called C18] was used as the stationary phase [64]. In the 1990s,

s alle pa ti les less tha μ a d highe p essu es as high as 100,000 psi) were applied in the

separation.

Most compounds can be separated by utilization of the four separation modes including reverse-

phase chromatography, normal-phase and adsorption chromatography, ion exchange

chromatography and size exclusion chromatography. The method of choice is determined by the

characteristics of chemical compounds to be separated: polarity, electrical charge and molecular

size. Many herbal compounds [65] and biological samples (involving protein and nucleotides) [66,67]

have been separated by reverse-phase chromatography (RPC). Regarding RPC, the column is packed

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with porous particles of silica gel in various shapes (spherics or irregular) of various diameters (1.8, 3,

5, 7, 10 µM etc.) with various pore sizes (such as 60, 100, 120, 300), and the surface are covered with

long covalently bonded alkyl chains (e.g. C18, C8, C3, phenyl, etc.) having a strong affinity for

hydrophobic compounds (non-polar). The mobile phase is composed of water and organic solvent

(methanol, acetonitrile). Generally, the water solvent strength is the highest and an initial gradient

run allows the organic solvent percentage to be lower, as a function of the time the organic solvent

content increases, thereby raising the elution strength with polar components eluting faster than

the less polar components [68].

The HPLC solvent delivery programme options include isocratic or gradient conditions. Regarding the

former conditions [69], the mixture of mobile phase is the optimized constant composition. The

delivery system remains at equilibrium with the column during the separation procedure since the

chemical constituents do not change. This method of chromatography has ideal reproducibility and

accuracy for some sensitive and routine sample analysis such as caffeine and garlic constituents [70].

Many herbal compounds are complex mixtures including both very hydrophilic and hydrophobic

compounds; but the HPLC system separates/resolves the individual constituents in reasonable

separation times. Obviously, the composition of the mobile phase determines the retention times

and resolution of the components. Therefore, the majority of chromatographic runs have been

based on linear gradient conditions in the mobile phase in which the solvent strength is increased

with time using a gradient elution separation. The retention factor (k or capacity) is usually improved

by altering the amount of organic solvent (modifier) composition.

The Isocratic separation is suitable for quality control studies because the elution data

reproducibility (e.g. retention time) by the separation conditions is stable. In contrast, the gradient

elution separation is suitable for the analysis of complex samples, applied to unknown mixtures and

linear gradients that are most commonly used. During the gradient elution the measured baseline is

less stable than in isocratic separations due to the frequent equilibrium changes between the mobile

and stationary phases. Therefore, it is advisable to choose high purity solvents as the mobile phase

to avoid contamination effects, run the pre-blank baseline at the start before any sequences of

injections to ensure the baseline and system is stable. It is also essential to perform blank sample

checks, performed by using the sample or standard solvent, to check the solvent purity. Some pH

modifiers are always added to the mobile phase, such as acetic acid, formic acid or ammonium salts,

and it is recommended to ensure that in blank samples there is a solvent UV absorption free to

target analyst interference before sample or standard injections are made [71]. When preparing

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samples, some analytes may be less soluble in the mobile phase, which is detrimental to the column

and decreases separation reproducibility. The mobile phase solvents are suggested also for

dissolving the sample and standards. All samples need to be filtered (e.g. 0.45um filter) prior to

injection into the flow stream.

The most common UV detection technique used is HPLC-DAD (diode-array detection), which

measures the absorbance of UV wavelength against the concentration of the analyst as shown with

the instrument [Fig.1.5 (c)].

Natural product analytes may o tai o o di g π* a d σ* o itals i the u satu ated atom

bonds, aromatic groups, or functional groups. The electrons absorb the incident energy and rise to

the higher energy state. The analyte UV energy absorbance is determined after the light passes

through the sample. Analyte concentrations can be measured by the Beer-Lambert law

equation: A = ε · c · l, where A is the a so a e, ε is the a so pti it oeffi ie t, is the analyst

concentration, and l is the length of the incident light through the flow cell containing the compound

elution from the column. The absorptivity coefficient can be measured via standard calibration

curves. So the same or similar substance concentrations in the unknown sample can be determined

from the instrument response (absorbance) [72].

In the DAD the tungsten lamp (visible range) and the deuterium lamp (UV range) radiation is

transmitted through the flow cell. The emission light is dispersed to individual wavelengths then

reaches the diode-array for detection. In this way, the analyte absorbance signal can be measured

for each component identification and quantitative analysis [73]. The detector sensitivity can be

determined by the lamp and the manufacture design factors and the measures range from 190nm to

700nm approximately. Concerns for sample detection involves the solvent cut-off value that is the

analyte approximate wavelengths (nm) when they are specified below the absorbance that may be

masked in the solvent. The solvents themselves absorb UV light in the lower wavelength range, such

as the UV cut off value (nm) of water, acetonitrile values are 190 nm and methanol for 205 nm.

Especially, under the condition of reverse-phase HPLC, the increased solvent in the mobile phase

might increase its UV absorbance that may mask the low wavelength sample elution absorbance.

1.3.2 Gas chromatography

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The technique of gas chromatography (GC) was invented by James and Martin in 1952 and became

the standard analytical method in many industries [74]. Separation of the components results from

the distribution (partitioning) of each component between the mobile phase (carrier gas) and the

stationary phase, but the system separation parameter control is mainly via the column temperature

programme and gas flows. The basic requirement of GC analysis is sample and solvent volatility.

Volatile samples generate gaseous mixtures containing the vapours of carrier gas, sample and

solvent prior to passing on to the column. In general, the required amount sample is small for

introduction onto the column and can be delivered by SPME fiber or microsyringe (about 1-20µl).

After the sample is injected into the inlet, there are two injection modes available, split and splitless.

The purpose of the two modes is to optimize the column separation. In the split mode, only a

percentage of the gas mixture (from 1:1 to 1:500) is directed into the column, the remainder is

expelled through the split outlet. This avoids column overloading and results in non-sharp and

fronting peaks. When the split outlet is blocked, all gaseous are exposed to the column, this is the

splitless mode and is mainly used in trace analysis due to sensitivity improvements. The carrier gas

most commonly used is helium. Capillary column gives better performance efficiency, high

resolution and are widely used [75]. The capillary column for GC consists of narrow tube coated on

the interior with the stationary phase such as polysiloxanes or polyethylene glycols (i.e., 0.25 μ fil

in a 0.32 mm tube). Under the same column length condition (almost 30m), the separation efficiency

can be determined by the column temperature (ranging from about 50°C to 250°C) and the gas flow

rate. Overall, temperature increases result in reduced retention time however the resolution quality

decreases. The priority separations method usually chooses temperature gradients starting with the

column relatively cool (about 40-50°C), and then gradually and constantly increasing the

temperature stepwise (temperature programming); as the volatile component can interact with the

stationary phase coating determined by polarity and boiling points. High gas flow rates reduce the

component retention time also. There are many different detectors used for GC, for example flame

ionization (FID), nitrogen–phosphorus (NPD) detection and others.

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Fig.1.6 Schematic of a typical simple GC-MS [reproduced from Restek Corporation website].

1.3.3 Mass spectrometry (MS) [76,77]

The basic configuration of the gas chromatograph and mass spectrometer (GC/MS), for which is used

to separate the components in an unknown sample and MS as GC detector is illustrated in Figure

1.6. Mass spectrometry is the most commonly used instrumental technique, for the identification

and quantification of components in the mixture. The computer controlled GC and MS parameters

such as separation programmes, injector temperature, MS scan mode and identification library

match is available for two or more hyphenated GC-MS systems, for example GCxGC-MS and GC-MS-

MS systems.

Mass spectrometers detect the ionized chemical species according to their mass to charge ratio

(m/z), which include ionization source, mass analyser and ion detection system. First, sample needs

to be ionized by various methods such as electron impact (EI), chemical ionisation (CI). EI is used in

this research project at the standard ionization energy of 70 eV. Once the electrons collide with the

molecular substrate (M), the loss one electron results in the molecular ion (M+) or it breaks into

various fragments that contain smaller ions or it involves neutral fragment losses (eg. H2O, NH3).

Then the ions are focused by a mass analyser for separation and resolution via their m/z value. The

ion process includes quadrupole, time-of-flight (TOF) analysers, magnetic sectors and so on. For the

Agilent HP 5973 Mass Spectrometer in the RMIT laboratory, quadrupole mass filters were used

[Fig.1.7]. This consists of four parallel metal rods, of which two opposite rods have an applied

potential of radio frequency(RF) voltage and the other two rods have a potential of direct current

(DC) offset voltage. The two pair applied voltage rods can select ions via m/z ratio. Only certain m/z

ratio ions can travel down the quadrupole filter and reach the detector. After signal amplification,

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the detector sends information to a computer recording all of the data produced and converts the

electrical impulses into visual displays (mass spectrum). The mass spectrum expresses the fragment

(ions) mass weight from the molecular with its characteristic relative abundances. The computer

operations allow data acquisition with two scan modes, total ion chromatogram (TIC) and selected

ion monitoring (SIM). The SIM mode utilizes selective monitoring of particular ions for example, a

unique ion of the compound (e.g.intact molecular fragment) and high abundance especially higher

mass. At present, data analysis programs run integration of chromatograms, and then compares the

obtained spectra to reference spectral databases in library search to generate reports including

identification and quality matches with reference library compounds.

Fig.1.7 Configuration of quadrupole mass filter [reproduced from reference 76]

Therefore, GC/MS can be used to analyse many low molecular weight compounds, applications that

require compounds to be chemically stable at high temperatures and volatile. Many samples from

complex matrixes (e.g. soils, herbal mixture, tissues etc.) contain many non-target ingredients that

may interfere with the data acquisition and analysis of the compounds of interest. It is essential to

treat these samples with solvent extraction or SPME techniques before GC/MS analysis.

1.4 Thioflavin T (ThT) fluorescence assays

The present methods to assay amyloid fibrils is to utilize thioflavin T (ThT) fluorescence, congo red

binding, transmission electron microscopy (TEM), and Fourier transform infrared spectroscopy

(FTIR); ThT dye is the most commonly used fluorescence detector for the identification and

quantification of amyloid fibrils in vitro.

The German physician scientist Rudolph Virchow oi ed a loid a d utilized iodine-sulphuric acid

treatment in 1854, as the first amyloid dyes to stain abnormal organs [78]. During the 20th century,

the amyloid fibrils were detected using congo red dye observed under polarized light microscopy,

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because the dye binding produces a birefringence pattern that differentiates amyloid fibrils from

other fibril proteins such as collagen and non amyloid deposits [79]. In 1959, the benzothiazole dye

ThT was first used as a fluorescent marker to study amyloid detection in the kidneys [80]. The

thiazole quaternary nitrogen of ThT selectively interacts with the hydroxyl groups of amyloid

deposits to form hydrogen bonds and produce the specific binding within the grooves between

solvent-exposed amino acid side chains of the amyloid fibril [81]. The florescence intensity increases

are recorded [Fig. 8 (a), (b) and(c)] [82]. ThT assays are routinely applied to study amyloid fibril

formation and the fluorescence intensity from the amyloid–ThT complex relates to the amount of

amyloid fibril present. ThT can avoid many issues compared with congo red birefringence staining

because amyloid deposits could be miscalculated due to sample thickness or orientation [83]. Also,

the congo red dye can result in low reproducibility due to the staining background after washing.

With the ThT assays, a phosphate buffer (10mM potassium phosphate, 150mM NaCl, or 50mM

phosphate; pH: 5.8–7.0) are used to prepare ThT stock solutions (0.8mg/mL) in the phosphate

buffer, needed to be covered with aluminium foil and kept at 4 °C refrigerator for less than 1 week;

the working solution should be diluted 50 times and made fresh daily [84].

1.4.1 Fluorescence spectrometry measurements

Fluorescence spectrometry is mostly used to identify or quantify an analyte in solution using a beam

of light with a wavelength between 180 and 800 nm being transmitted through the sample solution

in a cuvette. In fluorescence spectrometry [85,86] the light from an excitation lamp source that

passes through a filter or monochromator for selective specific spectral band (excitation spectrum),

is absorbed by the sample and measured. Some of this fluorescent light emitted by the sample

passes through a second filter or monochromator (emission spectrum) from an angle at 90° and the

detector measures the intensity of the emission, which is directly proportional to the analyte sample

[Fig.1.8 (d)]. The samples include amyloid fibrils such as β-sheet-rich deposits after ThT was added,

which fluoresces brightly with excitation and emission maxima at approximately 440-450 and 482-

490 nm, respectively; however other non-binding ThT in the aqueous solution, exhibit a weak

fluorescence intensity and lower (blue-shifted) excitation and emission maxima at 350 and 438-

440 nm, respectively [87]. Therefore, after ThT addition to the samples containing amyloid and

measurement of the fluorescence excitation at 440-450 nm the emission is detected at 482- 490 nm.

There are two common variations to this assay, (i) is a common measure with single time-point test,

that is, the sample containing fibrils are diluted in ThT buffered solutions and then recorded at

various time point readings (e.g. 24, 48, 72 hours); the other is fibrillation kinetics in real-time

measurement. The fibrils formation can be tracked immediately.

(a) (c)

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(b)

Fig.1.8 (a) The structure of Thioflavin T; (b)Magnified view of the ThT– i di g β-sheet of β-sheet-rich

deposits. The site near ThT-binding are shown as red sticks; (c) Essential component of a

fluorescence spectrometer; (d) Characteristic increase in ThT fluorescence upon binding to amyloid

fibrils. [reproduced from references 82,85,86]

1.5 Coffee herbal functional beverages

Coffee and herbs have a long human use history and have many benefits for human health according

to research studies. They are a combination of natural products to produce a functional beverage for

chronic consumption. In aim of this project is the development of an analytical method to identify

and quantify the major beverage ingredients and to explore the human taste perceptions of a

selection of coffee-herbal beverages.

1.5.1 Coffee aroma induces/invites functional beverage usage

Coffee is one of the most popular beverages in the world, that provides a stimulating effect on

human sensory organs. Progress in coffee chemical analysis utilizing GC-MS, has resulted in more

than eight hundred volatile compounds [88] being identified, many of which have been

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demonstrated to contribute to coffee aroma. The aroma profile of coffee is composed of the

following notes: sweet/caramel-like, earthy, roasty, smoky, fruity and spicy. The volatiles are from

the classes of compounds including furans, sulphur compounds, pyrazine, ketones, phenols, pyrroles

and comprise more than 60% of the total compounds. Kahweofuran, 2-furfurylthiol (roasty), 4-

vinylguaiacol (smoky), three alkylpyrazines (earthy), four furanones (sweet/caramel-like, spicy) and

five aliphatic aldehydes (fruity), are important determinants of coffee odour [88,89].

1.5.2 Herbs are widely used ingredients/nutrients to improve health

The documented benefits of liquorice and pharmacological studies indicate it is used to relieve

cough, pain, and antiviral activity, oxidative stress, inflammation and has neuro-protective

properties [90]. In the food industry, liquorice flavour is widely applied in candies or sweets and has

long been used to flavour some dishes, drinks, and candies, as liquorice-like flavour in Western

cuisines. Ginseng has a leading position in the best-selling natural supplementary food products in

the world. Ginseng and its extracts have been traditionally used to revitalize the body and mind,

increase physical strength, memory and prevent aging, particularly in China, Korea and Japan. Ginger

has a long and well document history consumed as a spice herb in cooking. Therapeutically it is

widely used to control fever, pain, indigestion and infection diseases.

1.5.3 Functional beverage sensory measuring

The sensory properties are a major determinant for the coffee-herbal functional beverage people

select and how much/often they are consumed. The functional beverage familiarity evaluations

apply a hedonic scale to predict the sensory acceptance, consumer behaviour and eventually modify

the beverage recipe further. The 9-point hedonic scales are applied to evaluate the aroma and

flavour of the functional beverage and investigate their contributing factors for the long term

functional beverage consumption. This is the one of most used methods to quantify consumer

acceptance, which was first published by Peryam and Pilgrim [91] and has been used in academic

and industrial consumer research in America and Europe. This hedonic scale describes an equal

number of positive and negative categories with intervals of equal size. For example, 1=not at all

liked, 3=slightly liked, 5=moderate liked, 7= like very much liked and 9=extremely likeable.

1.6 Project hypothesis and scope

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De e tia i ludi g Alzhei e s disease is a slo p og essi e eu odege e ati e disease

commencing in midlife and it is difficult to reverse the process of loss of cognitive ability. However,

we propose that if an effective treatment to reduce the rate of cognitive decline without any side

effects could be found to delay and protect the brain from the dementia process, this would be

highly desirable. Based on previous research, selected herbs and coffee to produce coffee herbal

functional beverages against dementia were chosen for this project. The selected herbal

compounds will be measured by analytical methods such as GC/MS and HPLC/UV in the coffee

herbal beverage [Fig.1.9]. Also, we will compare various herbal forms to ensure the highest

effective/best available sources of the herbs are used in our research. It is important to determine if

the herbal bioactive compounds can be extracted by safe and convenient methods (such as

decoction, percolation and microwave heating). A small group of volunteers will be invited to

provide their personal/subjective estimate of the coffee-herbal beverage sensory perception via

hedonic scales as feedback to improve the beverage recipe.

1.6.1 The selection criteria of the herbs used in functional beverages

Firstly, the herbs used should have a long history of consumption without any health side effects and

also be available as food supplements. Secondly, their selection is based on research evidence that

these herbal compounds can cross blood brain barrier (BBB) into the brain. Also that they are

effective in improving memory, are able to inhibit β-amyloid fibril formation or have anti-

inflammatory activity in the brain. Thirdly, the analysis of the main constituents present in the herbs

will be studied to determine whether they can be maximally extracted into the coffee herbal

beverage. Last but not the least, the herbs can produce favourable flavour and sensory perception,

which will encourage continuous consumption.

1.6.2 Key project questions

Although the core task of the project is to analyse the bioactive compounds in coffee and in the

herbs, the key questions addressed are not limited to their analysis. This is a multi-disciplinary

p oje t, hi h i ol es the edi i al k o ledge of Alzhei e s disease patholog , he al

pharmaceutical chemistry related to AD, natural product analysis (identification and quantitation)

and human research ethics.

36 | P a g e

1. Why may coffee herbal beverages be chemopreventive/neuroprotective against dementia?

2. What kinds of herbal compounds can be selected for preventing cognitive decline and what

are the herbal compound selection criteria?

3. What types of methods would be applied to measure the amount of target compounds in

coffee herbal beverages, including instrumentation, procedure and data analysis?

4. Can these effective bioactive compounds be extracted by a simple procedure such as a

coffee machine or microwave oven?

5. How can we measure the usto e s sensory responses to the new beverage?

Define the separation goal

Choose detector and

detector settings

Sample pre-treatment

(The analyst extraction, solubility

in solvent, purification and etal.)

Choose LC or GC method

(Including mobile phase, column type,

operation setting and preliminary run)

Retention time adjustment

(Including solvent strength, pH,

temperature, standards preparing)

Separation method validation

(Including calibration curve creating,

measuring accuracy, precision and etal.)

The analyte peak area record

and concentration calculation

Fig.1.9 Typical sample preparation and separation steps involved in GC/MS and HPLC

Sample Information

GC or HPLC

Sample preparation

Separation method

Optimize Separation

Routine test and

data analysis

Qualitative and quantitative

method

37 | P a g e

1.7 References

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2

Coffee Herbal Bioactive Compounds for the

Potential Prevention of Dementia

Chapter 2 is concerned with the cause and mechanism of Alzhei e s disease and herbal

bioactivities against the disease, of the compounds selected/utilised for this project. This chapter

describes the coffee herbal beverage compounds investigated.

2.1 Multiple targets agai st Alzhei er’s disease by natural products

The German physician Alois Alzheimer first recorded the disease s pto s of Alzhei e s disease i

1907, when he described the clinical case of Auguste Deter, in middle-age who had suffered serious

memory loss that worsened. He demonstrated the shrinking of the outer layer and dramatic

abnormal deposits around nerve cells in the patient s brain. The accumulation of amyloid

oligomers/plaques and neurofibrillary tangles are hypothesised to contribute to the

neurodegradation of neurons (nerve cells) in the brain and amyloidosis is hypothesised as the

central causative factor of Alzheimer's disease [1]. A framework divides the disease into four stages:

early dementia, mild dementia, moderate dementia and severe dementia. Recent research evidence

has i di ated that solu le Aβ oligo e s a e o e to i tha mature fibrils [2,3] suggesting that

oligomers should be a high priority target for designing potential antiamyloidogenic agents for AD.

44 | P a g e

Other AD hypotheses have been proposed including cholinergic, inflammatory /oxidative and

adenosine hypotheses. However, the most widely accepted theory is the amyloid hypothesis [4].

These hypotheses are also the targets for natural products to prevent neurodegeneration.

2.1.1 Target 1: A yloid β peptide is produ ed y proteases

Although the mechanism of AD is not fully understood, currently the a loid β peptide Aβ is the

main component of the amyloid plaques found in the brains of Alzheimer patients. Senile plaques

o tai oth Aβ a d Aβ [5] Fig.2.1. In particular, the i ease of Aβ o Aβ /Aβ atio leads

to amyloid oligomer formation, that causes subtle and then increasingly severe and permanent

changes of synaptic function [6]. Aβ is cleaved from the amyloid precursor protein (APP) by three

proteases: α-secretase (the non-a loigoge i path a sho i Figu e . a , β-secretase (cleaves

at the β-site of APP by the transmembrane enzyme aspartic protease BACE a d γ-secretase as

illustrated in Figure 2.2b. APP is a single transmembrane polypeptide and can undergo a variety of

proteolytic cleavages to secrete derivatives into the extracellular space [7]. β-Amyloid precursor

p otei βAPP is cleaved β-secretase [8]. The γ-secretase enzyme cleaves the remaining 99 amino

acid C-terminal fragment of APP C to Aβ o Aβ [7] and a C59 residue. The APP is cleaved by

α- se etase, eleasi g the solu le APPα peptide f ag e t a d C-terminal fragment further cleaved

γ-secretase, producing p3 nontoxic fragments [10]. The efo e, i easi g α-secretase activity can

de ease the le el of βAPP a d e ha e eu op ote ti e effe ts, hile β- se etase a d γ-secretase,

endoproteolyze APP to li e ate the Aβ peptide Fig. . . There is evidence that the i eased Aβ

p odu tio a d e ha ed depositio of β-sheet amyloid is linked to β- secretase

upregulation/activity in sporadic AD patients [11].

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Fig. 2.1 Amyloid cascade hypothesis [reproduced from reference 12]

Fig.2.2 Enzymatic processing of transmembrane APP: the non-amyloidogenic (A) and amyloidogenic

pathways (B). [reproduced from reference 13]

2.1.2 Target 2: Neurodegeneration is accelerated by inflammation and oxidative stress

It has long been known that the damage of brain tissue and the deposition of highly insoluble

a o al ate ials like da aged eu o s, eu itis, a loid β peptide deposits a d eu ofi illa

tangles) are powerful microglia activators [14]. Microglia are the immune cells of the brain where

local inflammatory responses (microgliosis and astrocytosis) are observed. As microglial activation in

particular, are core features of AD and secrete increased levels of multiple inflammatory

mediators[15], Aβ i du es the e p essio of p oi fla ato tokines including IL- β, IL-6, TNF,

oxygen free radicals and nitric oxide; also the induction of inflammatory enzyme systems such as the

nitric oxide synthase (iNOS) and the cyclooxygenase enzyme (markers of inflammation COX-2).

Overexpression of interleukin (IL)-1, IL-6 and tumour necrosis factor (TNF-α lead to dysfunction and

neuronal death [16,17]. Afte the o e sio of a esti g i oglia i to a a ti ated phe ot pe, it

can be observed that the increase of expression of amyloid precursor protein a d its β-secretase

lea ed p odu ts sAPPβs as ell as Aβ oligo e s lead to a u ulatio of Aβ. However, normally

this is reduced by the α-secretase cleaved products (sAPPas) in human neuronal cell lines [18]. There

is a correlation between diminished cognitive status and microglial activation in mild cognitive

impairmed patients [19]. Astrocytes, also known collectively as astroglia, are comprised of a highly

abundant population of glial cells and serve an array of important functions including the regulation

of extracellular ion concentrations, synaptic remodelling (addition and removal of synapses), and the

46 | P a g e

maintenance of protective barriers like the blood-brain barrier [20]. Senescent astrocytes were

found accumulated in brain tissue from aged individuals and AD patients [21]. Anti-inflammatory

approaches have been developed that may not cure AD directly but slow the progression or delay

the onset of this devastating disorder. More than 50% of the benefit from taking non-steroidal anti-

inflammatory drugs (NSAIDs) is to prevent the onset of dementia with long-term usage against AD

[22].

Oxidative stress is produced by the imbalance between production and removal of free radicals and

reactive oxygen species (ROS), such as superoxide anion (O2-), hydrogen peroxide (H2O2) and

h d o l adi al OH• ; the important sources of these are from glia cells inflammation and aging in

the brain. Proteins and lipids are targets for oxidative attack increase the risk of neurodegenerative

diseases occuring and result in protein misfolding [23]. During early dementia stage, it can be

observed that o idati e da age p e edes Aβ depositio due to ito ho d ial eta olis

dysfunction and apoptosis [24]. Many natural products that can eliminate free radicals and reactive

oxygen species (ROS), are antioxidants [25].

2.1.3 Target 3: Cholinesterase inhibition agai st Alzhei er’s disease

Cholinesterase inhibitor drugs are widely applied for treating the symptoms of AD and are licenced

AD drugs. Generally speaking, acetylcholine is an important neurotransmitter for cognition and

memory, and patients with dementia, AD have low levels of acetylcholine. If natural compounds can

sustain acetylcholine levels via cholinesterase inhibition, this sustains neurotransmission between

nerve cells, which in turn, may temporarily ameliorate or stabilise the symptoms of dementia.

2.1.4 Target 4: Adenosine receptor inhibition agai st Alzhei er’s disease

Adenosine, an endogenous nucleoside, plays important roles in synaptic transmission and neuronal

excitability in glia and neurons, which activate adenosine receptor including A1, A2A, A2B and A3 G-

protein coupled receptors to have effect on cognition, memory and neuronal degeneration [26].

Some research has suggested that the receptors of A1 and A2 were inhibited to improve cognitive

function or decrease cognitive impairment [27]. To prevent neuron damage, A1 adenosine

receptors activation tends to restrain glutamate release that hyperpolarize neurons by a

predominantly presynaptic action, while A2A adenosine receptors inhibition are more likely to

47 | P a g e

decrease transmitter release and postsynaptic depolarization and promote synaptic plasticity [28].

These two receptors are composed of dimers with different metabotropic receptors operated by

other neurotransmitters such as glutamate, NMDA and nitric oxide [29]. Therefore, adenosine

receptors could promote the inflammatory process regulation and pro-inflammatory cytokine

release such as interleukins and TNF and promote cognitive impairment during brain damage. It has

been observed that A1Rs expression is reduced in cortical and hippocampal regions but A2ARs

increase in limbic regions in aged animals. It is proposed that a new target for the development of

new therapies that A1 adenosine receptor (AR) agonists, A2AR antagonists and A3AR antagonists for

neuroprotection can be based on the adenosine neuromodulatory system. In the context of this

project, coffee is neuroprotective, as caffeine is a non-selective inhibitor of adenosine receptors and

is a protective agent against AD [30,31].

2.2 Coffee protects against dementia

There are over 25 varieties of coffee plants known, although only two are exploited in economically

significant quantities: Arabica (Coffee Arabica) and Robusta (Coffee by the canephora plant). Coffee

has long been demonstrated to possess various mechanisms against AD. We have focused on coffee

protection against dementia and Aβ i hi itio as affei e a cross the blood-brain barrier (BBB)

and reduce the symptoms of inflammation and oxidative stress in brain.

2.2.1 Caffeine and coffee compounds exert protective effects against AD

Caffeine is rapidly absorbed from the gastrointestinal tract in the stomach and small intestine, and

distributes rapidly in all tissues including the brain. In rodent studies aged cognitively-impaired AD

mice exhibited memory restoration and lower plasma a d ai Aβ levels following 1–2 months of

caffeine treatment (the human equivalent of 500 mg caffeine or 5 cups of coffee per day). It

suppresses oth β- a d γ-secretase levels [32,33]. Caffeine can i ease ai Aβ lea a e at the

BBB and decrease the inflammatory cytokine levels in the hippocampus [34,35]. It is a non-specific

adenosine antagonist, its neuroprotective effect is likely to be the blockade of adenosine A2A , which

play a role in the control of neuroinflammation [36,37]. Trigonelline, a coffee constituent and a

component of the popular Indian curry spice, [38]. Both caffeine and trigonelline are investigated as

anti-AD drug candidates [39]. Trigonelline docking ith Aβ42 interacts with key residues (His6, Tyr10,

His13 and His14) and alters its structure to inhibit its aggregation [40]. After 15 days of orally

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administered trigonelline (500 mg/kg) there was i p o ed e o i i e i du ed Aβ –35)

injection [41]. Chlorogenic acid (9mg/kg) a major polyphenolic component in coffee, significantly

improved the cognitive impairment of experimental models of AD induced by scopolamine

(0.5mg/kg) in mice and involved anti-AChE and anti-oxidative mechanisms [42]. The investigation

indicated that these coffee compounds including chlorogenic acid, quercetin, caffeine, can reduce

the release of TNF-α a d IL-6 from the activated microglia and astrocytes [43].

Fig.2.3 Structures of caffeine, trigonelline and caffeic acid

2.2.2 Human studies of neuroprotection of coffee against dementia

According to the Cardiovascular Risk Factors, Aging and Dementia (CAIDE) study data after an

average follow-up of 21 years (midlife visit), a total of 61 cases were identified as demented (48 with

AD) of 1409 individuals. Moderate coffee drinkers (3-5cups/day) at mild life had a 65–70% decreased

risk of dementia and AD compared with those drinking no or only little coffee (0-2 cups/day) [44]. Aβ

levels were not seen in human blood following acute caffeine administration. Other survey analysis

also concluded that coffee consumption is associated with a reduced risk of AD [45]. Interestingly,

Caffeine intake was associated with a better cognitive response to acetylcholinesterase inhibitors

(AChEIs) in AD women, according to northern Taiwan clinical trial that included 234 mild to

moderate AD patients studied from 2007 to 2010 [46].

2.3 Liquorice protects against dementia

Glycyrrhiza is a genus of about 18 accepted species in the legume family, of which Glycyrrhiza glabra

is a species native to the Mediterranean region called Licorice (liquorices); G. Echinata is a Russian

licorice and G. Uralensis are known as Chinese Licorice. More than 400 compounds have been

identified from Glycyrrhizin species; including flavonoids, glycyrrhizic acid (GA )and glabridin. All are

utilized for major medicinal purposes [47]. Glycyrrhizic acid contains both hydrophilic (glucuronic

http://en.wikipedia.org/wiki/Mediterranean

49 | P a g e

acid) and hydrophobic (glycyrrhetic acid) regions, which can form stable inclusion complexes with

various organic molecules because GA in solution can create cyclic structures [Fig.2.4] [48]. It may be

reasonable to propose that this molecular characteristic provides not only high stability but may

reduce side effects or improve the therapeutic efficiency.

Fig.2.4 β-Glycyrrhizic acid converts to a stable inclusion complex with other molecules (GA do ut

ring) [reproduced from reference 48]

2.3.1 Liquorice compounds that ay affe t Alzhei er’s Disease

Liquorice intake (150 mg/kg for 7 successive days) [49] inhibited acetylcholinesterase (AChE) in

brains of mice and prevents Aβ -35-induced (25uM) apoptosis in PC12 cells under 1mg/ml [50,51].

Glycyrrhizic acid (GA) (5,10 mg/kg) [Fig.2.5], which is the major active component of liquorice

suppressed the inductions of TNF-α, IL- β, COX-2, and iNOS to have the anti-inflammatory effects

[52] and protect neurons via anti-oxidative [53]. Some reports show that GA can be metabolized into

β-glyc heti i a id βGA , a othe ajor active compound in liquorice β-D-glucuronidase in

intestinal bacteria [54]. βGA can i d to i e alo o ti oid e epto s MRs a d β-

hydroxysteroid dehydrogenase type- β-HSD1) distributed mainly in neurons, moderately in

astrocytes of the hippocampus [55]. Diammonium glycyrrhizinate (DG) (0.01μg/μl) attenuates

eu o al i ju ia de easi g ROS p odu tio i du ed Aβ -42(2uM) and improved cognitive

i pai e t i Aβ -42 induced AD mice with 10mg/kg for 14 days [56]. Liquiritigenin (50μM)

effectively protects against glutamate-induced (5mM) hippocampus-derived neuronal cell line (HT22

cells) early apoptosis via inhibition of Ca2+ influx, intracellular reactive oxygen species (ROS)

production, and lipid peroxidation in mice [57]. It is similar to isoliquiritigenin (1- μM that

i hi ited Aβ -35)-induced (10μM) neuronal apoptotic death in relation with Ca2+ and ROS [51].

Liquorice-derived dehydroglyasperin C (DGC) has effects on inflammation-mediated

50 | P a g e

neurodegeneration and also reduces cytotoxicity and ROS generation [58].

Fig.2.5 Glycyrrhizic acid (GA) and Glabridin

2.3.2 Human studies of liquorice protection against dementia

Liquorice of 260ml hot water extract taken for 8 weeks, decreased in 20 healthy male smokers (aged

20-60) blood sample plasma levels of conjugated dienes (a maker for lipid peroxidation), that might

be ascribed to its antioxidant compounds [59]. According to the 2010 edition of the Chinese

Pharmacopoeia, liquorice is recorded at position 320 of 1062 prescriptions as a principal or assistant

drug [60]. It is reported that 10 mg glycyrrhizic acid intake daily is safe for most healthy adults [61].

2.4 Ginseng protects against dementia

Panax ginseng C. A. Meyer (Asian ginseng) and Panax quinquefolius L. (American ginseng) are the

two most recognized ginseng type around the world [62]. The key active ingredients of ginseng are

known as saponins or ginsenosides such as Ginsenoside-Rg1 (G-Rg1), Ginsenoside-Rg2 (G-Rg2) and

Ginsenoside-Rb1 (G-Rb1), Re, Rd [Fig.2.6a]. These compounds are known to improve attention,

processing, memory and preventing memory impairment [63]. Asian and American ginseng contain

similar ginsenoside profiles but Rb1 is richer in American ginseng [64.]

2.4.1 Ginseng compounds that ay affe t Alzhei er’s disease

Red ginseng is produced by steaming raw ginseng, the extract (100mg/kg) significantly ameliorated

young and aged rats with hippocampal lesions in the place-navigation deficits (PLT), because

expression of inflammatory cytokines such as TNF-α, IL- β, a d IL-6 were reduced in the serum level

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[65]. Ginsenoside composition from steamed ginseng also can inhibit acetylcholinesterase (AChE)

and butyrylcholinesterase (BChE) activities [66]. Ginsenoside Rg1 (10, 20 mg/kg) promoted spatial

learning and memory on ovariectomized rat model of AD after treatment 6 weeks; It can reduce

Aβ -42 generation by increasing ADAM 10 expression o e of α-secretase) and decreasing BACE1

expression o e of β-secretase) [67,68] ginsenoside Rb1 can inhibit liver cells fibrosis via decrease

plasma aminotransferase activities and inflammation [69]. The long-term pretreatment with

ginsenoside Rb1 attenuated the aged mice brain impairment via extensive anti-oxidant activity.

Ginsenoside Rb1 selectively inhibited the activity of L-type voltage-gated calcium channels to decline

the damage in hippocampal neurons [70,71]. In vitro, ginsenoside Rb1(0.1, 1mM) inhibited β-

secretase activity [72] and protected agai st Aβ -35 induced (25 or 50µM) cytotoxicity in PC12

cells, Ginsenoside Re(0.1-100uM) [73]. The inhibitory effect of ginsenoside Rg2 (0.2 mmol/L) against

the fo atio of Aβ –40 induced by glutamate in PC12 cells [74]. Rg2 (2.5, 5 and 10 mg/kg) also

prevented memory impairment in a rat model with vascular dementia via modulating the expression

of apoptotic related proteins [75].

(a) (b)

Fig.2.6 (a) Ginsenosides R , Rg ; α-Pi e e, β-Pinene and Camphene

2.4.2 Human studies of ginseng protection against dementia

Endogenous antioxidants in human plasma like glutathione peroxidase (GPx), catalase and

superoxide dismutase (SOD) activities were significantly increased after Ginseng (6 g/day)

supplement for 8 weeks because ginseng has effect on regulation of immune system to enhance

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resistance to illness [76,77]. In AD patients, red ginseng including Rb1 (1.96%), Rb2 (2.18%) Rg1

(0.49%), and Rg2 (0.13%) treated with 9 g/day the dose lasting 12 weeks, significantly improved the

Alzhei e s Disease Assess e t S ale ADAS , Cli ical Dementia Rating (CDR) scale [78]. Red Ginseng

is a cognitive enhancing saponin used as complementary anti-dementia medication. Panax ginseng

administration for 4 weeks was shown to be safe, tolerable, and free of any untoward toxic effect in

healthy male and female volunteers [79].

2.5 Ginger protects against dementia

Ginger (ginger root) is the rhizome of the plant Zingiber officinale and contains many potentially

bioactive compounds. Studies have shown that the volatile oil of ginger contains up to three percent

of fragrant essential oil including monoterpe oid f a tio α-pinene, camphene, geraniol, geranial

and zingiberene) [Fig.2.6b]. Overall, the in vitro evaluation of the ginger extract expressed high

antioxidant activity, inhibited AChE and BChE and activity against the fo atio of Aβ oligo e s ith

the ThT binding assay, which showed the neuroprotective functions [80].

2.5.1 Compounds in gi ger that ay affe t Alzhei er’s disease

The ginger component such as 6-gingerol has anti-inflammatory activities [81]. Furthermore, ginger

extract profoundly blocked expression of IL- β a d COX-2 by fibril Aβ -42) activation with THP-1

cell line preclinical studies, with properties similar to human microglial cells [82]. It should be

possible to reduce or delay the progression the pathogenesis of AD through anti-inflammatory

approaches. 6-Shogaol (10 mg/kg/day) for 5 consecutive days significantly decreased activation of

microglia and astrocyte visualized by rat monoclonal anti-CD11b (Mac-1) compared to after

stereotaxic injection of Aβ peptide –42 (10µM, 3µl). The same consequence was observed in the

hilar region of dentate gyrus (DG) in hippocampus. Meanwhile, 6-shogaol reduced memory

i pai e t ith Aβ peptide –42 hippocampus lesion in mice through the passive avoidance test.

This has also been confirmed in the scopolamine-induced memory impairment model by passive

avoidance test. Neuron growth factor and synaptophysin levels were significantly elevated in the 6-

shogaol treatment of mice hippocampal tissues [83]. In clinical trials, ginger is often advocated for

use for its beneficial effects against nausea and vomiting.

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3 The Analysis of Herbal-Coffee Beverages

We utilized coffee and herbs, including liquorice, ginseng and ginger, to produce herbal coffee

beverages. Quality control is one of the most important aspects for beverage usage and

modification. According to the effective bioactive compounds for preventing dementia described in

chapter 2, the bioactive compounds caffeine, trigonelline, glycyrrhizic acid ginsenoside Rb1 and

camphene were set as the analysis compounds. A herbal quality valuation method choose one or

two pharmacologically active components from the herbal mixtures [1]. The method has the

advantage to ensure the active compound concentrations are known and are reproducible providing

beverage quality assurance as the concentrations of the bioactive compounds determines the

potential pharmacological effects.

Chromatographic techniques have been developed to assess qualitative and quantitative analysis of

these herbal compounds over the past decades. For the analysis of the bioactive compounds in

beverages several different techniques have been applied, including liquid chromatography with UV

absorption detection (LC/UV), SPME-gas chromatography-mass spectrometry (SPME-GC/MS) as the

most commonly validated methods. In this chapter the results of the analysis of the coffee-herbal

beverages are described along with a preliminary test for natural product inhibition of amyloid fibril

formation.

3.1 The validation parameters of the chromatographic method

3.1.1 Quantitation using external standards

The reason analytical standards are employed is to identify the retention time of the substance of

interest and quantify its chemical peak area (or height), when compared with the pure reference

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substance in an unknown matrix /solution, using chromatography. The standard analysis methods

can be used in two ways, either as an internal standard or as an external standard. In the external

standard method a series of the pure chemical, with the sample solution in varying ratios, then run

the blank and the combination of standards and samples respectively. The standard absolute

response is then plotted against the ratio of the substance concentration to provide the calibration

curves [2]. Regression analysis is used to define the relationship between the variables, and the fit

assessed by the coefficient of determination (R2) of the amount of unknown samples can be

calculated using the regression.

With the external standard method, the reference standard (or standards) must have the same

retention time as the the substance in the sample using the same chromatographic conditions. In

addition, the substance accuracy (recovery rate) should be assessed and be in acceptable range

(100±2%) because errors could be induced by the standard concentrations preparing or injection-to-

injection variation in each test. The detector is assumed to supply the same response to the

standard and the samples and provide a linear response for the range of the standards measured.

The equation used to calculate target analysis is, C(u)=A(u)/A(std)×C(std) Assuming, the response

index of the detector is unity, where A(u) is the peak area of target analyte in unknown sample, A

(std) is the peak area of standard and C (u) and C (std) are the concentration of target analyte in the

unknown sample and standard solution respectively [3]. However, it is necessary to understand the

behavior of the response factor as the concentration or amount of target compound and external

standard are variable. The behavior of the response factor allows one to set limits on acceptance

range (similar to confidence interval) of the concentrations of the standards batch. These

parameters include identity confirmation, accuracy, precision, linearity and detection limit.

3.1.2 Peak identification [4,5]

In chromatography, the evidence provided for peak identification is that that the retention time of

the unknown peaks are the same as the known standard peaks. However some other compounds

could have similar retention times. Spectrometric methods are applied to confirm the detected

compound is the same as the analyte. For example, DAD detectors are classical spectrometric HPLC

detectors, which supply information about structural features of compounds from their UV spectra.

Mass spectrometry is also another powerful method for identity confirmation, which provides mass-

to-charge (m/z) values of ions fragment from the analyst molecules and the intact molecular masses

61 | P a g e

with little fragmentation can be observed as well. The mass spectrum is mass-to-charge (m/z) values

(x-axis) against intensity of the ions (y-axis) [6].

Many GC/MS instruments utilize electron ionization (EI) mode to form a radical cation via collision-

induced dissociation with standard ionization energy of 70 eV. The intensity ratio of ions derived

from the same compound should be consistent under the same EI, which result in fragmentation in a

characteristic and reproducible way. These fragments are able to be scanned via full scan mode or

selected ion monitoring (SIM) mode. The MS spectrum can then be compared to large reliable

reference libraries obtained with different instruments because of standardized EI, and the GC/MS

analysis programme can provide a with the reference substance [7]. Additionally, The MS spectra of

samples can compare the presence of these ions and their relative intensities with standard peaks.

Some guidelines recommend 2–4 ions be selected for compound identification, and the ions

intensity should be in the acceptable range. The intact molecular fragment or high m/z value ions

are selected as specific, but the low abundant ions (relative intensity <5%) are not usually used for

identification. The normal neutral losses (e.g. H2O, NH3) do not have a characteristic diagnostic value

and are also not used for identification [8].

3.1.3 Accuracy (recovery) [9,10]

The accuracy analysis is an analytical method in the matrix in which the targets compounds test

results obtained are compared to the the true value. The spike recovery analysis is the most used

method for determining in herbal compounds studies, in which the recovery is based on the known

amount of the analyte working in the linear range of detection. The evaluated constituent of interest

is placed in the blank matrix and then as a percentage of the true value increase in the matrix

(spiked). The assay analyses are performed over a range of 50-150% of the target concentration

calculation under the chromatographic conditions. Accuracy criteria for drug substances and drug

products are recommended to be performed in 100 ± 2% and at least in triplicate as stated in FDA

Guideline for Submitting Samples and Analytical Data for Methods Validation.

In the present analysis, the accuracy is measured by spike recovery with the ratio of standards

concentrations at the 50, 75 and 100% levels (the concentrations are 40ppm, 60ppm and 80ppm

each). A series of different mixed standard solutions (caffeine, trigonelline, GA and Rb1 in HPLC)

were prepared with known added amounts (20ppm) and injected in triplicate. The herbal target

62 | P a g e

compound peak area will increase by approximately 50, 75 and 100%, and correspond to the ratio of

concentration levels rising respectively. Regarding the semi-quantitative camphene analysis by

SPME-GC/MS for ginger, the higher standard range is also set at the 50, 75 and 100% levels. The

purity of standards can also affect accuracy. In the HPLC and GC/MS investigation, the five major

standards were purchased from a commercial supplier, Sigma-Aldrich at >97% except for GA (>70%).

3.1.4 Precision [11,12]

The precision of an analytical method assess the closeness of agreement between a set of replicate

measurements performed under the separation conditions. Precision may be discussed at the two

areas: one is repeatability (method precision), which carry out the analysis at the three levels (50%-

150%) and performed in triplicate; the other is intermediate precision which examines the analysis

on multiple days and maybe with a different operator, equipment or column. The agreement is

easu ed i te s of elati e sta da d de iatio RSD% a d the al ulatio e uatio : RSD% = σ/μ ×

%, he e the σ is the a al sis standard deviatio SD a d μ e p ess the ea . I ge e al, the

RSD% accepted criteria are less than 10% in the combined assay, which is contributed to by variation

in days of sample preparation, column or operator.

3.1.5 Linearity [13]

The Calibration curve is defined by the model equation. R2 measures how well the model fits the

data. The li ea it of the odel is dete i ed a la k-of-fit test hi h dete i es hethe a

linear equation is the best fit for the data. In chromatography, the detector linearity represents the

range of concentrations of substance that will give a linear response. This is also defined as the

specific concentration range.

In the project, the DAD standards concentration range was set between 20ppm to 100ppm. The

method chosen ensured that the output of the plot of peak area is proportional to the concentration

(or mass of solute passing though it per unit time) and that is the chemical standards injection (such

as caffeine, trigonelline, GA and Rb1) were eluted from the column in a reasonable time. The

calibration curves for HPLC utilized a five concentrations batch and represent the standard individual

performance to the DAD and measurement of goodness of fit (coefficient of determination (R2)).

Generally, these responses from each analytical method were matched to the linear regression

63 | P a g e

model, y = mx + b, where y refers to DAD response (peak area), x represents standard

concentrations batch and m and b relate to the best fit slope and y axis intercept for each calibration

curve respectively. Strictly, the quantitative method accepted a good R2 criteria of 0.99 or greater,

but in some semi-quantitative analysis, R2 > 0.97 can also be acceptance to be linear. The

measurement accurately should be validated by other analysis methods for comparison.

3.1.6 Limits of detection [14, 15]

The term Li it of Dete tio [LOD] refers to the lowest concentration of the analyte that can be

reliably distinguished from the noise (baseline). In chromatographic analysis, the common approach

to estimate LOD, via linear regression method calculation, assumes that the instrument response y is

linear against the standard concentration x over the range. The linear equation expressed as: y =

mx+b, where m is the slope of the calculation curve. LOD calculations relate to Signal to Noise ratio

(S/N), which is the analyte peak against the baseline noise and should be at least 3 times higher than

baseline as the threshold measurement caused by instrument parameters such as lamp aging, the

column, the mode of detection and even manufacture. The valuation analysis of LOD can be defined

by the e uatio : LOD= σ/ ; he e σ ep ese ts the standard deviation of the analyst response and

m also expresses the slope of the calculation curve. The SD of response is accepted to utilize the

lowest concentration in the standards range, which is the closest to the zero during the assessment.

Therefore, LOD is important to evaluate whether the analytical method is suitable for analytical

purposes.

3.1.7 Kováts index (retention index) [16,17,18]

Kováts index or Kováts retention index refers to normalization of the component retention times in

GC via logarithmic calculation with n-alkanes as standards. Retention time is essential for the

identification parameters of the component and should always be reproducible under the same

analysis conditions. However, the retention time is determined by various factors including the

characteristic component, column (e.g. length, diameter, stationary phase and thickness of film) flow

rate gas carrier and oven temperature programme. It is a widely accepted method to use relative

retention times to reduce the influence of these factors. The equation is RRT= standard RT/sample

RT, where the standard is one of the same/similar to the sample. The disadvantage is the standard

variability of different samples.

64 | P a g e

Swiss chemist Ervin Kováts who introduced this concept of the retention index system concept

during the 1950s. This indicates the retention behaviour of the various substance determined by a

uniform scale of a series related closely standards. In other words, the well-behaved series of

standards (such as n-alkanes) are measured on several instruments and the standards as reference

describe the relation with the elution sample in one instrument via system-independent constants.

The the similar constant can then be produced according to the association between the reference

(the standards e.g. n-alkanes) and the samples on another instrument. Therefore, the Kováts index

uses two standards (n-alkanes) elution times to adjust the elution behaviour of the component in

the unknown sample and create system-independent constants, which locate before and after the

component. The constant (Kováts index) has the advantage that the same component should have a

close index values on different GC instruments whatever the various samples or separation

conditions and overcomes the instrument parameters influences mentioned above. It is important

to choose the right n-alkanes as reference standards; it should be an increasing homologous series

for instance alkane (C8-C20) and in high purity (analytical grade).

The Kováts index can be calculated using the equation below, based on the log-linear relationship

between number of carbons (n) in an n-alkane homologous series and retention time.

Where: I is the Kováts index, n is the number of carbon atoms in the alkane (before the component),

t Nn is the lo e a o ato s ete tio ti e efo e the o po e t i the alka e, t N(n+1) is the

higher carbon atoms retention time in the alkane (after the component), and the component elutes

between Cn and Cn+1. There are software programs available that then generate I from the retention

times.

3.2 Introduction to the coffee and herbal compound analysis method

The heterocyclic components of coffee (trigonelline, nicotinic acid and caffeine) concentrations were

determined by RP-HPLC- diode-array detection of the brewed beverage sample [19,20]. The mobile

phase used was methanol or acetonitrile, timeline limit in one hour, the flow rates between 1.0 and

2 mL min− and thermostatically controlled columns normally kept at ambient or slightly above room

temperatures; trigonelline analysed at 266nm and caffeine at 275nm [21,22].

NnnN

NnNX

tt

ttnI

'log'log

'log'log100

)1(

65 | P a g e

Triterpene saponins and flavonoids are the major components in liquorice; the concentrations of

which are determined by plant growth, harvesting time and time of processing [23]. The main

bioactive constituent GA is a triterpene saponins, which can be separated on reversed-phase C18

eluted with isocratic methanol: MQ water: (0.2 M ammonium acetate or acetic acid) (67:33:1) and

detected at 254nm. Because of the acidity of saponins, modifiers (such as ammonium acetate or

acetic acid) were usually used to suppress their ionization and to achieve optimal separation [24,25].

The concentrations of seven major ginsenosides (Rb1, Rb2, Rc, Rd, Re, Rf, and Rg1) were analysed by

reversed-phase HPLC ith sta da d C olu s o × . ; μ a d the o ile

phase of acetonitrile and water with buffer was used with an optimized gradient elution program.

Diode-array detection (DAD) is set at 198–205 nm for weak UV absorption for ginseng saponin

analysis [26, 27].

In the present study polydimethylsiloxane (PDMS) 100uM fibre was used to separate and identify

essential oil volatile components, such as the five main components in ginger consisting of

a phe e, α-phella d e e, β-phellandrene, camphor and zingiberene, by SPME–GC–MS analysis

[28,29].

3.3 Coffee herbal compounds extraction procedure

3.3.1 Information on instruments and reagents

1. HPLC: HP Agilent series 1100 DAD system

2. GC: HP 6890 series GC system

3. MS: HP 5973 Mass Selective Detector

4. Fluorimetry: HORIBA Floromax-4 Spectrofluorometer

5. All coffee and herbs were purchased as food samples fas commercial products, which means

that these companies are responsible for quality control, evaluating the safety and their

products ingredients are stable. [Table 3.1]

Product Content Species Storage

condition

Expiry

date

Brand Product code

(Lot #)

Roast

Coffee

250g

(per bag)

Arabica °C 08/2017 Giancarlo

coffee

-

Liquorice 425mg Glycyrrhiza °C 11/2017 Natures 17036

66 | P a g e

Root (per capsule) glabra Sunshine

Liquorice

Root

250mg/ml

(in alcohol)

Glycyrrhiza

glabra

°C 07/2020 Eclectic

Institute

31382C

Ginseng

Root

100g

(per bottle)

Panax

quinquefolius L.

°C 01/2017 Naturex GA505003

Ginseng

Root

200mg

(per capsule)

Panax

ginseng

°C 01/2018 Biovea 5010641

Ginger

Dry root

250mg

(per capsule)

Ginger °C 02/2018 Biovea 5022161

Ginger

fresh root

50g Ginger °C Fresh use Supermarket

(Woolworths)

-

Table 3.1 Coffee and herbs products information list

3.3.2 Mode 1: Comparison of beverages with varying ratios of herbs, via HPLC and GC/MS

All herbs were stored separately in with well-closed containers with protection from light, the

laboratory temperature was around 20oC with low humidity. All herbal materials, coffee bean,

liquorice and ginseng were ground into powder form (size <2mm) and fresh ginger root was

prepared by cutting into small pieces (size <4mm). The coffee herbal (100mg) mixtures were

transferred to a vial with 2 mL of MQ water, with the coffee and herbal ratios as shown in Table 3.2.

The mixtures were well shaken and allowed to stand for 1 hour in a heat block at 90-100°C in sealed

vials. All samples were passed through a filtration membrane (0.45um) and the content of caffeine,

trigonelline, Rb1 and GA were determined by HPLC-DAD. For camphene, the SPME fiber was

inserted into the vials to obtain coffee herbal beverage aroma for 15min.

Sample

(100mg)

Coffee Ginseng Ginger Liquorice

1 10%(10mg) 20% (20mg) 30%(30mg) 40% (40mg)

2 20%(20mg) 10% (10mg) 40%(40mg) 30% (30mg)

3 30%(30mg) 40% (40mg) 10%(10mg) 20% (20mg)

4 40%(40mg) 30% (30mg) 20%(20mg) 10% (10mg)

Table 3.2 Coffee and Herbal Mixture Ingredients and Ratio lists

67 | P a g e

3.3.3 Mode 2: Comparison of different extraction methods for the coffee herbal beverage via

HPLC

For the microwave oven extraction procedure, the total mixtures of coffee and herb weight was 2g

and made up to a volume of 200 ml, which covered the sample powder thoroughly in the ceramic

cups. The series of beverage samples were put into the microwave cavity, and then heated by

microwave irradiation (0.5 min, 1min and 1.5min). The sample solutions were then transferred to

the 15ml flasks every 30 second and all samples were sto ed at − 20 °C until HPLC analysis was

performed.

For Soxhlet extraction, the ratios of coffee herbal samples (2 g) were weighed into the thimble. The

water solvent was made up to a volume of 200 ml. The Soxhlet extraction was performed by gentle

boiling for 8 hours, then the sample solution was collected in a 15ml volumetric flask. All samples

were stored at − 20 °C until HPLC analysis.

For the percolation extraction, coffee and herbal sample were prepared from a 2g ratio mixture

made up to a volume of 200mL, using a filter coffee machine. The extraction took around 5 min at

90 °C. The coffee herbal beverage samples were collected and transferred to 15mL volumetric flask

and sto ed at − 20 °C till HPLC-DAD analysis.

After all collections, the samples were well shaken in the flask. After shaking, the supernatant was

passed through a filtration membrane (0.45um) and the concentrations of caffeine, trigonelline and

Rb1 and GA were determined by HPLC-DAD.

3.4 The analysis methods of coffee-herbal beverage

3.4.1 HPLC protocol for coffee and Liquorice [30]

The HPLC protocol utilized measures the concentration of key coffee and liquorice constituents in

coffee-herbal beverage including caffeine, trigonelline and glycyrrhizic acid (GA). The mobile phases

68 | P a g e

constituted 4.1% Acetic Acid MQ water and methanol. Agilent C18 reverse-phase column (150 × 4.6

; μ as used and isocratic separation of 28% methanol within 1.5 min, then a linear gradient

of 28–95% methanol for next 10 min, post run 3 min at last. Separations were carried out at room

temperature with a flow rate of 2mL/min. The PAD detector was used in the range 200–320nm, and

quantitative analysis was performed at 254nm (GA), 266nm (trigonelline) and 275nm (caffeine).

3.4.2 HPLC protocol for ginseng compounds [31]

The HPLC protocol was used to measure the concentration of Rb1 in the coffee herbal beverage. The

mobile phases constituted 0.001% formic acid MQ water and acetonitrile (ACN). Agilent C18

reversed-phase olu × . ; μ as used a d iso ati sepa atio of % ACN ithi

min, then a linear gradient of 26-33% ACN at 10min, 33-45% ACN runs 35 min at last. Separations

were carried out at 35 oC with a flow rate of 0.8 mL/min. The PAD detector was operated in the

range 200–320nm, and quantitative analysis was performed at 203nm (Rb1).

3.4.3 GC/MS analysis of coffee-herbal beverage [32]

Extraction temperature and time are critical parameters in the SPME-GC/MS process. SPME of

coffee herbal beverage was achieved with a 100uM polydimethylsiloxane (PDMS) coating fiber from

Supelco at 90-100°C for 15 min. After the adsorption step, the fibre was transferred into GC injector

in which the analysts were thermally desorbed at 250°C and kept in the hot GC injection spot for 5

min before removal to obtain complete desorption. GC/MS column oven temperature 40 °C for 3

min, then programmed at a rate of 12 °C/min to 180 °C, kept at 180 °C for 5 min, and finally ramped

at a rate of 40 °C/min to 250 °C and kept for 2 min, He carrier gas at 1mL/min. Before use, the fiber

as o ditio ed a o di g to the supplie s i st uctions.

3.4.4 Analytical standards information

Analytical Standards of sufficient purity, stability and homogeneity were utilized to prepare the

calibration curve [Table3.3]. We obtained compounds from Sigma-Aldrich, who provided a

certificate of analysis (ISO system) for all analytical standards [25]. These standards can achieve the

accuracy and precision in the analytical results for qualitative and quantitative tests. Most standards

are of analytical grade except caffeine ( .0%) and glycyrrhizic acid ( %). During the preliminary

stage the coffee herbal beverage contents were not fixed; these should be developed for the

process using, for example, published data and the feedback from human sensory measurement.

Therefore, this grade reached the requirements for evaluation at the beginning stage and in an

economic way.

69 | P a g e

Product

code

Compound Grade Purity

Water Storage

condition

Expiry

date

Manufacture

04070 Alkane

(C8-C20)

Analytical

standard

neat None 4°C 2018 Sigma-

Aldrich

C0750 Caffeine Reagent Plus . % . 4°C 2018 Sigma-

Aldrich

T5509 Trigonelline

hydrochloride

Analytical

standard


Aldrich

G2137 Glycyrrhizic

acid

Non supply % None 4°C 2018 Sigma-

Aldrich

442505 Camphene Analytical

standard

% None 4°C 2017 Sigma-

Aldrich

Y0001347 Ginsenoside

Rb1

European

Pharmacopoeia

(EP) Reference

Standard


Aldrich

Table 3.3 Standards information list

Standard compounds were dissolved in methanol, glycyrrhizic acid (GA), caffeine and trigonelline at

2.0 mg/ml and camphene, Rb1 at 10mg/mL stock solutions then stored at -20°C until use. The

mixtures of standard compounds including glycyrrhizic acid (GA), caffeine and trigonelline were then

diluted to 1000ppm with 25% methanol solution, camphene diluted to 1000ppm and 10ppm

respectively via pure ethanol and Rb1 by 25% ACN to 1000ppm for usage.

3.5 Thioflavin T (ThT) spectroscopic assay materials and methods [33,34]

Thioflavin T (ThT), is a benzothiazolium salt dye that was first utilized as a fluorescence microscopy

probe to study amyloid fibril deposits in histological samples. Then, much later it was developed for

the in vitro spectrophotometric quantification/assay of amyloid fibrils by the detection of the

fluorescence emission of ThT because the emission intensity was found to correlate with the fibril

concentration. ThT has become one of the most widely used methods for selectively identifying and

analysing the formation of amyloid fibrils both in vivo and in vitro. When it binds to beta-sheet-rich

structures such as amyloid fibrils, ThT displays enhanced fluorescence and a characteristic blue shift

in the emission spectrum from approximately 510nm in the free state to 480nm when bound to

amyloid fibrils. In protein-only solutions, the interaction of ThT with amyloid fibrils is highly specific.

Neither amorphous aggregates nor soluble proteins in folded, unfolded, or partially folded states

enhance ThT fluorescence. However, ThT in situ also interacts with other biomolecules such as DNA

70 | P a g e

by intercalating between DNA base pairs and/or by binding to DNA grooves. Some polysaccharides,

including proteins such as acetylcholinesterase also induce the characteristic ThT fluorescence,

presumably through binding to structural cavities. These properties reduce its usefulness for the

analysis of amyloid fibrils in diagnostic histology.

Hen egg white lysozyme was purchased from Sigma-Aldrich. The protein concentration was

prepared at 50mg/ml then adjusted pH to 2 using aqueous HCl and an aliquot of 330ul was added to

the 1.5ml tube. All test compounds were dissolved in the MQ water including caffeine (10mg/ml),

trigonelline (10mg/ml), Vitamin B1 (10mg/ml) and caffeic acid (0.5mg/ml). These drugs (330ul) were

mixed in the protein tube separately and change pH to 2. All tubes were heated in the heat block

under 50 °C for 24 hours, 48 hours, 72 hours, 96 hours, 120 hours, 144 hours and 168 hours and the

fluorescence intensity changes can be measured daily.

Thioflavin T was purchased from Sigma-Aldrich. ThT (1 mg), dissolved in 1.25 mL phosphate saline

buffer (PBS, 10mM phosphate, 150mM NaCl, pH 7.0) and kept in the dark at 4 °C. The stock solution

was then diluted 50 times in the PBS buffer before daily usage. The diluted ThT fluorescence

intensity was measured at 440 nm (slit width 5 nm) excitation and emission measured at at 482 nm

(slit width 10 nm). All test tubes were classified into blank (base line), negative control (untreated

protein solution), positive control (treated protein solution), caffeine protein group (5mg/mL),

trigonelline protein group (5mg/ml), Vitamin B1 protein group (5 mg/mL) and caffeic acid protein

group (0.25 mg/ml). 5 µL of each solution was added to the ThT solution cuvette (about 1.5mL),

stirred for 1 min then the fluorescence intensity was measured. The measured fluorescence intensity

increase at 482 nm indicated the presence of aggregated protein accumulation in the solution.

3.6 The analysis of herbal-coffee beverages

3.6.1 Validation of the HPLC assay

The coffee herbal beverage analysis method was validated by the HPLC chromatogram of caffeine,

trigonelline, GA and Rb1 at 275nm, 266nm, 254nm and 203nm. [Fig.3.1, Fig.3.2]. Calibration curves

for all the components were obtained in 4-6 independent days using five batches of prepared

calibration standards containing 20, 40, 60, 80 and 100ppm. The slopes of the standard calibration

curves of the four compounds showed a good fit and for all least squares analyses; the R2 values

were greater than 0.999. The Limit of Detection (LOD) was estimated by considering a signal to noise

71 | P a g e

ratio (s/n) of 3:1. The the range of these LODs were around 3-4ppm except for GA of liquorice. The

results, calibration curves and linear ranges, together with the LOD values, are summarized in

Table3.4 and Fig.3.3. The percentage relative standard deviations (RSD%) and Mean Recovery

against 3 nominal concentrations (40ppm, 60ppm and 80ppm) were calculated in order to evaluate

precision (repeatability) and demonstrate the accuracy of the method during days, which RSD%

values of intra-day were less than 10% except GA of liquorice at the low concentration level

(20ppm), and the corresponding Mean Recovery values range were between 99.2% and 102.5%. The

RSD% and Mean Recovery are presented in Table 3.5. The results that the values noted above were

acceptable precision and accuracy, although the repeatability intra-days of this measurement

deteriorated at 20ppm of GA, the methods are still satisfactory.

Fig.3.1 HPLC chromatogram of a standard solution of Tigonelline, Caffeine and Glycyrrhizic acid (GA)

Caffeine

Trigonelline

GA

72 | P a g e

Fig.3.2 HPLC chromatogram of standard solution of Ginsenoside Rb1

Table 3.4 Regression equations and detection limits of components determined for

the presented HPLC assay

Components Calibration Curves Linear Ranger (ppm) R2 Detection Limit (ppm)

Caffeine y=30.181x+10.882 20-100 0.9997 3.334

Trigonelline y=14.856x-0.8158 20-100 0.9994 4.116

GA y=5.5154x+16.704 20-100 0.9998 12.313

Rb1 y=3.6325+3.6498 20-100 0.9992 3.674

Detection of Limit is defined as the concentration where the signal-to-noise ratio is ;

Fig.3.3 Calibration curves of Tigonelline, Caffeine and Glycyrrhizic acid (GA) and Ginsenoside Rb1 the

regression equations of the four compounds with R2 > . , espe ti el .

Caffeine

y = 30.181x + 10.882

R² = 0.9997

Trigonelline

y = 14.856x - 0.8158

R² = 0.9994

GA

y = 5.5154x + 16.704

R² = 0.9998 Rb1

y = 3.6325x + 3.6498

R² = 0.9992 0

500

1000

1500

2000

2500

3000

3500

0 20 40 60 80 100 120

Caffeine

Trigonelline

GA

Rb1

Standards of Caffeine, Trigonelline, GA and Rb1 callibration curves

mA

U

Standards Concentrations (ppm)

Rb

73 | P a g e

Table 3.5 Intra-day precision and accuracy for the HPLC assay of four

components

Nominal

Concentration (ppm)

Measured

Concentration R.S.D (%) Mean Recovery (%)

Caffeine

40 39.609±0.494 1.247 99.023

60 60.122±0.633 1.206 100.202

80 79.208±1.247 1.574 99.01

Trigonelline

40 39.555±1.602 4.051 98.887

60 59.915±3.014 5.036 99.859

80 78.963±4.883 6.184 98.703

GA

40 39.709±5.056 12.733 99.273

60 60.695±6.021 9.449 101.158

80 79.565±7.028 8.832 99.456

Rb1

40 39.162±1.585 4.046 97.905

60 61.473±2.357 3.835 102.456

80 80.109±1.756 2.192 100.13

N=4; R.S.D: Relative Standard Derivation; Mean value (± standard deviation)

3.6.2 Validation of the GC/MS assay

3.6.2.1 Method validation by Kováts index (I).

Kovats indices are used to measure/characterize compounds under isothermal column condition,

Where NX is the unknown analyte, Nn and N(n+1) are the number of carbon atoms of the reference

n-alka es hi h a ket the ete tio ti e of the u k o a al sts, t a e the ete tio ti e of the

analysts or references of n-alkanes. Here the n-alkanes retention time NC8 was set as t N , hile

a othe t NC as t N + . Values were calculated via the Kováts index (I) calculator.

From Fig.3.4, n-alkanes elution peaks were free of noise interference. Under the same three day

conditions, the n-alkanes (C8-C20) Kováts Indices relative standard deviation (RSD) values are <0.5%.

The method precision and accuracy is considered to be acceptable. The n-alkanes (C8-C20) mass

match quality received 95% on average [Table 3.6; Table 3.7]. The absorption amounts of

NnnN

NnNX

tt

ttnI

'log'log

'log'log100

)1(

74 | P a g e

monoterpenoid from coffee-ginger beverages relate to the absorption period. The amounts of

caffeine were obtained in 10min by the conditions, [Fig.3.5] however caffeine cannot be detected

less than 2min or 5min. The essential oils from ginger and caffeine Kováts Indices relative standard

deviation (RSD) values are also less than 0.5% after test three times and the analytes mass match

quality were 95% on average.

Table 3.6. Comparison of three times n-alkanes(C8-C20) Kovats Indices and the percent of quality

Mass Match.

Fig. 3.4 GC chromatogram of standard of n-alkanes(C8-C20)

Standard Kováts Indices Mass

Compounds Test1 Test 2 Test 3

Match

(%)

Octane C8H18 * * * 96

Nonane C9H20 931 930 931 95

Decane C10H22 1047 1047 1048 94

Undecane C11H24 1152 1151 1152 95

Dodecane C12H26 1247 1246 1248 95

Tridecane C13H28 1335 1335 1336 95

Tetradecane C14H30 1418 1417 1419 93

Pentadecane C15H32 1506 1503 1505 95

Hexadecane C16H34 1619 1611 1613 97

Heptadecane C17H36 1768 1758 1758 97

Octadecane C18H38 1882 1880 1883 98

Nonadecane C19H40 1944 1943 1944 97

Eicosane C20H22 * * * 99

C8

C10

75 | P a g e

Table 3.7 Comparison of three times monoterpenoid and caffeine Kováts Indices and the percent of

quality mass match. *was not detected

Fig.3.5 GC chromatogram of coffee-ginger beverage

3.6.2.2 Camphene calibration curves

The coffee herbal beverage quantitative analysis of camphene from ginger method was validated by

SPME-GC/MS chromatography. The linear ranger of the calibration curves for the GC-MS analysis

were 20-60 ppm(R2 =0.9686) and 0.2-5ppm(R2 =0.9998)[Fig.3.6 and Fig.3.7]; The limit of detection

(LOD) was measured using a a signal to noise ratio (s/n) of 3:1 and were 7.100 ppm and 0.055ppm,

using 20ppm and 0.2ppm camphene standard solution respectively.[Table 3.8] The results, shown in

Table 3.9, are expressed as the percentage relative standard deviations (RSD%) and Mean Recovery

against 3 nominal concentration (20, 40, 60ppm; or 0.2, 1, 5ppm) in both methods. The typical

precision range (RSD%) lies between 2 and 17%, which is acceptable considering the very low

concentrations of the analyte. The accuracy data, represented as mean recovery (%), suggested the

recovery was 91–112% with a mean of 100.364% and a RSD% of 8.382%.

Kováts Indices s

Compounds Test1 Test 2 Test 3

Match

(%)

Pinene<Alpha-> C10H16 975 976 976 96

Camphene C10H16 996 997 997 97

Myrcene C10H16 1036 1036 1037 96

Limonene C10H16 1083 1084 1084 97

Cineole <1,8-> C10H18O 1088 1089 1089 91

Linalool C10H18O 1152 1153 1153 90

Neral C10H16O 1283 1285 1285 95

Geranial C10H16O 1309 1310 1311 95

Zingiberene <Alpha->C15H24 * 1507 1505 95

Caffeine C8H10N4O2 1910 1910 * 95

C17

Camphene

Caffeine

Geranial

Cineole

Pinene

76 | P a g e

Table 3.8 Regression equations and detection limits of components determined for the

presented GC/MS assay

Components Calibration Curves

Linear Ranger

(ppm) R2

Detection

Limit (ppm)

Camphene y=7×106x+121186 0.2-5 0.9998 0.055

Camphene y=5×106x+2×10

7 20-60 0.9686 7.1

Detection of Limit is defined as the concentration when the signal-to-noise ratio is 3

Fig.3.6 Calibration curves of Camphene in the both the concentration ranges with R2 > 0.96

y = 5E+06x + 2E+07

R² = 0.9686

0

50000000

100000000

150000000

200000000

250000000

300000000

350000000

0 20 40 60 80

Camphene

Standards of Camphene calibration curve

Standard Concentration(ppm)

mA

U

y = 7E+06x + 121186

R² = 0.9998

0

5000000

10000000

15000000

20000000

25000000

30000000

35000000

0 1 2 3 4 5

Camphene

Standards of Camphene calibration curve

mA

U

Standard Concentration (ppm)

77 | P a g e

Table 3.9 Intra-day precision for the GC/MS assay of ginger component (n=4)

nominal

concentration (ppm)

Measured

concentration R.S.D (%)

Mean

Recovery (%)

Camphene

0.2 0.216±0.018 8.489 108.014

1 0.910±0.020 2.272 91.003

5 4.670±0.425 9.303 93.436

Camphene

20 22.403±2.366 10.564 112.014

40 38.296±1.552 3.016 95.741

60 61.188±10.546 17.236 101.98

R.S.D: Relative Standard Derivation; Mean value (± standard deviation)

Fig.3.7 GC/MS chromatogram of standards solution of Camphene and Caffeine

3.6.3 Mode 1: Compounds concentration in coffee-herbal beverage

In order to evaluate the applicability of the developed method, coffee-herbal beverage samples

were analysed, with ingredients content from 10% to 40%. Caffeine, trigonelline, GA and Rb1 peak

areas correspond to the concentration by the use of external calibration curves. The contents of

caffeine, trigonelline, GA and Rb1 in these samples are shown in Table 3.10, Table 3.11 and Table

3.12. Fig.3.8 and Fig.3.9 shows typical chromatograms that caffeine, trigonelline, GA and Rb1 were

separated in the beverage. Caffeine values ranged from 60.90 to 211.05 mg in a 350mL coffee cup.

Trigonelline contents in a 350mL cup are from 84.7mg to 170.8mg. Therefore, the contents of the

GA and Rb1 can be determined as well.

Various herbal source and extract methods lead to the contents variability. Clearly, the GA peak

areas in the chromatograms compare Liquorice Liquid extract (LL) with Liquorice Powder (LP),

indicating the greater mass GA obtained in Liquorice Liquid extract (LL). The results of the GA

analysis assays demonstrate that the amount is almost 2-3 fold greater than Liquorice Powder (LP) in

Camphene

Caffeine

78 | P a g e

coffee herbal beverage (350mL). Additionally, The GA concentration is more reproducible in

liquorice liquid extract (LL). The Rb1 assay results indicate that the amounts of the main ginsenosides

Rb1 in the extracts of American ginseng (AG) appears to be greater than that present in undefined

ginseng (UG), which are 3-4 times in coffee herbal beverage (350mL).

Table 3.10 Amounts peak area and the concentration of Caffeine, Trigonelline in coffee-herbal beverage (n=4)

Beverage Compounds 10% Coffee 20% Coffee 30% Coffee 40% Coffee

(100mg mixture in 2mL) Caffeine Trigonelline Caffeine Trigonelline Caffeine Trigonelline Caffeine Trigonelline

Peak Area

(mAU)

539.012±

68.454

357.964±

54.218

973.390±

65.820

536.507±

136.533

1446.192±

108.60

671.826

±84.73

1830.223

±105.92

836.880

±238.7

Con. In Analysis

(1:10) (ppm)

17.499±

2.268

24.150±

3.650

31.891±

2.181

36.169±

9.910

47.556±

3.6

45.277

±5.706

60.281

±3.510

48.767

±6.078

Con. In Beverage(mg/mL) 0.174 0.242 0.319 0.362 0.476 0.453 0.603 0.488

Contents in 350ml cup(mg) 60.900 84.700 111.650 126.700 166.600 158.550 211.050 170.800

Mean ± standard deviation.

Table 3.11 Comparison of peak area and the concentration of GA from liquorice Powder(LP), GA from

liquorice liquid extract(LL) in coffee-herbal beverage (n=3)

Beverage Compounds 10% Liquorice 20% Liquorice 30% Liquorice 40% Liquorice

(100mg mixture in 2mL) GA(LP)1:5 GA(LL)1:10 GA(LP)1:5 GA(LL)1:10 GA(LP)1:5 GA(LL)1:10 GA(LP)1:5 GA(LL)1:10)

Peak Area

(mAU)

88.694±

70.264

146.541

±32.621

194.040

±98.032

249.470

±33.588

379.820

±148.740

404.113

±17.131

493.410

±164.152

526.016

±10.133

Con. In Analysis

(ppm)

13.053±

12.740

23.541

±5.915

32.153

±17.774

42.203

±6.090

65.837

±26.968

70.241

±3.106

86.432

±29.762

92.344

±1.837


Contents in 350ml cup(mg) 22.750 82.250 56.000 147.700 115.150 245.700 151.200 323.050

Mean ± standard deviation

Table 3.12 Comparison of peak area and the concentration of Rb1 from American ginseng(AG), Rb1 from

unidentified ginseng source(UG) in coffee-herbal beverage (n=3)

Beverage Compounds 10% ginseng 20%ginseng 30% ginseng 40% ginseng

(100mg mixture in 2mL)

Rb1(UG)

1:5

Rb1(AG)

1:10

Rb1(UG)

1:5

Rb1(AG)

1:10

Rb1(UG)

1:5

Rb1(AG)

1:10

Rb1(UG)

1:5

Rb1(AG)

1:10

Peak Area

(mAU)

50.939

±10.684

91.124

±21.164

98.107

±6.935

181.080

±23.861

108.114

±31.436

241.621

±46.310

208.138

±35.923

312.918

±33.608

Con. In Analysis

(ppm)

13.018

±2.941

24.081

±5.826

26.003

±1.909

48.845

±6.569

28.758

±8.654

65.512

±12.749

56.294

±9.889

85.139

±9.252


mean ± standard deviation

79 | P a g e

Fig.3.8 Coffee herbal beverage sample HPLC chromatogram of trigonelline, caffeine and GA and

dilution (1:10) with 25% methanol.

Fig.3.9 Coffee herbal beverage sample HPLC chromatogram of Rb1(AG) and dilution (1:10) with 25%

Acetonitrile

3.6.4 Camphene concentration

Camphene concentrations in the coffee herbal beverage samples [Fig.3.10] were calculated from the

peak area with both standard calibration curves linear ranges defined. The camphene concentration

in fresh ginger (FG) was much greater, especially (30% and 40% ratio) in coffee herbal beverage

[Table.3.13] and were 16-26 fold higher than Ginger Capsule (GC). Therefore, Camphene is a highly

volatile compound in Fresh Ginger (FG) and it has been mentioned that Ginger Capsule (GC) should

be taken to account on rapid loss of the analyst.

Caffeine

GA Trigonelline

Rb1

80 | P a g e

Table 3.13 Comparison of peak area and the concentration of camphene from fresh ginger(FG), and from

ginger capsule(GC) in coffee-he al e e age

Beverage 10% ginger 20% ginger 30% ginger 40% ginger

Compounds FG GC FG GC FG GC FG GC

Peak Area

(Abundance)

4.197×107

±1.650×107

2.490×106

±7.029×105

2.224×107

±6.765×106

3.241×106

±5.925×105

1.533×108

±6.035×107

7.227×106

±2.158×106

1.808×108

±1.075×108

1.375×107

±3.099×106

Con. In Analysis

(ppm)

5.980

±2.358

0.339

±0.100

3.159

±0.966

0.445

±0.085

26.657

±12.070

1.015

±0.308

32.158

±21.490

1.947

±0.443

Contents in

350ml cup(mg) 2.093 0.118 1.105 0.155 9.329 0.355 11.255 0.681

Mean ± standard deviation.

Fig.3.10 Coffee herbal beverage sample GC/MS chromatogram of camphene and caffeine

3.6.5 Mode 2: Herbal-Coffee beverage extraction methods

Obviously, these compounds including caffeine, trigonelline, Rb1 and GA from coffee herbal

functional beverage, had a good yield obtained via pure water as the extraction solvent in

microwave-assisted, percolation (coffee machine) and Soxhlet extraction [Fig.3.11]. The recipe of

each component used a 25% ratio in the beverage, that is 0.5 each in 2g composites. Caffeine

(alkaloids), trigonelline (hydrochloride salt), glycyrrhizic acid (saponin) and ginsenoside Rb1

(protopanaxadiol and dammarane family) are all extracted via the excellent polar solvent water. It is

accepted coffee indigents are extracted via microwave heating and coffee machines and, especially,

Rb1 and GA from Liquorice and Radix ginseng are released at low temperature (90-100°C) and in a

short time (1min). However, there will be a shortage for Rb1 in the coffee machine extract in this

case. The coffee machine requires slow passage of a solvent through fine particles. Therefore, the

Camphene

Caffeine

81 | P a g e

sample mixture must be merged properly so that the solvent contacts these particles equally, which

sometimes is determined by the machine extract design. As Rb1 extraction can be seen in the

Soxhlet case, the longer the extracting time, the lower the yield. This phenomenon could be result

from the hydrolysis of one or two sugar from ginseng saponin (containing 4 carbohydrates) with long

heating time. On the other hand, because of water as the extract solvent, it carries along with lots of

other plant components such as sugar, starches proteins with longer heating time, that could adsorb

the effective constituents extracted. We considered that water as solvent and the microwave

heating and percolation (coffee machine) extraction can be used to produce coffee herbal functional

beverage. Hence Fig 3.11 shows that, except for Rb1, similar amounts of the compounds are

extracted by each method (the mean value for each method is within confidence intervals of the

other methods ie. they all overlap).

Fig.3.11 The coffee herbal beverage compounds concentration measurement via various extraction

methods (with 95% confidence intervals)

The microwave effect of coffee herbal functional beverage amount on the extraction efficiency was

also studied. Samples with the ratio of 75% coffee and 25% herbs the total mass 2 g in 200 ml water

were analysed by HPLC-DAD with these collected at 0.5 min, 1min and 1.5min. The above figure

shows the concentration of the four compounds (caffeine, trigonelline, GA and Rb1) in the functional

beverage. As seen from the above Figure 3.11, increasing the extraction time (from 0.5 min to 1.5

min) did not significantly enhance the extracted amount of the analysts in the beverage and the

extraction balance was achieved. Therefore, the mid extraction time for coffee herbal compounds

0

20

40

60

80

100

120

140

Trigoneline Caffeine GA Rb1

Microwave oven(1min)

Percolation (coffee machine)

Soxhlet extractor(>8 hours)

The coffee herbal beverage compounds concentration

via various extraction methods(n≥3)

Co

nce

ntra

tion

(pp

m)

82 | P a g e

with the microwave oven extraction set at 1 min. This extraction time shows that the maximum of

compounds are extracted and when preparing a coffee beverage with microwave heating, 1 min

would give a good temperature for herbal-coffee preparation [Fig.3.12].

Fig.3.12 The amount of compounds extraction via microwave heating

3.7 ThT assay measurements

Hen egg white lysozyme amyloid protein can spontaneously assemble into protein fibrils under the

experimental acid conditions of the in vitro studies. Green tea polyphenols [35], curcumin [36],

myricetin [37], have been reported as effective inhibitors of the fibrillogenesis of hen egg white

lysozyme (HEWL). A report demonstrated that curcumin can inhibit the aggregation and formation

of amyloid fibrillation of HEWL at pH 2.0 in a dose dependent manner [36]. Importantly, evidence

indicates that amyloid fibril inhibition of HEWL by phenolic compounds involves formation of

quinoprotein interactions [38]. HEWL was incubated with or without these compounds and ThT

fluorescence intensity increased in the presence of protein fibrils which were measured every 24

hours for each group. We monitored the temperature-dependence of ThT fluorescence intensity to

examine the ability of natural compounds to inhibit the aggregation of protein such as caffeine,

trigonelline and caffeic acid from coffee and Vitamin B1 as a resource from daily food. We will utilize

semi- ua titati e a al sis to ake the de isio es o o to o fi the possi ilit fo atu al

compounds to inhibit protein fibrillation. Another purpose of the experiment was to ensure

0

20

40

60

80

100

120

140

0.5min 1min 1.5min

Caffeine

Trigonelline

GA

Rb1

Cco

nce

ntra

tion

(pp

m)

83 | P a g e

measuring conditions including incubation temperature, test time point, drug concentration as

optimal and the reproducibility of all these parameters is important for further quantitative

assessment of more compounds at next stage. All ThT Fluorescence intensity normalized to the

blank and was plotted as a function of time for 5mg/ml or 0.25 mg/mL at 50 oC in PBS (pH=2)

without or with herbs. The normalization ThT fluorescence intensity incubated without herb

constituents (positive control) increased rapidly after the equilibration period (72 hours), which

demonstrated protein fibril formation. Caffeine, caffeic acid and vitamin B1, all led to a dramatically

decreased ThT fluorescence intensity normalization [Fig. 3.13], which was individually compared to a

positive control. This suggests that the compounds could affect the neuro-protection against protein

aggregation formation and this requires further investigation of the efficient concentration-

dependent inhibition. However, trigonelline, at concentrations up to 5mg/mL, had little effect on the

ThT fluorescence response and only a small decrease and this also needs further confirmation. The

negative control is untreated/blank protein solution as a measurement base line, which the protein

solution did not change the pH when heated. In addition, all test coefficient of determination (R2)

should improve further by more replicate test times during quantitative analysis.

R² = 0.7978

R² = 0.925

0

1

2

3

4

5

6

7

8

0 24 48 72 96 120 144 168

Negative

control

Positive control

Caffeine(5mg/

ml)

Expon. (Positive

control)

Caffeine Inhibition Curve for Fibrils Formation (caffeine n=3)

Hour

No

rma

lizatio

n to

Th

T b

lan

k

84 | P a g e

R² = 0.7978

R² = 0.9466

0

1

2

3

4

5

6

7

8

0 24 48 72 96 120 144 168

Negative control

Positive control

VitaminB1(5mg/ml)

Expon. (Positive

control)

Linear

(VitaminB1(5mg/ml))

Vitamin B1 Inhibition Curve for Fibril Formation (Vitamin B1 n=3)

Hour

No

rma

lizatio

n to

Th

T b

lan

k

R² = 0.7978

R² = 0.9737

0

1

2

3

4

5

6

7

8

0 24 48 72 96 120 144 168

Negative control

Positive control

Caffeic acid (0.25mg/ml)

Expon. (Positive control)

Expon. (Caffeic acid

(0.25mg/ml))

No

rma

lizatio

n to

Th

T b

lan

k

Caffeic acid Inhibition Curve for Fibrils Formation (caffeic acid n=3)

Hour

85 | P a g e

Fig.3.13 Semi-quantitative curve of protein fibrillation in the presence or absence of different nature

compounds. The values are means ± SD, n = 3.

3.6 References

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2004(812):53–70

[2]. Souza CA, Frederico AR. Internal Standard Versus External Standard Calibration: An Uncertainty

Case Study Of A Liquid Chromatography Analysis. Quim. Nova 2010(33):984-987

[3]. Karyn MU, Steven WH and et al. Precision of Internal Standard and External Standard Methods in

High Performance Liquid Chromatography. LCGC 2015(33):40–46

[4]. Peishan X, Sibao C. et al. Chromatographic fingerprint analysis—a rational approach for quality

assessment of traditional Chinese herbal medicine. Journal of Chromatography A 2006(1112):171–

180

[5]. Johan R and Karl-E. Analysis of acrylamide in cooked foods by liquid chromatography tandem

mass spectrometry. Analyst 2002(127):880-882

[6]. Chao Y, Derrick C. et al. Drug confirmation by mass spectrometry: Identification criteria and

complicating factors. Clinica Chimica Acta 2015(438):119–125

R² = 0.7978

R² = 0.9752

0

1

2

3

4

5

6

7

8

0 24 48 72 96 120 144 168

Negative control

Positive control

Trigonelline(5mg/ml)

Trigonelline Inhibition Curve for Fribils Formation (Trigonelline n=3)

Hour

No

rma

lizatio

n to

Th

T b

lan

k

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[7]. Robin MB, Raymond JC. et al. chromatography-tandem mass spectrometry to the analysis of

chemical warfare samples, found to contain residues of the nerve agent sarin, sulphur mustard and

their degradation products. Journal of Chromatography A 1994(662):301-321

[8]. U.S. Department of Health and Human Services Food and Drug Administration Center for

Veterinary Medicine. Guidance for Industry Mass Spectrometry for Confirmation of the Identity of

Animal Drug Residues. 2003

[9]. Ghulama. S. A Practical Approach to Validation of HPLC Methods Under Current Good

Manufacturing Practices. Equipment and Instrumentation Qualification 2004(5):29-37

[10]. Joseph MB, Paula NB. et al. Accuracy, Precision, and Reliability of Chemical Measurements in

Natural Products Research. Fitoterapia 2011(82): 44–52.

[11]. Maria PB and Federico M. Validation of a Reversed-Phase HPLC Method for Quantitative

Amino Acid Analysis. Journal of Biomolecular Techniques 2006(17):131–137

[12]. David MB. Validating Chromatographic Methods: A Practical Guide. John Wiley & Sons,

Inc.2006:178-180

[13]. Thomas K and Clark M. Calibration and validation of linearity in chromatographic

biopharmaceutical analysis. Journal of Pharmaceutical & Biomedical Analysis 1991(9):911-918

[14]. David A and Terry P. Limit of Blank, Limit of Detection and Limit of Quantitation. The Clinical

Biochemist Review 2008(29): S49-52

[15]. Marsin MS, Susie LL. et al. Comparison of Signal-to-noise, Blank Determination, and Linear

Regression Methods for the Estimation of Detection and Quantification Limits for Volatile Organic

Compounds by Gas Chromatography. Journal of AOAC INTERNATIONAL. 2009(92):1833-1838.

[16]. Karoly H, Miklos G. et al. Temperature dependence of Kováts indices in gas chromatography

revisited. Journal of Chromatography A 2002(973):135–142

[17]. Babushok VI, Linstrom PJ. et al. Development of a database of gas chromatographic retention

properties of organic compounds. Journal of Chromatography A 2007(1157):414–421

[18]. Girard B. Retention index calculation using Kovfits constant model for linear temperature-

programmed gas chromatography. Journal of Chromatography A 1996(721):279-288

[19]. Casal S, Beatriz O. et al. HPLC/diode-array applied to the thermal degradation of trigonelline,

nicotinic acid and caffeine in coffee. Food Chemistry 2000(68):481-485

[20]. Carla IR, Liliana M. et al. Application of solid-phase extraction to brewed coffee caffeine and

organic acid determination by UV/HPLC. Journal of Food Composition and Analysis 2007(20):440–

448

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[21]. Rostagnoa MA, Manchon R. et al. Fast and simultaneous determination of phenolic compounds

and caffeine in teas, mate, instant coffee, soft drink and energetic drink by high-performance liquid

chromatography using a fused-core column. Analytica Chimica Acta 2011(685):204–211

[22]. Casal S, Oliveira M. et al. Discriminate Analysis of Roasted Coffee Varieties for Trigonelline,

Nicotinic Acid, and Caffeine Content. Journal of Agricultural and Food Chemistry. 2000(48):3420-

3424

[23]. Qingying Z, Min Y. Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice). Journal

of Chromatography A 2009(1216):1954–1969

[24]Minglei T, Hongyuan Y. et al. Extraction of Glycyrrhizic Acid and Glabridin from Licorice.

International Journal of Molecular Sciences. 2008(9):571-577

[25]. Qingying Z, Min Y. Chemical analysis of the Chinese herbal medicine Gan-Cao (licorice). Journal

of Chromatography A, 2009(1216):1954–1969

[26]. Qin CJ, Martha RH. et al. Quantitative Determination of Ginsenosides by High-Performance

Liquid Chromatography-Tandem Mass Spectrometry. Phytochemical Analysis 2001(12):320–326

[27]. Lian WQ, Chong ZW and Chun SY. Isolation and analysis of ginseng: advances and challenges.

Natural Product Reports. 2011(28):467-495

[28]. Yajing S, Philip M. et al. Solid-phase micro-extraction—comprehensive two-dimensional gas

chromatography of ginger (Zingiber officinale) volatiles. Flavour and Fragrance Journal 2003(18):5–

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[29]. Yingjia Y, Taomin HG. et al. Development of gas chromatography–mass spectrometry with

microwave distillation and simultaneous solid-phase microextraction for rapid determination of

volatile constituents in ginger. Journal of Pharmaceutical and Biomedical Analysis 2007(43):24–31

[30]. Takuhiro U. et al. Interaction Analysis of Glycyrrhizin on Licorice Extract-Induced Apoptosis of

Human Leukemia Cells by Knockout Extract. Natural Products Chemistry & Research 2013(1):1-7.

[31]. Lie L, Jin-lan Z. et al. Simultaneous quantification of six major active saponins of Panax

notoginseng by high-performance liquid chromatography-UV method. Journal of Pharmaceutical and

Biomedical Analysis 2005(38):45–51

[32]. Akiyama M, et al. Analysis Of The Headspace Volatiles Of Freshly Brewed Arabica Coffee Using

Solid-Phase Microextraction. Food chemistry and toxicology 2007(72):388-396.

[33]. Wei W, Xun S, et al. TiO2 nanoparticles promote b-amyloid fibrillation in vitro. Biochemical and

Biophysical Research Communications 2008(373):315–318

[34]. Sean AH, Heath E. et al. The thioflavin T fluorescence assay for amyloid fibril detection can be

biased by the presence of exogenous compounds. FEBS Letters, 2009(276):5960–5972

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[35]. Ghosh S, Pandey NK. Dasgupta, S. − -Epicatechin gallate prevents alkali-salt mediated

fibrillogenesis of hen egg white lysozyme. International Journal of Biomolecular Macromolecules

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[36]. Wang SS, Liu KN, Lee WH. Effect of curcumin on the amyloid fibrillogenesis of hen egg-white

lysozyme. Biophysical Chemistry 2009(144): − .

[37]. He J, et al. Myricetin Prevents Fibrillogenesis of Hen Egg White Lysozyme. Journal of Agriculture

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89 | P a g e

4

Herbal-coffee beverage sensory analysis

Chapters 2 and 3 described the herbal-coffee beverage targeted bioactive compounds selection and

analysis. Chapter 4 describes the assessment of herbal-coffee sensory human perceptions of

potential/desirable/continuous consumption to sustain effective prevention against dementia.

4.1 Functional beverage sensory measurement

The European Functional Food Science Commission defined functional foods (which include

beverages) as:"...satisfactorily demonstrate to affect beneficially one or more target functions in the

body, beyond adequate nutritional effects, in a way which is relevant to either an improved state of

health and well-being and/or reduction of risk of disease." Functional foods became popular for

consumers who are seeking specific health effects to keep fit and feel good regardless as to whether

the food has been modified in some fashion [1]. Several herbs claim medicinal benefits due to their

preventative properties such as anti-inflammation, anti-oxidants that also includes the perception of

taste, aroma and the oral perception of texture [2,3,4]. The sensory properties determine peoples

selection and how much they consume [5]. The bioactives from natural herbs not only affect

function beverage performance but also the drinking habits and long term consumption. However,

undesirable flavours in functional beverages may decrease the sensory acceptance and eventually

may discourage continued consumption if perceived as bitter, acrid, astringent or salty off flavours.

The human study is designed to review results from investigation of human preferences and the

sensory perceptions to the new functional beverages that were sampled. Functional beverages of

o a ge fla ou ed a el β-glucan was assessed by 105 members of a panel through sensory

evaluation including sweetness, sourness, orange flavour and thickness to estimate overall market

acceptability [6].

4.2 Effect of herbal-coffee beverage constituents on sensory perception

90 | P a g e

Aroma is also central to a pleasurable eating/drinking experience and is one of the most labile

components of food, coffee is an outstanding example. There are more than eight hundred volatile

compounds that have been identified in coffee, many of which have been demonstrated to

contribute to its aroma. The aroma profile of coffee is composed of the following notes:

sweet/caramel-like, earthy, roasty, smoky, fruity and spicy [7]. Liquorice has a sweet flavour and is a

diet multi-therapy herb. Glycyrrhizinic acid, a chief sweet-tasting constituent extract from liquorice

root is about 50 times as sweet as cane sugar [8]. This product has been approved in the United

States for a wide range of applications [9]. Some applications claim liquorice root extract can be used

to make foamy food, to enhance foam stability or to impart aroma. Ginseng functional products

have been largely limited to functional beverages because of the undesirable sensory properties of

ginseng such as earthy, wood, astringent and bitter [10]. Ginger has been used as a spice for over

2000 years and has pleasurable aromas such as sweety, warm and also a pungency flavour.

4.3 Sensory assessment (9-point hedonic scale) applications in food science

Taste provides important sensory information of beverages and it also impacts hedonics, pleasure

and displeasure. Hedonics reflect the immediate experience or anticipation of pleasure from the

sensory stimulation of eating a food including taste, smell, and texture, [11] and promotes

behaviours of long term usage associated with the enhanced acceptance [12].

In human taste panel studies, subjects assess the taste quality and intensity such as overall intensity,

sweet, sour, salty, bitter, metallic, cooling, spicy, anaesthetic, astringent, irritating and other tastes

[13]. Twenty five assessors participated in sensory analysis of coffee colour, aroma and flavour

evaluation using a hedonic scale to determine the degree of likeness or not of the coffee samples

[14]. Sixty eight customers participated in a hedonic value characteristic of wild blueberry–soy

beverages by 9-point hedonic scale comprising colour, flavour, viscosity, customer attitudes and

overall acceptability [15]. Twelve persons using the sip-and-spit method, assessed the individual

theaflavins for the overall astringency of black tea [16].

Therefore, the o su e s se so pe eptio can be recorded through a 9-point hedonic scale

(sensory preferable level). And the sensory assessment form can be used to qualify or quantify the

pa ti ipa t s hedo i fa to s o le els of dist ess.

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4.4 Participant recruitment method

Any healthy males and females aged 18-70 years old, who like to drink coffee and are free of

allergies to herbs were potential participants in a sensory evaluation of coffee-herbal beverages at

RMIT University. The project was approved by the RMIT Human Research Ethics Committee. Eight

people were invited to participate in the project.

In the future the herbal beverage will be modified to satisfy the general public. All potential

participants were randomly selected. A possible way of random participant selection could be using

a lottery method. In practice, a Random Number Generator produces 2 random number orders

between the ranges 1 to 5 without duplicating numbers.

4.5 Herbal-coffee beverage preparation

The coffee-herbal samples (ginseng, ginger and liquorice) were boiled with hot water (90-100

degrees) for 45 min. The concentration used was 100mg of a mixture in 2 mL water.

The Testing Sample Ingredients are shown below.

Sample Coffee Ginseng Ginger Liquorice

1 10% (10mg) 20% (20mg) 30% (30mg) 40% (40mg)

2 20% (20mg) 10% (10mg) 40% (40mg) 30% (30mg)

3 30% (30mg) 40% (40mg) 10% (10mg) 20% (20mg)

4 40% (40mg) 30% (30mg) 20% (20mg) 10% (10mg)

0 Water Blank Blank Blank Blank

4.6 Herbal-coffee beverage sensory assessment procedure

Participants should follow a few steps when testing the samples. The whole procedure is expected to

take 30min. The test procedure was a "one off". No photographs or videos were recorded during the

assessment.

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Testing procedure

1. Every participant to mouth rinse with water at the beginning of the study.

2. The participants receive 5 taste samples about 1-2 ml per cup in 1 to 5 sequence.

3. These samples are in the mouth for at least three seconds; then the participant can choose to

swallow or expel sample.

4. The participants immediately record for each sample, their response to hedonic scale such as

flavour, tasty, texture.

5. Provide voluntary feedback on the coffee-herbal beverage for future modification (optional).

4.7 Sensory measurement protocol

Previously no adverse consequences or clinical reports were found after drinking 1g of the herb-

beverage. But undesirable flavours such as bitterness, astringent, earthy in some of the beverages

may decrease the sensory perception. This hedonic scale describes an equal number of positive and

negative categories with intervals of equal size; for example, 1=not at all like, 3=slight like,

5=moderate like, 7= like very much and 9=extremely likeable [Appendix 1].

4.8 Hedonic scale statistical analysis

Every participant tasted samples 1 to 5 once, so these sample items such as colour, sweetness and

astringent etc., were repeated measurement 8 times by 8 individuals. Then we compared the

difference of the items between the two sample groups via Paired Tests (the items of sample 1vs 2,

3, 4, 5; the items of sample 2vs 3, 4, 5; the items of sample 3vs 4, 5; and the items of sample 4vs 5).

Thus the null and alternative hypotheses are:

H : μD ea the ite of Hedo i S ale i this sa ple is less tha o e ual to that sample)

H : μD > ea the ite of Hedo i S ale i this sa ple is g eate that sa ple

Choosi g a le el of sig ifi a e of α= . a d assu i g that the diffe e es a e o all

distributed, for an item of n=8, there are n-1=7 degrees of freedom.

The reasons for assuming that the differences normally distribution:

1. There are no literature reviews to provide this information with regard to herbal-coffee hedonic

scale population distribution.

2. According to the Central Limit Theorem conclusion, if we do not know the specific shape of the

population distribution more than 30 samples would be selected. However, we want to improve the

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result accuracy as well as cost effectives of the procedure. This would be the best cost-effective

method of choice for the small group selection before any valued results obtained.

The conclusion data will reflect what sensory participant is preferable.

4.9 Hedonic scale record discussion

A small group of 8 healthy participants (4 males and 4 females) were randomly selected and

completed the Sensory Analysis Evaluation Form to evaluate the functional beverage flavor

acceptance and consumption. Overall, there were no significant average or distribution differences

in hedonic scale ratings between these samples, which could be induced by a small group selection

as one of reasons. All participants who tasted the beverage perceived the samples (1-4) between

strong and very strong, and the prepared herb concentrations were perceived as too high. The

normalized ati gs egati e o t ol efe to the ode of , p odu ed si ila fu tio s e ept the

sweetness tasty, which needs to be investigated further. The sample 3 and 4 (including 30% and 40%

coffee), texture (colour) number averages were greater than 30% of sample 1, 2 and had a

favourable colour of coffee brown [Fig.4.1]. Participants were more likely to enjoy spicy flavour,

where scale numbers exceeded nearly 40% in sample 1 and 2 containing 30-40% ginger. However,

ginseng root produced similar earthy scale ratings and modest equivalent associations with the

content, which also included roast smell sensations [Fig.4.2]. In the oral sensory perceptions,

participants were skewed toward higher intensities of astringent and bitterness, which indicates we

must remove astringent compounds such as ginseng, and particularly bitter compounds in coffee

because these decrease tasty acceptance and enjoyment and subtract from sweet sensory

perceptions [Fig.4.3]. Through this small group analysis some valuable data was obtained; we

consider that the elimination of the perceived negative sensory compounds that lead to earthy

astringent taste perceptions will enhance the perceived acceptable beverage taste including the

favored roasty and sweetness perceptions for future larger human taste perception studies.

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Fig.4.1. Texture of Hedonic Scales values in herbal-coffee beverage

Fig.4.2. Flavors of Hedonic Scales values in herbal-coffee beverage

Fig.4.3. Tasty of Hedonic Scales values in herbal-coffee beverage

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4.10 References

[1]. Martin H. Functional Foods: What Are They - How Are They Regulated - What Claims Can Be

Made. American Journal of law and Medicine 2005(31):305-340.

[2]. Drewnowski A. Taste preferences and food intake. Annual Review Of Nutrition 1997(17)::237–53

[3]. Perry R, Terry R, et al. Is Lavender An Anxiolytic Drug? A Systematic Review Of Randomised

Clinical Trials. Phytomedicine 2012(19):825-835.

[4]. Melissa H. Spicing Up Your Memory. Psychology Today 2003(11):1-1.

[5]. Sorensen LB, Moller P, et al. Effect of sensory perception of foods on appetite and food intake: a

review of studies on humans. International Journal of Obesity 2003(27):1152–1166

[6]. Feral T, Craig B, et al. Development of an orange-fla ou ed a el β-glucan beverage. Cereal

Chemistry 2004(81):499-503

[7]. Belitz H.-D, Grosch W, Schieberle P. Food Chemistry. 4th revised and extended ed.

[8]. Esra I and Senol I. Foaming behaviour of liquorice (Glycyrrhiza glabra) extract. Food Chemistry

2000(70):333-336

[9]. Faus I. Recent developments in the characterization and biotechnological production of sweet-

tasting proteins. Applied Microbiology and Biotechnology 2000(53):145-151

[10]. Hee SC, Young CL, et al. Consumer Acceptance of Ginseng Food Products. Journal of Food

Science 2011(76):516-522

[11]. Anna, B and Russell K. The influence of chemotherapy on taste perception and food hedonics: A

systematic review. Cancer Treatment Reviews 2012(38):152–163

[12]. Kelley AE, Bakshi VP, et al. Opioid modulation of taste hedonics within the ventral striatum.

Physiology & Behaviour 2002(76):365– 377

[13]. Vikas A, Mahesh K, et al. The latest trends in the taste assessment of pharmaceuticals. Drug

Discovery Today 2007(12):257-265

[14]. Luciane C., Hilary C, et al. Optimization of the roasting of robusta coffee (C. canephora

conillon) using acceptability tests and RSM. Food Quality and Preference 2001(12):153-162

[15]. Pottera RM, Dougherty MP, et al. Characteristics of wild blueberry–soy beverages. LWT-Food

Science and Technology 2007(40):807–814

[16]. Susanne S, Magnus J, et al. Evaluation of the taste contribution of theaflavins in black tea

infusions using the taste activity concept. European Food Research Technology 2004(218):442–447.

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5

Conclusion and suggestions for further work

The preceding chapters have illustrated the experimental design, instrumental analysis of coffee

herbal functional beverages. Sample extraction and analysis can be achieved with convenient

extraction methods that are compatible with existing HPLC-DAD and GC-MS detection. The

participant sensory evaluations were measured by a small group (8 people invited) with a 9-point

hedonic scale.

5.1 Conclusion

The strategy of this research project is focused on the potential prevention and treatment of

cognitive impairment by recommending the consumption of coffee herbal beverages as early as

possible in life.

Firstly, epidemiological studies suggest that dietary measures, physical exercise, and mental activity

may reduce the risk of cognitive impairment and AD in elderly subjects. However, these preventative

activities are not easy to qualify, quantify and to track their performance. As evidenced by the

effective constituents of coffee and herbs against the cognitive impairment (presented in Chapter 2)

they are an emerging area of interest. At the start of this project, we set the selection criteria and

chose suitable herbs and their constituent bioactives including caffeine, trigonelline, glycyrrhizic acid

(GA), Rb1 and camphene from coffee, liquorice, ginseng and ginger. These natural products are

active against dementia and have an acceptable human safety profile related to the amyloid

hypothesis, cholinergic hypothesis, inflammatory/oxidative hypothesis, Ca2+ and adenosine receptor

hypothesis, that contribute to the potential causes that lead to AD.

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Secondly, the purpose of the project was the analysis of the functional beverage to confirm the

concentration of the effective constituents that are conveniently extracted into the beverage, which

enables the analysis of the ingredients to evaluate their protective effects over long term

consumption (presented in Chapter 3). Water, a polar solvent, safe for the intake of the effective

constituents in coffee-herbal beverages. The target compounds were measured after the various

extraction methods such as decoction, percolation (coffee machine), microwave heating, Soxhlet

and SPME to evaluate if the compounds were effectively extracted by these various techniques.

After HPLC analysis, we concluded that the constituents (e.g. caffeine, trigonelline, GA and Rb1)

were released completely at 90-100°C (coffee machine) or 1min (microwave oven) that were much

better than the high temperature (100°C), or longer extraction times hou s by Soxhlet

extraction. In addition, we measured the increasing constituent ratios of the target compounds

concentration range from 10% to 40% by HPLC and GC/MS from decoction extraction methods. The

HPLC and GC/MS analysis methods were validated, by parameters including identification, accuracy,

precision, linearity and acceptable limit of detection. Therefore, the different ratio of the

components of these target compounds (caffeine, trigonelline, GA, Rb1 and camphene) is available

for evaluation for future work. We also compared the contents of these compounds from different

sources. The results indicated that American ginseng extracts contained the highest amount of Rb1

and fresh ginger containing camphene of ginger oil extracted the most GA. The result showed a

promising prospect for quality control of extracting the effective herb constituents to produce a

coffee herbal functional beverage.

We used in vitro Thioflavin T (ThT) fluorescence assays to confirm that the selected natural products

have the potential to inhibit β-sheet amyloid protein fibrillation. In vitro measurements of the pure

natural compounds including caffeine, caffeic acid and vitamin B1, inhibited the amyloid protein of

HEWL from fibrillation over extended heating times (50°C for 144 hours) in an acidic environment

(pH=2). Also, a small number of people (8 healthy adult participants) estimated the sensory

perception of the herbal-coffee beverages. The positive feedback from the participants indicated

that the sampled herbal-coffee beverages were too st o g , implying that the effective constituent

concentration were too high. In future a balance between neuroprotection and sensory acceptance

needs to be determined. This is particularly relevant to ginseng, as it has a characteristic earthy

astringent perception that strongly influences the taste of herbal-coffee beverages.

5.2 Suggestions for future work

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We used the in vitro model amyloid protein of HEWL to investigate whether some natural binding

effects were present in the coffee-herb constituents to inhibit amyloid fibril formation. Future work

could utilize native or synthetic a loid β protein or infected cells to assess the rate of inhibition of

natural products in vitro. The small [8] number of participants in human sensory perception study

was a limitation of this study and could be extended to a much larger cohort of panel members that

would more reliably determine the best acceptable herbal-coffee formulations. Future work should

include post-consumption/intake of the herbal beverages, the analysis of the detection,

measurement of relevant and specific herbal biomarker metabolites as evidence for their

benefits/presence, to encourage continuous consumer consumption whilst also providing/imparting

benefits against dementia.

In addition, it is important to measure the functional beverage-brain affects in vivo with animal

model studies in mice or rats, that can be used to monitor the beverage safety and efficiency. Also

for coffee-herbal beverages safe intake studies, the use of healthy animals would allow the

investigation and analysis of any affects on various organs extended over months or years.

Additionally, AD transgenic mice can be used to evaluate the beverage brain protection performance

and efficacy.

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Appendix 1

Table.1. Herbal Coffee Beverage Sensory Analysis Evaluation

This template was used for data collation to help assess the herbal coffee beverage acceptance.

Code: Gender: Age ranges: ~40 40~60 60~

0=none of above, 1=not at all like, 3=slight like, 5=moderate like, 7= like very much and 9=extremely

likeable

Number the items in order of your taste

Sample 1 2 3 4 5

A Texture

Hedonic Scale

0,1,3,5,7,9 0,1,3,5,7,9 0,1,3,5,7,9 0,1,3,5,7,9 0,1,3,5,7,9

Colour

B Odour Flavour

Roasty

Spicy

Root/Earthy

C Tasty

Sweetness

Bitterness

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Astringent

D Expected cost

(350ml/bottle)

~$3 $3~$5 $5~

E Opinion(optional)

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