Final thesis pdf (a4)

IDENTIFICATION OF COMPONENTS IN EXTRACTS
OF HERICIUM ERINACEUS (BULL.: FR.) PERS THAT
STIMULATE IN VITRO NEURITE OUTGROWTH OF
NG108-15

WONG YUIN TENG

FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR

2012

IDENTIFICATION OF COMPONENTS IN EXTRACTS
OF HERICIUM ERINACEUS (BULL.: FR.) PERS THAT
STIMULATE IN VITRO NEURITE OUTGROWTH OF
NG108-15

WONG YUIN TENG

DISSERTATION SUBMITTED IN FULFILLMENT OF THE
REQUIREMENT FOR THE DEGREE OF
MASTER OF SCIENCE

INSTITUTE OF BIOLOGICAL SCIENCES
FACULTY OF SCIENCE
UNIVERSITY OF MALAYA
KUALA LUMPUR

2012

ABSTRACT

Hericium erinaceus, locally known as cauliflower mushroom, and elsewhere as

lion’s mane mushroom, Houtou (monkey head mushroom), Yamabushitake and

Harisenbon (balloon fish), is an edible mushroom. It is well known for its medicinal and

nutritional values. Hericium erinaceus is reported to have good anti-tumor properties

and nerve tonic effects. Although H. erinaceus is a temperate mushroom, it has been

successfully cultivated in Malaysia. However, there are very few reported studies on the

chemical constituents that stimulate the neurite outgrowth for the locally cultivated

species.

The crude aqueous ethanol extract of H. erinaceus and its fractionated extracts

(hexane, ethyl acetate and water) were evaluated for their effect in stimulating the

neurite outgrowth using neural cell line NG108-15 whilst the Nerve Growth Factor

(NGF) was used as the positive standard. The crude aqueous ethanol extract of H.

erinaceus showed 15.0 % increase in neurite outgrowth at the concentration of 10.0

µg/ml. However, the crude aqueous ethanol extract showed decreased neurite growth as

the dose was increased. The hexane, ethyl acetate and water fractions showed an

increase in neurite outgrowth when the dose was increased exponentially (10.0, 25.0,

50.0 and 100.0 µg/ml). Maximum stimulation of neurite outgrowth was recorded with

ethyl acetate fraction with 68.5 % increase compared to negative control followed by

hexane fraction with 65.2 % increase.

The combined fraction of hexane and ethyl acetate was further subjected to flash

column chromatography. Among the 7 isolated fractions (fraction E1-E7), fraction E1

and fraction E2 show relatively higher neurite stimulation activity compared to other

fractions. Maximum stimulation was recorded as 160.6 % increase and 149.1 %

ii

increase compared to negative control at the concentration of 100 µg/ml for fraction E1

and fraction E2 respectively.

The chemical compositions of the fraction E1 of H. erinaceus were analayzed by

GCMS. Four components were identified from fraction E1 comprising about 80.5 % of

the total. Fraction E1 was made up of ethyl palmitate (29.8 %), ethyl stearate (2.3 %),

ethyl oleate (18.6 %) and ethyl linoleate (29.9 %). Further isolation of fraction E2 using

preparative TLC and HPLC gave subfraction sub4b_4 and subfraction sub4b_6.

Subfraction sub4b_4 showed better neurite stimulation activity compared to subfraction

sub4b_6 with 187.1 % increase in comparison.

The chemical compositions of subfraction sub4b_4 and sub4b_6 were analyzed

by NMR and LC/MS/MS. The components identified from subfraction sub4b_4 were

hericenone C (and its isomer) and 4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-formyl-

3-hydroxy-5-methoxylbenzyl oleate (and its isomer). On the other hand, subfraction

sub4b_6 comprised of hericenone C, 4-(3’,7’-dimethyl-5’-oxo-2’,6’-octadienyl)-2-

formyl-3-hydroxy-5-methoxylbenzyl oleate and a phenolic component attached to the

fatty ester side chain contained 26 carbons with 3 double bonds.

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ABSTRAK

Hericium erinaceus, dikenali sebagai cendawan bunga kobis di Malaysia dan

cendawan “lion’s mane”, Houtou (cendawan kepala monyet), Yamabushitake dan

Harisenbon di tempat lain. Cendawan ini boleh dimakan dan ia terkenal dari segi nilai

perubatan dan nutrisi. Kebelakangan ini, laporan saintifik menunjukkan H. erinaceus

mempunyai nilai anti-tumor yang baik dan sebagai tonik terhadap saraf. Walaupun H.

erinaceus merupakan cendawan yang ditanam di kawasan sederhana tetapi kini berjaya

ditanam di Malaysia yang beriklim tropika. Walaubagaimanapun, tidak banyak terbitan

laporan termpatan yang melaporkan tentang komposisi kimia dalam H.erinaceus yang

ditanam secara tempatan dalam rangsangan pertumbuhan saraf.

Ekstrak mentah akueus etanol dan fraksi-fraksi (heksana, etil asetat dan air) dari

H.erinaceus telah diselidik dalam rangsangan pertumbuhan saraf pada sel saraf NG108-

15 dan NGF digunakan sebagai kawalan positif. Ekstrak mentah akueus ethanol

menunjukkan peningkatan sebanyak 15.0 % dalam pertumbuhan saraf pada kepekatan

10.0 µg/ml. Walaubagaimanapun, peningkatan kepekatan ekstrak mentah akueus

ethanol akan menyebabkan penurunan dalam pertumbuhan saraf. Fraksi hexana, etil

asetat dan air akan menyebabkan peningkatan dalam pertumbuhan saraf apabila

kepekatan fraksi-fraksi ditingkatkan secara eksponen (10.0, 25.0, 50.0 and 100.0 µg/ml).

Pertumbuhan maksimum saraf direkodkan oleh fraksi etil asetat dengan 68.5 %

peningkatan berbanding dengan kawalan negatif dan diikuti oleh fraksi hexana dengan

65.2 % peningkatan berbanding dengan kawalan negatif.

Pengasingan komponen daripada gabungan fraksi heksana dal etil asetat

menggunakan kaedah kromatografi kolum kilat menghasilkan 7 fraksi (fraksi E1-E7).

Fraksi E1 dan E2 menunjukkan aktiviti pertumbuhan saraf yang lebih tinggi jika

berbanding dengan fraksi-fraksi lain. Peningkatan pertumbuhan maksimum sebanyak

iv

160.6 % dan 149.1 % direkodkan oleh fraksi E1 dan E2 pada kepekatan 100µg/ml

berbanding kawalan negatif.

Komposisi kimia fraksi E1 bagi H. erinaceus dianalisis dengan menggunakan

GCMS. Empat komponen telah dikenalpasti daripada fraksi E1 dan komponen-

komponen tersebut adalah terdiri daripada 80.5 % daripada keseluruhan fraksi E1.

Komponen yang terkandung dalam fraksi E1 adalah etil palmitat, etil stearat, etil oleat

dan etil linoleat. Subfraksi sub4b_4 dan subfraksi sub4b_6 adalah hasil daripada isolasi

fraksi E2 dengan menggunakan preparatif TLC dan HPLC. Subfraksi sub4b_4

menunjukkan aktiviti pertumbuhan saraf yang lebih baik daripada subfraksi sub4b_6

iaitu 187.1 % peningkatan pada kepekatan 100 µg/ml jika dibandingkan dengan

kawalan negatif.

Komposisi kimia subfraksi sub4b_4 dan sub4b_6 dianalisis dengan

menggunakan NMR dan LC/MS/MS. Komponen-komponen yang dikenalpasti daripada

subfraksi sub4b_4 termasuk hericenone C (dan isomernya) dan 4-(3’,7’-dimetil-5’-oxo-

2’,6’-octadienil)-2-formil-3-hidroksi-5-metoksibenzil oleat (dan isomernya). Identifikasi

subfraksi sub4b_6 menunjukkan kehadiran hericenone C, 4-(3’,7’-dimetil-5’-oxo-2’,6’-

octadienil)-2-formil-3-hidroksi-5-metoksibenzil oleat dan satu komponen fenolik yang

mengandungi rantai ester asid lemak yang mempunyai 26 karbon dan 3 ikatan dubel.

v

ACKNOWLEDGEMENT

Writing a significant scientific thesis is hard work and it would be impossible

without support from various people. First of all, I wish to express my greatest

appreciation towards my supervisor Professor Datin Dr. Sri Nurestri Abdul Malek, my

co-supervisor, Professor Dr. Noorlidah binti Abdullah, the project leader of this research,

Professor Dr. Vikineswary Sabaratnam from Mushroom Research Centre and Dr.

Murali Naidu from the Faculty of Medicine for the intellectual guidance, valuable

advices and help that was given to me during my research. The thesis would not have

been written successfully without their continuous supervision and guidance. I would

like to thank to University Malaya for the grant (PS191/2009A) and fellowship support.

My special appreciation to my labmates and friends’ enthusiasm and support in

providing relevant assistance and help to complete this study. Thanks to Wong Kah Hui,

Lai Puei-Lene, Priscilla Ann, Joanna Eik Lee Fang, Hong Sok Lai, Lee Guan Serm,

Phang Chung Weng, Sujatha Ramasamy, Sharifah Nur Syed Abdul Rahman, Gowri

Kanagasabapathy, Jaime Stella Richardson, Ong Kia Ju and Mamalay. A special thank

to Madam Chang May Hing for her kind gesture in helping me especially in the

laboratory system operation procedures.

Last but not least, I would like to express my appreciation to my parents, Mr

Wong Wai Yew and Madam Chia Saw Meng, and other members of my family for their

emotional, financial support and providing a lovely environment for me.

vi

CONTENTS

PAGE
ABSTRACT ii
ABSTRAK iv
ACKNOWLEDGEMENTS vi
LIST OF FIGURES x
LIST OF TABLES xii
LIST OF APPENDICES xiii
LIST OF SYMBOLS AND ABBREVIATIONS xv

CHAPTER I
INTRODUCTION 1

CHAPTER II
LITERATURE REVIEW
2.1 Medicinal mushrooms and its usages 5
2.2 Hericium erinaceus
2.2.1 Origin 7
2.2.2 Classification 8
2.2.3 Medicinal properties, nutritional and bioactive components derived 8
from Hericium erinaceus
2.3 Nervous system and neurodegenerative diseases
2.3.1 Neurite 12
2.3.2 Neurodegenerative diseases (factors, therapies to cure and prevent) 12
2.4 Neuroprotective, neurotrophic, neuronal differentiation and neurite 16
stimulation effects of Hericium erinaceus
2.5 Neurite outgrowth bioassay system of neural hybrid cell line NG108-15
2.5.1 Formation of NG108-15 hybrid cell 19
2.5.2 Characteristics of NG108-15 hybrid cell 19

CHAPTER III
MATERIALS AND METHODS
3.1 Extraction
3.1.1 Preparations of fruitbodies 21

vii

3.1.2 Preparation of aqueous ethanol crude extract 21
3.1.3 Solvent-solvent extraction (fractionation) 21
3.2 Neurite outgrowth activity assay
3.2.1 Preparation of stock solutions 23
3.2.2 Cell culture 23
3.2.3 Preparation of medium and buffer for cell culture
3.2.3.1 Dulbecco’s Modified Eagle’s Medium (DMEM) 23
3.2.3.2 Phosphate buffer saline 24
3.2.4 Cell culture techniques
3.2.4.1 Revival of frozen cells 24
3.2.4.2 Subculture of cells 25
3.2.4.3 Medium renewal 25
3.2.4.4 Cryopreservation of cells 26
3.2.5 Effect of Hericium erinaceus on stimulation of neurite outgrowth 26
activity of NG108-15
3.2.6 Scoring of neurites 26
3.2.7 Statistical analysis 27
3.3 Isolation of bioactive constituents
3.3.1 Column chromatography 28
3.3.2 Analytical thin layer chromatography 28
3.3.3 Preparative thin layer chromatography 29
3.3.4 High performance liquid chromatography (HPLC)
3.3.4.1 HPLC samples and mobile phase preparation 29
3.3.4.2 Analytical HPLC 30
3.3.4.3 Semipreparative HPLC 30
3.4 Identification
3.4.1 Gas chromatography-mass spectrometry (GCMS) 32
3.4.2 Nuclear magnetic resonance spectroscopy (NMR) 32
3.4.3 Liquid chromatography- mass spectrometry (LC/MS/MS) 32

CHAPTER IV
RESULTS & DISCUSSION
4.1 Extraction, fractionation and isolation
4.1.1 Extraction, fractionation and isolation of aqueous ethanol extract of 33
Hericium erinaceus

viii

4.2 Neurite outgrowth activity
4.2.1 Effect of aqueous ethanol extract and fractions of Hericium 37
erinaceus on the neural cell line NG108-15
4.2.2 Effect of the fraction E1-E7 of Hericium erinaceus on the neural 45
cell line NG108-15
4.2.3 Effect of the subfraction sub4b_4 and sub4b_6 of Hericium 57
Erinaceus on the neural cell line NG108-15
4.3 Overall comparison of aqueous ethanol extract, hexane fraction, ethyl 62
acetate fraction, water fraction, fraction E1-E7, subfraction sub4b_4 and
subfraction sub4b_6
4.4 Identification of chemical constituents
4.4.1 Identification of chemical constituents of the fraction E1 64
4.4.2 Identification of chemical constituents of the subfraction sub4b_4 66
4.4.3 Identification of chemical constituents of the subfraction sub4b_6 70
4.5 Overall comparison of the identified compounds and the neurite 75
stimulation activity in fraction E1, fraction E2 (subfraction sub4b_4 and
subfraction sub4b_6) of Hericium erinaceus

CHAPTER V
GENERAL DISCUSSION & CONCLUSION 78

REFERENCES 83

APPENDICES 91

ix

LIST OF FIGURES
FIGURE TITLE PAGE
2.1 Hericium erinaceus (Bull.: Fr.) Pers. 7

2.2 HeLa cell growth inhibitory substances isolated from Hericium 10
erinaceus

2.3 Hericenones isolated from fruiting body of Hericium erinaceus 16
which showed NGF synthesis promoting activity

2.4 Erinacines isolated from fruiting body of Hericium erinaceus 18
which showed NGF synthesis promoting activity

3.1 A schematic diagram showing the extraction and fractionation 22
procedures, process of biological investigations and isolation of
active fractions of Hericium erinaceus

3.2 A schematic diagram showing the isolation of active fractions, 31
process of biological investigations and identification of the
active fractions

4.1 Aqueous ethanol extraction of Hericium erinaceus 33

4.2 Fractionation of aqueous ethanol extract of Hericium erinaceus 34

4.3 Isolation of combined hexane and ethyl acetate extract of 35
Hericium erinaceus obtained through flash column
chromatography

4.4 Isolation of fraction E2 of Hericium erinaceus by using 36
preparative thin layer chromatography and high performance
liquid chromatography

4.5 Percentage of neurite bearing cells incubated with varying 37
concentrations of aqueous ethanol crude extract, hexane
fraction, ethyl acetate fraction and water fraction of Hericium
erinaceus

4.6 The morphology of the NG108-15 cells treated with various 39
concentrations of crude aqueous ethanol extract of Hericium
erinaceus

concentrations of hexane fraction of Hericium erinaceus

concentrations of ethyl acetate fraction of Hericium erinaceus

concentrations of water fraction of Hericium erinaceus

x

concentrations of fraction E1 of Hericium erinaceus







4.17 Percentage of neurite bearing cells incubated with varying 57
concentrations of subfractions sub4b_4 and sub4b_6 of
Hericium erinaceus

concentrations of subfraction sub4b_4 of Hericium erinaceus

concentrations of subfraction sub4b_6 of Hericium erinaceus

4.20 Compounds (I and II) identified in subfraction sub4b_4 68

4.21 Compounds (I, II and III) identified in subfraction sub4b_6. 73

xi

LIST OF TABLES

TABLE TITLE PAGE
2.1 Neuronal properties of neuroblastoma x glioma hybrid cells 20
NG108-15

4.1 Stimulation of neurite outgrowth activity in the NG108-15 cells 38
with varying concentrations of aqueous ethanol extract and
fractions of Hericium erinaceus

with varying concentrations of fractions (E1-E4) of Hericium
erinaceus

with varying concentrations of fractions (E5-E7) of Hericium
erinaceus

4.4 Stimulation of neurite outgrowth activity of the NG108-15 cells 58
with varying concentrations of sub4b_4 and sub4b_6 of Hericium
erinaceus

4.5 Identified constituents of fraction E1 of Hericium erinaceus 64
1
4.6 H- and 13C-NMR for subfraction sub4b_4 in CDCl3 69
1
4.7 H- and 13C-NMR for subfraction sub4b_6 in CDCl3 74

xii

LIST OF APPENDICES
APPENDIX TITLE PAGE
1 Calculation for sample yield 91

2 Statistical analysis for percentage of neurite bearing cells of 92
aqueous ethanol extract by using one way ANOVA

hexane fraction by using one way ANOVA

ethyl acetate fraction by using one way ANOVA

water fraction by using one way ANOVA

fraction E1 by using one way ANOVA







subfraction sub4b_4 by using one way ANOVA

subfraction sub4b_6 by using one way ANOVA

15 The total ion chromatogram (TIC) of fraction E1 of 105
Hericium erinaceus

16 Mass spectrum of fraction E1 of Hericium erinaceus 106-107
1
17 H-NMR spectrum of subfraction sub4b_4 108- 110
13
18 C NMR spectrum of subfraction sub4b_4 111-113

xiii

19 DEPT NMR spectrum of subfraction sub4b_4 114-116
1
20 H-NMR spectrum of subfraction sub4b_6 117-119
13
21 C NMR spectrum of subfraction sub4b_6 120-122

22 DEPT NMR spectrum of subfraction sub4b_6 123-126

23 Chromatogram and mass spectrum data of peak in 127
LC/MS/MS at retention time 5.76 min in subfraction
sub4b_4 with [M+H]+ of 148.8









xiv

LIST OF SYMBOLS AND ABBREVIATIONS

Ac Acetone

AD Alzheimer’s disease

ADFM Alzheimer’s Disease Foundation Malaysia

ADI Alzheimer's Disease International

α Alpha

ANOVA Analysis of variance

ApoE4 Apolipoprotein E4

ATCC American Tissue Culture Collection

β beta

Ca2+ Calcium ion

CO2 Carbon dioxide

CHCl3 Chloroform

cm Centimeter

°C Degree celcius

CDCl3 Deuterated chloroform

Da Dalton

DLPE Dilinoleoyl-phosphatidylethanolamine

DMSO Dimethyl sulfoxide

DMEM Dulbecco’s Modified Eagle’s Medium

EDTA Ethylenediaminetetraacetic acid

ER Endoplasmic reticulum

GC-MS Gas Chromatography-Mass Spectroscopy

g Gram

g/l Gram per litre

HMG-CoA 3-hydroxy-3-methyl-glutaryl-CoA

xv

HPLC High-performance liquid chromatography

hr Hour

HIV Human immunodeficiency virus

HCl Hydrochloric acid

HAT Hypoxanthine- aminopterine- thymidine

kg Kilogram

λ Lambda

< Less than

LC/MS/MS Liquid chromatography-mass spectrometry

L Litre

LDL Low-density lipoprotein

m/z Mass-to-charge ratio

MHz Megahertz

mRNA Messenger RNA

MeOH Methanol

µg/ml Microgram per mililitre

µM Micromolar

mg/ml Miligram per mililitre

ml Mililitre

mm Milimetre

min Minute

ng/ml Nanogram per mililitre

nm Nanometer

NGF Nerve Growth Factor

NO Nitric oxide

N Normality

xvi

NMR Nuclear magnetic resonance spectroscopy

% Percentage

PTFE Polytetrafluoroethylene

KH2PO4 Potassium hydrogen phosphate

psi Pounds per square inch

± Plus-minus

RP Reverse phase

rpm Rotation per minute

Na2HPO4 Disodium hydrogen orthophosphate

NaHCO3 Sodium bicarbonate

NaCl Sodium chloride

NaOH Sodium hydroxide

Na+ Sodium ion

TMS Tetramethylsilane

TLC Thin layer chromatography

USP-NF The United States Pharmacopeia–National Formulary

UPLC Ultra pure liquid chromatography

UV Ultraviolet

v/v Volume per volume

w/v weight per volume

xvii

CHAPTER I

INTRODUCTION

Neurodegenerative diseases can be defined as hereditary and sporadic conditions

which are characterized by progressive nervous system dysfunction. Alzheimer’s

disease (AD) is one of the major neurodegenerative diseases. According to a report from

Alzheimer's Disease International (ADI), it is estimated that there are currently about 18

million people with AD worldwide. According to Alzheimer’s Disease Foundation

Malaysia (ADFM), approximately 50,000 Malaysians are currently diagnosed with the

illness. The production of reactive oxygen species during oxidative stress is speculated

to be pathologically important in neurodegenerative diseases. Degeneration of

cholinergic neurons and concomitant impairment of cortical and hippocampal

neurotransmission lead to cognitive and memory deficits (Schorderet, 1995). Therefore,

the characterization of neurite formation, maturation and collapse/ resorption is an area

of interest because these cellular processes are essential for the interconnection of

neuronal cell bodies.

Choline supplementation (lecithins) and/ or acetylcholinesterase inhibitors

(Tacrine) have been used to attenuate the cognitive and memory deficits. However,

these agents have showed several side effects such as gastrointestinal troubles, hepatitis

and reversible hepatotoxicity. The use of neurotrophin NGF (nerve growth factor) has

been initiated to treat neurodegenerative diseases. However, NGF cannot pass through

the blood-brain barrier. Therefore, it needs to be injected directly into the brain to be

effective (Kawagishi et al., 2002). If a substance can permeate the membrane and

stimulates the NGF synthesis in brain, this may result in the repair of the damaged

nervous functions.

1

Mushrooms, belonging to the kingdom fungi, are well-known for their medicinal

and therapeutic values for centuries, since every culture has a written or oral tradition of

using mushroom for their healing powers (Hobbs, 1995). There are over 1.5 million

species of fungi on earth, but mushrooms only constitute 14,000 species (Hawksworth,

2001). However, the well investigated known species of mushrooms are still very low.

Only 700 species are eaten as food and 50 species are poisonous (Halpern, 2007).

In recent years, studies on the medicinal values of the edible mushrooms have

gained a great deal of interests from researchers, as there is demand for more natural

remedies for life's ailments. Mushrooms are valuable health food - low in calories, high

in vegetable proteins, chitin, iron, zinc, fiber, essential amino acids, vitamins and

minerals. Besides that, mushrooms have been used as bioengineering resources in the

development of food materials (functional foods) as well as starting materials in the

production of drugs. For example, the hot water extracts from dried fruitbodies of H.

erinaceus are used as health drink (Yang and Jong, 1989). It has been pickled in brewed

wine to give a health drink (Mizuno, 1999).

Mushrooms possess many medicinal properties, pharmacological effects and

physiological properties such as bioregulation, maintenance of homeostasis, regulation

of biorhythm, prevention and improvement in cancer, cerebral stroke and heart diseases,

decreasing blood cholesterol, antifungal, anti-inflammatory, antiviral, antibacterial and

antiseptic, antidiabetic, serve as kidney and nerve tonic, hepatoprotective and sexual

potentiator (Wasser and Weis,1999).

Hericium erinaceus, belonging to the Basidiomycetes class, is an edible

mushroom occurring widely in Japan and China. These mushrooms grow on dead or

dying wood. Hericium erinaceus, known as Yamabushitake (mountain hidden

mushroom), Jokotake (drinker fungus), Usagitake (rabbit fungus) and Harisenbon

2

(balloon fish) in Japan; Houtou (monkey head mushroom) and Hedgehog mushroom in

China and cauliflower mushroom (cendawan ‘bunga kobis’) in Malaysia. As a culinary

delicacy, H. erinaceus is one of the few mushrooms imparting the flavor of lobster and

shrimp when cooked.

Hericium erinaceus has served as traditional medicines in many regions. In

China, it is prescribed for stomach disorders, ulcers and gastrointestinal ailments. In

North American, native Americans used H. erinaceus as a styptic, applied as a dried

powder to cuts and scratches to stop them from bleeding.

Some compounds have been successfully isolated from the fruiting bodies and

mycelia of H. erinaceus which showed NGF stimulation. Hericenones isolated from the

fruiting bodies of H. erinaceus have been shown to promote NGF synthesis (Kawagishi

et al., 1991). Erinacines isolated from mycelium of H. erinaceus have been identified as

stimulators of nerve growth factor (NGF) synthesis (Kawagishi et al., 1996; Shimbo et

al., 2005). Dilinoleoyl-phosphatidylethanolamine (DLPE) isolated from the fruiting

bodies of H. erinaceus may reduce the risk of neurodegenerative diseases by reducing

the endoplasmic reticulum (ER) stress (Nagai et al., 2006).

The screening for neurite outgrowth activity by H. erinaceus in an in vitro model

provides important preliminary data to select mushroom extracts for isolation purposes.

The neural hybrid clone, NG108-15 cell line is most widely used as an in vitro model of

neuronal differentiation because of its high proliferative activity and rapid elaboration

of neurites (Smalheiser, 1991). The advantages of this bioassay is that it uses a

continuous cell line, thus avoiding the need for dissection.

Hericium erinaceus is a temperate mushroom reported to fruit in cool

temperature. Currently, it is successfully cultivated in Malaysia. The mushroom now

grown in tropical climate, may have bioactive profiles different from temperate grown

3

H. erinacius. However, Wong et al. (2007, 2009) have shown that the cultivation

temperature did not affect this. Both the ethanol and water extract of H. erinaceus

grown locally displayed stimulation of the neurite outgrowth using an in vitro model.

Further, antioxidant and antimicrobial activities have been reported (Wong et al., 2009).

It was reported that extracts of H. erinaceus enhanced nerve regeneration (Wong et al.,

2009; 2011). It was therefore of interest to identify the chemical constituents in the

mushroom extract which may be responsible for stimulating neurite outgrowth.

Objectives of study

The objectives of the study were to:

(a) evaluate the crude and fractionated extracts of H. erinaceus for their effects in

stimulating the neurite outgrowth using the neural cell line NG 108-15.

(b) identify the most active fraction.

(c) identify the components present in the most active fraction .

4

CHAPTER II

LITERATURE REVIEW

2.1 MEDICINAL MUSHROOMS AND ITS USAGES

Fleshy mushrooms (members of the class basidiomycetes) have long been used

for their medicinal and therapeutic values. The term ‘medicinal mushroom’ is now

increasingly gaining worldwide recognition due to its value in the prevention and

treatment of diseases. Furthermore, it can be easily obtained from the natural

environment.

Medicinal properties of mushrooms have been widely studied. It was recorded

that mushrooms can exert a number of beneficial physiological effects. Auricularia

auricula-judae has been identified as a mushroom with reducing effect on the risk

factors of cardiovascular diseases. It has been reported to lower down the total

cholesterol and low density lipoprotein (LDL) level in hypercholesterolemic rats

(Cheung, 1996; Chen et al., 2008) and reduced blood platelet binding which will cause

arterial thromboses (Fan et al., 1989). Cordyceps sinensis, Grifola frondosa and

Lentinus edodes were effective in reduce the triglyceride level (Francia et al., 1999).

Besides that, Ganoderma lucidum and G. frondosa reduced blood pressure in

spontaneously hypertensive rats (Kabir et al., 1988; 1989). There were few species of

mushrooms which possessed hypoglycemic action such as Agaricus bisporus

(Swanston-Flatt et al., 1989), Agrocybe aegerita (Kiho et al., 1994), C. sinensis (Kiho

et al., 1996) and G. frondosa (Kubo et al., 1994). Ergosterol, an antitumor compound

which has been isolated out from the mushroom Agaricus blazei, reduced the tumor

growth with no side effects (Takaku et al., 2001).

Mushrooms are also good candidates for promoting neuronal differentiation and

survival. For example, polysaccharides in aqueous extract of G. lucidum induce

5

neuronal differentiation of rat pheochromocytoma PC12 cells and prevent NGF-

dependent PC12 neurons from undergoing apoptosis (Cheung et al., 2000; Silva, 2004).

Cyathane diterpenoid, termed scabronines, have been isolated from Sarcodon

scabrosus, a bitter mushroom (Ohta et al., 1998), and have been reported to stimulate

neurite outgrowth in rat pheochromocytoma cells (PC12) cultivated with the

conditioned medium of human astrocytoma cells (1221N1) (Obara et al., 1999). Water

extract of Tremella fuciformis induced neurite outgrowth in PC12 cells and improved

the memory deficit in rats by increasing the central cholinergic activity (Kim et al.,

2007).

Wu Ri, a famous Chinese physician from the Ming Dynasty (A.D. 1368- 1644),

claimed that L. edodes contain the ability to increase energy, cure colds, eliminate

worms and improve blood circulation. In “Shen Nong Ben Cao Jing”, G. lucidum, is

ranked under the superior medicine reported to be effective for multiple diseases and

mostly responsible for maintaining and restoring the body balance with no unfavorable

side effects. In the Taoist tradition, G. lucidum is said to enhance spiritual receptivity

and it was used by monks to calm the spirit and mind. It is also considered a symbol of

feminine sexuality as it refines the beauty and complexion.

6

2.2 HERICIUM ERINACEUS

2.2.1 Origin

Hericium erinaceus (Bull.: Fr.) Pers. (Figure 2.1), a member of the

basidiomycetous fungus, is well known as a traditional medicine or food in Japan and

China. In Japan, H. erinacues is called Yamabushitake because it resembles the

ornamental cloth worn by Yamabushi. It is also called Jokotake (drinker fungus),

Usagitake (rabbit fungus), Harisenbon (balloon fish) due to its shape.

Figure 2.1: Hericium erinaceus (Bull.: Fr.) Pers.

This mushroom is called Houtou (monkey head mushroom) in China due to the

close resemblance of fruiting body to the head of a baby monkey. It also known as

Hedgehog mushroom according to its shape. A Chinese traditional drug prepared by

drying this mushroom is also called Houtou. The hot water extracts from dried

fruitbodies are used as health drink (Yang and Jong, 1989). It can be pickled in brewed

wine to give a health drink (Mizuno, 1999).

Hericium erinaceus is a wood destroying fungus and grows in standing and

decayed broadleaf trees such as oak, beech, and walnut. The cultivation of H. erinaceus

7

has been established using artificial logs made with agricultural residues in either bottles

or polypropylene bags (Mizuno, 1999; Chang and Miles, 2004).

2.2.2 Classification

Kingdom : Fungi

Phylum : Basidiomycota

Class : Basidiomycetes

Order : Russulales

Family : Hericiaceae

Genus : Hericium

Species : erinaceus

2.2.3 Medicinal properties, nutritional and bioactive components derived from

Hericium erinaceus

Medicinal properties of H. erinaceus have been widely studied. Both the fruiting

bodies and mycelia of H. erinaceus contain bioactive polysaccharides which exhibit

various pharmacological activities including immunomodulatory effect, as well as anti-

tumor, hypoglycemic and anti-aging properties (Zhang et al., 2007). Fifteen

polysaccharides have been successively extracted out with hot water. Five types of

polysaccharides which showed relatively strong antitumor activity and a good life

prolongation effect were glucoxylan, xylan, heteroxyloglucan, glucoxylan protein

complex and galactoxyloglucan protein complex (Zhang et al., 2007).

Besides the polysaccharides, an ergosterol derivative was also isolated from H.

erinaceus. This compound showed cytotoxic effects on the cervical carcinoma HeLa

cells (Mizuno, 1999), antitiumor activity against Walker carcinosarcoma and human

mammary adenocarcinoma cell lines in vitro (Jong and Donovick, 1989), human gastric
8

tumor cell line, human hepatoma cell line, human colorectal tumor cell line and murine

sarcoma-180. It also showed antivenom, anti-inflammatory (Keyzers and Davies-

Coleman, 2005) and antimicrobial activity (Lu et al., 2000).

Hericenones, erinacines, hericerin and hericenes, the aromatic compounds that

identified in H. erinaceus, showed a wide range of in vitro and in vivo bioactivities

(Shang et al., 2012). The novel oxyketo acid, Y-A-2, cytotoxic phenols, hericenone A

and hericenone B (Kawagishi et al., 1990), two novel γ- pyrones, erinapyrone A and

erinapyrone B (Figure 2.2) (Kawagishi et al., 1992) extracted from the fruiting body of

H. erinaceus using ethanol or acetone, showed inhibition against the proliferative

activity of HeLa cells.

Besides that, various acidic phenol-like and neutral fatty acid-like compounds

such as hericenones and hericerins found in H. erinaceus (Kim et al., 2000) were

effective against pathogenic microorganisms and showed antibacterial activity at low

concentrations against S. aureus, B. subtilis and E. coli respectively. Two novel and a

known chlorinated orcinol derivaties were also isolated from the mycelium of H.

erinaceus. These three compounds exhibited antimicrobial activities against Bacillus

subtilis, Saccharomyces cerevisiae, Vetticillium dahlia and Aspergillus niger (Okamato

et al., 1993).

Ethanol extract of mycelia or fruitbodies promoted better antimutagenic effects

than water extract examined with the Ames test (Wang et al., 2001). On the other hand,

methanol extract of fruitbodies was found to have hypoglycemic effect and reduce

elevation rates of serum triglyceride and total cholesterol levels when administered to

streptozotocin-included diabetic rats (Wang et al., 2005). Yang et al., (2003)

investigated the hypolipidemic effect of an exo-biopolymer produced from a submerged

culture of H. erinaceus in dietary-included hyperlipidemic rats. The exo-biopolymer

reduced the level of plasma total cholesterol, low density lipoprotein cholesterol,

9

triglyceride, phospholipids, atherogenic index and hepatic HMG-CoA reductase

activity; and preserving the high density lipoprotein at relatively high level. These

effects would help to reduce the risk of atherosclerosis.

OH O

COOH
OH
Y-A-2 (Novel oxyketo acid)

O OH O

O
H3CO

Hericenone A

O OH O

N
H3CO

Hericenone B

O O

HO OH
O O

Erinapyrone A Erinapyrone B

Figure 2.2: HeLa cell growth inhibitory substances isolated from Hericium erinaceus

10

This mushroom has been reported to exhibit significant antioxidant activity

which might help to reduce the oxidative damage caused by the uncontrolled production

of oxygen-derived free radicals (Mau et al., 2001). The reduction of free radicals might

lower the risk in the onset of many diseases such as cancer, rheumatoid arthritis,

artherosclerosis, degenerative processes and deterioration of physiological functions

associated with aging. Besides that, total polyphenols were the major natural antioxidant

components found in the methanol extract from dried H. erinaceus fruit bodies (Mau et

al., 2002).

In Chinese traditional medicine, it is used for the treatment for neurasthenic

gastritis and gastroduodenal ulcer. In recent year, cultures or their extracts processed in

tablets have been produced in large scale for curing gastric ulcer and chronic gastricism.

Nitric oxide (NO) is a pleiotropic biological molecule involved in a myriad of

physiological and pathological processes such as regulation of blood pressure,

neurotransmission, signal transduction, anti-microbial defense, immunomodulation,

cellular redox regulation and apoptosis. The water extract of H. erinaceus activated the

macrophages and induce NO production in peritoneal macrophages and RAW 264.7 cell

line through the activation of transcription factor NF-KB (Son et al., 2006).

A 63kDa laccase, with a novel N-terminal sequence isolated from the water

extract of H. erinaceus dried fruiting bodies showed inhibitory effect towards HIV-1

reverse transcriptase (Wang and Ng, 2004). HIV-1 reverse transcriptase was involved in

HIV replication; inhibitors of this enzyme are potential therapeutic agents in the battle

against HIV (Sarafianos et al., 2009).

11

2.3 NERVOUS SYSTEM AND NEURODEGENERATIVE DISEASES

2.3.1 Neurite

The characterization of neurite formation, maturation and collapse/ resorption is

an area of interest because these cellular processes are essential for the interconnection

of neuronal cell bodies. Neurites are particularly interesting in relation to

neuropathological disorders, neuronal injury/ regeneration and neuropharmacological

research and screening (Smit et al., 2003). Neurites emerging from cloned neural cell

lines have been studied extensively over the past 15 years (Smalheiser & Schwartz,

1987). It was appreciated very early that some clones can express neurites

spontaneously, even without inducing them to differentiate, but most neurobiologists

have ignored this class of neurites in favor of studying clones such as the PC12 cell line,

whose neurites are under inducible control and contain characteristics of axons in

differentiated neurons. Bioassay which uses the PC12 cell line of rat

pheochromocytoma was described by Greene (1977) and Greene & Tischler (1982).

The matured neurite, called neuron, is responsible for receiving stimuli, producing and

transmiting electrical signal called nerve impulses, or action potentials. It also

synthesizes and releases neurotransmitters.

2.3.2 Neurodegenerative diseases (factors, therapies to cure and prevent)

Neurodegenerative diseases can be defined as hereditary and sporadic conditions

which are characterized by progressive nervous system dysfunction. These disorders are

often associated with atrophy of the affected central or peripheral nervous system

structures. Neurological disorders are quite diverse, chronic, challenging to treat, and

often disabling. They can be caused by many different factors, including (but not

limited to): inherited genetic abnormalities, problems in the immune system, injury to

12

the brain or nervous system, or diabetes. Many mental illnesses are believed to be

neurological disorders of the central nervous system, but they are classified separately.

The production of reactive oxygen species during oxidative stress is speculated

to be pathologically important in neurodegenerative diseases which include Alzheimer’s

disease, Parkinson’s disease, amyotrophic lateral sclerosis, and Huntington’s disease

(Halliwell and Gutteridge, 1999). Alzheimer’s disease is the most common form of

senile dementia. Alzheimer’s disease could be caused by both environmental and

genetic factors. This has been proved by the genetic linkage studies on the

chromosomes 14, 21 (early-onset) and 19 (late-onset). Trisomy and mutations of the β-

amyloid precursor protein gene on chromosome 21 are the causes that lead to the early-

onset of the Alzheimer’s disease (Goate et al., 1991). Early-onset familial forms of

Alzheimer’s disease could be caused by genetic mutations which may affect

chromosome 14 (Mullan et al., 1993). Mutations on chromosome 19 and the

concomitant expression of variant apolipoprotein E4 (ApoE4) from ApoE gene (ε4

allele) are associated with sporadic and late-onset familial forms of Alzheimer’s disease

(Uterman, 1994). However, less than 1% of patients who suffer from the disease are due

to these genetic causes. It is probable that the majority of the cases is caused by a

variety of environmental factors which may be either sufficient to trigger disease by

themselves, or sufficient when acting synergistically with the patient genotype. A direct

or indirect role has been attributed to normal or structurally altered amyloid β-protein

(concentrated in senile plaques) and/or excessively phosphorylated tau protein (located

in neurofibrillary tangles) (Schorderet, 1995). Degeneration of cholinergic neurons and

concomitant impairment of cortical and hippocampal neurotransmission lead to

cognitive and memory deficits (Schorderet, 1995).

Neuronal cell death is an essential feature of neurodegenerative disease. Many

types of neuronal cell death, for example, these which are associated with amyloid-β,

13

glutamate and nitric oxide are thought to be caused by endoplasmic reticulum stress.

Glutamate toxicity is a major contributor to pathological cell death within the nervous

system and appears to be mediated by reactive oxygen species (Lee et al., 2003). Thus,

it is reasonable to suspect that molecules which are able to attenuate endoplasmic

reticulum stress might reduce both the risk for and the extent of the damage in

neurodegenerative disease (Nagai et al., 2006).

Treatments for reducing neuronal cell death are important for preventing as well

treating neurodegenerative disease, including dementia and motor dysfunction.

However, because neurodegenerative diseases have, typically, a long incubation period

prior to diagnosis and are symptom-free; hence, there is a late diagnosis of the disease.

This is a severe problem because once neurons are dead or neuronal circuits destroyed,

lost of brain function associated with the neurons or neuronal circuits is almost

impossible to restore.

Attenuation of the cognitive deficits by using choline supplementation

(lecithins) and/ or acetylcholinesterase inhibitors might enhance the cholinergic activity

if the cognitive deficits are due to the loss of cholinergic activity. Tacrine

(tetrahydroaminoacridine, Cognex®), a potent, centrally active and reversible

acetylcholinesterase inhibitor, was used together with morphine to lessen respiratory

depression without affecting analgesia in the mid 1940s. Tacrine has been used alone, or

in combination with lecithin, to treat symptoms of the Alzheimer’s disease (Chatellier et

al., 1990; Farlow et al., 1992). It showed a slight but statistically significant

improvement in the physician's score on the visual analogue scale (Chatellier et al.,

1990). Only a small percentage of patients, moderately affected or treated at an early

stage of Alzheimer’s disease, seem to benefit from the drug (Farlow et al., 1992).

However, it showed several side effects such as gastrointestinal troubles, hepatitis and

reversible hepatotoxicity.

14

Investigation on the projection of neurotrophin NGF (Nerve Growth Factor)

which could counter the degeneration of cholinergic neurons to the hippocampus, a

recognized memory center, were recently initiated. NGF is a protein that is essential for

supporting the growth and maintenance of peripheral sympathetic neurons as well as

facilitating the development of some sensory neurons for a brief period during early

development (Shimbo et al., 2005). Infusions of NGF into the brain of a patient can

improve performance in memory test and prevent or stabilize the processes of

cholinergic pathway degeneration (Schorderet, 1995).

Alternatively, antioxidants, free redical scavengers and/ or non-steroidal anti-

inflammatory agents such as α-tocopherol (vitamin E), ubiquinols (coenzyme Q),

retinoic acid (vitamin A), and ascorbic acid (vitamin C), may be screened as potential

therapies for neurodegenerative disease induced by multiple endogenous and/ or

exogenous factors (Schorderet, 1995).

15

2.4 NEUROPROTECTIVE, NEUROTROPHIC, NEURONAL

DIFFERENTIATION AND NEURITE STIMULATION EFFECTS OF

HERICIUM ERINACEUS

Hericenone C, D, E, F, G, H have been successively isolated from H. erinaceus

(Kawagishi et al., 1991, 1993). Among them, hericenone C, D, E (Figure 2.3) have been

proven to show NGF synthesis promoting activity (Kawagishi et al., 1991).

O OH

CHO

16"
O
H3CO

O

Hericenone C

O OH

CHO

18"
O
H3CO

O

Hericenone D

O OH

CHO

18"
O
H3CO
9" 12"
O

Hericenone E

Figure 2.3: Hericenones isolated from fruiting body of Hericium erinaceus which

showed NGF synthesis promoting activity.

16

This mushroom also produces erinacines A (Shimbo et al., 2005), B, C

(Kawagishi et al., 1994), D (Kawagishi et al., 1996a), E, F, G (Kawagishi et al., 1996b)

which have been identified as stimulators of nerve growth factor (NGF) synthesis

(Figure 2.4). Stimulators of NGF synthesis have been used as medicines for

degenerative neuronal disorders such as Alzheimer’s disease and peripheral nerve

regeneration. NGF is a protein that is essential for supporting the growth and

maintenance of peripheral sympathetic neurons as well as facilitating the development

of some sensory neurons for a brief period during early development (Shimbo et al.,

2005). NGF cannot pass through the blood-brain barrier, the semi-permeable membrane

between the blood and brain. Only small and lipid soluble molecules can pass through

the membrane. NGF is too large to permeate it. Therefore, it needs to be injected

directly into the brain to be effective (Kawagishi et al., 2002). If a substance can

permeate the membrane and stimulates the NGF synthesis in brain, this may result in

repairing the damaged nervous functions.

Dilinoleoyl-phosphatidylethanolamine (DLPE), an endoplasmic reticulum (ER)

stress- attenuating molecule which might reduce the ER-stress, has been isolated from

the fruit bodies of H. erinaceus and these may reduce the risk of getting

neurodegenerative diseases (Nagai et al., 2006). ER stress is the major cause of the

neuronal cell death which leads to the neurodegenerative diseases.

17

O O
O O

H OH
OH O
HO
OH OH
CHO CHO

Erinacine A Erinacine B

O O O
O

H
H OH
OH
O HO
OH OH
CH2OH C2H5O CHO

Erinacine C Erinacine D

H
O O
O O
H H OH H H OH
H H

OH OH

HO OH HO OH

Eriancine E Erinacine F

O
O
O O
H H OH
H

OH

HO OH

Erinacine G

Figure 2.4: Erinacines isolated from mycelium of Hericium erinaceus which showed

NGF synthesis promoting activity.

18

2.5 NEURITE OUTGROWTH BIOASSAY SYSTEM OF NEURAL HYBRID

CELL LINE NG108-15

2.5.1 Formation of NG108-15 hybrid cell

6-thioguanine-resistant clonal mouse neuroblastoma cells N18TG2 and the

bromodeoxyuridine-resistant rat glioma cells C6-BU-1 were fused with the aid of

inactivated Sendai virus to generate the neuroblastoma x glioma hybrid cell clone,

NG108-15 (Hamprecht et al., 1985). Cells were grown in selective hypoxanthine-

aminopterin-thymidine (HAT) medium, which was known to select for the wild-type

hybrid cells and against the parental cell lines and their corresponding homokaryocytes

(Littlefield, 1964).

2.5.2 Characteristics of NG108-15 hybrid cell

The hybrid cell is used as model neurons because every characteristic generally

ascribed to neurons has been observed with the hybrid cell. The properties of NG108-15

are summarized in Table 2.1 (Hamprecht et al., 1985). Due to the complexity of the

mammalian nervous system, it is hard to assign a certain effect observed to a certain cell

type. Problems that are difficult to solve with animal or tissue experiments can be

tackled with the aid of cultured cells. Then, it is like having at one’s disposal the

numerous cell types as homogenous cell populations for studying their individual

differentiated functions and their mechanisms of intercellular communication.

19

Table 2.1: Neuronal properties of neuroblastoma x glioma hybrid cells (NG108-15)

Neuronal Properties

Extension of long processes

Clear and dense core vesicles

Excitable membranes (inward current of action potentials carried by Na+ or Ca2+

Formation of functional synapses

Neurotransmitter enzymes

- Choline acetyltransferase
- Dopamine-β-hydroxylase

Synthesis of neurohormones

- Acetylcholine
- Leu- and Met- enkephalin
- Dynorphine-(1-8), α-neoendorphine
- β-Endorphine
- Vasoactive intestinal peptide
- Angiotensin
- Hydra head activator- like activity

Uptake system for

- Catecholamines
- Taurine

Depolarization- induced Ca2+- dependent release of acetylcholine

Receptors for neurohormones

- Acetylcholine
- Noradrenaline
- Opioids (Morphine, enkephaline)
- Prostaglandin E1
- Adenosine

20

CHAPTER III

MATERIALS AND METHODS

3.1 EXTRACTION

3.1.1 Preparation of fruitbodies

Hericium erinaceus fresh fruitbodies were obtained from Ganofarm Sdn. Bhd. in

Tanjung Sepat, Selangor. The fruitbodies were freeze-dried at 50± 2 C for 48 hours.

The dried fruit bodies were blended in the commercial Waring blender and stored in

airtight containers prior to assay.

3.1.2 Preparation of aqueous ethanol crude extract

The powdered freeze-dried fruit bodies of H. erinaceus was soaked in 80 %

(v/v) aqueous ethanol for 3 days. The residue was then resoaked in 80 % (v/v) aqueous

ethanol and the extraction and filtration process was repeated three times. The solvent

containing extract was then concentrated under vacuum using a rotary evaporator.

3.1.3 Solvent- solvent extraction (fractionation)

The crude extract obtained was successively fractionated with hexane, ethyl

acetate and water using a separating funnel. All the fractions (hexane, ethyl acetate and

water) were filtered and concentrated under vacuum using a rotary evaporator to give

hexane, ethyl acetate and water fractions

Figure 3.1 shows the flow chart of the extraction and fractionation procedures,

process of biological investigations and isolation of active fractions of H. erinaceus.

21

Fresh Hericium erinaceus

Freeze-dried and ground to fine powder

Dried and ground H. erinaceus

i. Extraction with 80% ethanol (3 times)
ii. Concentration under reduced pressure

Aqueous ethanol crude extract

i. Extraction with hexane
ii. Concentration under reduced
pressure

Hexane soluble fraction Hexane insoluble fraction

i. Partition (v/v) between
ethyl acetate and water
(Ratio 1:2)
ii. Concentration under
reduced pressure

Ethyl acetate fraction Water fraction

In vitro neurite outgrowth assay by
using NG108-15 hybrid clone

Isolation of active fractions

Figure 3.1: A schematic diagram showing the extraction and fractionation procedures,

process of biological investigations and isolation of active fractions of Hericium

erinaceus

22

3.2 NEURITE OUTGROWTH ACTIVITY ASSAY

3.2.1 Preparation of stock solutions

Each extract and fractions were dissolved in dimethylsulfoxide (DMSO) to form

stock solutions 20 mg/ml for neurite outgrowth assay and kept at -20 ºC for future use.

The concentration of samples was prepared according to the requirements for the assay

by serial dilutions using the media or media with Tween 80.

3.2.2 Cell culture

The neural hybrid clone NG108-15 was chosen for this purpose. NG108-15 cells

were purchased from American Type Culture Collection (ATCC) and cultured in

Dulbecco’s Modified Eagle’s Medium (DMEM).

3.2.3 Preparation of medium and buffer for cell culture

3.2.3.1 Dulbecco’s Modified Eagle’s Medium (DMEM)

Basic medium

Final volume cell culture grade water (80 - 90 %) was measured. Water

temperature should be 15-30 C. Dry powder medium (13.38 g/l) was added slowly and

allowing mixing time between additions (original package was rinsed with small

amount of water to remove all traces of powder and added to solution). The solution

was mixed for 30 minutes. 3.7 g/l of sodium bicarbonate (NaHCO3) and hypoxanthine-

aminopterine- thymidine (HAT) were added and stirred until dissolved. While mixing,

the pH of the medium was adjusted to 6.9-7.1 using 1N NaOH or 1N HCl. Additional

water was added to bring the solution to final volume and continue mixing for at least

30 minutes. The medium was sterilized by filtration using a membrane with a pore size

of 0.22 microns and aseptically dispensed into sterile container.
23

Complete growth medium

Basic medium described above supplemented with 10 % (v/v) of fetal bovine

serum, 100units/ml penicillin and 100 µg/ml streptomycin.

Revival medium

Complete growth medium described above supplemented with 20 % (v/v)

instead of 10 % (v/v) fetal bovine serum.

Cryoprotectant medium

Basic growth medium described above supplemented with 10 % (v/v) dimethyl

sulfoxide (DMSO) and 50 % (v/v) fetal bovine serum.

3.2.3.2 Phosphate Buffer Saline

1.52 g of sodium hydrogen phosphate (Na2HPO4), 0.58 g of potassium hydrogen

phosphate (KH2PO4) and 8.5 g of sodium chloride (NaCl) were dissolved in 1L distilled

water and pH was adjusted to 7.2. The solution was filtered with filter paper and

autoclaved for 15 minutes at 121 C, 15 psi. The solution was stored at room

temperature.

3.2.4 Cell culture techniques

3.2.4.1 Revival of frozen cells

The vial containing frozen cells was thawed by gentle agitation in a 37 C water

bath. To reduce the loss of viability, the vial was thawed rapidly. Once the contents

were thawed, the contents were transferred aseptically into a centrifuge tube by

pipetting. 1 ml of revival medium was added and centrifuged at 1000 rpm for 5 minutes.

24

Supernatant was discarded and pellet was resuspended in 1 ml of revival medium. The

suspended cells were pipetted into a cell culture flask containing 5- 10 ml revival

medium. The culture was incubated at 37C in a 5% CO2 incubator at atmospheric

pressure.

3.2.4.2 Subculture of cells

The culture was examined with an inverted microscope to check for any

evidence of microbial contamination and to determine whether the majority of the cells

were attached to the bottom of the flask. When the color of the medium changed from

reddish to yellowish (the medium become acidic) or every 2- 3 days, the culture

medium was removed and discarded. Trypsin-EDTA (1 ml) and phosphate buffer saline

solution (3 ml) were added to the flask and cells were observed under inverted

microscope until cell layer was detached from the bottom of the flask. The contents of

the flask were transferred aseptically into a centrifuge tube containing 2 ml of complete

growth medium by pipetting. The cells were centrifuged at 1000 rpm for 5 minutes.

Supernatant was discarded and pellet was resuspended in 3- 4 ml of complete growth

medium. Appropriate amounts of suspended cell were added to a new culture flask

containing complete growth medium. Cultures were incubated at 37 C in a 5 % CO2

incubator at air atmosphere.

3.2.4.3 Medium renewal

The medium was changed 3 to 4 times weekly.

25

3.2.4.4 Cryopreservation of cells

Cryopreservation of cells was carried out in accordance with subculturing

procedure except that the pellet obtained after centrifugation was suspended in

cryoprotectant medium instead of complete growth medium. The suspended cells were

then transferred to a cryogenic vial and stored in liquid nitrogen vapor phase.

3.2.5 Effect of Hericium erinaceus on stimulation of neurite outgrowth activity of

NG108-15

The neural hybrid cell NG 108-15 was cultured until 60-70 % confluent prior to

assay. Cells were detached from the flask with 0.25 % (w/v) solution of trypsin in

phosphate buffer saline solution and washed in culture medium. The cell pellet was

obtained by centrifugation at 1000 rpm for 5 minutes. The density of the cells was

counted by 0.4 % (w/v) of tryphan blue exclusion method in a haemocytometer. The

cells were plated into 6 well plates coated with 2 x 10-5 % (w/v) of poly-D-lysine at a

cell density of 10000 cells per well in medium containing 4 various concentrations (10,

25, 50, 100 µg/ml) of extract and fractions and subfractions of fruit bodies. Negative

control is the well with the untreated cells whilst positive control is the well with the

cells treated with Nerve Growth Factor (NGF). Plates were incubated at 37 C in a 5 %

CO2 humidified incubator. Cells were observed for neurite outgrowth, branching of

neurites after 24 hours.

3.2.6 Scoring of neurites

A cell was considered as positive for bearing neurites if it had at least one thin

extension longer than one full diameter of its cell body. Specifically excluded were

extensions associated with clearly different patterns of cell responses, such as broad,

26

sheet-like spreading of cells or the rare radially oriented processes apparently arising by

“shrinkage” (Smalheiser and Schwartz, 1987). Cell clumps containing more than five

cells were also not included in the results. If more relaxed criteria had been used, many

short extensions would have been counted as neurites, and the assay would not have

been useful to detect stimulatory effects upon neurite formation (Smalheiser and

Schwartz, 1987). Duplicates were set up for each concentrations tested. Approximately

300 cells in each well were evaluated. Neurite formation was quantified by scoring the

number of cells processing neurites and expressed as a percentage of the total number of

cells counted.

Neurite bearing cells (%) = number of cells processing neurites x 100 %
total number of cells counted

The result was also expressed in percentage increase in neurite bearing cells in

comparison to negative control.

Percentage increase compared to negative control

= neurite bearing cells of extract – neurite bearing cells of negative control x 100 %
neurite bearing cells of negative control

3.2.7 Statistical analysis

The means of data were subjected to a one way analysis of variance (ANOVA)

and the significance of the difference between means was determined by the Duncan’s

multiple range test at 95 % least significance difference (P<0.05).

27

3.3 ISOLATION OF BIOACTIVE CONSTITUENTS

3.3.1 Column chromatography

The combined fraction of hexane and ethyl acetate was subjected to silica gel

flash column chromatography. Column chromatography was performed by using Merck

silica gel. The gel was packed onto the column. After the sample was introduced to the

column, solvent with increasing polarity gradient was used to elute the column

[developing solvent: chloroform (100 % chloroform) → chloroform-acetone mixtures

(20 % Ac/CHCl3, 40 % Ac/CHCl3) → chloroform-methanol mixtures (10 %

MeOH/CHCl3, 30 % MeOH/CHCl3) → methanol (100 % MeOH)]. Fractions were

monitored by thin layer chromatography (TLC) and fractions that possess the same

spots/bands on the TLC were combined and where necessary subjected to further

separation. Nine fractions were obtained (E1-E9) from the flash column

chromatography which monitored by TLC.

Figure 3.2 shows the flow chart of the isolation of active fractions of H.

erinaceus.by using different types of chromatographic technique, process of biological

investigations and identification of the active fractions.

3.3.2 Analytical thin layer chromatography

TLC was routinely used to detect and separate the various compounds. The

fractions from column chromatography were examined by TLC using precoated glass

plates, 0.25 mm thickness, silica gel F254 (Merck, Darmstadt, G.F.R). The TLC plates

were spotted with a piece of fine glass capillary tube and then developed in saturated

chromatography tanks with various solvent systems at room temperature. The spots

were visualized by examination of the TLC plates under UV light, followed by applying

iodine vapor.

28

3.3.3 Preparative thin layer chromatography

Subfraction E2 was successively subjected to preparative thin layer

chromatography. The sample was dissolved by using chloroform. The TLC precoated

(silica gel F254) glass plates (Merck, Darmstadt, G.F.R) with 0.25 mm thickness, 10 cm

(width) x 10 cm (height) were used. A line was drawed (about 1 cm) from the bottom of

the plate. ). The line on the TLC plates was spotted with a piece of fine glass capillary

tube and then developed in saturated chromatography tanks (100 % CHCl3) at room

temperature. After the solvent reach the solvent front, the plates were taken out from the

chromatography tanks. When the plates were dried enough, the bands were visualized

by using UV light. The bands were marked lightly by using pencil. The bands were

scrapped off onto a clean, white paper by using the edge of a spatula. The compounds

were washed off from the silica gel by using chloroform. The solvent containing

samples were filtered and concentrated under vacuum using a rotary evaporator.

3.3.4 High performance liquid chromatography (HPLC)

3.3.4.1 HPLC samples and mobile phase preparation

The samples were dissolved in methanol/acetonitrile mixture. The samples were

filtered through 0.45 µm Sartorius minisart PTFE-membrane syringe filter to remove

any particular matter that might clog the column. The mobile phase that used was

acetonitrile. The mobile phase was filtered by using 0.45 µm Sartorius PTFE-membrane

and degassed before introduced to the system. All the solvents that used were in HPLC

grade.

29

3.3.4.2 Analytical HPLC

Analytical HPLC analysis were carried out by using the instrument Waters Delta

Prep consists of water Prep LC controller, quaternary pump, vacuum degasser, UV

detector (water 2487, Dual λ Absorbance Detector). The separation profiles of the

samples can be improved by changing the solvent system of the mobile phase, flow rate

and column. The column that used for analytical HPLC was performance RP-18

encapped column 100-4.6 mm purchased from Merck. The analysis was carried out in

isocratic mode at a flow rate of 1 ml/min, with column effluent being monitored at the

wavelength of 214 nm and 254 nm.

3.3.4.3 Semipreparative HPLC

The samples were then further isolated out by using Chromolith Semiprep RP-

18 column encapped 100-10 mm purchased from Merck. The separation was carried out

in isocratic mode by using 100 % acetonitrile as mobile phase at a flow rate of 3

ml/min, with column effluent being monitored at the wavelength of 214 nm and 254

nm. The separated subfraction was collected manually.

30

Combined fraction of
hexane and ethyl acetate

Flash column chromatograhy

E1 E2 E3 E4 E5 E6 E7


Identification of the Preparative thin layer
chemical constituents chromatography
by GC-MS

sub4b

HPLC

sub4b_4 sub4b_6

Identification of the
chemical constituents
by NMR

Identification of the
chemical constituents
by LC/MS/MS


Figure 3.2: A schematic diagram showing the isolation of active fractions, process of

biological investigations and identification of the active fractions.

31

3.4 IDENTIFICATION

3.4.1 Gas chromatography-mass spectrometry (GCMS)

GCMS analysis was performed on fraction E1 using Network Gas

Chromatography System (Agilent Technologies 6890) and Inert Mass Selective

Detector (Agilent Technologies 5975) (70eV direct inlet) on a HP-5MS (5 % phenyl

methyl siloxane) capillary column (30 m x 250 µm x 0.25 µm) initially set at 150 ºC,

then programmed to 300 ºC at 5 ºC min-1 and held for 10 minutes at 300 ºC using

helium as the carrier gas. The total ion chromatogram obtained was autointegrated by

chemstation and the constituents were identified by comparison with the accompanying

mass-spectra database (NIST 05 Mass Prectral Library, USA) wherever possible.

3.4.2 Nuclear magnetic resonance spectroscopy (NMR)

All the NMR experiments were performed on a JEOL 400MHz NMR

spectrometer that install with the JEOL Delta software. Subfractions sub4b_4 and

sub4b_6 were dissolved in deuterated chloroform and 1H, 13C and 1H-1H-COSY spectra

were collected. The internal standard for 1H NMR was TMS (δ: 0.00) and 13
C was

CDCl3 (δ: 77.00).

3.4.3 Liquid chromatography-mass spectrometry (LC/MS/MS)

LC/MS/MS analysis were carried out on subfractions sub4b_4 and sub4b_6 by

using the instrument Applied Biosystems 3200Q Trap LCMS/MS with Shimadzu ultra

pure liquid chromatography (UPLC) system. Full scan with MS/MS data collection was

used. Positive ionization mode was set. The column that was used is Phenomenex Aqua

C-18 with dimension 50.0 mm x 2.0 mm x 5.0 µM. Rapid screening was performed

with 10 min run time.

32

CHAPTER IV

RESULTS & DISCUSSION

4.1 EXTRACTION, FRACTIONATION AND ISOLATION

4.1.1 Extraction, fractionation and isolation of aqueous ethanol extract of Hericium

erinaceus

The flow chart shows the extraction and fractionation procedures for H. erinaceus

(Figure 4.1 and 4.2)

Fresh H. erinaceus (1.33 kg)

Dried and ground to fine powder

Dried and ground H. erinaceus (200.00 g)

i. Extraction with 80 % ethanol (3 times)
ii. Concentration under reduced pressure

Aqueous ethanol extract (52.29 g)

Figure 4.1: Aqueous ethanol extraction of Hericium erinaceus.

Fresh H. erinaceus (1.33 kg) yielded 200.00 g of dried and ground H.erinaceus.

The dried H. erinaceus sample (200.00 g) yielded 52.29 g of crude aqueous ethanol

extract.

33

Aqueous ethanol extract

i. Extraction with hexane
ii. Concentration under reduced
pressure

Hexane soluble fraction Hexane insoluble fraction
(3.85 g, 7.36 %)

i. Partition (v/v) between
ethyl acetate and water
(Ratio 1:2)

ii. Concentration under
reduced pressure

Ethyl acetate fraction Water fraction

(0.77 g, 1.47 %) (44. 34 g, 84.80 %)

Figure 4.2: Fractionation of aqueous ethanol extract of Hericium erinaceus.

The aqueous ethanol extract was further extracted with hexane to give hexane-

soluble fraction (3.85 g, 7.36 %) and hexane-insoluble residues. The hexane-insoluble

residues were further partitioned between ethyl acetate-water (ratio 1:2) to give the ethyl

acetate-soluble fraction (0.77 g, 1.47 %) and water fraction (44. 34 g, 84.80 %).

34

The combined hexane and ethyl acetate fraction were subjected to flash column

chromatography to yield 7 fractions, which were E1 (384.0 mg, 0.73 %), E2 (780.8 mg,

1.49 %), E3 (438.2 mg, 0.84 %), E4 (62.4 mg, 0.12 %), E5 (39.7 mg, 0.08 %), E6

(183.1 mg, 0.35 %), E7 (1068.2 mg, 2.04 %) (Figure 4.3). The percentage yields were

calculated based on the crude aqueous ethanol extract.

Combined fraction of
hexane and ethyl acetate

Flash column chromatography
(developing solvent: CHCl3 →
CHCl3/Ac → CHCl3/MeOH →MeOH)

E1 (384.0mg, E3 (438.2mg, E5 (39.7mg, E7 (1068.2mg,
0.73 %) 0.84 %) 0.08 %) 2.04 %)

E2 (780.8mg, E4 (62.4mg E6 (183.1mg,
1.49 %) 0.12 %) 0.35 %)

Figure 4.3: Isolation of combined hexane and ethyl acetate extract of Hericium

erinaceus obtained through flash column chromatography.

35

Fraction E2 obtained from flash column chromatography were further subjected

to preparative thin layer chromatography to yield subfraction sub4b (187.7 mg, 0.36 %)

(Figure 4.4). Sub4b was then subjected to high performance liquid chromatography

(HPLC) to give sub4b_4 (68.5 mg, 0.13 %) and sub4b_6 (38.5 mg, 0.07 %) (Figure 4.4).

E2

Preparative thin layer
chromatography

Sub4b (187.7 mg,
0.36 %)

High perfomance liquid chromatography
(HPLC)

Sub4b_4 (68.5 mg, Sub4b_6 (38.5 mg,
0.13 %) 0.07 %)

Figure 4.4: Isolation of fraction E2 of Hericium erinaceus using preparative thin layer

chromatography and high performance liquid chromatography.

36

4.2 NEURITE OUTGROWTH ACTIVITY

4.2.1 Effect of aqueous ethanol extract and fractions of Hericium erinaceus on the

neural cell line NG108-15

Aqueous ethanol extract and fractions of H. erinaceus were screened for the in

vitro neurite outgrowth activity on the neural hybrid cell line NG108-15 at various

concentrations (µg/ml) (Figure 4.5; Table 4.1). Cells were observed under a phase

contrast microscope for the neurite outgrowth and branching of neurites. The effect of

the various extracts on the morphology and neurite extension of the NG108-15 cells are

given in Figure 4.6 (crude aqueous ethanol extract), Figure 4.7 (hexane fraction), Figure

4.8 (ethyl acetate fraction) and Figure 4.9 (water fraction).

50
45
Neurite- bearing cells (%)

40
35 ethanol crude extract
30 Hexane fraction
25 Ethyl acetate fraction
40.6

20 Water fraction
34.5
34.1
32.3

27.8
27.4

15
26.5

25.6
25.4
24.6

24.2
24.1

24.0
23.7

23.5
23.0

22.9

22.5
21.9

20.9
20.5
19.9

19.0

18.6

10
5
0
Negative NGF 10 25 50 100
control (20ng/ml)
Concentrations (µg/ml)

Figure 4.5: Percentage of neurite bearing cells incubated with varying concentrations of

aqueous ethanol crude extract, hexane fraction, ethyl acetate fraction and water fraction

of Hericium erinaceus (nerve growth factor, 20 ng/ml, used as positive control).

37

Table 4.1: Stimulation of neurite outgrowth activity in the NG108-15 cells with varying concentrations of aqueous ethanol extract and

fractions of Hericium erinaceus. NG108-15 cells without extract was negative control. 20 ng/ml of nerve growth factor (NGF) was used as

positive control.

Ethanol crude extract Hexane fraction Ethyl acetate fraction Water fraction

Treatment Neurite Increase Neurite Increase Neurite Increase Neurite Increase
concentration bearing cells compared to bearing cells compared to bearing cells compared to bearing cells compared to
(µg/ml) (%) control (%) (%) control (%) (%) control (%) (%) control (%)

Negative
control 19.9±1.5ab - 24.6±1.5a - 20.5±1.5a - 19.0±1.7a -

Positive
control (NGF) 23.0±1.8c 15.5 28.9±0.9bc 11.3 24.1±0.2b 17.8 24.2±0.2b 27.1

10 22.9±0.5c 15.0 25.4±1.3ab 3.3 23.7±0.8b 15.9 21.9±1.8ab 14.9

25 22.5±0.7bc 13.2 32.3±2.6bc 31.4 26.5±2.0bc 29.4 24.0±1.6b 26.3

50 20.9±0.9abc 4.8 34.1±0.1c 38.7 27.8±1.6c 35.4 23.5±1.9b 23.3

100 18.6±1.3a -6.4 40.6±2.5d 65.2 34.5±0.9d 68.5 25.6±1.3b 34.4

Note: Data are expressed as means ± standard deviation (n = 2). Means with different letters in the same column are significantly different
(P < 0.05), one-way analysis of variance/ANOVA)

38

A B
neurite

C D

E F

neurite

Figure 4.6: The morphology of the NG108-15 cells treated with various concentrations
of crude aqueous ethanol extract of Hericium erinaceus [24hr of incubation at 37 ºC in a
5 % CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20
ng/ml) was negative and positive control, respectively.]

A: negative control (cells without extract); B: positive control - NGF (20 ng/ml);
C: 10 µg/ml of crude aqueous ethanol extract;
D: 25 µg/ml of crude aqueous ethanol extract;
E: 50 µg/ml of crude aqueous ethanol extract;
F: 100µg/ml of crude aqueous ethanol extract
39

A B

neurite

C D
neurite

neurite

E F

neurite neurite

of hexane fraction of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 % CO2
humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml)
was negative and positive control, respectively.]

C: 10 µg/ml of hexane fraction; D: 25 µg/ml of hexane fraction;
E: 50 µg/ml of hexane fraction; F: 100 µg/ml of hexane fraction

40

A B

neurite

neurite

C D

neurite
neurite

E F neurite

neurite

neurite

of ethyl acetate fraction of Hericium erinaceus [24 hr of incubation at 37 ºC in a 5 %
CO2 humidified incubator. NG108-15 cells without extract or treated with NGF (20
ng/ml) was negative and positive control, respectively.]

C: 10 µg/ml of ethyl acetate fraction; D: 25 µg/ml of ethyl acetate fraction;
E: 50 µg/ml of ethyl acetate fraction; F: 100 µg/ml of ethyl acetate fraction

41

A B

neurite

C D
neurite

E F

neurite

neurite

of water fraction of Hericium erinaceus [24hr of incubation at 37 ºC in a 5 % CO2
humidified incubator. NG108-15 cells without extract or treated with NGF (20 ng/ml)
was negative and positive control, respectively.]

C: 10 µg/ml of water fraction; D: 25 µg/ml of water fraction;
E: 50 µg/ml of water fraction; F: 100 µg/ml of water fraction

42

Table 4.1 and Figure 4.2 showed the effects of various concentrations of crude

aqueous ethanol extract and fractions of H. erinaceus on neurite outgrowth of the

NG108-15 cells after 24 hr of incubation. Aqueous ethanol extract at 10 µg/ml cause

maximal stimulation of neurite outgrowth. The percentage of neurite bearing cells was

significantly higher in extract concentration of 10 µg/ml compared to negative control.

Furthermore, increasing the concentration of the extract has a minimal effect on the

number of neurite bearing cells. Extract concentration of 100 µg/ml showed 6.4 %

decreased of neurite bearing cells when compared to negative control.

When the aqueous ethanol extract was further fractionated, three fractions were

obtained (hexane, ethyl acetate and water fraction). Hexane fraction showed significant

stimulation of neurite outgrowth at the concentration of 25 µg/ml. When the

concentration was increased (10, 25, 50 and 100 µg/ml), there was an increase in the

percentage of neurite bearing cells (25.4 %, 32.3 %, 34.1 % and 40.6 % respectively)

for the hexane fraction. Hexane fraction showed 65.2 % increased in neurite bearing

cells when compared to negative control at the highest tested concentration of 100

µg/ml.

Ethyl acetate fraction showed significant stimulation (p ˂ 0.05) of neurite

outgrowth at the concentration as low as 10 µg/ml. There was an increase in the

percentage of neurite bearing cells for the ethyl acetate fraction when the concentration

is increased (23.7 %, 26.5 %, 27.8 %, 34.5 % for 10, 25, 50 and 100 µg/ml respectively).

Ethyl acetate fraction showed 68.5 % increased in neurite bearing cells compared to

negative control at the highest tested concentration of 100 µg/ml.

At 25 µg/ml, water fractions caused significant stimulation (p ˂ 0.05) of neurite-

bearing cells compared to negative control. However, water fraction did not show a

significant difference of neurite bearing cells at concentration above 25 µg/ml.

43

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