anti-viral activities of centella asiatica...
TRANSCRIPT
UNIVERSITI PUTRA MALAYSIA
ANTI-VIRAL ACTIVITIES OF CENTELLA ASIATICA L., CURCUMA LONGA L. AND STROBILANTHES CRISPUS L. AGAINST
PSEUDORABIES VIRUS IN ANIMAL CELL LINES
HANISA BINTI HOSNI
FPV 2006 6
ANTI-VIRAL ACTIVITIES OF CENTELLA ASIATICA L., CURCUMA LONGA L. AND STROBILANTHES CRISPUS L. AGAINST PSEUDORABIES VIRUS IN
ANIMAL CELL LINES
By
HANISA BINTI HOSNI
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Master of Science
July 2006
DECLARATION
I hereby declare that the thesis is based on my original work except for quotations and citations which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any other degree at UPM or other institutions.
_____________________
HANISA BINTI HOSNI
Date: 28 November 2006
11
To my husband, Mohd Azhar,
For encouraging and sustaining To my children, Athirah and Azeem, For making it all worthwhile To my parents, Hosni and Patimah, For making it possible
2
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirement for the degree of Master of Science
ANTI-VIRAL ACTIVITIES OF CENTELLA ASIATICA L., CURCUMA LONGA L. AND STROBILANTHES CRISPUS L. AGAINST PSEUDORABIES VIRUS IN
ANIMAL CELL LINES
By
HANISA BINTI HOSNI
July 2006
Chairman : Professor Mohd Azmi Mohd Lila, PhD
Faculty : Veterinary Medicine
Herpes simplex virus (HSV) is a major opportunistic pathogen in immunosuppressed
patients and a serious disease in high human immunodeficiency virus (HIV)/ acquired
immunodeficiency syndrome (AIDS) prevalence areas. The existence of resistant strains
to the available drug is therefore an urgent need to identify new alternative agents for
HSV. The aim of this study is to investigate anti-viral activities of the plants. Assays
were developed to determine the characteristics of anti-viral activities, as anti-viral
attachment, anti-prophylactic and virucidal. The pseudorabies virus (PrV) has been used
as representative of HSV. Three medicinal plants have been used, which is Centella
asiatica L. (C. asiatica), Strobilanthes crispus L. (S. crispus) and Curcuma longa L. (C.
longa). The plants are reputed in traditional medicine for many treatments of diseases.
Firstly, the potential cytotoxicity was evaluated for plant methanol extracts (ME) and
aqueous extracts (AE) in cell line by using MTT assay. All three plant extracts were
3
4
found significantly (P < 0.05) non cytotoxic towards African Green Monkey Kidney
(Vero) cells, Baby Hamster Kidney (BHK) cells and Rabbit Kidney (RK) cells. The
plant ME was generally more cytotoxic than AE, showed lower (76 µg/ml) non-toxic
limit concentration (NTLC50) than plant AE (82 µg/ml). The least cytotoxic was the
extract of S. crispus followed by C. asiatica and C. longa. The resistance of three
different cell lines was also compared and it showed the BHK cells were the most
toughest and resistant to the plant extracts. In anti-viral analysis, it was discovered that
all plant extracts showed marked prophylactic activity, considerable anti-attachment and
virucidal abilities up to 75% inhibition of cytopathic effect (CPE) formation. The C.
longa extract was found as a potent anti-viral agent followed by S. crispus and C.
asiatica. It exerted better prophylactic activity against PrV compared to the other two
plants. Whereas S. crispus was found very effective as virucidal agent while C. asiatica
as anti-viral attachment agent. Based on the results, it was also found that plant ME
possessed better anti-viral activity than plant AE. These results also showed each of
three plants possessed different anti-viral activities. The anti-viral activities were also
varies in different cell lines tested. All three plants were analysed by liquid
chromatography-electrospray ionisation-mass spectrometry (LC-ESI-MS) to identify
plant compounds. The analysis revealed several compounds in C. asiatica, S. crispus and
C. longa ME. This study discovered the promising anti-viral activities of C. asiatica, S.
crispus and C. longa but not any identified plant compound against PrV in vitro. These
results suggest that all three plant extracts have potent anti-viral agents against PrV as
representative of HSV that can be exploited for development of an alternative medicine
to prevent HSV infections.
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
ANTIVITI ANTI-VIRUS OLEH CENTELLA ASIATICA L., CURCUMA LONGA L. DAN STROBILANTHES CRISPUS L. TERHADAP VIRUS PSEUDORABIES DI
DALAM BARISAN SEL
Oleh
HANISA BINTI HOSNI
Julai 2006
Pengerusi : Profesor Mohd Azmi Mohd Lila, PhD
Fakulti : Perubatan Veterinar
Herpes simplex virus (HSV) adalah patogen oportunis utama kepada pesakit-pesakit yang
sistem imunnya ditekan dan adalah satu penyakit serius di kawasan yang lazimnya
mempunyai perebakan Virus Kurang Daya Tahan Penyakit (HIV) atau Sindrom Kurang
Daya Tahan Penyakit (AIDS) yang tinggi. Kewujudan strain virus yang rintang kepada
ubatan anti-virus menyebabkan keperluan mendesak untuk mencari agen alternatif bagi
merawat HSV. Tujuan kajian ini adalah untuk mengkaji aktiviti anti-virus oleh
tumbuhan. Kaedah-kaedah untuk menentukan sifat dan ciri aktiviti anti-virus
pseudorabies (PrV) iaitu anti-perlekatan, anti-pre-rawatan dan membunuh virus telah
dijalankan. PrV telah digunakan sebagai mewakili HSV. Tiga tumbuhan ubatan telah
digunakan iaitu Centella asiatica L. (C. asiatica), Strobilanthes crispus L. (S. crispus)
dan Curcuma longa L. (C. longa). Tumbuhan-tumbuhan ini mempunyai reputasi yang
baik dalam merawat pelbagai penyakit di dalam perubatan tradisional. Pertama, potensi
toksik ekstrak methanol (EM) and ekstrak akuas (EA) tumbuhan telah dianalisa ke atas
5
barisan sel dengan menggunakan kaedah MTT. Kesemua ekstrak tumbuhan dijumpai
signifikan (p < 0.05) tidak menyebabkan toksik kepada sel Buah Pinggang Monyet Hijau
(Vero), sel Buah Pinggang Anak Hamster (BHK) dan sel Buah Pinggang Arnab (RK).
Secara amnya, EM tumbuhan lebih toksik ke atas sel berbanding AE tumbuhan,
menunjukkan kepekatan tidak toksik pada 50% yang lebih rendah (76 µg/ml) daripada
AE tumbuhan (85 µg/ml). Kerintangan ketiga-tiga barisan sel terhadap ekstrak tumbuhan
juga dibandingkan dan menunjukkan sel BHK adalah paling kuat dan rintang kepada
ekstrak tumbuhan. Kesemua ekstrak tumbuhan menunjukkan kebolehan yang jelas
aktiviti pre-rawatan manakala anti-perlekatan dan kebolehan membunuh virus yang boleh
dipertimbangkan sehingga 75% penekanan ke atas pembentukan Kesan Saitopatik (CPE).
Ekstrak C. longa telah dijumpai sebagai agen anti-viral yang sangat berpotensi, diikuti
ekstrak S. crispus dan C. asiatica. Ia menunjukkan aktiviti pre-rawatan yang lebih baik
ke atas PrV berbanding dua tumbuhan tersebut. Manakala ekstrak S. crispus telah
dijumpai menunjukkan kesan efektif sebagai agen membunuh virus dan ekstrak C.
asiatica sebagai agen anti-perlekatan. Berdasarkan kepada keputusan, ia menunjukkan
ME tumbuhan memberikan aktiviti anti-virus yang lebih baik berbanding AE tumbuhan.
Keputusan juga menunjukkan bahawa setiap tumbuhan memberikan aktiviti anti-virus
yang berbeza. Aktiviti anti-virus juga berbeza di dalam barisan sel yang berbeza.
Ketiga-tiga tumbuhan telah dianalisa dengan menggunakan kromatografi cecair-ionisasi
elektrospray-spektrometer jisim (LC-ESI-MS) untuk mengenalpasti bahan tumbuhan.
Analisa tersebut mendedahkan bahan-bahan yang terkandung di dalam C. asiatica, S.
crispus dan C. longa EM. Kajian ini menemui aktiviti anti-virus bagi C. asiatica, S.
crispus dan C. longa tetapi bukan daripada mana-mana bahan tumbuhan yang
6
7
dikenalpasti terhadap PrV secara in vitro. Keputusan kajian juga mencadangkan bahawa
ketiga-tiga tumbuhan mempunyai potensi sebagai agen anti-virus ke atas PrV sebagai
mewakili HSV yang boleh dieksploitasikan bagi pembangunan ubatan alternatif bagi
menghalang jangkitan HSV.
ACKNOWLEDGEMENTS
In The Name Of Allah, The Beneficent, The Merciful
All gratifications are referred to ‘Allah’, the Omnipotent and Omnipresent. Without His
Permission and Gifts for every strength, I just think that I could not be able to complete
this thesis.
Especially supervisors, Prof. Dr. Mohd Azmi Mohd Lila, Prof. Madya Dr Mohd Nazrul
Hakim bin Abdullah and Prof. Dr. Suhaila Mohamed for unrelenting guidance, and wise
suggestions. My thankful also for providing me with a very fine place and satisfactory
laboratory facilities. Not to forget to Prof Madya Dr Saidi Abdul Moin for his
supervision for the statistical analysis.
I would also like to express my unforgettable appreciation to Mr. Kamarudin Awang Isa,
all staffs of Bacteriology Laboratory, my friends, Suria, Radhiah, Mat Lip and Kak Sam,
who have truly made this thesis possible.
Last but not least, the foremost, profound and heart-felt gratitude is to my extraordinary
husband, Mr. Mohd Azhar Abdul Rahman for his unremitting love and constantly
supports which motivated me to accomplish this worthwhile thesis. I express my sincere
to my beloved parents, Hj Hosni Jalaludin and Hajjah Patimah Baharudin for their
everlasting support and encouragement.
8
I certify that an Examination Committee has met on 6th July 2006 to conduct the final examination of Hanisa Binti Hosni on her Master of Science thesis entitled Anti-viral Activities of Centella Asiatica L., Strobilanthes Crispus L. And Curcuma Longa L. Against Pseudorabies Virus in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981. The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:
Dato’ Sheikh Omar Abdul Rahman, PhD Professor, Department of Veterinary Pathology and Microbiology Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman)
Noordin Mohamed Mustapha, PhD Associate Professor, Head, Department of Veterinary Pathology and Microbiology Faculty of Veterinary Medicine Universiti Putra Malaysia (Internal Examiner)
Zamberi bin Sekawi, MPath Head, Department of Medical Microbiology and Parasitology Faculty of Medicine and Science Health Universiti Putra Malaysia (Internal Examiner)
Mustafa Ali Mohd, PhD Deputy Dean, Infrastructure and Development, Faculty of Medicine Universiti of Malaya (External Examiner)
_________________________________
HASANAH MOHD GHAZALI, PhD Professor/ Deputy Dean School of Graduate Studies Universiti Putra Malaysia
Date:
9
10
This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfilment of the requirement for the degree of Master of Science. The members of the Supervisory Committee are as follows:
Mohd Azmi Mohd Lila, PhD Professor Faculty of Veterinary Medicine Universiti Putra Malaysia (Chairman)
Suhaila Mohamed, PhD Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)
Mohd Nazrul Hakim Abdullah, PhD Associate Professor Faculty of Medicine and Health Sciences Universiti Putra Malaysia (Member)
________________________
AINI IDERIS, PhD Professor/ Dean School of Graduate Studies Universiti Putra Malaysia.
Date: 16 January 2007
TABLE OF CONTENTS Page
DEDICATION 2 ABSTRACT 3 ABSTRAK 5 AKNOWLEDGEMENTS 8 APPROVAL 9 DECLARATION 11 LIST OF TABLES 15 LIST OF FIGURES 18 LIST OF ABBREVIATIONS 20 CHAPTER I INTRODUCTION 24 Viral Infections 24 Herpes Virus Infection 25 Anti-Viral Drugs 26 The Effects of Antiviral on Viruses 26 II LITERATURE REVIEW
The Virus 30 Herpes Virus 30 Viral Pathogenesis 32 Molecular Consideration of Anti-herpes Drugs 33 Medicinal Plants 38 Traditional Medicine 39 C. asiatica Plant Description 42 Active Constituents 44 Traditional Medicine 46 Modern Medicine 48 S. crispus Plant Description 50 Active Constituents 52 Traditional Medicine 52 Modern Medicine 53 C. longa. syn: C. domestica Plant Description 54 Active Constituents 57 Traditional Medicine 57 Modern Medicine 59
12
Liquid Chromatography-Electrospray Ionisation
-Mass Spectrometry 60
III POTENTIAL CYTOTOXICITY OF PLANT EXTRACTS Introduction 62
Materials and Methods 64 Preparation of Plant Extracts 64 Plant Extractions Aqueous Extract 65 Methanol Extract 65 Cell Lines Preparation 66 Cell Lines Media Preparation 66 Haemocytometer Cell Counting 70 Plating Out Cells 71 Plant Extract Addition 71 MTT Solution Preparation 74 Estimation of Cell Viability 74 Statistical Analysis 75
Results General 76 Plant Extracts in BHK cells 78 Plant Extracts in Vero cells 83 Plant Extracts in RK cells 88 Discussion and Conclusion 92 IV ANTI-HERPES ACTIVITY OF PLANT EXTRACTS Introduction 97 Material and Methods 99
Preparation of Plant Extracts 99 Plant Extractions Aqueous Extract 100 Methanol Extract 100 Cell Lines Preparation 101
Cell Lines Media Preparation 101 Virus 103 Cell Culture Inoculation 103 Harvesting of Virus 104 Virus Titration by TCID50 104 Antigen Detection-Immunoperoxidase 105 Anti-viral Assays 107 Prophylaxis Study 107 Attachment Assay 108 Virucidal Assay 108 Estimation of Cell Viability 109
Statistical Analysis 109
13
14
Results 110 Antigen Detection-Immunoperoxidase 110 Anti-viral Activity of C. asiatica in BHK, Vero
and RK Cells 112 Anti-viral Activity of S. crispus in BHK, Vero
and RK Cells 119 Anti-viral Activity of C. longa in BHK, Vero
and RK Cells 127 Discussion and Conclusion 133 V LC-ESI-MS ANALYSIS OF PLANT EXTRACTS Introduction 140 Material and Methods 141 Preparation of Methanol Plant Extracts 141 Solvents and Chemicals 142 LC-MS Instrumentation 142 LC Conditions 142 Results 144 C. asiatica Leaf Extracts 144 S. crispus Leaf Extracts 148
C. longa Rhizome Extracts 150 Discussion and Conclusion 154 VI GENERAL DISCUSSION AND CONCLUSION 165 BIBLIOGRAPHY 171 APPENDICES 187 BIODATA OF THE AUTHOR 191
LIST OF ABBREVIATIONS
ACV Acyclovir
ADV Adenovirus
ADV-3 Adenovirus type 3
AE Aqueous Extract
AIDS Acquired Immunodeficiency Syndrome
ATCC American Type Cell Culture
ATV Antibiotic Trypsin-Versine
BDMC Bisdemethoxycurcumin
BHK Baby Hamster Kidney cells
BSA Bovine Serum Albumin
BVdU E-5-(2-bromovinyl)-2’-deoxyuridine+
BVdU-TP Brivudin-5’-triphosphate
CMV Cytomegalovirus
CPE Cytopathic effect
ddI Dideoxyinosine
DAB 3,3’-diaminobenzidine
DLA Dalton’s Lymphoma Ascites
DMC Demethoxycurcumin
DMSO Dimethylsulphoxide
DNA Deoxyribonucleic Acid
EAC Ehrlich Ascites Cells
20
EBV Epstein Barr Virus
EMEM Eagle’s Minimal Essential Medium
ESI Electrospray Ionisation
FCS Fetal Calf Serum
GCV Ganciclovir
GLM General Linear Model
HBB 2-(hydroxybenzyl)-benzimidazole
HeLa Human Epithelioid Cervical Carcinoma
HHV Human Herpes Virus
HHV-1 Human Herpes Virus Type 1
HIV Human Immunodeficiency Virus
HIV-1 Human Immunodeficiency Virus Type-1
HPLC High Performance Liquid Chromatography
HSV Herpes Simplex Virus
HSV-1 Herpes Simplex Virus Type 1
HSV-2 Herpes Simplex Virus Type 2
HZV Herpes Zoster Virus
IUdR Iododeoxyuridine
LC-MS Liquid Chromatography-Mass Spectrometry
LTR Long Terminal Repeat
ME Methanol Extract
MG Maximum Growth; Cell treated with media only
21
MTT 3-(4, 5-dimethylthiazol-2-yl)-2, 5
diphenyltetrazolium bromide
MW Molecular weight
NMR Nuclear Magnetic Resonance
NTLC Non-Toxic Limit Concentration
NTLC50 Non-Toxic Limit Concentration at 50 percent
PAA Polyacrylic acid
PBS Phosphate Buffer Saline
PI Post Infection
PrV Pseudorabies Virus
PVAS Polyvinylalcohol sulphate
PVS Polyvinyl sulphonate
RK Rabbit Kidney cells
RNA Ribonucleic Acid
RRV Ross River Virus
RSV Respiratory Syncytial Virus
SAH S- adenosylhomocysteine
SI Selective Index
TE Tris-EDTA
TECA Titrated extract of C. asiatica
TCID50 Tissue Culture Infective Dose at 50
TK Thymidine kinase
TLC Thin Layer Chromatography
22
23
TNF Tumor Necrosis Factor
TSP Thermospray
USA United States of America
UV Ultraviolet
VCV Vanciclovir
VERO African Green Monkey Kidney cells
VSV Vesicular Stomatitis Virus
WHO World Health Organization
LIST OF FIGURES
Figure Page
2.1 Herpes virus replication cycle. 33
2.2 Medicinal plant used in this study, C. asiatica leaves. 43
2.3 Medicinal plant used in this study, S.crispus leaves. 51
2.4 C. longa leaves. 55
2.5 Medicinal plant used in this study, C. longa rhizome 56
3.1 Cell culture used in this study, Vero cells. 67
3.2 Cell culture used in this study, BHK cells. 68
3.3 Cell culture used in this study, RK cells 68
3.4 The 96-well plate for cytotoxicity assay 73
4.1 Infected vero cells with PrV 110
4.2 Infected vero cells with PrV treated with immunoperoxidase stain 111
5.1 a) Chemical structures of asiatic acid. 145
5.1 b) Chemical structures of asiaticoside. 145
5.1 c) Chemical structures of madecassic acid. 146
5.1 d) Chemical structures of madecassoside. 146
5.2 LC-ESI-MS chromatogram of C. asiatica ME 147
5.3 Chromatographic peak of compound 1 147
5.4 Chemical structure of verbascoside 148
5.5 LC-ESI-MS chromatogram of S. crispus ME 149
5.6 Chromatographic peak of compound 2 149
18
19
5.7 a) Chemical structures of curcumin. 151
5.7 b) Chemical structures of demethoxycurcumin 151
5.7 c) Chemical structures of bisdemethoxycurcumin. 151
5.7 d) Chemical structures of dihydrocurcumin 151
5.8 LC-ESI-MS chromatogram of C. longa ME 152
5.9 Chromatographic peak of compound 3, 4 and 5. 153
5.10 Positive-ion ESI-MS of compound 1 155
5.11 Proposed positive-ion ESI dissociation of compound 1 157
5.12 Positive-ion ESI-MS of compound 2 159
5.13 Proposed positive-ion ESI dissociation of compound 2 160
5.14 Proposed positive-ion ESI dissociation of compound 3, 4 and 5 162
LIST OF TABLES
Table Page
2.1 Folk uses of C. asiatica around the world. 47
2.2 Folk uses of C. longa around the world. 58 3.1 Means ± SD of cell viability of BHK cells treated with
plant ME. 78 3.2 The NTLC50 of plant ME and controls in BHK cells. 79 3.3 Means ± SD of cell viability of BHK cells treated with
plant AE. 80 3.4 The NTLC50 of plant AE and controls in BHK cells. 81 3.5 Means ± SD of cell viability of Vero cells treated with
plant ME. 83 3.6 The NTLC50 of plant ME and controls in Vero cells. 84 3.7 Means ± SD of cell viability of Vero cells treated with
plant AE. 85 3.8 The NTLC50 of plant AE and controls in Vero cells. 86 3.9 Means ± SD of cell viability of RK cells treated with
plant ME. 88 3.10 The NTLC50 of plant ME and controls in RK cells. 89 3.11 Means ± SD of cell viability of RK cells treated with
plant AE. 90 3.12 The NTLC50 of plant AE and controls in RK cells. 91 4.1 Percent inhibition of virus-induced CPE in BHK cells
treated with C. asiatica in different anti-viral assays. 112 4.2 The assessment of anti-viral activity of ME and AE of
C. asiatica in BHK cells. 113
15
4.3 Percent inhibition of virus-induced CPE in Vero cells treated with C. asiatica in different anti-viral assays. 114
4.4 The assessment of anti-viral activity of ME and AE of
C. asiatica in Vero cells. 115 4.5 Percent inhibition of virus-induced CPE in RK cells
treated with C. asiatica in different anti-viral assays. 116 4.6 The assessment of anti-viral activity of ME and AE of
C. asiatica in RK cells. 117 4.7 Percent inhibition of virus-induced CPE in BHK cells
treated with S. crispus in different anti-viral assays. 119 4.8 The assessment of anti-viral activity of ME and AE of
S. crispus in BHK cells. 120
4.9 Percent inhibition of virus-induced CPE in Vero cells treated with S. crispus in different anti-viral assays. 121
4.10 The assessment of anti-viral activity of ME and AE of
S. crispus in Vero cells. 123 4.11 Percent inhibition of virus-induced CPE in RK cells
treated with S. crispus in different anti-viral assays. 124 4.12 The assessment of anti-viral activity of ME and AE of
S. crispus in RK cells. 125 4.13 Percent inhibition of virus-induced CPE in BHK cells
treated with C. longa in different anti-viral assays. 127
4.14 The assessment of anti-viral activity of ME and AE of C. longa in BHK cells. 128
4.15 Percent inhibition of virus-induced CPE in Vero cells treated with C. longa in different anti-viral assays. 129
4.16 The assessment of anti-viral activity of ME and AE of
C. longa in Vero cells. 130 4.17 Percent inhibition of virus-induced CPE in RK cells
treated with C. longa in different anti-viral assays. 131
16
17
4.18 The assessment of anti-viral activity of ME and AE of S. crispus in RK cells. 132
5.1 LC-MS gradient program for C. asiatica, S. crispus and
C. longa analysis. 143 5.2 Relative abundance of compound 1 and fragment ions 158 5.3 Relative abundance of compound 2 and fragment ions 161 5.4 tR, MS data and peak assignments for the analysis
of C. longa ME. 163 5.5 Relative abundance of compound 3, 4 and 5 and
fragment ions 163
CHAPTER I
INTRODUCTION
Viral Infections
Viral infections are major health problem all over the world due to their morbidity and
mortality. There are many types of transmissions of viral infections in man. They are
respiratory or salivary spread, which is not readily controllable; faecal-oral spread that is
controllable by public health measures and venereal spread, which is socially difficult to
control. An example is HSV (Dawn and Robert, 1997).
The immune system appears as the controlling factor within the host that maintains
beneficial microbes at harmless levels and prevents infections by dangerous agents. It is
able to combat a variety of infections from birth on. Evolutionary pressure has forced
most viruses to develop many strategies to subvert the immune system. Some of them
are production of antigenic variants; adopted by influenza virus and HIV, avoiding
recognition by lymphocytes; for example adenovirus, manipulate cytokine pathways to
avoid destruction by the host immune response, the production of materials that interfere
with the production of pro-inflammatory cytokines; for example vaccinia and cowpox
viruses, and interfering with the alternate pathways of complement activation; for
example HSV (Hans et al., 2000; Bruce, 2002).