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UNIVERSITI PUTRA MALAYSIA

STANDARDISED Andrographis paniculata BURM. NEES EXTRACTS AND

ANDROGRAPHOLIDE PREVENT AIRWAY INFLAMMATION IN HOUSE DUST MITE AND DIISOCYANATE- INDUCED

ASTHMA MODELS

IBRAHIM SULAIMAN

FPSK(P) 2018 34

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PMSTANDARDISED Andrographis paniculata BURM. NEES EXTRACTS AND

ANDROGRAPHOLIDE PREVENT AIRWAY INFLAMMATION IN HOUSE

DUST MITE AND DIISOCYANATE- INDUCED

ASTHMA MODELS

By

IBRAHIM SULAIMAN

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfillment of the Requirements for the Degree of Doctor of Philosophy

May 2018

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COPYRIGHT

All material contained within the thesis, including without limitation text, logos, icons,

photographs, and all other artwork, is copyright material of Universiti Putra Malaysia

unless otherwise stated. Use may be made of any material contained within the thesis

for non-commercial purposes from the copyright holder. Commercial use of material

may only be made with the express, prior, written permission of Universiti Putra

Malaysia.

Copyright © Universiti Putra Malaysia

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfilment

of the requirement for the degree of Doctor of Philosophy

STANDARDISED Andrographis paniculata BURM. NEES EXTRACTS AND

ANDROGRAPHOLIDE PREVENT AIRWAY INFLAMMATION IN HOUSE

DUST MITE AND DIISOCYANATE- INDUCED

ASTHMA MODELS

By

IBRAHIM SULAIMAN

May 2018

Chairman : Professor Johnson Stanslas, PhD

Faculty : Medicine and Health Sciences

The therapeutics of asthma is commonly based on the use of steroids and

bronchodilators. However, steroid-sensitive asthmatics are at the risk of debilitating

side effects associated with persistent use of steroids. There is, therefore, the need for

discovery and development of better and safer alternatives. Andrographis paniculata

(AP) is traditionally used as a herbal remedy to a wide range of inflammatory

conditions. Its anti-inflammatory activity is attributable to its major diterpenoid,

andrographolide (AGP). Anti-asthma activity of AGP was previously reported in

ovalbumin mouse asthma model. This study investigated the anti-asthma potential of

standardised Andrographis paniculata aqueous extract (APAE) and aqueous ethanolic

extract (APEE50) in house dust mite (HDM) induced asthma. In addition, the efficacy

and mechanism of action of AGP in the prevention of airway inflammation and

oxidative stress in toluene diisocyanate (TDI)-induced occupational asthma (OA)

model were evaluated. The extracts were standardised based on percentage

distribution of AGP, neoandrographolide (NAG) and 14-deoxy-11,12-

didehydroandrographolide (DDAG). The AGP, NAG and DDAG contents of

standardised APAE were approximately 3.4%, 1.1% and 0.1% (w/w), while that of

APEE50 were 8.7%, 1.4% and 0.3% (w/w) respectively. APAE was proven to inhibit

NF-κB p65 signalling pathway in a TNF-α-exposed A549 bronchial epithelial cell line

without inducing cytotoxicity. The inhibition of p65 signalling pathway occurred by

preventing IKK phosphorylation, IĸB-α activation, p65 nuclear translocation and

DNA binding activity of p65. In vivo analysis of APAE and APEE50 activity in 14

days HDM-induced asthma model recorded substantial improvement in asthma

markers. Treatments were administered following a prophylactic regimen. Both

treatments significantly decreased bronchoalveolar lavage fluid (BALF) total and

differential leukocyte count at a dose range of 50 – 200 mg/kg. A significant reduction

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in BALF IL-4, IL-5, IL-13, and eotaxin, as well as HDM-specific IgE, total serum IgE

and IgG were recorded. Histopathological analysis of lung samples showed a

remarkable reduction in perivascular and peribronchial inflammation, as well as

suppression of mucus production by both APAE and APEE50. A dose-dependent

decrease in airway resistance and a slight increase in dynamic lung compliance were

witnessed. Notably, the expression of NF-κB transcribed genes, Th2 inducible genes

and eosinophil modulating genes were decreased, while Nrf2 gene was concomitantly

upregulated. Diisocyanate-induced asthma presented airway inflammation of

neutrophilic endotype. The administration of AGP (0.1, 0.5 and 1 mg/kg) produced

progressive decreased in airway inflammation parameters. In addition to suppression

of airway cellular infiltration and mucus production, collagen deposition was deterred

by the treatments. TDI exposure induced an aberrant distribution of E-cadherin and β-

catenin in airway epithelia. AGP ameliorated the loss of airway integrity by restoring

normal distribution of E-cadherin and β-catenin. The mechanism of action of AGP in

TDI-induced asthma occurred through ROS scavenging and up-regulation of the

pulmonary HO-1 level via p38/Akt/GSK-3β/Nrf2 dependent pathway. The expression

of both Th1 and Th2-related genes were downregulated in AGP treated animals.

Furthermore, TDI-induced airway hyperreactivity was improved by AGP

administration. These findings strongly suggest that AGP and AP extracts could serve

as potential alternative treatments for chemical and nonchemical-induced asthma

respectively.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

EKSTRAK Andrographis paniculata BURM. NEES YANG

DISELARASKAN DAN ANDROGRAPHOLIDE DAPAT MENCEGAH

KERADANGAN SALURAN PERNAFASAN DI DALAM MODEL

PENYAKIT ASMA AKIBAT KUTU HAMA DAN DIISOCYANATE

Oleh

IBRAHIM SULAIMAN

Mei 2018

Pengerusi : Profesor Johnson Stanslas, PhD

Fakulti : Perubatan dan Sains Kesihatan

Perubatan teraputik yang lazim digunakan untuk penyakit asma berdasarkan

penggunaan steroid berserta bronkodilator. Selain itu, pesakit asma yang sensitif

terhadap penggunaan steroid berisiko untuk mengalami kesan sampingan berikutan

penggunaan steroid dalam jangka masa yang panjang. Oleh itu, penemuan dan

perkembangan perubatan alternatif yang lebih baik dan selamat diperlukan.

Andrographis paniculata (AP) digunakan untuk melegakan pelbagai jenis keradangan

dalam rawatan tradisional. Andrographolide (AGP), sebagai bahan utama diterpenoid

dapat mengakibatkan aktiviti antikeradangan berlaku. AGP dilaporkan mempunyai

kesan pencegahan penyakit asma dalam model mencit dengan penggunaan ovalbumin.

Tujuan penyelidikan ini adalah untuk menyiasat keupayaan anti-asma dengan

menggunakan ekstrak cecair AP (APAE) dan ekstrak cecair-etanol AP (APEE50) yang

diselaraskan pada model asma yang diakibatkan oleh kutu hama. Tambaha n pula,

keberkesanan dan tindakan mekanisma AGP dalam pencegahan keradangan di saluran

pernafasan dan tekanan pengoksidaan didorong oleh toluene diisocyanate (TDI) telah

dinilai. Bahan-bahan ekstrak tersebut diselaraskan berdasarkan pada peratusan

pengagihan AGP, neoandrographolide (NAG) dan 14-deoxy-11,12-

didehydroandrographolide (DDAG). Kandungan AGP, NAG dan DDAG dalam

APAE yang telah diselaraskan adalah 3.4%, 1.1% dan 0.1% (w/w) manakala APEE50

adalah 13.6%, 1.2% dan 0.2% (w/w). APAE terbukti dapat menghalang laluan isyarat

NF-κB p65 dalam sel epitelium bronkial A549 yang didedahkan kepada TNF-α

(20ng/ml). Penghalangan laluan isyarat p65 adalah disebabkan oleh penghalangan

fosforilasi IKK, pengaktifan IĸB-α, translokasi p65 ke dalam nukleus dan pengikatan

p65 ke DNA. Penganalisaan APAE dan APEE50 selama 14 hari ke atas model in vivo

yang dijangkiti oleh kutu hama menunjukkan penambahbaikan yang ketara dalam

penanda penyakit asma. Secara profilaktik, rawatan telah disuapkan. Kedua-dua

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APAE dan APEE50 pada dos 50-200mg/kg dapat mengurangkan bilangan keseluruhan

dan perbezaan leukosit dalam cecair bronkoalveolar lavage (BALF) secara ketara.

Pengurangan IL-4, IL-5, IL-13, dan eotaxin di dalam BALF dan spesifik IgE HDM,

keseluruhan IgG dan IgE dalam serum dapat dikurangkan secara mendadak. Analisa

histopatologi dari sampel paru-paru mencit menunjukkan bahawa pengurangan

keradangan di persekitaran vaskular dan bronkial malah pengurangan bendalir oleh

APAE dan APEE50. Dos berkurang dalam rintangan saluran pernafasan dan dynamic

compliance meningkat. Selain itu, ekspresi gen hasilan NF-κB, gen dorongan Th2 dan

gen pengawalan eosinophil telah berkurang manakala gen hasilan Nrf2 meningkat.

Asma yang disebabkan oleh TDI menunjukkan keradangan pada saluran pernafasan

jenis neutrophil. Penggunaan AGP (0.1, 0.5 dan 1mg/kg) dapat mengurangan

keradangan saluran pernafasan secara berperingkat. Selain daripada mencegah

kemasukan sel ke dalam saluran pernafasan dan penghasilan bendalir, perawatan juga

menghalang dari pemendapan kolagen. Pendedahan TDI mengakibatkan pengagihan

E-cadherin dan β-catenin yang tidak normal pada tisu epitel saluran pernafasan. AGP

memulihkan intergriti saluran pernafasan dengan mengembalikan pengagihan E-

cadherin dan β-catenin secara normal. Tindakan mekanisma AGP pada asma jenis TDI

adalah melalui proses neutralisasi ROS dan peningkatan tahap HO-1 pulmonari

melalui laluan p38/Akt/GSK-3β/Nrf2. Ekspresi gen berkaitan dengan Th1 dan Th2

telah menurun bagi haiwan yang dirawat dengan AGP. Hiperaktiviti saluran

pernafasan akibat TDI juga bertambah baik dengan rawatan AGP. Penemuan ini

menunjukkan bahawa ekstrak AP dan AGP berupaya digunakan sebagai rawatan

alternatif untuk merawat penyakit asma yang berpunca dari bahan bukan kimia

mahupun kimia.

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ACKNOWLEDGEMENTS

All thanks to Allah for taking me this far in life, satisfying my quest for higher

education and for making Professor Johnson Stanslas my supervisor, advisor and

mentor. Apart from giving me the opportunity to conduct my research under his

supervision, Prof Johnson impacted positively on my scientific reasoning,

interpersonal relationship, and student supervision skills. I learnt a lot from his ethical

standards and conflict resolution potentials. Furthermore, his ability to treat his

students equally irrespective of gender, race, nationality or religion was so comforting.

Prof’s ability to give second chances to his students has shaped my understanding of

the impact of interpersonal variations and how to accommodate my future students.

Most importantly, thank you for being available whenever I needed your attention

with my work, thank you for pushing so hard and thank you for the befitting advice

you regularly give, especially during the monthly progress meeting.

I thank the Malaysian Ministry of Higher Education for offering me Malaysian

International Scholarship (MIS), without which my dream of completing a PhD could

have been shattered. I will also like to extend my appreciation to Malaysian Ministry

of Agriculture and Agro-Based Industry, for supporting this research through the

National Key Economic Area Research Grant Scheme (NRGS/NH1014D026).

To my co-supervisors, Dr Lim Chee Woei and Associate Professor Dr Norhafizah

Mohtarrudin, I truly appreciate your contributions to the design and presentation of

my work. Dr Lim, being an expert in asthma research, he ensured I started my work

on the right path, by successfully guiding me through easy take-off steps, which could

have taken a long time to optimise. He guided me through numerous asthma research

hands-on technique and I appreciate that a lot. My esteem gratitude to Dr Norhafizah,

who despite her tight clinical and administrative schedules, she ensured my

histopathology and immunohistochemistry data acquisition, analysis and

interpretation were done properly.

This thesis is not only a representation of my work on a script, it is a milestone in three

years of relentless effort that was achieved by consistently working day and night,

with the support of my co-students, research assistants and laboratory science officers.

My gratitude to Mrs Juita Chupri, a senior medical laboratory technologist who

thought me numerous histopathological techniques. To my ex-research assistants, Mr

Mohd Fauzan and Mohd Faiz bin Mat Saad, I appreciate your assistance especially

with the animal care and husbandry. I also thank Mr Soo Hon Liong and Jonathan

Teng Yi Chuon for their commitment towards the growing and propagation of AP

plant in Universiti Putra Malaysia (UPM). Mr Soo also assisted in numerous ways,

especially in the procurement of some vital research materials through a research grant

we both worked under. The assistance of Mr Tan Khaishin, during a multitasking in

vivo analysis of intracellular reactive oxygen species, will forever be appreciated.

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Furthermore, I thank UPM graduates, Dr Isa Abubakar Ali, Dr Usman Bala and Dr

Sani Dahiru for guiding through my first immunofluorescence, western blotting and

plant extraction techniques respectively. I also thank Dr Audrey Yong, Prof Johnson’s

ex-student and a lecturer in MAHSA University Malaysia, for the free comprehensive

theoretical and hands-on training she offered me on high-performance liquid

chromatography technique. I extend my gratitude to Ms Pri Chaskar and Noarin Islam

for ensuring that this thesis is clearly written with minimal spelling and grammatical

errors. My gratitude to the rest of my Lab mates for their moral and brotherly support

during my entire stay in the Lab.

My acknowledgement will be incomplete without appreciating the persistent support

of my parents, siblings, wife, in-laws, children and friends. My mother, Hajiya Maryan

Usman, has been the pillar of my successful life endeavours, she has been supportive

and optimistic, thank you for being my best friend. The understanding from my wife

is indescribable, been a scientist herself, she understands and encouraged me to put in

days and nights toward achieving my dream. Thank you for the sacrifices, thanks for

been my life partner and a shoulder to lie on.

Finally, I’ll like to extend a vote of thanks to my state, Kano State Government and

my employer, Bayero University Kano, for giving me the opportunity to proceed on

502 “Kwankwasiyya” MSc fellowship scheme at Universiti Sultan Zainal Abidin,

Malaysia. Without which, I might not have been to Malaysia, nor will I have met Prof

Johnson, be his student or even secure MIS scholarship for my PhD.

Thank you all, God bless Universiti Putra Malaysia, God bless Malaysia and God

Bless Nigeria my country.

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This thesis was submitted to the Senate of the Universiti Putra Malaysia and has been

accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The

members of the Supervisory Committee were as follows:

Johnson Stanslas, PhD

Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Chairman)

Norhafizah Mohtarrudin, MBBS, M.path

Associate Professor

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Member)

Lim Chee Woei, PhD

Senior Lecturer

Faculty of Medicine and Health Sciences

Universiti Putra Malaysia

(Member)

___________________________

ROBIAH BINTI YUNUS, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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Declaration by graduate student

I hereby confirm that:

this thesis is my original work;

quotations, illustrations and citations have been duly referenced;

this thesis has not been submitted previously or concurrently for any other degree

at any institutions;

intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Research and innovation) before thesis is published (in the form

of written, printed or in electronic form) including books, journals, modules,

proceedings, popular writings, seminar papers, manuscripts, posters, reports,

lecture notes, learning modules or any other materials as stated in the Universiti

Putra Malaysia (Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Research) Rules 2012. The thesis has undergone plagiarism detection software

Signature: ________________________ Date: __________________

Name and Matric No.: Ibrahim Sulaiman, GS42010

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Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the writing of this thesis was under our supervision;

supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate Studies) Rules 2003 (Revision 2012-2013) were adhered to.

Signature:

Name of Chairman

of Supervisory

Committee:

Professor Dr. Johnson Stanslas

Signature:

Name of Member

of Supervisory

Committee:

Associate Professor Dr. Norhafizah Mohtarrudin

Signature:

Name of Member

of Supervisory

Committee:

Dr. Lim Chee Woei

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vii

DECLARATION ix

LIST OF TABLES xvi

LIST OF FIGURES xvii

LIST OF APPENDICES xxi

LIST OF ABBREVIATIONS xxii

CHAPTER

1 INTRODUCTION 1 1.1 Background 1 1.2 Statement of Research Problem 2 1.3 Justification for the Study 3 1.4 Hypothesis 4 1.5 Objective of the Study 5

1.5.1 General Objective 5 1.5.2 Specific Objectives 5

2 LITERATURE REVIEW 6 2.1 Asthma 6 2.2 Prevalence of asthma 8

2.3 Asthma phenotypes and endotypes 10 2.4 Pathophysiology of asthma 14

2.4.1 Eosinophilic asthma (allergic/atopic/child-onset/steroid-sensitive/mild-to-moderate Asthma) 14

2.4.2 Neutrophilic Asthma (Non-allergic/Non-atopic/Adult-

onset/Steroid-resistant/Moderate-to-Severe Asthma) 16 2.5 Role of NF-κB signalling in airway inflammation 20 2.6 Role of Nrf-2 signalling in airway inflammation 22

2.7 Asthma Drugs and Targets 23 2.7.1 Bronchodilators 24

2.7.2 Corticosteroids 25 2.7.3 Mast cell stabilisers and leukotriene modifiers 26

2.7.4 Biologics 27 2.8 Experimental asthma models 29

2.8.1 In vivo models 29 2.8.2 In vitro and ex vivo models 30

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2.9 Role of natural products in drug discovery and development 31 2.10 Botanical drugs used in management of asthma and other

diseases 31 2.11 Andrographis paniculata 33

3 AP EXTRACTS PREPARATION AND HIGH-PERFORMANCE LIQUID CHROMATOGRAPHY (HPLC) STANDARDISATION 35 3.1 Introduction 35 3.2 Materials and method 36

3.2.1 Chemicals 36 3.2.2 Equipment 36 3.2.3 Propagation and collection of plant material 36 3.2.4 Plant extraction 37

3.2.5 High-performance liquid chromatography (HPLC) analysis 41 3.2.5.1 HPLC instrumentation 41 3.2.5.2 Method validation 41 3.2.5.3 Determination of AGP, NAG and DDAG

content in AP extracts 43 3.2.5.4 Determination of aqueous solubility of AP

extracts 43 3.2.6 Statistics 44

3.3 Results 44 3.3.1 HPLC method validation 44

3.3.1.1 Selectivity 44 3.3.1.2 Linearity 45 3.3.1.3 Precision 46

3.3.1.4 Limits of detection and quantitation 48 3.3.1.5 Accuracy 49

3.3.2 AGP, NAG and DDAG content in AP extracts 49 3.3.3 Aqueous solubility 52

3.4 Discussion 54 3.5 Conclusion 57

4 APAE INHIBIT NF-κB SIGNALING IN TNF-α EXPOSED A549 CELLS 58 4.1 Introduction 58 4.2 Materials and method 60

4.2.1 Drugs and chemicals 60

4.2.2 Cell culture reagents and consumables 60

4.2.3 Equipment 60

4.2.4 Human adenocarcinoma lung epithelial cells (A549 cells) 61

4.2.4.1 Cell maintenance 61 4.2.4.2 Cell plating 61

4.2.5 Cell viability 62

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4.2.6 TNF-α stimulation and treatment 62 4.2.6.1 Protein Extraction 63 4.2.6.2 Immunoblotting 64 4.2.6.3 Confocal immunofluorescence-

immunohistochemistry 64 4.2.6.4 NF-κB Transcription Factor Assay

(TransAM Assay) 65 4.2.6.5 Statistics 66

4.3 Results 66 4.3.1 Cytotoxic effect of AP extracts on A549 cells 66 4.3.2 APAE suppressed TNF-α-induced activation of

IKK-IκB-p65 signalling pathway in A549 cells 67 4.3.3 APAE prevented nuclear translocation of NF-κB p65

subunit in TNF-α stimulated A549 cells 68

4.3.4 APAE inhibits NF-κB DNA binding in a dose-dependent manner 70

4.4 Discussion 72 4.5 Conclusion 75

5 APAE AND APEE50 PREVENTED ASTHMA SYMPTOMS IN HDM-INDUCED ASTHMA MODELS 77 5.1 Introduction 77 5.2 Materials and method 78

5.2.1 Reagents 78 5.2.2 Equipment 79 5.2.3 Animals 79 5.2.4 Asthma Induction 79

5.2.5 Experimental design 80 5.2.6 BALF collection and analysis 81

5.2.6.1 Total cell count 82 5.2.6.2 Cell staining and differential leukocyte count 82

5.2.6.3 BALF enzyme-linked immunosorbent assay 83 5.2.7 Serum preparation and analysis 83 5.2.8 Lung tissue collection and processing 83

5.2.8.1 Histological analysis 84 5.2.8.2 Gene expression analysis 85

5.2.9 Airway hyperresponsiveness test 86 5.2.10 Statistics

86

5.3 Results 87 5.3.1 APAE and APEE50 inhibited recruitment of inflammatory

cells into the BALF 87 5.3.2 APAE and APEE50 prevented airway accumulation of

inflammatory cell infiltrates and reversed airway mucus

secretion in HDM exposed mice 93

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5.3.3 Effects of APAE and APEE50 on BALF cytokines and serum immunoglobulins 107 5.3.3.1 IL-4 107 5.3.3.2 IL-5 107 5.3.3.3 IL-13 110 5.3.3.4 Eotaxin 110 5.3.3.5 IFN-γ 113 5.3.3.6 IgE and IgG 113

5.3.4 Effect of APAE and APEE50 on pulmonary expression of inflammatory and oxidative stress-related genes 118 5.3.4.1 Oxidative stress-related genes 118 5.3.4.2 Th2-related genes 118 5.3.4.3 Th2-related genes 121 5.3.4.4 Macrophage-related genes 121

5.3.4.5 Extracellular matrix and mucus-related genes 124 5.3.5 Administration of APAE and APEE50 inhibits airway

resistance and improve compliance in HDM exposed

mice 124 5.4 Discussion 129 5.5 Conclusion 131

6 AGP AMELIORATES TDI-INDUCED AIRWAY INFLAMMATION 134 6.1 Introduction 134 6.2 Materials and method 135

6.2.1 Reagents 135 6.2.2 Equipment 135

6.2.3 Animals 135 6.2.4 Chemical induction of asthma and experimental design 136 6.2.5 BALF collection and analysis 138 6.2.6 Serum preparation and analysis 138

6.2.7 Lung tissue collection and processing 138 6.2.7.1 Histological analysis 138 6.2.7.2 Nrf2-HO-1 immunoblotting 139 6.2.7.3 Gene expression analysis 139

6.2.8 Airway hyperresponsiveness test 139

6.2.9 Statistics 139 6.3 Results 140

6.3.1 Inhibition of airway inflammatory cell recruitment

in AGP treated TDI-induced mouse asthma models 140 6.3.2 Administration of AGP downregulated BALF

intracellular reactive oxygen species level in TDI

exposed mice 143

6.3.3 Effects of AGP on BALF cytokines and serum immunoglobulins 144

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6.3.4 AGP attenuated pulmonary inflammation, airway mucus secretion and collagen deposition in the lungs of TDI

exposed mice 144 6.3.5 Effect of AGP on the distribution of E-cadherin and

β-catenin in bronchial epithelia of TDI-exposed mice 153 6.3.6 AGP induced p38 MAPK-dependent Nrf2 upregulation 160 6.3.7 Effects of AGP administration on expression of

inflammatory and oxidative stress-related genes in TDI

induced asthma models 162 6.3.7.1 Oxidative stress-related genes 162 6.3.7.2 Th2-related genes 163 6.3.7.3 Th1-related genes 164 6.3.7.4 Extracellular matrix and mucus-related genes 164

6.3.8 Effect of AGP on airway resistance and dynamic

compliance 165 6.4 Discussion 167 6.5 Conclusion 171

7 SUMMARY, CONCLUSION AND RECOMMENDATION FOR FUTURE RESEARCH 172 7.1 Research summary 172 7.2 General conclusion 173 7.3 Recommendation for future research 174

REFERENCES 175 APPENDICES 207

BIODATA OF STUDENT 233 LIST OF PUBLICATIONS 234

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LIST OF TABLES

Table Page

3.1 HPLC method selectivity validation 45

3.2 Intraday precision analysis for retention time 46

3.3 Intraday precision analysis for peak area 47

3.4 Interday precision analysis for retention time 47

3.5 Interday precision analysis for peak area 48

3.6 LOD and LOQ of AGP, NAG and DDAG as calculated using

calibration data obtained from the intraday precision analysis. 48

3.7 Recovery of reference compounds 49

3.8 Quantity of AGP, NAG and DDAG in the differential AP extracts 50

3.9 Aqueous solubility of differential AP extracts 53

5.1 Experimental design for HDM study 81

5.2 Histology scoring system 84

6.1 Experimental design for TDI study 136

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LIST OF FIGURES

Figure Page

1.1 Research concept map 4

2.1 Cytokine network in asthma 7

2.2 Prevalence of asthma among 13 – 14 year olds 8

2.3 Clinically observed characteristics used to describe asthma phenotype 11

2.4 Linking phenotype to endotypes 13

2.5 Dendritic cell and Th2 activation 15

2.6 Allergic and non-allergic eosinophilic airway inflammation in asthma 17

2.7 Neutrophilic airway inflammation in asthma 19

2.8 The canonical (classical) and non-canonical (alternative) NF-ĸB

Signalling pathways 21

2.9 Nrf2 Activation Pathway 23

2.10 Stepwise control of asthma symptoms 27

2.11 Potential of biologics in asthma control 28

2.12 Year to year percentage of approved therapeutic agents derived from

natural product origin 32

2.13 Structure of three major diterpenoids isolated from Andrographis

paniculata 34

3.1 AP at the point of harvest 38

3.2 AP propagation, collection and extract preparation 40

3.3 HPLC chromatogram for AGP, NAG and DDAG before and after

spiking AP material 45

3.4 Typical Chromatogram of (A) APAE, (B) APEE50 and (C) the three

reference standards 51

4.1 TNF-α induced NF-ĸB activation protocol 63

4.2 Viability of A549 cells following administration of APAE and APEE50 67

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4.3 Suppression of NF-κB signalling pathway by APAE 69

4.4 APAE inhibited nuclear translocation of NF-κB p65 in TNF-α

induced A549 cells 71

4.5 DNA-binding activity of p65 and p50 NF-κB subunits in TNF-α

stimulated A549 cells 72

4.6 Proposed inhibition of NF-κB signalling pathway by APAE 76

5.1 HDM-asthma induction protocol 80

5.2 Treatment regimen for APAE, APEE50 and PRED 81

5.3 Schematic illustration of AHR test 87

5.4 Photomicrograph of BALF leukocyte distribution in APAE treated

HDM sensitised and challenged mice 89

5.5 Photomicrograph of BALF leukocyte distribution in APEE50 treated

HDM sensitised and challenged mice 90

5.6 Total and differential leukocyte counts in BALF of (A) APAE and

(B) APEE50-treated HDM sensitised and challenged mice 91

5.7 Percentage BALF leukocyte distribution in (A) APAE and

(B) APEE50-treated HDM sensitised and challenged mice 92

5.8 Photomicrograph of lung parenchyma inflammation 94

5.9 Semi-quantitative histological analysis of hematoxylin and eosin

(H&E) stained lung sections from HDM-induced animals 99

5.10 Photomicrograph of goblet metaplasia 101

5.11 Dose-dependent decrease in mucus secretion among APAE and

APEE50 treated HDM induced mice 106

5.12 Level of BALF IL-4 in (A) APAE and (B) APEE50-treated mice 108

5.13 Level of BALF IL-5 in (A) APAE and (B) APEE50-treated mice 109

5.14 Level of BALF IL-13 in (A) APAE and (B) APEE50-treated mice 111

5.15 Level of BALF Eotaxin in (A) APAE and (B) APEE50-treated mice 112

5.16 Level of BALF IFN-γ in (A) APAE and (B) APEE50-treated mice 114

5.17 Level of total serum IgE in (A) APAE and (B) APEE50-treated mice 115

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5.18 Level of serum HDM-specific IgE in (A) APAE and

(B) APEE50-treated mice 116

5.19 Level of total serum IgG in (A) APAE and (B) APEE50-treated mic 117

5.20 Expression of three oxidative stress-related genes in HDM-induced

asthma 119

5.21 Pulmonary expression of four Th2-related genes in HDM-induced

asthma 120

5.22 Pulmonary expression of three eosinophil-related genes in

HDM-induced asthma 122

5.23 Pulmonary expression of four macrophage modulating genes in

HDM-induced asthma 123

5.24 Pulmonary expression of three extracellular matrix-related genes

in HDM-induced asthma 125

5.25 Pulmonary expression of Muc5ac and Chil4 in HDM-induced asthma 126

5.26 Airway resistance of mechanically ventilated (A) APAE and

(B) APEE50-treated mice 127

5.27 Dynamic lung compliance of mechanically ventilated (A) APAE

and (B) APEE50-treated mice 128

5.28 Proposed mechanism of action of AP extracts in HDM-induced

asthma 133

6.1 TDI sensitisation and challenge 137

6.2 TDI asthma induction protocol 137

6.3 BALF total and different inflammatory leukocyte counts in

AGP- treated TDI-sensitised and challenged mice 140

6.4 BALF leukocyte distribution in AGP-treated TDI-sensitised and

challenged mice 141

6.5 Photomicrograph of leukocyte distribution in AGP-treated

TDI-sensitised and challenged mice 142

6.6 AGP decreased BALF ROS production in TDI sensitised and

challenged mice 143

6.7 BALF cytokine and serum immunoglobulins levels in TDI study group 145

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6.8 Photomicrograph of lung parenchyma from TDI-sensitised and

challenged animals 148

6.9 Semi-quantitative histopathological analysis of H&E stained lung

sections from TDI-exposed animals 149

6.10 AGP-mediated suppression of airway mucus secretion in TDI

sensitised and challenged animals 150

6.11 Semi-quantitative analysis of airway mucus secretion by goblet cells

in TDI-exposed animals 151

6.12 AGP-mediated reduction in airway collagen deposition in TDI

induced asthma 152

6.13 AGP inhibited collagen deposition in TDI exposed mice 153

6.14 Immunoreactivity of airway epithelial AJ proteins 155

6.15 Upregulation of total pulmonary E-cadherin and β-catenin in AGP

treated animals 159

6.16 AGP mediated upregulation of Nrf2 and HO-1 161

6.17 Expression of three oxidative stress-related genes in TDI exposed

animals 162

6.18 Pulmonary expression of five Th2-related genes in TDI exposed

animals 163

6.19 Pulmonary expression of four Th1-related genes in TDI exposed

animals 164

6.20 Pulmonary expression of extracellular matrix and mucous related

genes in TDI-exposed animals 165

6.21 Airway resistance of mechanically ventilated TDI-exposed animals 166

6.22 Dynamic lung compliance of mechanically ventilated TDI-exposed

animals 167

6.23 The proposed mechanism of action of AGP in TDI-induced occupational

asthma 171

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LIST OF APPENDICES

Appendix Page

3Ai Calibration curve for AGP 207

3Aii Calibration curve for NAG 207

3Aiii Calibration curve for DDAG 208

3B Summarised HPLC method validation report 209

3Ci Chromatogram of APEE 210

3Cii Chromatogram of APME 210

3Ciii Chromatogram of APDE 211

3Civ Chromatogram of APMDE 211

3D Yield of extraction 213

3E Sample aqueous solubility HPLC report interface 213

4A Grouped Graph for three biological replicates of APAE cytotoxicity

test 214

4B Cell viabilities for co-administration of treatment with 20 ng/mL of

TNF-α in A549 cells. 215

4C Recipe for 16 mL of 10 % Polyacrylamide Gel Mix 216

5A Animal ethics approval 217

5B Calibration curves for BALF cytokine and serum immunoglobulin

ELISA 218

5C Nanodrop RNA concentration and purity 220

5D RNA integrity assessed by gel electrophoresis 221

5E Gene list for custom RT2 qPCR array 222

5F RT2 profiler PCR quality control results 223

5G Melting curve analysis and signal quantification peaks of some of

the genes analysed using RT2 profiler PCR 224

5H An unsupervised hierarchical cluster displaying a heat map for (A)

APAE (B) APEE50 treated animals and (C) AGP-TDI study group 225

5I Comparison of APAE and APEE50 activity at 200 mg/kg dose 227

6A Uncropped images of immunoblots 227

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LIST OF ABBREVIATIONS

%RSD Relative standard deviation

15-HETE 15-Hydroxyicosatetraenoic acid

APEE50 Andrographis paniculata ethanolic aqueous extract

5-LOX 5-lipoxygenase

Å Armstrong

ACN Acetonitrile

AGP Andrographolide

AHR Airway hyperresponsiveness

AIR Asthma Intervention Research

AJ Adherens junction

AL-1 Andrographolide-lipoic acid conjugate

ANOVA Analysis of variance

AOO Acetone olive oil

AP Andrographis paniculata

AP-1 Activator protein-1

APAE Andrographis paniculata aqueous extract

APDE Andrographis paniculata dichloromethane extract

APEE Andrographis paniculata ethanolic extract

APM Andrographis paniculata plant material

APMDE Andrographis paniculata methanol-dichloromethane extract

APME Andrographis paniculata methanolic extract

ASM Airway smooth muscle

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BAFF B-cell activating factor

BALF Bronchoalveolar lavage fluid

BCA Bicinchoninic acid assay

BSA Bovine serum albumin

CAT Catalase

CCL C-C motif ligand

Ccl C-C motif chemokine ligand gene

CDC Center for Disease Control

Cdyn Dynamic lung compliance

CFA Complete Freund’s adjuvant

Chil4 Chitinase-like 4 gene

Clca3a1 Chloride Channel Accessory 3a1 gene

Cldn1 Claudin 1 gene

CLR C-type lectin receptor

COPD Chronic Obstructive Pulmonary Disorder

CRTH2 chemoattractant receptor-homologous molecule expressed on

Th2 cells

CT Threshold cycle

CXCL C-X-C motif ligand

Cxcl C-X-C motif chemokine ligand gene

DAB Diaminobenzidine

DALYs Disability-adjusted life years

DAPI 4',6-diamidino-2-phenylindole

DCFDA 2’,7’-dichlorofluorescein diacetate

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DDAG 14-deoxy-11,12-didehydroandrographolide

DEX Dexamethasone

DMSO Dimethyl sulfoxide

DPX Distyrene plasticiser xylene

dsRNA Double-stranded RNA

DTT Dithiothreitol

Duox1 Dual oxidase-1 gene

DW Dry weight of plant material

ECL Enhanced chemiluminescence

EDTA Ethylenediaminetetraacetic acid

ELISA Enzyme-linked immunosorbent assay

EMTU Epithelial-mesenchymal trophic unit

EPR Expert Panel Report

Erk Extracellular signal-regulated kinases

ERK Extracellular-regulated kinase

EtOAc Ethyl acetate

FBS Fetal bovine serum

FDA Food and Drug Administration

FENO Fractional exhaled nitric oxide

FEV1 Forced expiratory volume in the first second

GAPDH Glyceraldehydes-3-phosphate dehydrogenase

GBD Global Burden of Disease

GCP Good Clinical Practice

GERD Gastroesophageal reflux disease

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GINA Global Initiative for Asthma

GM-CSF Granulocyte macrophage-colony stimulating factor

GMP Good manufacturing practices

GOI Gene of interest

GPx Glutathione peroxidase

GR Glutathione reductase

GR0-a Growth-regulated protein-alpha

GSH Glutathione

GSK-3β Glycogen synthase kinase 3β

H&E Haematoxylin and Eosin

HBSS Hank’s balanced salt solution

HDAC Histone deacetylase

HDAC2 Histone deacetylase 2

HDI Hexamethylene diisocyanate

HDM House-dust mite

HEK293 Human embryonic kidney 293

Hmox1 Heme Oxygenase-1 gene

HO-1 Heme Oxygenase-1

HPLC High-performance liquid chromatography

HRP Horseradish peroxidase

HRV Human rhinovirus

i.d Internal diameter

i.n Intranasal

i.p Intraperitoneal

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IBM International Business Machines Corporation

IC50 Concentration at which 50% inhibition is achieved

Icam1 Intercellular Adhesion Molecule 1 gene

ICH International Conference on Harmonisation of Technical

Requirements for Registration of Pharmaceuticals for Human

Use

ICS Inhaled corticosteroids

IEC International Electrotechnical Commission

Ifng Interferon-gamma gene

IFN-γ Interferon-gamma

IgE Immunoglobulin E

IgG Immunoglobulin G

IKK Inhibitor of kappa B kinase

IL Interleukin

ILC2 Type 2 innate lymphoid cells

ISAAC International Study of Asthma and Allergies in Childhood

ISO International Organisation for Standardisation

IVC Individually ventilated cages

IκBα Inhibitor of kappa B

JNK c-Jun amino-terminal kinases

JNK c-Jun N-terminal kinase

LABA Long-acting beta-2 agonists

LC-MS Liquid chromatography-mass spectroscopy

LD50 Lethal dose for 50% of animals

LOD Limit of detection

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LOQ Limit of quantitation

LPS Lipopolysaccharide

LTC Cysteinyl leukotrienes

LTRA Leukotriene receptor antagonist

mAb Monoclonal antibody

MAPK Mitogen-activated protein kinase

MBP Major basic protein

MDCK Madin-Darby canine kidney cells

MDI diphenyl-methane diisocyanate

MGDC Mouse genomic DNA contamination

MHC Major histocompatibility complex

Mmp9 Matrix metalloproteinase 9 gene

MT Masson’s trichrome

mTOR Mechanistic target of rapamycin

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

Muc5ac Mucin 5AC gene

MUC5AC Mucin 5AC

MUC5B Mucin 5B

n Number per group

NAG Neoandrographolide

ND Not detected

NDA New drug application

NEA Non-eosinophilic asthma

NEMO NF-κB essential modulator

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NES Nuclear export sequence

Nfe2l2 Nuclear Factor Erythroid-2-Like-2 gene

NF-κB Nuclear factor kappa-light-chain-enhancer of activated B cells

NHMS National Health Morbidity Survey

NIH National Institutes of Health

NLS Nuclear localisation sequence

NOAEL Non-observable adverse effect level

NQO-1 NAD(P)H:Quinone Oxidoreductase 1

Nrf2 Nuclear factor erythroid-2-related factor 2

NVDS Non-vitamin dietary supplements

OA Occupational asthma

OCS Oral corticosteroids

OECD The Organisation for Economic Co-operation and

Development

OVA Ovalbumin

p Statistical significance level

p.o Per oral

PA Peak area

PAF Platelet-activating factor

PAMP Pathogen-associated molecular patterns

PAR2 Protease-activated receptor 2

PAS Periodic acid Schiff

PBS Phosphate buffered saline

PDA Photodiode array

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PDE Phosphodiesterase

PI3K Phosphatidylinositol-3-kinases

PIC/S Pharmaceutical Inspection Cooperation Scheme

PPC Positive PCR control

PRR Pattern recognition receptors

PTK Protein tyrosine kinase

QC Quality control

r2 Coefficient of determination of linear regression

RANK Receptor activator of NF-κB

RI Airway resistance

RNS Reactive nitrite species

ROS Reactive oxygen species

rpm Revolution per minute

RPMI 1640 Roswell Park Memorial Institute Media

RSV Respiratory syncytial virus

RT Retention time

RTC Reverse transcription control

SABA Short-acting beta-2 agonists

SD Standard deviation

SDS-PAGE Sodium dodecyl sulfate polyacrylamide gel electrophoresis

SEM Standard error of mean

Serpinb2 Serpin Family B Member 2

SOD Superoxide dismutase

SPSS Statistical package for social sciences

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Syk Spleen tyrosine kinase

TBST Tris-buffered saline Tween-20

TCF T-cell factor

TDI Toluene diisocyanate

TEA Triethylamine

TEMED N,N,N′,N′-tetramethylethylenediamine

TGF-b Transforming growth factor-beta

TGF-βRII Transforming growth factor, beta receptor II

Th T-helper cells

Timp tissue inhibitor of matrix metalloproteinase gene

TLR Toll-like receptor

TNBS Trinitrobenzenesulfonic acid

Tnf Tumor necrosis factor gene

TNFR1 Tumour necrosis factor-alpha receptor

TNFSF13B Tumour necrosis factor ligand superfamily member 13B

TNF-α Tumour necrosis factor-alpha

T-PER Tissue protein extraction reagent

TSLP Thymic stromal lymphopoietin

UHPLC Ultra-high pressure liquid chromatography

UPM Universiti Putra Malaysia

USP United States Pharmacopoeia

v/v Volume per volume

VCAM-1 Vascular cell adhesion molecule 1

VSMCs Vascular smooth muscle cells

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w/v % weight per volume

w/w % weight per weight

WHO World Health Organisation

WW Wet weight of plant material

YLLD Years of life lived with disability

γ-GCL γ- Glutamate Cysteine Ligase

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CHAPTER 1

1 INTRODUCTION

1.1 Background

Asthma is a chronic airway inflammatory disorder characterised by persistent airway

inflammation, airway obstruction, airway hyperresponsiveness (AHR) and

remodelling ( Hill & Wood, 2009; Chung et al., 2014). Exposure of tissues or organs

to destructive stimuli such as irritants, microbial pathogens or toxic cellular elements

triggers inflammatory responses (Brennan & Bowie, 2010). This entails a cascade of

activities involving various inflammatory cells such as neutrophils, basophils,

eosinophils, dendritic cells, monocytes, macrophages, mast cells, B-cells as well as T-

cells. Inflammatory processes are regulated in such a way that appropriate leukocytes

are recruited to the site of inflammation (Turner et al., 2014). Similarly, the interaction

of several cellular elements in response to trigger, sensitiser and irritants has been the

bedrock of asthma pathogenesis. These capture the heterogeneity of the disorder when

it comes to its presentation, aetiology and pathophysiology, thereby contributing to its

complexity and difficulty in treatment.

Clinically, airway inflammatory complications in asthma lead to recurrent airway flow

limitations associated with wheezing, coughing, shortness of breath and chest

tightness. Uncontrollable and life-threatening episodic asthma exacerbations or “flare-

ups” could occur as a result of severe expiratory airway flow limitations (Keglowich

& Borger, 2015; GINA, 2017). Moreover, the pathobiology of asthma is

heterogeneous and genetically complex, thereby making the discovery of more

effective treatments a challenging task (Anderson, 2008).

Andrographis paniculata (AP) is a herbaceous plant belonging to the Acanthaceae

family, it is often cultivated in India, China, and Malaysia for medicinal purposes. The

plant is extremely bitter, and it is often referred to as the “the king of bitters”. In recent

decades, several reports have revealed the diverse therapeutic potentials of AP extracts

in properly controlled clinical trials (Mishra et al., 2007; Chao & Lin, 2010; Thakur,

et al., 2014). Andrographolide (AGP), a labdane diterpenoid, is the major bitter-tasting

secondary metabolite contained in the plant, and it is often considered to be the major

bioactive constituent of the plant (Hidalgo et al., 2005; Lim et al., 2012; Jayakumar et

al., 2013). In addition to anti-inflammatory potential, AP herb possesses analgesic,

antimalarial, anti-oxidant, antineoplastic, antiulcerogenic, antibacterial, antiplatelet,

antidiarrhoeal and antithrombotic properties (Jarukamjorn & Nemoto, 2008).

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1.2 Statement of Research Problem

Asthma is a global health concern affecting about 334 million people across all age

groups (Global Asthma Network, 2014).Although the number is little when compared

to that of diseases such as cancer and cardiovascular disorders, asthma is among thirty

most prevalent diseases in the world (Wang et al., 2017). Its prevalence, especially

among children, has consistently been on the rise especially in developing countries

(Asher & Pearce, 2014). The alarming incidence of asthma necessitated the

establishment of asthma control and relief guidelines such as the Global Strategy for

Asthma Management and Prevention by Global Initiative for Asthma (GINA); Expert

Panel Report (EPR) Guidelines for the Diagnosis and Management of Asthma; and

British Guideline for Management of Asthma. In spite of these efforts, asthma control

is still at sub-optimal level (GINA, 2017). Furthermore, environmental and lifestyle

risk factors have contributed to the rise in the prevalence of asthma.

Urbanisation is considered an important environmental risk factor for asthma. Rapid

industrialisation among the low-income and middle-income countries further exerts

the burden of urbanisation on residents of such communities (Robinson et al., 2011;

Gaviola et al., 2016). Although Malaysia is gradually advancing towards international

high-income threshold, the country is not spared from the devastating burden of

industrialisation on respiratory health (Qureshi et al., 2015). According to the

Malaysian National Health Morbidity Survey (NHMS) 2011, the prevalence of asthma

among Malaysians rose from 4.5% in 2006 to 6.3% in 2011 (Liam et al., 2014; Chan

et al., 2015). Whereas, epidemiological studies on the distribution of non-

communicable diseases in Malaysia reported 13.2% asthma prevalence among older

Malaysians (Teh et al., 2014).

Although some countries have recorded an appreciable decline in hospitalisation and

deaths due to asthma, the disease is still a major source of global socio-economic

burden. It creates a substantial burden on patients and their families, resulting in

significant morbidity and even mortality. Depending on the symptoms and state of

airway limitations, treatments are stepped-up when adequate control is not achieved

and stepped-down upon reaching stability. This form of step-wise treatment is strongly

advocated for asthma management (Holgate & Thomas, 2017). Management of

intermittent asthma requires an initial administration of short-acting beta-2 agonists

(SABA). Stepping up of this treatment involves the administration of low dose inhaled

corticosteroids (ICS) in combination with SABA (Reddel et al., 2015). Depending on

the severity, the doses of ICS could be increased to middle or high dose in combination

with long-acting beta-2 agonists (LABA), whereas SABA could be administered only

when needed (Reddel et al., 2015). Due to variation in responses, add-on medications

such as cromolyn, leukotriene receptor antagonist (LTRA), omalizumab or even short

courses of oral corticosteroids (OCS) are administered to achieve the desired response

(GINA, 2017). Nevertheless, asthma remains inadequately controlled in some patients

especially the severe endotype. Most of these medications have also shown some

detrimental adverse effects, therefore, this necessitates the need to develop better

treatments for asthma.

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1.3 Justification for the Study

The traditional use of AP in the treatment of inflammatory diseases, diabetes and

infections in Asian (e.g. China, India and Thailand) and Scandinavian countries has

long been documented (Jayakumar et al., 2013). Asthma as an inflammatory and

oxidative stress disorder affecting lower respiratory tract could be treated with the

anti-inflammatory and antioxidant activity of AP. Although the plant is traditionally

used in the management of upper respiratory tract infection and the common cold,

there is no evidence for its anti-asthma activity and mechanism of action in the

prevention of asthma symptoms. Persistent activation of nuclear factor kappa-light-

chain-enhancer of activated B cells (NF-κB) and disruption of nuclear factor

erythroid-2-related factor 2 (Nrf2) activity have been associated with asthma

pathogenesis (Williams et al., 2008; Schuliga, 2015). Major bioactive molecules

contained in AP, namely AGP and 14-deoxy-11,12-didehydroandrographolide

(DDAG) were shown to ameliorate asthma symptoms in mouse asthma models by

inhibiting NF-κB signalling and stimulating heme oxygenase-1 (HO-1) and Nrf2

activity (Bao et al., 2009; Guan et al., 2011). This research is conceptualised based on

the possible attenuation of asthma by Andrographis paniculata aqueous extract

(APAE) and Andrographis paniculata aqueous ethanolic extract (APEE50) via the

inhibition of NF-κB activation by AP extracts (Figure 1.1). The study further explored

the potential of AGP in ameliorating asthma through the upregulation of HO-1

production and Nrf2 activation in mouse asthma models.

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Figure 1.1 : Research concept map. Hypothetical inhibitory effect of (A) APAE and

APEE50 on NF-κB signalling pathway and (B) the up-regulatory effect of AGP on

HO-1 and Nrf2 activation.

1.4 Hypothesis

AP extracts possess anti-asthma activity and inhibit inflammation through the

inactivation of the NF-κB signalling pathway.

AGP prevents chemical-induced asthma via activation of Nrf2 pathways.

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1.5 Objective of the Study

1.5.1 General Objective

The main objective of this study is to investigate the therapeutic potential of AP

extracts and AGP in experimental asthma models.

1.5.2 Specific Objectives

i. To prepare, standardise and estimate the aqueous solubility of six solvent AP extracts.

ii. To determine the effect of APAE on the NF-κB cell signalling pathway in A549 cells.

iii. To assess the efficacy of APAE and APEE50 in house-dust mite (HDM)-induced mouse asthma model.

iv. To determine the efficacy and elucidate the mechanism of action of AGP in a toluene diisocyanate (TDI)-induced asthma model.

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