Clinacanthus Nutans Lindau Extracts on Hyperuricemic Mice

EFFECTS OF CLINACANTHUS NUTANS LINDAU EXTRACTS ON HYPERURICEMIC MICE

MAJED BIN ABD GHAFFAR ABD HALIM MAETALONG

A thesis submitted in fulfilment of the requirement for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Chemistry)

Kulliyyah of Pharmacy International Islamic University Malaysia

NOVEMBER 2017

ABSTRACT

Clinacanthus nutans or Sabah Snake Grass (Acanthaceae) has been commercialised locally as an anti-gout, and anti-hyperuricemia. However, the scientific evidences that supports its efficacy in treating these illnesses is still insufficient. The study aims to investigate the effect of different extracts and doses of C. nutans leaf in the normalisation of uric acid levels in hyperuricemic mice, conduct a metabolite fingerprinting on the mice serum through an LC-MS-based metabolomics approach, and to evaluate the acute toxicity effect of the C. nutans extract on kidney and liver samples of the mice of the selected doses. The dried leaves were extracted by soaking in different solvents with different concentrations of ethanol in water (0, 20, 50, 70, 100%, v/v). These extracts were tested in vivo using a potassium oxonate-induced hyperuricemia mice model. The results showed that the administration of the aqueous and 50% ethanolic extract with the dose 800 mg/kg b.w. for 4 weeks significantly (P<0.05) reduced and normalised the serum uric acid level of the mice starting from week 3. The same effect was also noticed for allopurinol whereby uric acid levels were potently normalised throughout the experiment (week 1 to 4). However, the data obtained from the LC-MS based fingerprinting showed that the metabolite profile of the mice serum treated with allopurinol was affected negatively in comparison with the healthy group starting from week 2. Interestingly, the metabolite profile of the mice treated with the C. nutans aqueous extracts at 800 mg/kg b.w. dose was better than that of allopurinol whereby they normalised the metabolic profile of the mice in week 4. The treatment with allopurinol, C. nutans aqueous and 50% ethanol extracts did not cause any histopathological or behavioural side effects to the mice with zero mortality up to 4 weeks of treatment. Moreover, the identification of the serum metabolites involved in the treatment is necessary to understand the mechanism of action of the C. nutans extracts. This study concludes that the C. nutans leaf extract has a potential in treating hyperuricemia and gout.

خالصة البحث

تسوق نبتة كليناكانتوس نوتانس (Clinacanthus nutans) أو عشبة األفعى الصباحية )فصيلة األقنتيات( حمليا كدواء لعالج داء النقرس وفرط محض اليوريك يف الدم. ولكن األدلة العلمية اليت تثبت فعاليتها يف معاجلة هذه األمراض غري كافية. هدفت الدراسة للتحقيق يف أثر اجلرعات املختلفة ملستخلصات نبتة الكليناكانتوس نوتانس يف عالج فرط محض اليوريك يف الدم يف الفئران املصابة بفرط يف محض اليوريك، وإجراء التبصيم األيضي ملصل دماء الفئران باتباع طريقة امليتابولوميات املعتمدة على الطيف الكتلي-االستشرا السالل(LC-MS) ، ولتقييم التأثري السمي احلاد جلرعات خمتارة من املستخلصات. مت استخالص األوراق اجملففة هلذه النبتة بالنقع يف حماليل اإليثانول املالي املختلفة الًتكيز )v/v ،%100 ،00 ،00 ،20 ،0(. مت اختبار هذه املستخلصات يف النموذج احليواين لفرط محض يوريك احملدث مبركب أوكسونيك البوتاسيوم. أظهرت النتالج أن إعطاء املستخلصات املالية ومستخلصات 00% إيثانول جبرعة 000 مغ/كج من وزن الفأر ملدة 4 أسابيع خفض بشكل كبري (p<0.05) مستويات محض اليوريك املرتفعة يف الدم إىل مستواها الطبيعي بدءا من األسبوع الثالث. مت مالحةة نفس األثر مع األلوبيورينول حيث خفض بشكل فعال مستويات محض اليوريك املرتفعة يف الدم طيلة مدة العالج )من األسبوع األول إىل الرابع(. على الرغم من ذلك، أظهرت بيانات امليتابولوميات املعطاة من التبصيم املعتمد على الــ LC-MS أن التوصيف األيضي لدماء الفئران املعاجلة باأللوبيورينول قد تأثر بشكل سليب بدءا من األسبوع الثاين مقارنة باجملموعة الضابطة. كان من املثري لالهتمام أن التوصيف األيضي للفئران املعاجلة باملستخلصات على جرعة 000 مغ/كج من وزن الفأر كان أفضل من تلك املعاجلة باأللوبيورينول حيث قامت بارجاع التوصيف األيضي لدماء الفئران يف األسبوع الرابع. مل حيدث العالج باأللوبيورينول، واملستخلصات املالية، مستخلصات 00% إيثانول أي حالة موت أو أثر جانيب من الناحية السلوكية أو اهليستولوجية لغاية 4 أسابيع من العالج. باإلضافة إىل ذلك، من الواجب القيام بعملية التعرف على املستقلبات الىت هلا دور يف العالج وذلك لفهم آلية عمل املستخلصات. تستنتج هذه الدراسة أن مستخلصات الكليناكانتوس نوتانس عالج واعد لالستعمال يف مداواة الفرط يف مستوى محض اليوريك يف الدم.

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APPROVAL PAGE

I certify that I have supervised and read this study and that in my opinion, it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Chemistry).

………………………………….. Alfi Khatib Supervisor

………………………………….. Bisha Fathamah binti Uzir Co-Supervisor

I certify that I have read this study and that in my opinion it conforms to acceptable standards of scholarly presentation and is fully adequate, in scope and quality, as a thesis for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Chemistry).

………………………………….. Irna Elina binti Ridzwan Internal Examiner

………………………………….. Mohd Zaini Asmawi External Examiner

This thesis was submitted to the Department of Pharmaceutical Chemistry and is accepted as a fulfilment of the requirement for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Chemistry).

………………………………….. Siti Zaiton binti Mat So'ad Head, Department of Pharmaceutical Chemistry

This thesis was submitted to the Kulliyyah of Pharmacy and is accepted as a fulfilment of the requirement for the degree of Master in Pharmaceutical Sciences (Pharmaceutical Chemistry).

………………………………….. Juliana binti Md. Jaffri Dean, Kulliyyah of Pharmacy

DECLARATION

I hereby declare that this thesis is the result of my own investigations, except where otherwise stated. I also declare that it has not been previously or concurrently submitted as a whole for any other degrees at IIUM or other institutions.

Majed bin Abd Ghaffar Abd Halim Maetalong

Signature………………..….……. Date …......

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

EFFECTS OF CLINACANTHUS NUTANS LINDAU EXTRACTS ON HYPERURICEMIC MICE

I declare that the copyright holders of this dissertation are jointly owned by the student and IIUM.

No part of this unpublished research may be reproduced, stored in a retrieval system, or transmitted, in any form or by any means, electronic, mechanical, photocopying, recording or otherwise without prior written permission of the copyright holder except as provided below

1. Any material contained in or derived from this unpublished research may be used by others in their writing with due acknowledgement.

2. IIUM or its library will have the right to make and transmit copies (print or electronic) for institutional and academic purposes.

3. The IIUM library will have the right to make, store in a retrieved system and supply copies of this unpublished research if requested by other universities and research libraries.

By signing this form, I acknowledged that I have read and understand the IIUM Intellectual Property Right and Commercialisation policy.

Affirmed by Majed bin Abd Ghaffar Abd Halim Maetalong

……..…………………….. ……………………….. Signature Date

ACKNOWLEDGEMENT

Alhamdulillah, I thank Allah subhanahu wa ta’ala for giving me the strength in carrying out this work, and the motivation to finish it.

My love and gratitude for my adored parents for supporting me in everything I do, and for my beloved wife, Kevser Irfan Unal, for accompanying me in this journey, her nonstop support, and her sacrifice to bear and take care of our child Hanan.

I would like to thank Dr. Alfi Khatib for accepting me as his student, his patience with me, and his priceless expertise, care and guidance. I would like to thank Dr. Nik Mohd Idris bin Nik Yusoff and Dr. Bisha Fathamah binti Uzir for their invaluable knowledge, wisdom, and moral support.

I would also like to thank the Ministry of Agriculture for their generous support of this work through the research grant NRGS SP15-060-0182.

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TABLE OF CONTENTS

Abstract in English ...... ii Abstract in Arabic ...... iii Approval page ...... iv Declaration ...... iv Copyright Page ...... vi Acknowledgement ...... vii List of Figures ...... x List of Tables ...... xiii List of Abbreviations ...... xiv

CHAPTER ONE: INTRODUCTION ...... 1 1.1 Background of the Study ...... 1 1.2 Problem Statement ...... 6 1.3 Research Objectives...... 7 1.4 Research Questions ...... 7 1.5 Research Hypothesis ...... 7

CHAPTER TWO: LITERATURE REVIEW ...... 8 2.1 Clinacanthus nutans Lindau ...... 8 2.1.1 Botanical Aspects and Geographical Distributions ...... 8 2.1.2 Ethnobotanical Uses and Toxicity of Clinacanthus nutans ...... 10 2.1.3 Chemical Constituents of Clinacanthus nutans ...... 11 2.2 Hyperuricemia and Gout...... 23 2.2.1 Uric Acid’s Physiology and Biochemistry ...... 25 2.2.2 Uric Acid’s Degradation and Disposal ...... 28 2.2.3 Treatment of Hyperuricemia and Gout ...... 31 2.3 Role of Medicinal Plants and Natural Products in Hyperuricemia ...... 35 2.4 Experimental Hyperuricemia in Rodents...... 37 2.5 Metabolomics ...... 42 2.6 Toxicity and Adverse Effects of Herbal Medicines ...... 46

CHAPTER THREE: METHODOLOGY ...... 48 3.1 Chemicals and Reagents ...... 48 3.2 Instrumentation ...... 48 3.3 Preparation of C. nutans Aqueous Ethanol Extracts ...... 48 3.3.1 Plant Material ...... 48 3.3.2 C. nutans Aqueous and Ethanol Extraction Process ...... 49 3.4 In Vivo Preliminary Experiments on Hyperuricemia Mice Model ...... 50 3.4.1 Preparation of Mice ...... 50 3.4.2 Effect of Different Blood Sampling Technique on Serum Uric Acid ...... 50 3.4.3 Effect of Potassium Oxonate Administration on Serum Uric Acid and Optimisation of the Hyperuricemia Mouse Model...... 51 3.5 Effects of C. nutans Leaf Aqueous Ethanol Extracts on Hyperuricemic Mice ...... 52

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3.6 Sample Preparation of Collected Mice Serum for LC-MS/MS Analysis ... 56 3.7 LC-MS/MS Based Fingerprinting of Mice Serum Samples ...... 57 3.8 Toxicity Evaluation ...... 58 3.8.1 Behavioural Observation ...... 58 3.8.2 Histopathological Examinations ...... 59 3.8.3 Tissue Processing ...... 59 3.8.4 Sectioning Process ...... 60 3.8.5 Fishing and Drying of Tissue Sample ...... 61 3.8.6 Hematoxylin-Eosin (H&E) Staining ...... 61 3.9 Statistical Analysis...... 64

CHAPTER FOUR: RESULTS AND FINDINGS ...... 65 4.1 Extraction of Clinacanthus nutans ...... 65 4.2 In Vivo Preliminary Experiment on Hyperuricemia Mice Model ...... 66 4.2.1 Effect of Different Blood Sampling Technique on Serum Uric Acid ...... 66 4.2.2 Effect of Potassium Oxonate Administration on Serum Uric Acid and Optimisation of the Hyperuricemia Mouse Model...... 66 4.2.3 Effect of C. nutans Ethanol Extracts on Serum Uric Acid of Hyperuricemic Mice...... 68 4.3 Fingerprinting of Mice Serum Using LC-MS/MS-Based Metabolomics... 73 4.4 Toxicity Evaluation ...... 79 4.4.1 Mortality Rate and Sign of Toxicity ...... 79 4.4.2 Body Weight ...... 80 4.4.3 Weight of Organs ...... 80 4.4.4 Gross Observation of Organs ...... 81 4.4.5 Histopathological Examination ...... 81

CHAPTER FIVE: DISCUSSION ...... 90 5.1 Extraction of Clinacanthus nutans ...... 90 5.2 In Vivo Preliminary Experiment on Hyperuricemia Mice Model ...... 91 5.2.1 Effect of Different Blood Sampling Technique on Serum Uric Acid ...... 91 5.2.2 Effects of Potassium Oxonate Administration on Serum Uric Acid and Optimisation of The Hyperuricemia Mouse Model ...... 90 5.3 Effect of C. nutans Ethanol Extracts on Serum Uric Acid of Hyperuricemic Mice ...... 93 5.4 LC-MS/MS Based Fingerprinting of Mice Serum Samples ...... 96 5.5 Conclusion ...... 98 5.6 Future Recommendations ...... 98

REFERENCES ...... 99

APPENDIX I: IACUC-IIUM Ethical Approval Letter ...... 118 APPENDIX II: Mean Body Weights of Animals Measured Daily for 28 Days . 120 APPENDIX III: Behavioural Observation Chart ...... 121 APPENDIX IV: LC-qTOF-MS Spectra of Healthy Mice Serum ...... 122 APPENDIX V: Loading Column Plots ...... 123

LIST OF FIGURES

Figure No. Page No.

2.1 ‎Clinacanthus nutans farm in Malaysia 8

2.2 Physical chemistry of uric acid and monosodium urate formation. 26

2.3 Purine degradation pathway 27

2.4 Uric acid transportasome. 31

3.1 Experimental mice grouping for the 7-day treatment experiment (n=6). 53

3.2 ‎The administration procedure of the treatment in the experimental 54 animals

3.3 ‎Experimental mice grouping for the 28-day treatment experiment (n=6). 55

3.4 Flowchart of mice serum sample preparation for LC-MS/MS. 56

3.5 Protocol of tissue fixation in paraffin wax 61

3.6 Protocol procedure for H&E staining 61

4.1 ‎Effects of different doses of P.O. (50, 100, and 250 mg/kg b.w) on serum 67 uric acid of mice.

4.2 ‎Serum uric acid concentration after 7 days of treatment (n=6) 69

4.3 ‎The increment of the serum uric acid level (mg/dL) of mice during 28 73 days of different treatments.

4.4 ‎The score scatter plot from principal component analysis of the serum of 75 the healthy mice (H), potassium oxonate-induced hyperuricemic mice (P.O.), and allopurinol-treated mice (10 mg/kg b.w.) (ALP) analysed using LC-MS 1 week after the treatment (n=6).

4.5 ‎The score scatter plot from principal component analysis of the serum of 76 the healthy mice (H), potassium oxonate-induced hyperuricemic mice (P.O.), and allopurinol-treated mice (10 mg/kg b.w.) (ALP) analysed using LC-MS 2 weeks after the treatment (n=6).

4.6 ‎The score scatter plot from principal component analysis of the serum of 76 the healthy (H), potassium oxonate-induced hyperuricemic mice (P.O.), and allopurinol (10 mg/kg b.w.) treated mice (ALP) analysed using LC- MS 3 weeks after the treatment (n=6).

4.7 ‎The score scatter plot from principal component analysis of the serum of 77 the healthy (H), potassium oxonate-induced hyperuricemic mice (P.O.),

and allopurinol (10 mg/kg b.w.) treated mice (ALP) analysed using LC- MS at 4 weeks after the treatment (n=6).

4.8 ‎The score scatter plot from principal component analysis of the serum of 77 the healthy (H), potassium oxonate-induced hyperuricemic mice (P.O.), and C. nutans 50% ethanol extract (800 mg/kg b.w.) treated mice (50CN800) analysed using LC-MS at 3 weeks after the treatment (n=6).

4.9 ‎The score scatter plot from principal component analysis of the serum of 78 the healthy (H), potassium oxonate-induced hyperuricemic mice (P.O.), and C. nutans 50% ethanol extract (800 mg/kg b.w.) treated mice (50CN800) analysed using LC-MS at 4 weeks after the treatment (n=6)

4.10 ‎The score scatter plot from principal component analysis of the serum of 78 the healthy (H), potassium oxonate-induced hyperuricemic mice (P.O.), and C. nutans water extract (800 mg/kg b.w.) treated mice (0CN800) analysed using LC-MS 3 weeks after the treatment (n=6).

4.11 ‎The score scatter plot from principal component analysis of the serum of 79 the healthy (H), potassium oxonate-induced hyperuricemic mice (P.O.), and C. nutans water extract (800 mg/kg b.w) treated mice (0CN800) analysed using LC-MS 4 weeks after the treatment (n=6).

4.12 ‎Mean body weights of animals measured daily for 28 days. 80

4.13 ‎Healthy group representative H&E staining of liver tissue (×20 83 magnification).

4.14 ‎ALP group representative H&E staining of liver tissue (×40 84 magnification).

4.15 ‎0CNE800 group representative H&E staining of liver tissue (×40 84 magnification).

4.16 ‎0CNE800 group representative H&E staining of liver tissue (×4 85 magnification). 4.17 ‎0CNE800 group representative H&E staining of liver tissue (×40 85 magnification).

4.18 ‎50CNE800 group representative H&E staining of liver tissue (×20 86 magnification).

4.19 ‎ALP group representative H&E staining of kidney tissue (×40 86 magnification).

4.20 ‎Healthy group representative H&E staining of kidney tissue (×4 87 magnification).

4.21 ‎0CNE800 group representative H&E staining of kidney tissue (×4 87 magnification).

4.22 ‎0CNE800 group representative H&E staining of kidney tissue (×40 88 magnification).

4.23 ‎50CNE800 group representative H&E staining of kidney tissue (×4 88 magnification).

4.24 50CNE800 group representative H&E staining of kidney tissue (×40 89 magnification).

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LIST OF TABLES

Table No. Page No.

2.1 Constituents of Clinacanthus nutans 13

2.2 Previous studies that used the potassium oxonate-induced 40 hyperuricemia model. 4.1 Average yields of C. nutans extraction 65

4.2 Serum uric acid levels measured 3 times over 14 days from two 66 different blood sampling techniques.

4.3 The serum uric acid concentration of mice at different time points 71 of the treatment

4.4 The increments of serum uric acid concentration of mice at 72 different time points of the treatment

4.5 Relative weights (g) of organs of mice 81

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LIST OF ABBREVIATIONS

COPCORD Community oriented program for the control of rheumatic disease

DDD Defined daily dose

NSAID Non-steroidal anti-inflammatory drug

COX-2 Cyclooxygenase-2

WHO World Health Organisation b.w. Body weight

N/A Not available cPLA2 Cytosolic phospholipase A2

TLR-4 Toll-like receptors-4

ATP Adenosine triphosphate

PRPP 5-phosphoribosyl 1-pyrophosphate

HGPRT Hypoxanthine-guanine phosphoribosyltransferase

XO Xanthine oxidase

XD Xanthine dehydrogenase

XOR Xanthine oxidoreductase

NAD+ Nicotinamide adenine dinucleotide

FAD Flavin adenine dinucleotide

MRP4 Multidrug resistance-associated protein 4

SLC22A6 Solute Carrier Family 22 Member 6.

URAT1 Urate transporter 1

ABCG2 ATP Binding Cassette Subfamily G Member 2

GLUT9 Glucose transporter 9

US FDA United States Food and Drug Administration

SPF Mice Specific-pathogen-free mice

ICR Mice Institute for Cancer Research mice

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ddy Mice Deutschland, Denken, and Yoken mice

1H-NMR Hydrogen-1 nuclear magnetic resonance

PCA Principal component analysis

HPLC High performance liquid chromatography

UPLC-MS Ultra-performance liquid chromatography, mass spectrometry

LC-MS Liquid chromatography–mass spectrometry

UV Ultraviolet

GC-MS Gas chromatography–mass spectrometry

Liquid Chromatography Quadrupole Time-of-Flight Mass LC-qTOF-MS Spectrometry w/w Weight per weight w/v Weight per volume v/v Volume per volume

CNE Clinacanthus nutans extracts

ALP Allopurinol

ESI Electrospray ionization

CMC Carboxymethyl cellulose

CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Hyperuricemia, or high uric acid, is clinically a common metabolic abnormality.

Around 10% of men and women are recorded with hyperuricemia not less than once in their lives with a reported prevalence between 5% and 30% in the general population globally, and it is elevated in certain ethnicities (Kuo et al., 2015). For example, hyperuricemia is more common in the Maori aborigines (27.1%) than the

Europeans (9.4%) in New Zealand, and in the Taiwan aborigines the prevalence was around 41.4% (Reginato et al., 2012). Moreover, it was found that the urban population had a higher prevalence of hyperuricemia than the rural population in

Beijing (Li et al., 1997). Hyperuricemia is considered to be more prevalent globally because of the improvements in living standards (Lawrence, 1964), prolonged life span, and the side effects of particular medications in few cases (Demartini, 1965;

Scott, 1991). This trend has caused a significant high morbidity and has burdened the health care system (Wertheimer et al., 2013).

According to an article by Kuo et al. (2015) in Nature Reviews, gout is reportedly rare in Malaysia. Moreover, Malaysia was categorised by the COPCORD

(Community Oriented Program for the Control of Rheumatic Diseases) as having low gout prevalence (<0.5%). However, despite that claim, the use of anti-gout preparations in Malaysia, measured by DDD, the assumed average maintenance dose per day for a drug used for its main indication in adults (WHO, 2004), was ranked second (1.5554 DDD/1000 population/day) in 2010, and the three most used anti-gout preparations were allopurinol, colchicine, and probenecid (Ministry of Health, 2014).

In the same report, anti-inflammatory and anti-rheumatic products, which are also prescribed in cases of gout (to relief inflamed joints during attacks due to crystal deposition), ranked first (11.7605 DDD/1000 population/day) in 2010.

Gout was known centuries ago, and a better comprehension about its pathophysiology is already developed (Pai, Raslan, and Schlesinger, 2015). The metabolic disorder results from the deposition of monosodium urate or uric acid crystals in joint structures and in other sites in the form of tophi. Impaired renal uric acid excretion alone, or in combination with genetic malfunctions that regulate uric acid could lead to gout. Elevated serum uric acid or hyperuricemia is a prerequisite for the formation of the crystals. The distinct resembling features of gout are acute attacks of joint inflammation, which often occur at the first metatarsophalangeal joint (the big joint of the big toe), although their incidence in other joints and in bursae is also very common (Pascual and Sivera, 2007). Gout is the most common type of inflammatory arthritis in adults. Gout affects 8.3 million Americans (Zhu, Pandya, and Choi, 2011) and 5% of US veterans (Singh, Hodges, Toscano, and Asch, 2007). It leads to frequent emergency room visits (Garg et al., 2013) and costs 20 billion dollars annually

(Wertheimer et al., 2013).

According to a survey, in Malaysia, anti-inflammatory agents are the drugs that are mostly used for the alleviation of acute and chronic gout, with a low prescription of corticosteroid (Yeap et al., 2009). In that study, for the treatment of acute gout,

68.0% of doctors use non-selective non-steroidal anti-inflammatory drugs (NSAIDs),

53.9% use selective COX-2 inhibitors, 66.4% use colchicine and 10.2% use allopurinol (Yeap et al., 2009). In the treatment of chronic gout, 36.7% use NSAIDs,

44.5% use COX-2 inhibitors, 19.5% use colchicine and 93% use allopurinol. In both acute and chronic gout, corticosteroids are not used by over 90% of respondent

doctors. Xanthine oxidase inhibitors lower the uric acid concentration in blood by inhibiting the xanthine oxidase enzyme which is the cause for the generation of uric acid from the purines. As a result of the inhibition, uric acid concentrations in blood and urine are lowered, and there will be a concurrent rise in the elimination of xanthine and hypoxanthine that are the more soluble precursors of uric acid (Angstadt,

1997). Patients who are characterised with persistent elevation of uric acid or those with insufficient renal excretion are best prescribed with xanthine oxidase inhibitors such as allopurinol (Wright and Pinto, 2003).

Traditional remedies are consumed in the primary health care by about 75 to 80

% globally, particularly in developing countries. The consumption of herbal products is also popular in some developed nations such as Germany, United States of

America, and France (Ekor, 2014). The WHO has estimated the demand for medicinal plants is approximately $14 billion per annum (2006) and the demand is growing at the rate of 15–25% annually. The WHO estimates that by 2050 the trade will be up to

US$ 5 trillion (Zhang et al., 2012). The herbs and herbal products sales in the United

States of America and the European Union are estimated to be worth more than US $

8 billion and 20 billion annually, respectively, while the worldwide herbal medicine market is appraised to be from 30 to 60 billion dollars annually (Vunnava et al., 2014).

Previously, therapeutic plants were the source for a number of clinically significant drugs such as morphine, digoxin and atropine, and in the search for new drugs these plants are excellent sources of lead compounds.

Malaysia is a fast developing country with a population that approaches 31.7 million (Department of Statistics Malaysia, 2015). The prevalence of traditional and complementary medicine usage in Malaysia is moderately high (Aziz and Tey, 2009;

Hasan et al., 2009), particularly for adults in their middle-ages who consume these

medicines to improve their well-being, which includes sexual libido (Hassali et al.,

2012). Studies in Malaysia have also shown that patients with chronic diseases (Hasan et al., 2009), and cancer (Farooqui et al., 2012) also choose traditional and complementary medicines as an alternative source of treatment. The search for novel anti-hyperuricemic compounds from medicinal plants and herbal products is continuing. Currently, the largest underexplored rainforest for the discovery of novel drugs is in tropical and subtropical provinces of the world (Balick et al., 1996).

Malaysia having located in this area is well recognised for its diverse nature and forests. Malaysians also adopt traditional remedies as an alternative choice for the prevention and treatment of diseases, including gout and rheumatism, as indicated by the overserved abundance of herbal supplements in the Malaysian market. However, the validity of most of these claims has not been scientifically proven and therefore, it is of interest to evaluate the anti-hyperuricemic effect of the local plants in Malaysia.

Clinacanthus nutans Lindau is a plant species that is categorised under the

Acanthaceae family. It is a small shrub generally found in South East Asia, specifically in Malaysia and Thailand (Tuntiwachwuttikul et al., 2004).

In Malaysia, the dried leaf of C. nutans, in a specific amount, is traditionally boiled with water and consumed as a herbal tea. The fresh leaves of C. nutans are usually consumed raw, or mixed with other juices, such as apple juice, green tea, or sugarcane juice, and served as a fresh drink or an energising beverage, moreover, there are many products on the market that are based on C. nutans products, such as herbal teas, capsules, coffee sachets, tablets, and concentrated plant extracts (N.

Shakir, personal communication, June 14, 2016). It has been traditionally used to treat cancer, diabetes, fever, diarrhoea, and dysuria (Chu, 2013). It has also been established that this plant has heat and stasis-reducing effects, and regulation of

menstruation, and liver and gallbladder cleansing, (Shim et al., 2013). Leaves of C. nutans has been also widely used as an anti-hepatitis, analgesic, antioxidant, antidiabetic, and anti-herpes agent. In Thailand and Indonesia, it has been used as anti-inflammatory agents for the treatment of insect bites and allergic responses

(Yoosook et al., 1999).

Metabolomics resemble a snapshot picture of a particular organism, which displays the compounds available and in what quantities at a certain point of time.

Analysing several samples (i.e. several snapshots) allows metabolites marker identification or patterns that are distinguishing for a species, a cultivar, or a particular stage of development (Worley and Powers, 2013). It also enables the identification of conditions, such as stress, disease state, or daily and seasonal changes. In other words, the number of variables in a metabolomics study is almost unlimited. To organise these variables, they can be separated to three types: a genotype’s metabolomic characterisation; states of development; and changes due to environmental or external factors (Kim et al., 2011).

Recently, novel methods are developing, which could assist in offering a better understanding of the mechanism of action of medicinal plants, and could guide to the likelihood of getting evidence and assurance of the pharmacological activities over variable raw materials batches. These approaches are based on the holistic consideration of the natural products, i.e. the majority of phytochemical components, instead of concentrating on particular distinct components or a sets of active compounds (Yuliana et al., 2011). On the other hand, the triggered changes of a particular sample on the, and/or the metabolomic patterns, transcriptome, or proteome can be compared with the changes seen after treatment with identified drugs, instead of only observing explicit changes.

1.2 PROBLEM STATEMENT

Conventional medications for the treatment of hyperuricemia, such as allopurinol and probenecid, are mostly used to inhibit the production of uric acid and promote the excretion of uric acid, and they are clinically effective. However, these drugs have significant side effects, such as liver and kidney function damage, bone marrow suppression, gastrointestinal reactions, and relapse after medication withdrawal, thus, long-term administration of these medications is not recommended (Fam et al., 2001;

Khoo & Leow, 2000; Pan et al., 2013). Therefore, the development of novel hypouricemic agents with greater efficacy and a broader safety profile is greatly needed. Traditional uses of C. nutans leaf suggest that it possesses anti-inflammation and anti-hyperuricemia properties which relate to suppression of gout with low side effects. However, these claims lack scientific evidence which will be explored in this study.

1.3 RESEARCH OBJECTIVES

This study embarks on the following objectives:

1. To investigate the effect of different extracts and doses of C. nutans leaf in

the normalisation of uric acid levels in hyperuricemic mice.

2. To investigate the effect of oral administration of C. nutans leaf in

hyperuricemic mice based on metabolite fingerprinting of their serum

following the metabolomics approach.

3. To evaluate the acute toxicity effect of C. nutans extract on the kidney and

liver at the selected doses.

1.4 RESEARCH QUESTIONS

1. Which extracts and doses of C. nutans leaf extracts is effective in

normalising uric acid levels in hyperuricemic mice?

2. Will the most effective extract and dose of C. nutans leaf be able to

normalise hyperuricemia mice based on metabolite fingerprint of its

serum?

3. Do the effective doses have toxic effects?

1.5 RESEARCH HYPOTHESIS

C. nutans leaf extract is effective in treating hyperuricemia.

CHAPTER TWO

LITERATURE REVIEW

2.1 CLINACANTHUS NUTANS LINDAU

2.1.1 Botanical Aspects and Geographical Distributions

Clinacanthus nutans Lindau (Figure 2.1) is known as Sabah Snake Grass, or Pokok

Belalai Gajah in Malaysia, Dandang Gendis or Ki Tajam in Indonesia, twist of flowers or alligator flower in China, and phaya yo, phaya plong thong, or saled pangpon tua mea (saliva of female mongoose) in Thailand (Aslam et al., 2015; Kosai et al., 2016; Nadiah et al., 2016). It belongs to the family of Acanthaceae, and it is a well-known South East Asian medicinal plant. Taxonomically this plant can be classified as the following: Kingdom: Plantae; phylum: Magnoliophyta; class:

Magnoliopsida; subclass: Asteridae; order: Lamiales; family: Acanthaceae; genus:

Clinacanthus Lindau; and species: C. nutans (Burm. f.) Lindau (Wu et al., 2001).

Figure 2.1 Clinacanthus nutans farm in Malaysia

This plant is a small shrub that can be found throughout South East Asia and

China, and it is primarily indigenous to Thailand, Indonesia and Malaysia. The

Acanthaceae family is a principal dicotyledonous flowering group of plants that includes about 2,500 species and 250 genera. The majority of these plants are shrubs, twining vines, or tropical herbs; and a number of them are epiphytes. Species from the

Acanthaceae family are mainly distributed in Africa, South and Central America, and

South-East Asia. The species of this family can strive in most habitats, including dense or open forests, damp soils and valleys, sea shores, swamps, and mangroves.

This family is believed to be among the main origins of therapeutic herbs that yields effective traditional remedies against particular diseases. C. nutans is one of the significant species of the Acanthaceae family and it is being utilised extensively in

South East Asia. At present, C. nutans has been widely studied regionally for its numerously acclaimed medicinal properties (Yahaya et al., 2015). This plant is known among traditional healers in Thailand as an antidote against snake venom. In Thailand,

Malaysia, and Indonesia, C. nutans is for treating a number of diseases, for instance fever, skin rashes, diabetes mellitus, insect bites, and they work as diuretics

(Kongkaew & Chaiyakunapruk, 2011; Shim et al., 2013)

This perennial herb can reach a height of 1 m with the growth of pubescent branches. The leaves are structurally simple, narrowly elliptic-oblong or lanceolate in shape, opposite in direction (3.5–14.0 cm long × 1.0–2.0 cm wide), and pale green to dark green in colour. The stems of these shrubs are cylindrical, striate and glabrescent, which resembles the curve of an elephant’s trunk, hence, the name Pokok Belalai

Gajah (elephant’s trunk plant) in Malay. The petioles are 0.5–1.5 cm in length, and they are sulcated, bifarious. The leaf blades are structurally lanceolate-ovate, lanceolate, or linear-lanceolate. The tips of the leaves are acute or acuminate; and with