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Clinacanthus Nutans Lindau Extracts on Hyperuricemic Mice

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Clinacanthus Nutans Lindau Extracts on Hyperuricemic Mice EFFECTS OF CLINACANTHUS NUTANS LINDAU EXTRACTS ON HYPERURICEMIC MICE BY 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. ii خﻻصة البحث تسوق نبتة كليناكانتوس نوتانس (Clinacanthus nutans) أو عشبة اﻷفعى الصباحية )فصيلة اﻷقنتيات( حمليا كدواء لعﻻج داء النقرس وفرط محض اليوريك يف الدم. ولكن اﻷدلة العلمية اليت تثبت فعاليتها يف معاجلة هذه اﻷمراض غري كافية. هدفت الدراسة للتحقيق يف أثر اجلرعات املختلفة ملستخلصات نبتة الكليناكانتوس نوتانس يف عﻻج فرط محض اليوريك يف الدم يف الفئران املصابة بفرط يف محض اليوريك، وإجراء التبصيم اﻷيضي ملصل دماء الفئران باتباع طريقة امليتابولوميات املعتمدة على الطيف الكتلي-اﻻستشرا السالل(LC-MS) ، ولتقييم التأثري السمي احلاد جلرعات خمتارة من املستخلصات. مت استخﻻص اﻷوراق اجملففة هلذه النبتة بالنقع يف حماليل اﻹيثانول 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 اﻷسبوع الثالث. مت مﻻحةة نفس اﻷثر مع اﻷلوبيورينول حيث خفض بشكل فعال مستويات محض اليوريك املرتفعة يف الدم طيلة مدة العﻻج )من اﻷسبوع اﻷول إىل الرابع(. على الرغم من ذلك، أظهرت بيانات امليتابولوميات املعطاة من التبصيم املعتمد على الــ LC-MS أن التوصيف اﻷيضي لدماء الفئران املعاجلة باﻷلوبيورينول قد تأثر بشكل سليب بدءا من اﻷسبوع الثاين مقارنة باجملموعة الضابطة. كان من املثري لﻻهتمام أن التوصيف اﻷيضي للفئران املعاجلة باملستخلصات على جرعة 000 مغ/كج من وزن الفأر كان أفضل من تلك املعاجلة باﻷلوبيورينول حيث قامت بارجاع التوصيف اﻷيضي لدماء الفئران يف اﻷسبوع الرابع. مل حيدث العﻻج باﻷلوبيورينول، واملستخلصات املالية، مستخلصات 00% إيثانول أي حالة موت أو أثر جانيب من الناحية السلوكية أو اهليستولوجية لغاية 4 أسابيع من العﻻج. باﻹضافة إىل ذلك، من الواجب القيام بعملية التعرف على املستقلبات الىت هلا دور يف العﻻج وذلك لفهم آلية عمل املستخلصات. تستنتج هذه الدراسة أن مستخلصات الكليناكانتوس نوتانس عﻻج واعد لﻻستعمال يف مداواة الفرط يف مستوى محض اليوريك يف الدم. iii 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 iv 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 ….............................. COPYRIGHT PAGE v 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. Copyright © 2017 Majed bin Abd Ghaffar Abd Halim Maetalong and International Islamic University Malaysia. All rights reserved. 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 vi 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. vii 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 .............................................................................
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