EFFECT OF CUCURBITACEAE EXTRACTS ON GLUCOSE AND LIPID METABOLISM IN 3T3-L1 ADIPOCYTES BY SASSI ASSIA A thesis submitted in fulfillment of the requirement for the degree of Master of Health Sciences (Nutrition Sciences) Kulliyyah of Allied Health Sciences International Islamic University Malaysia FEBRUARY 2017 ABSTRACT The use of herbal remedies in the prevention of disease conditions has been the focus of many studies due to their multiple health benefits. One such condition is a state of hyperglycemia which may be altered with the use of natural products originating from herbs, fruits or vegetables. Three of the proclaimed vegetables were used in this study, a commonly consumed gourds belonging to the Cucurbitaceae family, namely: Momordica charantia (also known as Bitter gourd, Karela or Peria Katak), Trichosanthes cucumerina (known as Snake gourd or Labu Ular) and Lagenaria siceraria (known as Bottle Gourd, Opo squash or Calabash). Water and ethanol extracts from whole vegetable, peels and seeds were assessed for antioxidant activities using 2, 2-diphenyl-l-picrylhydrazyl assay (DPPH assay), total phenolic assay (TPC assay) and total flavonoid assay (TFC). The total phenolic contents were highest in the ethanol extract of Lagenaria Siceraria peels (LSPe), the water extract of Momordica charantia seeds (MCSw) and the ethanol extract of Trichosanthes cucumerina peels (TCPe) being 35.58±1.2, 33.03±1.4 and 20.70±1.26 mg GAE/g, respectively. Total flavonoid contents were highest in the seeds of all three vegetables for the ethanol extract of Lagenaria Siceraria seeds (LSSe), the water extract of Trichosanthes cucumerina seeds (TCSw) and the ethanol extract of Momordica charantia seeds (MCSe) being 0.73±0.01, 0.67±0.02 and 0.65±0.02 mg QE/g, respectively in accordance with the high total phenol content. The extracts exhibited high free radical scavenging activity with the highest inhibition for DPPH being 79.80, 73.92 and 71.97% for LSPe, TCPe and MCSw, respectively. Cell viability assay for water and ethanol extracts revealed that 3T3-L1 adipocytes maximum toleration concentration was 0.063 mg/ml. The extracts were further tested on adipocytes’ differentiation and showed a stimulation of lipid droplets formation during adipogenesis. The extracts significantly (p<0.001) increased glycerol release levels during adipolysis compared to the control and were higher in all three sources for the ethanol extracts of Trichosanthes cucumerina whole vegetable (TCWe), Lagenaria siceraria whole vegetable (LSWe) and Momordica charantia peels (MCPe), respectively. The extracts also significantly (p<0.001) promoted the uptake of glucose in the cells for the ethanol extract of Lagenaria siceraria whole vegetable (LSWe), ethanol extract of Trichosanthes cucumerina whole vegetable (TCWe) and water extract of Momordica charantia seeds (MCSw) for up to 81.31, 71,24 and 56.18%, respectively. The effect of the extracts was further tested on adiponectin and leptin concentrations in adipocytes using ELISA kit assay where it indicated a significant (p<0.01) increase in adiponectin concentrations for the water extract of Lagenaria siceraria peels (LSPw), the water extract of Momordica charantia peels (MCPw) and the water extract of Trichosanthes cucumerina seeds (TCSw) with 5405±1557, 4966±1097 and 3593±225 ng/ml, respectively and a decrease in leptin concentrations for the extracts LSPw and MCSe with 14633±612 and 17578±1536 pg/ml, respectively. The present study data showed that there is a beneficial effect of the extracts on adipogenesis, adipolysis and glucose uptake in 3T3-L1 adipocytes. Furthermore, the observed adipocytes concentrations of adiponectin and leptin could be of clinical importance in energy regulation which is a key factor in diabetes, obesity and metabolic syndrome. ii خﻻصة البحث استخدام العﻻجات العشبية يف الوقاية من احلاﻻت املرضية يعترب حمورا للعديد من الدراسات بسبب فوائدهم الصحية املتعددة. أحد هذه احلاﻻت هو حالة ارتفاع السكر يف الدم اليت ميكن أن تتغري مع استخدام املنتجات الطبيعية اليت تستخلص من اﻷعشاب والفواكه أو اخلضروات. ثﻻثة من اخلضروات مت استخدامها يف هذه الدراسة، القرع الذي يشيع تناوله ينتمي للعائلة القرعية، وهي: قرع مر Momordica charantia )املعروف أيضا ابسم القرع املر، كاريﻻ أو برياي كااتك، Trichosanthes cucumerina)املعروفة ابسم اﻷفعى القرع أو ﻻبو أوﻻر( وLagenaria siceraria )املعروفة ابسم زجاجة القرع، ﻻبو سكواش أو كاﻻابش(. مت اﻻستخﻻص ابستخدام املاء واﻻيثانول )اﻻستخﻻص من اخلضروات كلها، القشور والبذور( لﻷنشطة املضادة لﻷكسدة ابستخدام فحص 2، 2-ثنائي-ل picrylhydrazyl فحص (DPPH(، وجمموع الفينول )فحص TPC) وجمموع فحص الفﻻفونويد (TFC). بلغ إمجايل حمتوايت الفينول اﻷعلى يف مستخلص اﻹيثانول من القشور(Lagenaria siceraria (LSPe ، ومستخلص املياه من بذور القرع املر (MCSw) ومستخلص اﻹيثانول من القشور Trichosanthes cucumerina (TCPe)كانت 35.58 ± 1.2، 33.03 ± 1.4 و 20.70 ± 1.26 مليجرام GAE/ جرام على التوايل. وبلغ إمجايل حمتوايت الفﻻفونويد أعلى يف بذور مجيع اخلضروات الثﻻثة ملستخلص اﻹيثانول من البذور(Lagenaria siceraria (LSSe ، ومستخلص املياه من البذور (Trichosanthes cucumerina (TCSw ومستخلص اﻹيثانول من بذور القرع املر (MCSE) كانت 0.73 ± 0.01، 0.67 ± 0.02 و 0.65 ± 0.02 مليجرام QE / جرام على التوايل. تأظهر املستخلصات ارتفاعا عاليا يف نشاط الكسح للجذور احلرة وفقا ﻷعلى تثبيط DPPHحيث كانت 79.80، 73.92 و 71.97٪ للTCPe ،LSPe و MCSwعلى التوايل. كشف فحص من و اخلﻻاي للمستخلصات املائية واﻻيثانول أن تركيز اخلﻻاي الدهنية أقصى تركيز للتحمل 0.063 مليجرام / مليلرت. مت اختبار مستخلصات أخرى على متايز اخلﻻاي الدهنية وأظهرت حتفيز تشكيل قطرات الدهون أثناء تكون الشحم. ارتفعت مستوايت تركيز اجللسرول ارتفاعا ملحوظا (P <0.001) خﻻل حتلل الدهون مقارنة ابجملموعة الضابطة وكانت أعلى يف مجيع املصادر الثﻻثة للمستخلص اﻹيثانول من Trichosanthes cucumerina اخلضار كلها(Lagenaria ، (TCWe Sicerariaاخلضار كلها (LSWe) وقشور القرع املر (MCPe ) على التوايل. املستخلصات أيضا اظهرت ارتفاعا بشكل ملحوظ )P <0.001( لتعزيز امتصاص اجللوكوز يف اخلﻻاي ملستخلص اﻹيثانول من Lagenaria Siceraria اخلضار كلها(LSWe) ، ومستخلص اﻹيثانول من Trichosanthes cucumerina اخلضار كلها (TCWe) ومستخلص املياه من بذور القرع املر (MCSw)تصل إىل 81.31، 71،24 و 56،18٪على التوايل. مت اختبار أتثري املستخلصات برتكيزات أخرى لﻻديبونيكتني وهرمون الليبتني يف اخلﻻاي الدهنية ابستخدام فحص ال ELISA حيث أشارت إىل ارتفاع ملحوظ (p <0.01) يف تركيزات اﻻديبونيكتني ملستخلص املياه من القشور(Lagenaria Siceraria (LSPw ، ومستخلص املياه من مومورديكا القشور (Momordica charantia (MCPwومستخلص املياه من بذور (Trichosanthes cucumerina (TCSw بنسب 5405 ± 1557، 4966 ± 1097 و 3593 ± 225 اننوجرام / مليلرت على التوايل واخنفاض يف تركيزات هرمون الليبتني ملستخلصات LSPw بنسب 14633 ± 612 و 17578 ± 1536 جزء من اجلرام / مليلرت على التوايل. وأظهرت بياانت الدراسة أن هناك أتثري مفيد من املستخلصات على تكوين الدهون، حتلل الدهون وامتصاص اجللوكوز يف اخلﻻاي الدهنية .وعﻻوة على ذلك، قد لوحظ أن تركيز اخلﻻاي الدهنية لﻻديبونيكتني واللبتني قد تكون ذات أمهية سريرية يف تنظيم الطاقة الذي يشكل عامﻻ رئيسيا يف مرض السكري، والسمنة ومتﻻزمة التمثيل الغذائي. 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 of Health Sciences (Nutrition Sciences). ………………………………………….. Muhammad Muzaffar Ali Khan Khattak 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 of Health Sciences (Nutrition Sciences). ………………………………………….. Zaildul Islam Sarker Internal Examiner ………………………………………….. Loh Su Peng External Examiner This thesis was submitted to the Department of Nutrition Sciences and is accepted as a partial fulfillment of the requirements for the degree of Master of Health Sciences (Nutrition Sciences). ………………………………………….. Nor Azwani Binti Mohd Shukri Head, Department of Nutrition Sciences This thesis was submitted to the Kulliyyah of Allied Health Sciences and is accepted as a partial fulfillment of the requirements for the degree of Master of Health Sciences (Nutrition Sciences). ………………………………………….. Wan Azdie Bin Mohd Abu Bakar Dean, Kulliyyah of Allied Health Sciences iv DECLARATION I hereby declare that this thesis is the result of my own investigation, 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. Sassi Assia Signature…………………. Date ….................. v INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH I declare that the copyright holders of this dissertation are jointly owned by the student and IIUM. Copyright © 2017 Sassi Assia and International Islamic University Malaysia. All rights reserved. EFFECT OF CUCURBITACEAE EXTRACTS ON GLUCOSE AND LIPID METABOLISM IN 3T3-L1 ADIPOCYTES 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 retrieval 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 Commercialization policy. Affirmed by Sassi Assia ……..……..…………… ………………….. Signature Date vi ACKNOWLEDGEMENT “No pain that we suffer, no trial that we experience is wasted. It ministers to our education, to the development of such qualities as patience, faith, fortitude and humility. All that we suffer and all that we endure, especially when we endure it patiently, builds up our characters, purifies our hearts, expands our souls and it is through sorrow and suffering, toil and tribulation, that we gain the education that we come here to acquire.” O.F.W.