PHYTOCHEMICAL CONSTITUENTS OF X PARADISIACA ABB FLOWER EXTRACTS AND ITS GALACTAGOGUE EFFECTS ON LACTATING RATS

BY

AZIZAH MAHMOOD

A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy (Biotechnology)

Kulliyyah of Science International Islamic University Malaysia

JUNE 2014 ABSTRACT

Banana, which is native to Southeast Asia, is considered as one of the most important contribution to the international fruit industry and being a very important source of income, employment and export earnings in some developing countries. The present research focused on the agricultural by product in which the flower of Musa x paradisiaca ABB was selected to study for its phytochemical constituents and galactagogue effects on lactating rats. Initially, varieties of musa species were screened for the presence of secondary metabolites, followed by quantitative analysis and antioxidant activities of the phytochemicals in the selected species. The potential use of the flower in the form of crude and aglycone form of aqueous extract (AFAE) were then extensively study for its effects on galactagogue, growth rate of the suckling pups, mammary glands development and nutritional quality of the milk produced. Chemical compounds that responsible to the galactagogue activities were isolated and characterized for its chemicals structure determination at the end of the study. Result of phytochemicals screening on Musa extracts showed the presence of alkaloids, glycosides, steroids, saponins, tannins, flavanoids and terpenoids. DPPH free radical scavenging assay indicated potential use of the extracts as antioxidant agent. Galactagogue measurement with the crude by weight-suckle-weight method showed that the aqueous extract gave significant milk production compared to petroleum ether and ethanol extracts. Comparable amount of milk was also identified in the rats treated with AFAE (4.65± 1.67 g/pup/day) and the aqueous crude extracts (4.86 ± 2.36 g/pup/day). These values are equivalent to 28.03% and 24.87% higher of milk production in the aqueous and AFAE extracts, respectively, than the control group. AFAE and aqueous extract showed higher weight gain of pups than condensed tannin (CT) and metoclopramide within the experimental period. Pups growth rate was significantly improved for the groups treated with the aqueous and AFAE extract. Analysis on mammary gland showed that AFAE was capable to significantly increase the weight of mammary gland (31.73 ± 0.44 mg/g body weight), protein content of the gland (11.66 ± 0.44 g/100g mammary gland) and size of alveoli (124.49 – 139.91 µm) compared to CT (106.83 – 112.85 µm). Consistency of the results from observations on mammary gland and measurement of milk production in vivo confirmed the role of AFAE in promoting galactagogue activities. AFAE was also found to provide more wholesome of milk production because analysis showed it contains higher protein (34.29 ± 0.30%) and lower fat (43.71± 0.90%) than the control group. Spectroscopy data of 1H-NMR, 13C-NMR, IR and MS spectra indicated that bioassay guided isolation of the AFAE afforded four types of compounds; stigmasterol, β-sitosterol, caryophyllene and ophiobolin. In conclusion, the bioactive compounds exist in the flower were identified as the compounds responsible to the increased of milk production in lactating rats. This study provides novel information regarding phytochemical compounds from Musa x paradisiaca ABB species which influenced galactagogue activities. The benefits of this work could be extended for pharmacological use of the extract compound for mothers of inadequate milk production and/or could be exploited for commercialization for pets and livestock.

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ABSTRAK

Pisang, yang berasal dari Asia Tenggara, adalah salah satu penyumbang utama industri buah global dan menjadi sumber penting pendapatan, pekerjaan dan eksport sesetengah negara membangun. Kajian ini memberi tumpuan kepada hasil sampingan pertanian di mana jantung pisang daripada Musa x paradisiaca ABB dipilih untuk dikaji kandungan fitokimia dan kesannya sebagai agen peningkatan susu (galactagogue) pada tikus yang menyusu. Peringkat awalnya, pelbagai spesis Musa disaring berdasarkan kehadiran fitokimia sekunder, diikuti analisis kuantitatif dan aktiviti antioksidan fitokimia berkaitan untuk spesis terpilih. Potensi kegunaan jantung pisang dalam bentuk ekstrak kasar dan ekstrak air tanpa glukosida (AFAE) dikaji secara intensif dari aspek kesan galactagogue, kadar pertumbuhan anak susuan, perkembangan kelenjar susu dan nilai nutrisi susu. Bahan kimia yang bertanggungjawab terhadap aktiviti galactagogue ditentukan di akhir kajian. Keputusan pemeriksaan fitokimia ekstrak Musa menunjukkan kehadiran alkaloid, glikosida, steroid, saponin, tanin, flavanoid dan terpenoid. Radikal bebas scavenging assay dengan DPPH, menunjukkan ekstrak ini berpontensi sebagai agen anti pengoksidaan. Pengukuran pengeluaran susu menggunakan kaedah timbang-susu- timbang menunjukkan ekstrak air dapat meningkatkan susu secara ketara berbanding ekstrak petroleum eter dan etanol. Kesetaraan jumlah susu dikenalpasti bagi tikus yang dirawat dengan AFAE (4.65± 1.67 g/anak/hari) dan ekstrak air (4.86 ± 2.36 g/anak/hari). Ukuran ini adalah bersamaan dengan peningkatan susu sebanyak 28.03% dan 24.87% lebih tinggi secara relatif bagi kumpulan ekstrak air dan AFAE berbanding kawalan. Ekstrak AFAE dan air menunjukkan peningkatan berat anak yang lebih tinggi berbanding condensed tannin (CT) dan metoclopramide dalam tempoh eksperimen. Kadar pertumbuhan anak meningkat secara ketara bagi kumpulan yang dirawat dengan ekstrak air dan AFAE. Analisis ke atas kelenjar susu menunjukkan bahawa AFAE dapat meningkatkan secara ketara berat kelenjar (31.73 ± 0.44 mg/g berat badan), kandungan protein kelenjar (11.66 ± 0.44 g/100g kelenjar susu) dan saiz alveoli (124.49 – 139.91 µm) berbanding CT (106.83 – 112.85 µm). Keputusan yang konsisten dari pemerhatian ke atas kelenjar susu dan pengukuran pengeluaran susu secara in vivo mengesahkan peranan AFAE dalam merangsang aktiviti galactagogue. AFAE juga menyumbang kepada pengeluaran susu yang sihat kerana analisis menunjukkan ia mengandungi protein yang lebih tinggi (34.29 ± 0.30%) dan lemak yang lebih rendah (43.71 ± 0.90%) berbanding kumpulan kawalan. Data spektroskopi menggunakan spektrum 1H-NMR , 13C-NMR , IR dan MS menunjukkan pengasingan bioesei berpandu ke atas AFAE menghasilkan empat kompoun; stigmasterol, β-sitosterol, caryophyllene dan ophiobolin. Secara kesimpulannya, sebatian bioaktif yang wujud dalam bunga telah dikenal pasti sebagai sebatian yang bertanggungjawab kepada peningkatan pengeluaran susu bagi tikus yang menyusu. Kajian ini memberikan novel maklumat mengenai sebatian fitokimia dari spesis Musa x paradisiaca ABB yang mempengaruhi aktiviti galactagogue. Manfaat dari hasil kerja ini boleh dimajukan untuk kegunaan farmakologi bagi kegunaan ibu-ibu yang ketidakcukupan pengeluaran susu dan/atau seterusnya boleh dieksploitasi untuk dikomersialkan bagi kegunaan haiwan peliharaan dan ternakan.

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خالصة البحث

املوز، واليت تعود اصوهلا اىل مناطق جنوب شرق اسيا، وتعترب من اهم املسامهات لصناعة الفاكهة الدولية ومصدرا مهما للدخل والعمالة والصادرات يف بعض الدول النامية. منط منو املوز يؤدي اىل وفرة الزهور. مت اختيار موز الربا سيديا ABB لدراسة الرتكيب الكيميائي والفعالية احليوية كمدر للحليب عند الفئران. بداية، مت فحص بعض أصناف املوز للبحث عن املستقلبات الثانوية مث التحليل الكمي والفعالية املضادة لألكسدة للمواد الكيميائية النباتية يف االنواع املختارة. متت دراسة امكانية استخدام خالصة االزهار وكذلك الشكل الالسكري للخالصة املائية )FAFA( كمدر للحليب وكذلك معدل منو اجلراء الرضيعة، وتطور الغدد الثديية واجلودة الغذائية للحليب املنتج. مت عزل وتوصيف البنية الكيميائية للمركبات الكيميائية املسؤولة عن ادرار احلليب حيث أظهرت النتائج وجود قلويدات، جليكوسيدات، واملنشطات، الصابونني, العفص، فالفونيدات وتريبينوئيدات. بينت نتائج فحص اجلذور احلرة DPPH فعالية عالية للخالصات كمضادات لألكسدة. استخدمت طريقة الوزن-الرضاعة- الوزن وقد بينت النتائج إنتاج كمية حليب كبرية مقارنة لدى استخدام اخلالصة املائية مقارنة مع خالصة االيرت واإليثانول. وقد مت احلصول على كميات معتربة من احلليب أيضا يف الفئران املعاجلة ب AFAE)1.67 ±4.65غ/اجلرو/يوم( واخلالصة املائية )68.4± 68.4 ملغ/اجلرو /يوم(. هذه القيم تعادل .6.88 ٪ و 668.2 ٪ أعلى من جمموعة الشاهد للخالصة املائية و AFAE، على التوايل. أظهرت AFAE و املستخلص املائي أعلى زيادة وزن يف اجلراء من مستخلص التانني املكثف )TC( و ميتوكلوبراميد وكذلك معدل منو اجلراء. وأظهر حتليل الغدة الثديية أن AFAE قادرة على زيادة كبرية يف وزن الغدة الثديية ).782.± 8866 ملغ / غ من وزن اجلسم ( حمتوى الربوتني )77844 ± 8866 غ/ 788 غ الغدة الثديية( وحجم احلويصالت اهلوائية 766861-7.1817ميكرون( مقارنة مع )112.85 – 106.83 ميكرون( لل CT. أكدت نتائج الدراسة على يف االنسجة دور AFAE يف تعزيز ادرار احلليب وبني حتليل AFAE أنه حيتوي على أعلى نسبة بروتني )6861. ± 88.8٪( واقل نسبة دهون )6.827 ± 8818٪( من اجملموعة الشاهد. وأظهرت نتائج التحليل الطيفي 1H-NMR, 13C-NMR ، اشعة حتت احلمراء و الكتلة عزل اربعة مركبات من AFAE باسلوب العزل احليوي وهي ستيغماستريول، β سيتوستريول، ، كريوفيلني و اوفيوبولني. يف اخلتام، أثبتت هذه الدراسة وجود مركبات فعالة حيويا كمدرات للحليب يف زهور املوز. من املمكن عزل هذه املركبات واستخدامها لألمهات اللوايت يعانني عن عدم كفاية إنتاج احلليب ميكن استغالهلا للتسويق وزيادة احلليب لدى ا اتحليوان االليفة واملواشي.

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APPROVAL PAGE

The thesis of Azizah Binti Mahmood has been approved by the following

...... Muhammad Nor Omar Supervisor

...... Nurziana Binti Ngah Co-Supervisor

...... Jamaluddin Bin Mohd Daud Internal Examiner

...... Muhamad Bin Zakaria External Examiner

...... Nasr el-Din Ibrahim Ahmed Chairman

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DECLARATION PAGE

I hereby declare that this dissertation is the results 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.

Azizah Mahmood

Signature ...... Date......

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COPYRIGHT

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

Copyright@2014 by Azizah Mahmood. All right reserved.

PHYTOCHEMICAL CONSTITUENTS OF MUSA X PARADISIACA ABB FLOWER EXTRACTS AND ITS GALACTAGOGUE EFFECTS ON LACTATING RATS

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 only 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.

Affirmed by Azizah Mahmood.

Signature...... Date......

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ACKNOWLEDGEMENTS

First of all, I would like to acknowledge my sincere thanks and deepest respect to my supervisor, Associate Professor Dr. Muhammed Nor bin Omar for giving me the opportunity to pursue my doctorate in the Kulliyyah of Sciences and for his uncountable supporting and kindness. I also indebted to my co-supervisor Assistant

Professor Dr. Nurziana binti Ngah for her full guidance and helpful advice for this dissertation. Special thanks are to Professor Dr. Ahmed Jalal Khan Chowdhury, the

Deputy Dean of Post-graduate for the Kulliyah of Science, for giving me supporting and consideration throughout this study.

My sincere thanks are to brother Ahmad Muzammil on providing the facilities for my work, brother Zulkifli for helping on animal caring and treatments along the study and also other laboratory members for their technical assistance. My special thanks go to my colleagues at the Research Laboratory of Biotechnology, Maryana

Mohammad Nor, Husna Hawa Mohd Hassan and other graduate or undergraduate students for useful scientific hints and friendship.

My thanks would go to my beloved husband, Rozi Said for his never ending support and motivation and my childrens Alya, Arfa, Alfi and Amri for their loving considerations all through these years.

Finally, I would like to acknowledge my sincere thanks to Malaysian Ministry of Higher Education for their periodically funding the scholarship during my doctorate. Without their financial supporting I would not have met to this dissertation.

Azizah Mahmood

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TABLES OF CONTENT

Abstract ...... ii Abstract in Bahasa Malaysia ...... iii Abstract in Arabic …………………………………………………………...... iv Approval Page...... v Declaration Page...... vi Copyright...... vii Acknowledgements...... viii List of Tables...... xiv List of Figures...... xvi List of Symbols and Abbreviations ...... xix

CHAPTER ONE: INTRODUCTION, STATEMENT OF THE PROBLEM AND LITERATURE REVIEW ...... 1 1.1 INTRODUCTION AND STATEMENT OF PROBLEM ...... 1 1.2 LITERATURE REVIEW...... 3 1.2.1 The Family of ...... 3 1.2.1.1 Synonym Used Species of ...... 3 1.2.1.2 Other Common Names of Banana Plants ...... 3 1.2.2 The Earliest History of Musaceae Family ...... 4 1.2.3 Classification of Edible ...... 4 1.2.4 Musa acuminata x balbisiana hybrids (ABB Group) ...... 5 1.2.5 The Emerging of Banana Flower and its Physical Characteristics ...... 6 1.2.6 The Utilization of Musa sp. and its Flower ...... 8 1.2.7 Phytomedicinal Values of Musa sp...... 9 1.2.8 Ethnophamacological Studies on Musa sp...... 12 1.2.9 Clinical Tests of Bioactive Compounds of Musa sp...... 13 1.2.10 Clinical Tests Correlate to Bioactive Compounds in the Flower of Musa ...... 14 1.2.11 Galactagogue and Its Definition ...... 15 1.2.12 Breastfeeding Recommendation ...... 15 1.2.13 Significance of the Needs for Galactagogues ...... 15 1.2.14 Adverse Effects of Modern Drug ...... 16 1.2.15 Plants as Potential Alternative Therapies ...... 17 1.2.16 Herbal Galactagogues ...... 17 1.2.17 Indicated Increased of Milk Yield after Intervention of Galactagogue ...... 18 1.2.18 Chemical Constituents of Galactagogue herbs ...... 19 1.2.19 The Effects of Mechanism of Action ...... 20 1.3 HYPOTHESIS OF THE STUDY ...... 21 1.4 OBJECTIVES OF THE STUDY ...... 21

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CHAPTER TWO: SCREENING OF PHYTOCHEMICALS IN BANANA FLOWERS ...... 23 2.1 INTRODUCTION ...... 23 2.1.1 Phytochemical Screening of Extracts ...... 24 2.1.2 Bioactive Phytochemicals ...... 25 2.1.3 Phytochemicals and Antioxidant Activity ...... 27 2.1.4 Types of Secondary Phytochemicals ...... 28 2.1.4.1 Saponin ...... 28 2.1.4.2 Phenolic compounds ...... 29 2.1.4.3 Flavanoids ...... 30 2.1.4.4 Alkaloids ...... 33 2.1.4.5 Tannin ...... 34 2.1.4.6 Terpenes ...... 35 2.1.5 DPPH Free Radical Scavenging Activity ...... 37 2.1.6 Phytochemicals Content in Banana Plant ...... 38 2.1.7 Selection of Appropriate Extraction Solvents ...... 39 2.2 EXPERIMENTAL PROCEDURE ...... 41 2.2.1 Chemicals and Instruments ...... 41 2.2.2 Methods of Phytochemicals Screening ...... 43 2.2.2.1 Collecting and Processing of Plant Materials for Phytochemicals Screening ...... 43 2.2.2.2 Extraction of Plant Materials ...... 44 2.2.2.3 Phytochemicals Screening of the Extract ...... 44 2.2.2.3.1 Test for Flavanoids ...... 44 2.2.2.3.2 Test for Tannins ...... 45 2.2.2.3.3 Test for Alkaloids ...... 45 2.2.2.3.4 Test for Saponins ...... 45 2.2.2.3.5 Test for Glycoside ...... 46 2.2.2.3.6 Test for Terpenoids and Steroids ...... 46 2.2.3 Methods of Phytochemicals Quantitative Analysis ...... 46 2.2.3.1 Determination of Total Phenolic Content ...... 46 2.2.3.2 Determination of Flavonoid Content ...... 47 2.2.3.3 Determination of Tannin Content ...... 47 2.2.3.3.1 Preparation of Reagents ...... 48 2.2.3.3.2 Preparation of Standard Curve of Tannic Acid48 2.2.3.3.3 Preparation of Sample ...... 48 2.2.3.4 Determination of Alkaloid Content ...... 49 2.2.3.5 Determination of Saponin Content ...... 49 2.2.3.6 Determination of Antioxidant Activity ...... 50 2.3 RESULTS AND DISCUSSION ...... 51 2.3.1 Extraction of Raw Material ...... 51 2.3.2 Phytochemical Screening of M. x paradisiaca Flower Extracts ...... 52 2.3.3 Phytochemicals Content in M. x paradisiaca ABB Flower Extract ...... 57 2.3.4 Antioxidants Activity in M. x paradisiaca Flower ...... 62 2.4 CONCLUSION ...... 64

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CHAPTER THREE: GALACTAGOGUE EFFECTS OF CRUDE EXTRACTS OF MUSA X PARADISIACA ABB FLOWER ...... 66 3.1 INTRODUCTION ...... 66 3.1.1 Significance of Breastfeeding ...... 67 3.1.2 Lactogenic Effects of Plants ...... 68 3.1.3 Milk Yield Estimation ...... 68 3.1.4 Pups Growth in Relation to Milk Supply ...... 70 3.1.5 Factors Affected Milk Production ...... 70 3.2 EXPERIMENTAL PROCEDURES ...... 72 3.2.1 Selection of Plant Species ...... 72 3.2.2 Preparation of the Plant Extract ...... 74 3.2.3 Chemicals and Rats Feed ...... 74 3.2.4 Animal Study ...... 75 3.2.5 Intervention Procedures ...... 75 3.2.6 Statistical Analysis ...... 76 3.3 RESULT AND DISCUSSION ...... 77 3.3.1 Milk Production ...... 77 3.3.2 Growth Rate and Weight Gain of Pups ...... 84 3.4 CONCLUSION ...... 88

CHAPTER FOUR: THE EFFECTS OF CRUDE FRACTIONATION ON MILK PRODUCTION IN VIVO ...... 90 4.1 INTRODUCTION ...... 90 4.1.1 Biological Activities of Phytochemicals toward Galactagogue ...... 91 4.1.1.1 Isolation of Condensed Tannins ...... 93 4.1.2 Lactation Induction with Metochlopramide Hydrochloride (reglan® tablet) ...... 94 4.2 EXPERIMENTAL PROCEDURE ...... 95 4.2.1 Extraction of Aglycone Form of Aqueous Extract (AFAE) ...... 95 4.2.2 Extraction of Condensed Tannin (CT) ...... 96 4.2.3 Animal Test ...... 97 4.2.4 Chemicals and Rats Feed ...... 97 4.2.5 Statistical analysis ...... 98 4.3 RESULT AND DISCUSSION ...... 99 4.3.1 Milk Production ...... 99 4.3.2 Growth Rate and Weight Gain of Pups ...... 102 4.3.3 Correlation Between Milk Production and Pups Weight Gain ...... 107 4.3.4 Factors to Consider in Measuring Milk Production ...... 111 4.3.5 Effect of Treatments on Maternal weight ...... 112 4.3.6 Expected Mechanism of Action Toward Galactagogue Effects ...... 112 4.3.6.1 Effects of AFAE on Lactation Performance ...... 113 4.3.6.2 Effects of Condensed Tannin on Lactation Performance ...... 115 4.3.6.3 Effects of Metochlopramide on Lactation Performance ...... 116

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4.4 CONCLUSION ...... 117

CHAPTER FIVE: EFFECTS OF AGLYCONE FORM OF AQUEOUS EXTRACT (AFAE) ON MAMMARY GLAND DEVELOPMENT AND NUTRITIONAL COMPOSITION OF MILK ...... 119 5.1 INTRODUCTION ...... 119 5.1.1 Mammary Gland and Its Development ...... 121 5.1.2 Mammary Glands of Rat ...... 123 5.1.3 Mammary Gland and Milk Production ...... 124 5.1.4 Effects of Herbal Galactagogue on Mammary Gland ...... 126 5.1.5 Protein Content in Mammary Gland ...... 126 5.1.6 Transformation of Nutrients ...... 127 5.1.7 Milk Composition ...... 129 5.2 METHODOLOGY ...... 131 5.2.1 Mammary Gland Histology ...... 131 5.2.1.1 Preparation of Alcoholic Bouin's (Gendre's Solution) ...... 133 5.2.1.2 Method of Tissue Processing ...... 133 5.2.1.3 General Staining Procedures for Microscope slides ...... 133 5.2.1.4 Measurement of mammary alveoli diameter...... 134 5.2.2 Determination of Protein in Mammary Gland ...... 134 5.2.3 Milking Procedure ...... 135 5.2.4 Determination of Nutrients in Rats Milk ...... 137 5.2.4.1 Determination of Proximate Composition in Milk ...... 137 5.2.4.2 Determination of Minerals in Milk ...... 137 5.2.4.2.1 Samples Digestion ...... 137 5.2.4.2.2 Mineral Analysis ...... 137 5.2.5 Chemicals and Equipments ...... 138 5.3 RESULT AND DISCUSSION ...... 140 5.3.1 Mammary Gland Histology ...... 140 5.3.2 Estimation of mammary alveoli diameter ...... 143 5.3.3 Weight of Mammary Gland ...... 147 5.3.4 Protein Content in Mammary Gland ...... 148 5.3.5 Nutrient Composition of Milk ...... 14 5.3.5.1 Proximate composition ...... 149 5.3.5.2 Minerals Composition ...... 152 5.4 CONCLUSION ...... 153

CHAPTER SIX: STRUCTURAL IDENTIFICATION OF COMPOUND ...... 155 6.1 INTRODUCTION ...... 155 6.1.1 Chemical Characterization of Musa x paradisiaca ABB...... 155 6.1.2 Thin Layer Chromatography (TLC) ...... 156 6.1.3 Gas Chromatography/Mass Spectrometry (GC/MS) ...... 156 6.1.4 Infrared (IR) Spectroscopy ...... 157 6.1.5 Nuclear Magnetic Resonance (NMR) ...... 157 6.2 EXPERIMENTAL PROCEDURES ...... 158

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6.2.1 Chemicals and Instruments ...... 158 6.2.2 Sample Isolation ...... 159 6.2.3 Chromatotron Chromatography ...... 159 6.2.4 Preparation of Silica Coating and Handling Procedure of Chromatotron ...... 160 6.2.5 Preparation of Vanillin Sulfuric Acid ...... 161 6.2.6 Isolation of Compounds Using Chromatotron ...... 161 6.2.7 Thin Layer Chromatography (TLC) ...... 162 6.2.8 Nuclear Magnetic Resonance (NMR) ...... 162 6.2.9 GC/MS Analysis ...... 163 6.2.10 Reagents and chemicals ...... 163 6.3 RESULTs AND DISCUSSION ...... 163 6.3.1 Compound MP1 (Stigmasterol) ...... 164 6.3.2 Compound MP2 (β-sitosterol) ...... 168 6.3.3 Compound MP3 (Cryophyllene) ...... 172 6.3.4 Compound MP4 (Ophiobolin) ...... 176 6.4 CONCLUSION ...... 179

CHAPTER SEVEN: CONCLUSION AND SUGGESTION FOR FUTURE WORKS ...... 180 7.1 CONCLUSION ...... 180 7.2 SUGGESTIONS FOR FUTURE RESEARCH ...... 182

BIBLIOGRAPHY …………………………………………………………..….….205 APPENDIX A: List of Conferences and Publications……………………… 203 APPENDIX B: Publication in European Journal of Scientific Research…….. 204 APPENDIX C: Publication in Asian Pacific Journal of Tropical Medicine…. 212 APPENDIX D: Certificate of Approval from Animal Ethics Committee……. 217 APPENDIX E: IR spectrum of PMP1 (stigmasterol)...... 218 APPENDIX F: Mass Spectrum of MP1 (stigmasterol)...... 219 APPENDIX G: IR spectrum of MP2 (β-sitosterol)...... 220 APPENDIX H: Mass spectrum of MP2 (β-sitosterol)...... 221 APPENDIX I: IR spectrum for MP3 (cryophyllene)...... 222 APPENDIX J: MS spectrum of MP3 (cryophyllene)...... 223 APPENDIX K: NMR Spectrum H-H COSY of MP3 (cryophyllene)...... 224 APPENDIX L: IR spectrum of MP4 (ophiobolin)...... 225 APPENDIX M: NMR Spectrum H-H COSY of MP4 (ophiobolin)...... 226 APPENDIX N: One Way Analysis of ANOVA of Milk Production using Crude Extract………………………………………………... 227 APPENDIX O: One...... Way Analysis of ANOVA of Milk Production using Crude Fractionation…………………………………………. 229

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LIST OF TABLES

Table No. Page No.

2.1: Types of phytochemicals extracted by different sovents 40 2.2: Types of chemicals and manufacturers 42 2.3: Instruments used with their model and company 43 2.4: Selected species of Musa flowers for screening of phytochemicals 43 2.5: Percentage of yield extracted by each of the four different extractants 51 from 15 g of dried flower. 2.6: Phytochemicals constituents present in petroleum ether extract 53 2.7: Phytochemicals constituents present in chloroform extract 54 2.8: Phytochemicals constituents present in ethanol (95%) extract 55 2.9: Phytochemicals constituents present in aqueous extract 56 2.10: Total phytochemicals content of banana flower (M. x paradisiaca) 57

2.11: Radical scavenging activity (IC50) of the extracts of M. x paradisiaca flower and BHT 63 3.1: Composition of animal diets as stated on the food label 75 3.2: Milk production of various solvent extracts during 10 days of lactation 77 3.3: Data of milk yield measurement for different treatment groups 83 3.4: Comparison between initial and final weight, and weight gain of pups 86 4.1: List of chemicals used and manufacturer 98 4.2: Milk production of various treatments of extracts groups during 10 days lactation 100 4.3: Comparison between initial and final weight, percentage of body increment and weight gain of pups 103 5.1: Classification of essential trace elements, its functions and deficiency 130 5.2: Chemicals used with their brand and manufacturer 139 5.3: Instruments used with their model and company 139 5.4: Means of alveoli diameter (µm) between different positions of mammary glands 145 5.5: Mammary tissue mass and protein content during lactation based on wet tissue weight 147

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5.6: Nutrients composition of rats milk for various treatments (dry weight basis) 150 5.7: Minerals content of rats milk for various treatments 153 6.1: Instruments used with their model and manufacturer 158 6.2: Types of chemicals and manufacturer 159 6.3: Preparation of silica for preparative TLC of chromatotron glass plate 161 6.4: Comparison of IR spectroscopic data of MP1 with stigmasterol 164 6.5: 1H- and 13C-NMR chemical shift of compound MP1 (stigmasterol) (δ ppm), 500 MHz in d1 - CDCl3 167 6.6: 1H- and 13C-NMR chemical shift of compound MP2 (β-sitosterol) (δ ppm), 500 MHz in d1 - CDCl3 171 6.7: 1H- and 13C-NMR chemical shift of compound of MP3 (cryophyllene) (δ ppm), 500 MHz in d1-CDCl3 175 6.8: 1H- and 13C-NMR chemical shift of compound MP4 (ophiobolin) (δ ppm), 500 MHz 177

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LIST OF FIGURES

Figure No. Page No.

1.1: Geographical distribution of the main species and subspecies ancestral to common edible banana 5

1.2: Banana male bud showing parts of bract and floret 7

1.3: Characters of banana flower used in determining species and genome groups of edible banana 8 1.4: Banana trees and its parts of the plant 10 1.5: An example of banana plant of Musa x paradisiaca ABB 11 1.6: Review of pharmacological activities found in Musa paradisiaca plant 14 2.1: Class of plant bioactive substances 26 2.2: An example of molecular structure of saponin 29 2.3: Scavenging of ROS (R*) by flavanoid 31 2.4: Structures of the six main classes of flavanoids 32 2.5: A molecular structure of Paclitaxel 33 2.6: Basic structure of tannin 35 2.7: The molecule of free radical and reduced form of DPPH 37 2.8: Standard curve for total phenolic content as gallic acid equivalents 58

2.9: Standard curve for total phenolic content as gallic acid equivalents 60 2.10: Standard curve for tannin content as tannic acid equivalents 61 3.1: Banana plant selected in the study 73 3.2: Mean of milk production per day for various treatment groups of lactating 79 3.3: Effect of various solvent extracts of Musa flower on milk production during 10 days of experimental period 81 3.4: Changes in body weight (g) of pups in treated and control groups throughout the experimental period 85 3.5: Percentage of body weight increment of pups for various treatment groups 86 4.1: Chemical structure of metochlopramide HCl (reglan®tablet) 94

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4.2: Effect of treatment on milk production of lactating rats up to 15 days of lactation 101 4.3: Mean of milk produced (g/pup/day ± SD) by rats treated with different samples 105 4.4: Mean of weight gain of suckling pups for various treatment groups 105 4.5: Changes in body weight (g) of pups in the treated and control groups throughout the experimental period 107 4.6: Correlation between the milk production and weight gain for negative control group 108 4.7: Correlation between the milk production and weight gain for metochlopramide 108 4.8: Correlation between the milk production and weight gain for AFAE 109 4.9: Correlation between milk production and weight gain for aqueous extract 109 4.10: Correlation between milk production and weight gain for CT 110 4.11: Correlation between the mean of milk produced by rats and weight gain of the pups 110 5.1: The anatomical location of the rat mammary glands and their numbering 123 5.2: Branching network found in mammary glands 124 5.3: Steps involve in dissection process of mammary gland of rat 132 5.4: A modified milking apparatus 136 5.5: Process of collecting milk using a modified milking apparatus 136 5.6: Histological section of inactive mammary gland of rat (10x) 141 5.7: Histological section of inactive mammary gland of rat (20x) 141 5.8: Histological section of lactating rats mammary gland (20x) 142 5.9: Histological section of lactating rats mammary gland (10x) 142 5.10: Histological section of a single alveolus of mammary gland at higher magnification (100x) 144

5.11: Comparison of the alveoli diameter of mammary glands between control (distilled water), AFAE and CT 146 6.1: Principle of chromatotron technique 159

1 6.2: H-NMR spectrum for MP1 in d1-CDCl3 165 13 6.3: CNMR spectrum for MP1 in d1 - CDCl3 166

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6.4: Chemical structure of stigmasterol 168

1 6.5: H-NMR spectrum for MP2 in d1-CDCl3 169 13 6.6: C-NMR spectrum for MP2 in d1 - CDCl3 170 6.7: Chemical structure of β-sitosterol 172

1 6.8: H NMR spectrum of MP3 in d1-CDCl3 173 13 6.9: C NMR spectrum of MP3 in d1-CDCl3 174 6.10: Chemical structure of caryophyllene 175

1 6.11: H NMR spectrum of MP4 in d1-CDCl3 178 13 6.12: C NMR spectrum of MP4 in d1-CDCl3 178

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LIST OF SYMBOLS AND ABBREVIATIONS

1D One dimentional 2D Two dimentional µL 10-6 litre (s) or microlitre (s) µg 10-6 gram (s) or microgram(s) AA Amino acid ABs Alveolar buds AFAE Aglycon form of aqueous extract ANOVA Analysis of variance BST Bovine growth hormone CP Crude protein cps Counts per second CNS Central nervous system COSY Correlated Spectroscopy CT Condensed tannins DMSO Dimethyl sulfoxide DNA Deoxyribonucleic acid DPPH 1, 1-diphenyl-2-picryl-hydrazyl DSHEA Dietary Supplement Health and Education Act EAA Essential amino acids EGF Epidermal growth factor EtOAc Ethyl acetate EtOH Ethanol FI-O* aroxyl radical FI-OH Alavanoid FDA Food and Drug Administration GC Gas Chromatography GH Growth hormone HCl Hydrochloric acid H&E Haematoxylin and eosin HNO3 Nitric acid HPLC High Performance Liquid Chromatography H2SO4 Sulfuric acid HT Hyroysable tannins Hz Hertz IACUC Institutional Animal Care and Use Committee IC50 Concentration that inhibit a response by 50% relative to positive control ICP-MS Inductively Coupled Plasma Mass Spectroscopy L Liter(s) LSD Least square difference L/min Liter per minute λ (nm) Wavelength (in nanometers) M Molar MAPK Mitogen activated protein kinase MeOH Methanol

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MG Ratio of milk protein (grams per day) mg 10-3 gram (s) or milligram (s) mg/g Miligram per gram ml 10-3 liter (s) or milliliter (s) ml/min Mililiter per minute mbar Milibar m.p. Melting point MΩ cm Milliohm (definition of purified water quality) MS Mass Spectroscopy or Mass Spectrum m/z Mass to charge ratio ND Not determined N Normality NAN Non-ammonia nitrogen NIR Near Infrared Reflectance nm Nanometer NMR Nuclear Magnetic Resonance P Probability PEG Polyethylene glycol Pet ether Petroleum ether PMS Premenstrual syndrome ppb Part per billion ppm Parts per million PRL Prolactin r2 correlation coefficient R* Radicals Rf Retention factor; migration distance of analyte as a fraction of distance to solvent front in thin-layer chromatography RNA Ribonucleic acid ROS Reactive oxygen species rpm Round per minute SHBG Sex hormone binding globulin STAT Signal transducers and activators of transcription Si Silica Sp Species SPSS Statistical Package for the Social Sciences TA Tannic acid TDLUs Terminal ductule lobular units TEBs Terminal end buds TLC Thin Layer Chromatography TMS Tetramethylsilane UV Ultraviolet VFI Voluntary feed intake W Watt WHO World Health Organization

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CHAPTER ONE

CHAPTER 1: INTRODUCTION, STATEMENT OF THE PROBLEM AND LITERATURE REVIEW

1.1 INTRODUCTION AND STATEMENT OF PROBLEM

Bananas and plantains are among the popular and cheapest foods throughout the

tropical and sub-tropical regions of the world. Globally in 2009, approximately 4.9

million hectares of the harvested area planted with bananas and plantains, with an

annual production of around 97.3 million metric tonnes (Evans & Ballen, 2012). A

tremendous increase of banana yield production (49%) was recorded from 2000 to

2009. According to the Food and Agriculture Organization of the United Nations

(FAO) Statistics estimation, world total exports of banana accounted for 18.3 million

tonnes in 2009. In some developing countries of Latin America, the Caribbean,

Southeast Asia and West Africa, banana industry being a very important source of

income, employment and export earnings. Although banana is one of the most

important commercial crops in the world, it is estimated that 87% of the production is

purposely for local consumption (Dzomeku et al., 2007).

The flower of banana is mostly removed from the center of the pseudostem

before harvesting to prompt the growth of fruits thus gain higher yield. Most flowers

have only been used as organic material and fertilizer in plantations and only little of

them were consumed. Although, recently more attention has been focused on the

utilization of agricultural by-products, banana flowers have received little attention

from the world of science toward favorable utilization of the resources. As reported by

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Alisi (2008), banana family has some traditional medicinal value even though it is more of interest for its nutrients than for its medicinal properties.

It is believed that the flowers of musa sp have tremendous pharmacological value based on ethnomedicinal surveys around the world supported by limited bioactivities and clinical research. Banana flowers have been used traditionally to alleviate menorrhagia, dysentery, diabetes mellitus, asthma, heart pain, diarrhea and stomach cramps (Imam & Akter, 2011; Sumathy et al., 2011). Sheng et al. (2010) mentioned that “further studies are needed to document the biological effect of banana flower in physiology and healthy benefits of human being. These may include the investigation of its effect of tannin, phytate, total phenolics flavonoid, alkaloid and sterol in human health as daily diet components”.

Most of banana flowers are acknowledged remarkably astringent in taste.

Because of its astringency, not all flowers of banana species can be eaten and many were recognized to be taboo for post-partum mothers due to its “cooling” properties. As a consequence, the flowers have limited uses in cooking and food products. There is no research reported concerning medicinal uses, isolated substances or pharmacological effects of banana flower means for galactagogue. Therefore, it is meaningful to search for alternative utilizations of the flower to make it more appreciable and valuable.

Through the development of standardized extracts, as well as well-controlled clinical trials, it is hope to increase public interest for products of banana flowers. Thus, this research suggests the alternative therapy for its potential efficacy of bioactive compounds in the management of inadequate breast milk supply (hypogalactia) of breastfeeding women. The utilization of banana flower as lactogenic food could provide additional benefits in reducing its waste, and increasing its uses in pharmaceutical and nutraceutical as a therapeutic agent.

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1.2 LITERATURE REVIEW

1.2.1 The Family of Musaceae

Bananas and plantains derive from the family of Musaceae. They are classified as perennial, monocotyledonous herbs with 2-9 m (6.6-30 ft) in height that arise from subterranean rhizomes. Musaceae family is composed of the genera Musa, and, possibly a third, Musella. Musa is the largest genus in the family and all edible banana fruits are produced by plants belonging to the genus. The word Musa, thought to be derived either from “mouz” (the Arab word for banana), or from Antonia Musa, a

Roman physician to Augustus Caesar (Vàlmayor et al., 1991). Historically, Musa has been classified into five sections based on their chromosome number and moephological characters i.e., Musa x=11, Rhodochlamys x=11, Australimusa x=10,

Callimusa x=10 and Ingentimusa x=7 (x is the number of chromosome in haploid genome) (Daniells et al., 2001).

1.2.1.1 Synonym Used Species of Banana Plants

Scientifically, banana plants can be recognized by many other names as the following;

Ensete glaucum Musa rubra. M. balbisiana. M. basjoo. M. cocciniea. M. uranoscopos.M. feji. M. lasiocarpa. M. nana. M. cavendishii. M. sinensis. M. sapientum. M. troglodytarum. M. wilsonii. Musella lasiocarpa.

1.2.1.2 Other Common Names of Banana Plants

Banana plants can be easily recognized based on their common or local names such as; cooking banana, dessert banana, plantain [English], Mai’a [Hawai’i], ba jiao, gan jiao, xiang jiao, ba jiao gen (stump), kan-lu (nectar), jiao yu (sap) [China]; guinea [Spanish], box haaz. Mopan [Maya]; jaina [Fiji], kera [Nepal]; mei’a [Tahiti]; meika [Cook

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Island]; fa’i. soa’a [Samoa]; kadali [India]; pisang, jantung pisang (flower)[Malaysia]; pisang, getang [Indonesia].

1.2.2 The Earliest History of Musaceae Family

The earliest agriculture in the Pacific region has been dated at around 8,000 BC, in which the Fe’i bananas were of some importance (Sharrock & Frison, 1998). Banana plants in the earliest cultivation have been described as ‘proto-agriculture’ and were collected from the wild rather than planted (Price, (1995). The first scientific term given to banana was Musa paradisiaca Linn. by Karl Linnaeus, in his book Species

Plantarum in 1753. Later Musa sapientum Linn. was introduced in 1759 to describe a dessert banana (Valmayor et al., 2000).

1.2.3 Classification of Edible Bananas

Simmonds & Shepherd (1955) developed a classification system to classify all the edible bananas cultivars systematically. They concluded that the edible bananas originated from two wild and seedy species, Musa acuminata Colla and Musa balbisiana Colla which are native to Southeast Asia. Figure 1.1 shows geographical distribution of both the edible bananas in the region (De Langhe et al., 2009). Musa sapientum were the scientific term given to dessert bananas which stand for sweet fruits that were eaten fresh. Musa paradisiaca, on the other hand was applied to plantains which require cooking before they are eaten.

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