Acrostichum Aureum and A

WOUND HEALING PROPERTIES OF ACROSTICHUM AUREUM AND A. SPECIOSUM ON NIH/3T3 FIBROBLASTS CELL LINE AND EXCISIONAL WOUND ON RABBITS

HENDY PUTRA BIN HERMAN

A thesis submitted in fulfillment of the requirement for the degree of Master of Science (Biosciences)

Kulliyyah of Science International Islamic University Malaysia

JUNE 2014

ABSTRACT

Acrostichum aureum or Piai raya has been known for its medicinal value for wound healing treatment among Malays in Malaysia. In this study, the traditional use of this plant together with A. speciosum (Piai lasa) was proven for the first time in vitro and in vivo. Sampling of both plants have been done in Matang mangroves, Taiping, Perak on February 2011. They were dried, ground into fine powder, extracted with water and ethanol to produce extracts and they were fractionated by Vacuum Liquid Chromatography (VLC) to produce fractions. For the first part of study, screening was done on the extracts to identify their phytochemicals content. From the present study, it was reported that both plants have various potential compounds such as tannins, saponins, alkaloids, and flavonoids. Later, antimicrobial study showed that only Staphylococcus aureus was susceptible to the extract treatment and this led to the second part of study which was in vitro wound scratch assay. From MTT assay, the best concentration for the proliferative effect of fibroblasts was 200 µg/mL and hence it was used for the wound scratch assay. There were eights extracts and fractions that showed better mean migration rate but only four were selected to be tested on rabbits. For in vivo study, three groups of four rabbits each were tested using four different extracts with two different concentrations (5 % and 10 %). The excisional wounds were inflicted at the back of the rabbits and treatments were applied once daily until complete healing. Based on wound contraction, epithelization period and histopathological study, it was found that aqueous extract of rhizomes (10 %) and leaves A. aureum (5 %) showed the best wound healing properties with more collagens and fibroblasts proliferation and complete epithelized cells. Therefore, this study has added some novel findings in exploring the use of A. aureum and A. speciosum as wound healing treatement. In conclusion, this study has successfully justified the medicinal use of rhizomes A. aureum in wound healing treatment. In addition to that value, A. speciosum which was not well-studied and other parts of the plant, stems and leaves, also have been reported to possess similar medicinal properties for the first time.

خ ال صة ال بحث

عُرف نبات الـ )Acrostichum aureum( أو ما يُعرف زللياً عند ادلاليزين بـ ) Piai raya( أنو ذو قيمة طبية وعالجية لتطبيب اجلروح ومعاجلتها. يف ىذه الدراسة وألول مرة مت استكشاف الفعالية الطبية ذلذا النبات باإلضافة إىل نبات الـ)A. speciosum( أو الـ(Piai lasa) يف ىذه الدراسة مت الكشف عن الفعالية الطبية ذلذان النباتان على أنسجة األرانب مباشرة باإلضافية إىل جتربتها على اخلاليا النامية على األطباق ادلخربية. ُُجعت العينات النباتية خالل شهر شباط 2011من منطقة متانج وتايبنج وبرياك يف ماليزيا حيث انتشار األشجار اإلستوائية ادلستهدفة يف ىذه الدراسة. مت جتفيف النبات وطحنو للحصول على مسحوق دقيق احلجم ، ومن مث مت استخالصة بإستخدام الكحول اإليثيلي وادلاء، وبعد ذلك مت جتزأة ادلستخلص بإستخدام تقنية فصل السوائل بالتفريغ (VLC) للحصول على مستخلصات رلزأة من ادلستخلص اإلساسي. يف اجلزء األول من الدراسة ُكشف عن ادلكونات الكيميائية النباتية الفعالة ادلتوفرة يف ادلستخلصات ، ومت التعرف واثبات وجود تنوع من ادلركبات احليوية اذلامة مثل أنواع التانني ، وأنواع السابونني ، وأنواع األلكلويد ، وأنواع الفالفينويد ، وبينت الدراسة أن وجود أنواع التانني ظهر يف مستخلصات الكحول اإليثيلي أكثر من وجوده يف ادلستخلصات ادلائية. حتليل واختبار الـ (MTT) كشف أن 200 ميكروجرام من ادلستخلص ىي أفضل تركيز لزيادة عملية تراكم وتكون الفيربوبالست وىو الرتكيز الذي مت اإلعتماد عليو واستخدامو لتجارب اخلدش فيما بعد. خالل الدراسة تبني أن ىناك مثانية مستخلصات وأجزاء أظهرت معدل ىجرة للخاليا بشكل فعال ، يف ادلقابل فقط مت استخدام أربعة منها يف التجارب على األرانب وذلك حملدودية الكميات ادلتوفرة من باقي ادلستخلصات )أقل من 100 ملجرام(. قُسمت األرانب إىل ثالث رلموعات كل رلموعة حتتوي على أربعة أرانب وكل رلموعة مت تعريضها ألربع مستخلصات سلتلفة برتكيزين سلتلفني ) 5% و 10 %( باإلضافة إلستخدام مادة الـ (Solcoseryl jelly) ك ُمحكم إجيايب باإلضافة إلستخدام كرمي (Aqua) ُزلكم سليب. اجلُروح اصطُنعت يف ظهر األرانب ومت تعريضها للعالج يومياً حيت مت تطبيبها متاماً واختفاء اجلروح ، وطبقاً لرتكيز اجلروح وادلدة الزمنية لتكون النسيج اإلبثيلي والدراسة النسيجية ُوجد أن ادلستخلص ادلائي للسيقان اجلذعية ) 10%( وأوراق نبات الـ ) .A aureum ( )5%( أظهروا نتائج وخصائص أفضل من غريمها يف معاجلة اجلروح ، حيث كان ىناك زيادة يف تكون الكالجني والفيربوبالست واخلاليا اإلبثيلية. وبناءاً على ما سبق فإننا نستطيع القول بأن ىذه الدراسة أضافت معلومات جديدة ومشجعة لإلىتمام هبذان النباتان ادلستهدفني يف ىذه الدراسة إلستكشاف أمهيتهما واستخدامها يف معاجلة اجلُروح وتطبيبها. وأخرياً نستخلص أن ىذه الدراسة بيُّنت أمهية استخدام السيقان اجلذعية ونبات الـ ) A. aureum( يف عالج اجلروح. باإلضافة دلا سبق كشفت الدراسة عن القيمة الطبية لنبات الـ ) A. speciosum( وأمهية دراسة باقي أجزاءه من سيقان وأوراق يف عالج اجلروح حيث أهنم ىذا النبات مل يلقى ادلزيد من اإلىتمام ومل يُدرس بشكل ُمكثف ذلذا اذلدف.

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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 of Science (Biosciences).

...... Deny Susanti Supervisor

...... Muhammad Taher Co-Supervisor

...... Shahbudin Saad 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 of Science (Biosciences).

...... Suzanah Abdul Rahman Internal Examiner

...... Mahmood Ameen Abdulla External Examiner

This thesis was submitted to the Department of Biotechnology and is accepted as a fulfilment of the requirements for the degree of Master of Science (Biosciences).

...... Noor Faizul Hadry Nordin Head, Department of Biotechnology

This thesis was submitted to the Kulliyah of Science and is accepted as a fulfilment of the requirements for the degree of Master of Science (Biosciences).

...... Kamaruzzaman Yunus Dean, Kulliyyah of Science

DECLARATION PAGE

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.

Hendy Putra Bin Herman

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

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

WOUND HEALING PROPERTIES OF ACROSTICHUM AUREUM AND A. SPECIOSUM ON NIH/3T3 FIBROBLASTS CELL LINE AND EXCISIONAL WOUND ON RABBITS

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.

Affirmed by Hendy Putra Bin Herman

……………………….. ………………………..

Signature Date

Dedicated to my family and friends

For outstanding supports and loves

Thank You Allah

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ACKNOWLEDGEMENTS

A great journey shall begin with the very first step.

First of all, I would like to praise Allah S. W. T for giving me perseverance and strength to finish my Master study. I would like to extend my utmost appreciation and grateful to my main supervisor Assoc. Prof. Dr. Deny Susanti and two co- supervisors Assoc. Prof. Dr. Shahbdudin Saad and Assoc. Prof. Dr. Muhammad Taher for their constructive guidance and supervision. Not forgetting also Madam Norazsida Ramli for being a cooperative principal researcher. I am so grateful for the opportunity to be a research assistant for this project, thus special thanks to Ministry of Higher Education for providing FRGS grant for this project and providing MyBrain scholarship for me to pursue my Master study.

Million thanks to all staff of Kulliyah of Science and Kulliyyah of Pharmacy for assisting me to carry out the research until the end especially Br. Muzammil, Br. Halim, Br. Zul, Br. Pok Ya, Br. Azizul, Sr. Yanti from Kulliyyah of Science and Sr. Yanti and Sr. Sri from Kulliyyah of Pharmacy. Not forgetting also staff admin office especially Sr. Lin, Sr. Siti, and Sr. Adah.

For my beloved parents (Haji Herman and Hajjah Yusniarti) and six siblings, four sister-in-laws, and 15 nieces and nephews, thank you so much for all your unconditional love and supports during my two years research. I am blessed to have all my loving ones by my side through thick and thin. Special thanks to Dr. Azrul Safuan and my younger sister Haryanti for assisting me in lab work. I am also in debt with Br. Helmi for the Arabic translation of the abstract. May Allah reward you in hereafter. For all my friends, my fellow postgraduates; Jazli, Fuad, Adah, Ija, Mir, Min, Nisah, Rahima, Putri, Sihin, Kak Yan, Kak Husna, Kak Azizah, and Br. Surairi especially my labmates; Hassan, Nissad, and Zaffar. Jazakallahu khairan kathira. May Allah bless our effort to disseminate scientific knowledge for the ummah. Amin.

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TABLE OF CONTENTS

Abstract ...... ii Abstract in Arabic ...... iii Approval Page ...... iii Declaration ...... vi Copyright ...... vii Dedication ...... vii Acknowledgements ...... viii Table of Contents ...... ix List of Tables ...... xii List of Figures ...... xiii List of Plates ...... xiv List of Appendices ...... xv List of Abbreviations ...... xvi

CHAPTER ONE: INTRODUCTION ...... 17 1.1 Background of Research ...... 17 1.2 Objectives ...... 19 1.3 Research Questions ...... 19 1.4 Hypotheses ...... 20 1.5 Overview of The Study ...... 20

CHAPTER TWO: LITERATURE REVIEW ...... 21 2.1 Medicinal Plants for Wound Healing ...... 21 2.2 Mangroves Ecosystem ...... 23 2.2.1 Use of Mangroves as a Medicinal Plant...... 24 2.2.2 Acrostichum species as a threat to mangroves plants ...... 25 2.2.3 Acrostichum species ...... 26 2.2.3.4 Traditional use of Acrostichum species ...... 29 2.3 Wound Healing Process ...... 31

CHAPTER THREE: EXTRACTION, PHYTOCHEMICALS SCREENING, AND ANTIMICROBIAL PROPERTIES OF ACROSTICHUM SPECIES.36 3.1 Summary ...... 36 3.2 Introduction ...... 37 3.3 Methodology ...... 38 3.3.1 Plants Extraction ...... 39 3.3.2 Phytochemicals Screening ...... 40 3.3.2.1 Alkaloid testing/ Mayer’s test ...... 40 3.3.2.2 Terpenoid and Steroid testing/ Libermann-Burchard test ...... 40 3.3.2.3 Flavonoid testing ...... 41 3.3.2.4 Tannin testing ...... 41 3.3.2.5 Saponin testing ...... 41 3.3.3 Total tannin content ...... 42 3.3.3.1 Preparation of standard calibration curve ...... 42

3.3.4 Antimicrobial study of extracts A. aureum and A. speciosum ...... 43 3.3.4.1 Microbial strain ...... 43 3.3.4.2 Disc diffusion method...... 43 3.4 Results and Discussion ...... 44 3.4.1 Extraction of A. aureum ...... 44 3.4.2 Extraction of A. speciosum ...... 45 3.4.3 Fractionation of A. aureum...... 47 3.4.4 Fractionation of A. speciosum ...... 48 3.4.5 Phytochemicals screening ...... 48 3.4.6 Total tannin content ...... 52 3.4.7 Antimicrobial study of Acrostichum species ...... 55 3.5 Conclusion ...... 58

CHAPTER FOUR: IN VITRO STUDY OF ACROSTICHUM SPECIES ON NIH/3T3 FIBROBLASTS CELL LINE ...... 59 4.1 Summary ...... 59 4.2 Introduction...... 60 4.3 Methodology ...... 62 4.3.1 MTT Assay ...... 62 4.3.2 Scratch Wound Assay...... 63 4.4 Results and Discussion ...... 65 4.4.1 MTT assay ...... 65 4.4.2 Mean migration rate in scratch wound assay ...... 66 4.5 Conclusion ...... 72

CHAPTER FIVE: IN VIVO WOUND HEALING STUDY OF ACROSTICHUM SPECIES ON RABBITS ...... 73 5.1 Summary...... 73 5.2 Introduction ...... 74 5.3 Methodology ...... 76 5.3.1 Rabbit as animal model ...... 76 5.3.2 Excision wound creation ...... 78 5.3.3 Grouping of animals ...... 79 5.3.4 Treatment preparation ...... 79 5.3.5 Topical application of vehicles ...... 79 5.3.6 Determination of wound contraction ...... 80 5.3.7 Histopathological examination ...... 82 5.3.8 Statistical analysis ...... 83 5.4 Results and Discussion ...... 84 5.4.1 Wound Contraction ...... 87 5.4.2 Epithelization Period ...... 90 5.4.2 Histopathological Evaluation ...... 93 5.5 Conclusion ...... 101

CHAPTER SIX: CONCLUSION AND FUTURE WORK ...... 103 6.1 Conclusion ...... 103 6.2 Future Work ...... 104

REFERENCES ...... 105

APPENDIX A ...... 119 APPENDIX B ...... 120 APPENDIX C ...... 121 APPENDIX D ...... 122 APPENDIX E ...... 124 APPENDIX F ...... 125 APPENDIX G ...... 127

LIST OF TABLES

Table No. Page

2.1 Details of plants used for wound healing in Malaysia 22

2.2 Functions of growth factors and cytokines in wound healing 35

3.1 Observation of froth formation for saponin testing 4 2 3.2 Preparation of tannin standard calibration curve 43 3.3 Weight of powder, crude extract, and yield of extract A. aureum 44 3.4 Weight of powder, crude extract, and yield of extract A. speciosum 45 3.5 Weight of crude extract, fractions, and yield of extract A. aureum 47 3.6 Weight of crude extract, fractions, and yield of extract A. speciosum 48 3.7 Results of phytochemicals screening 49 3.8 Total tannin content 53 3.9 Antimicrobial properties of Acrostichum spp. 55 4.1 In vitro scratch assay suitability 60 4.2 Details of description NIH/3T3 fibroblasts cell line 61 4.3 Result of MTT assay 65 4.4 Concentration-dependant (low and high) for extracts 70 5.1 Differences between rodents and human 76 5.2 Three treatment groups of white New Zealand rabbits 80 5.3 Scoring method for histopathological findings 83 7.1 Percentage wound contraction (%) of different extracts 124

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LIST OF FIGURES

Figure No. Page

2.1 Scientific classification of A. aureum and A. speciosum 26 2.2 Detail characteristics of A. aureum 27 2.3 Detail characteristics of A. speciosum 28 2.4 Inflammation phase in wound healing 31 2.5 Proliferation phase in wound healing 32 2.6 Remodelling phase in wound healing 33 3.1 Satellites image of Matang mangroves, Taiping, Perak 38 3.2 Standard curve of tannic acid 52 4.1 Mean migration rate (µm/hour) vs Treatment group (A. aureum) 67 4.2 Mean migration rate (µm/hour) vs Treatment group (A. speciosum) 68 4.3 Mean migration rate (µm/hour) vs Treatment group (fractions) 69 5.1 Treatment group for in vivo study 77 5.2 Percentage wound contraction vs day 86 5.3 Epithelization period (days) vs Treatment group 90

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LIST OF PLATES

Plate No. Page

3.1 Example of A. aureum in Matang mangroves 39 4.1 NIH/3T3 fibroblast cells 63 4.2 A wound scratch pre-treatment 64 4.3 Measurement of wound scratch 12 hour post-treatment 64 5.1 Photomicrographic observation of wound contraction 85 5.2 5 % aqueous extract of rhizomes A. aureum/Aqua cream 93 5.3 10 % aqueous extract of rhizomes A. aureum/Aqua cream 94 5.4 5 % aqueous extract of leaves A. aureum/Aqua cream 94 5.5 10 % aqueous extract of leaves A. aureum/Aqua cream 95 5.6 5 % aqueous extract of stems A. aureum/Aqua cream 95 5.7 10 % aqueous extract of stems A. aureum/Aqua cream 96 5.8 5 % aqueous extract of stems A. speciosum/Aqua cream 96 5.9 10 % aqueous extract of stems A. speciosum/Aqua cream 97 6.0 Solcoseryl jelly (Positive control) 97 6.1 Aqua cream (Negative control) 98 7.1 Voucher specimen A. aureum 120 7.2 Voucher specimen A. speciosum 121 7.3 Saponin testing 122 7.4 Terpenoid testing 122 7.5 Alkaloid testing 123 7.6 Flavonoid testing 123 7.7 Tannin testing 123

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LIST OF APPENDICES

Appendix Page

A IREC approval meeting No 3/2011 119 B A. aureum voucher speciment 120 C A. speciosum voucher speciment 121 D Phytochemical screening observations 122 E Percentage wound contraction 124 F Recipes of modified Masson’s Trichrome staining 125 G Publications and achievement 127

LIST OF ABBREVIATIONS

Abbreviation Full name

ATCC American Type Culture Collection CAM Chorioallantoic Membrane DMEM Dulbecco’s Modified Eagle Medium DMSO Dimethyl Sulfoxide DPPH 2, 2-Diphenyl-1-picrylhydrazyl ECM Extracellular Matrix FBS Fetal Bovine Serum HIV Human Immunodeficiency Virus H & E Haematoxylin & Eosin MTT Methylthiazol Tetrazolium OH Hydroxyl Radical PDGF Platelet Derived Growth Factor SOR Superoxide Radical VLC Vacuum Liquid Chromatography WHO World Health Organization m metre cm centimetre mm millimetre g gram mg milligram µg microgram ng nanogram L litre mL millilitre µL microlitre

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CHAPTER ONE

INTRODUCTION

1.1 BACKGROUND OF RESEARCH

Commercial drugs are widely available in health industry but in some developing countries, medicinal plants still capture the interest of many researchers. Recently, the interest in medicinal plants is fuelled by the continuous rising costs of prescription drugs in the maintenance of personal health and well-being, and the bioprospecting of new plant-derived drugs (Hoareau and Dasilva, 1999). World Health Organization

[WHO], (2002) reported that global market for traditional medicine is around US$ 60 billion. In USA, there are more than 1500-1800 botanical products sold and medicinal herbs is a major segment in their pharmaceutical market (Matthews, Lucier, and

Fisher, 1999). The use of herbal medicine was inherited from our ancestors thousand years ago. For example, in old civilization like India, herbal medicine was the key component in their ancient traditional medicine such as Ayurveda and Unani system of medicine used about 700 plant species while Siddha and Amchi used about 600 plant species (Joy, Thomas, Mathew, and Skaria, 1998).

Malaysia as a tropical country is blessed to be a home for some household name of medicinal plants such as Tongkat Ali (Eurycoma longifolia), Pegaga

(Centella asiatica) and Misai Kucing (Orthosiphon stamineus) (Rajen, 2004). The herbal industry in Malaysia is blooming annually and it triggers the researchers to develop Malaysian Herbal Pharmacopoeia and Pharmacopoeia Commission in order to assure the quality and reproducibility of the herbal product in the country (Zhari,

2010). This effort is in conformance with one of the strategies by National Policy of

Traditional and Complementary Medicine which is to support efficacy of traditional/ complementary medicinal products by the availability of scientific evidence (Ministry of Health Malaysia, 2001). As a comparison, even though many types of herbal products were consumed by American people but their safety, toxicity, and side effects were yet to be established by scientific research (Bent, 2008).

Historically, Malacca as a renowned port in Southeast Asia played a vital role in herbal trade between foreign traders and Malays. This has resulted in the introduction of foreign herbs into Malay peninsular such as Mentha arvensis from Sri

Lanka, Piper sarmentosum from Indonesia, and Centella asiatica from India (Joseph,

Sugumaran, and Lee, 2005). Nowadays, many researchers in Malaysia are interested to do various kinds of research on medicinal plants found in its rainforest and mangroves parks since this country is listed as one of twelve megadiversity countries in the world, with 1100 species of ferns and fern allies (Ministry of Science,

Environment and Technology, 1998). For that purpose, ethnobotanical studies have provided a very vital information for researchers around the world to explore more on ferns medicinal properties (Singh, Dixit, and Sahu, 2005; Karthik, Raju, Ayyanar,

Gowrishankar, and Sekar, 2011; Benniamin, 2011; Singh, S. and Singh, R., 2012).

Pteridaceae is a fern family that consists of 50 genera and 950 species worldwide (Smith, Pryer, Schuettpelz, Korall, Schneider, and Wolf, 2008). One of the prominent characters of this family is the presence of long to short creeping and ascending rhizomes (Smith et al., 2008). Acrostichum aureum and Acrostichum speciosum are two examples of species under Pteridaceae family that possess medicinal properties in wound healing (Bandaranayake, 1998). These plants are commonly found in mangroves in tropical region especially Malaysia, hence Malays are known to use its rhizome for wound healing (Bandaranayake, 1999). Thus, this

study aimed to test scientifically the effects of the both plant extracts for wound healing treatment. Moreover, to date there is no study ever done on the plant efficacy for wound healing.

Since people in rural area are far away from medicinal facilities, they heavily rely on traditional medicine for wound healing. For example, due to its medicinal value for wound healing treatment, Aloe vera is very common herb planted in

Malaysia (Joseph et al., 2005). As a comparison, Acrostichum spp. could be found in abundance in mangroves area and compared to A. vera, these ferns giants need minimum care to grow. Apart from that, since Malays are consuming its young leaves as ulam, therefore they could take the rhizomes to be used as a paste for wound healing treatment. Therefore, in this study, A. aureum and A. speciosum were chosen to test their wound healing properties due to their abundance in mangroves, lacking of scientific study, and traditional use among Malays in Malaysia.

1.2 OBJECTIVES

1. To identify the phytochemical compounds in A. aureum and A. speciosum.

2. To test antimicrobial properties of A. aureum and A. speciosum.

3. To evaluate wound healing properties of ethanol, aqueous extracts and their

fractions from leaves, stems, and roots of A. aureum and A. speciosum in vitro

and in vivo and to compare their effectiveness as a wound healing agent.

1.3 RESEARCH QUESTIONS

1. Do extracts of A. aureum and A. speciosum exhibit wound healing properties

in vitro and in vivo?

2. Which extracts (A. aureum and A. speciosum) exhibit better wound healing

properties in vitro and in vivo?

1.4 HYPOTHESES

Extracts of A. aureum and A. speciosum exhibit different effectiveness in wound healing properties in vitro and in vivo.

1.5 OVERVIEW OF THE STUDY

For the first phase of study, phytochemical compounds of both A. aureum and A. speciosum were screened and total tannin content was done to know their quantitative amount of tannins as a target compound. Next, antimicrobial study was done to assess their properties to enhance wound healing process.

Second phase of the study was in vitro study using NIH/3T3 fibroblasts cell line. MTT assay was done to know optimum concentration of the extracts that had proliferative effect on fibroblasts. After that, scratch wound assay was done as a screening of their wound healing potential. Of eight crude extracts and fractions tested that showed better mean migration rate compared to negative control, only four were tested further on rabbits due to their limited amount of fractions.

Third phase of study began with excisional wound inflicment on dorsal part of rabbits. The wounds were treated once daily until complete healing on day 15 with four types of crude extracts with different concentration (5 % and 10 %), Solcoseryl jelly served as a positive control and Aqua cream served as negative control. Next, histopathological study was done on day 15 and the slides were assed individually on three parameters which were collagen, fibroblasts, and epithelization.

CHAPTER TWO

LITERATURE REVIEW

2.1 MEDICINAL PLANTS FOR WOUND HEALING

The treatment of wound healing became human’s interest since a long time ago. From east to the west, it became a universal subject when human started to use various kinds of medicinal plants as a wound healing treatment. For example, a bread mold is used in Chinese folk medicine to treat small burns which could be found in written records at least for 2000 years (Brown, 1992). Ibn Sina explained that papyrus is beneficial for haemorrhage to cease and fresh wounds to scar over. This practice is parallel to a hadith narrated by Abu Hazim who had heard Sahl b. Sa’d asked about the treatment for Prophet Muhammad (PBUH)’s wound on the day of Uhud. Fatimah, his daughter burnt a piece of matting to ashes and applied it to the wound. The matting is made from papyrus that contains a strong caustic property that useful to heal wounds (Ibn

Qayyim, trans. 2001).

When we trace back the Greek physician practice during Hippocrates (460-370

B. C. E.), the concept of combating and curbing bacterial infection in wound is still a major concern for wound healing treatment until nowadays. Previously, the physicians applied wine into the wound and later it was discovered that polyphenols, malvoside in wine was the compound responsible for bactericidal activity (Eaglstein, 2005). This practice is similar with the use of Calendula succus containing fresh juice of

Calendula officinalis after surgery for enhancing wound healing process. This preparation is included in The Complete German Commission E Monographs for wound healing treatment (MacKay and Miller, 2003). Therefore, various plants have

been studied for wound healing properties worldwide including Malaysia. Various medicinal plants in Malaysia have been proven beneficial in accelerating wound healing process (Table 2.1). The abundance of fauna in Malaysia has been used by many ethnic groups for traditional medicine. Thus, it is a big interest to search for the other potential Malaysian plants and Acrostichum spp. is selected in this study due to its traditional Malay practice in wound healing.

Table 2.1 Details of plants have been tested for wound healing study in Malaysia (2006-2012). Plant name Extract used Model of study Author

1. Plantago Leaves aqueous extract Excision/ Sprague Mahmood and major Dawley male rats Phipps (2006) 2. Allium Garlic bulbs aqueous Excision/ Sprague Sidik et al. sativum extract Dawley male rats (2006) 3. Rafflesia Flowers methanolic Excision/ Sprague Mahmood et al. hasseltii extract Dawley male rats (2009) 4. Ficus Whole plants aqueous Excision/ Sprague Mahmood et al. deltoidea extract Dawley male rats (2010) 5. Elaeis Leaves methanolic Excision/ Sprague Sreenivasan et guineensis extract Dawley male rats al. (2010) 6. Tamarindus Seed Phosphate Excision/ ICR Mohd Yusof et indica Buffered Saline (PBS), female mice al. (2011) aqueous, ethanolic, methanolic extract 7. Phyllanthus Leaves aqueous extract Excision/ Sprague Khaled et al. niruri Dawley male rats (2012) 8. Terminalia Stem bark aqueous and Excision/ Wistar Mohammed coriacea methanolic extract albino male rats Safwan et al. (2012)

2.2 MANGROVES ECOSYSTEM

Mangroves are salt tolerant forest ecosystems found mainly in the tropical and subtropical intertidal regions of the world largely confined to the region between 30° north and south of the equator (Bandaranayake, 2002). In total, there are 84 mangrove plant species in the world, out of which 70 species are true mangroves and 14 species are semi-mangroves tides (Jun, Qiang, Xing, Min, Jian, and Mei-hua, 2008). There are

55 mangrove species from 22 genera and 18 families could be found along Indian

Ocean region. This showed that the region has a great diversity of mangrove species with Indonesia has 45 species, followed by Malaysia (40 spp.), India (39 spp.),

Thailand (34 spp.) and Singapore (31 spp.) (Kathiresan and Rajendran, 2005).

Basically, mangrove plants are categorized into two groups, true mangrove and semi-mangrove plants. The true mangrove plants are restricted to the typical intertidal mangrove habitats whereas semi-mangrove plants grow on the landward fringe mangrove habitat or in terrestrial marginal zones subjected to irregular high tides. On the other hand, there is a third group called mangrove associated plants which are salt tolerant terrestrial plants occasionally found in landward edge of mangrove habitat and are irregularly flushed by high tide (Jun et al., 2008). There was a controversy regarding Acrostichum spp. in which some scientists classified it as true mangroves and others had classified it as associate mangroves. However, a recent study conducted by Wang, Ruan, Cai, Luo, Xu, and Wu (2010) indicated that both A. aureum and A. speciosum are associate mangroves due to higher specific leaf area

(SLA), lower leaf succulence, lower sodium content and chlorine concentration.

2.2.1 Use of Mangroves as a Medicinal Plant

Mangrove plants have been used in folklore medicine to treat various diseases for centuries. They are a great resource for a new pharmaceutical discovery based on the traditional practice (Ashriq, 2013). Bandaranayake (1998) has listed a list of medicinal use of mangrove plants. For instance, they are used as aphrodisiacs (Acanthus ilicifolius, Xylocarpus moluccensis), antitumor (Avicennia africana, Bruguiera parviflora) and diabetes treatment (Bruguiera rumphii, Nypa fruiticans). Meanwhile, associate mangrove like Acrostichum spp. is used traditionally to treat boils and wounds (rhizome) and rheumatism (leaves) (Polunin, 1988; Bandaranayake, 1999).

The use of medicinal mangrove plants has triggered the interest of scientist worldwide to investigate them scientifically. Many mangrove species have been proven to possess medicinal value. For example, Ravikumar, Gnanadesigan, Suganthi, and Ramalakshmi (2010) reported that ethanolic extract of Rhizophora mucronata and

Avicenna marina plant parts (hypocotyls, collar, bark and flower) had shown antibacterial activity against isolated urinary tract infectious bacterial pathogens.

Besides, Premanathan, Chandra, Bajpai, and Kathiresan (1992) demonstrated that five extracts of mangrove plants (bark of R. mucronata and leaves of Excoecaria agallocha, Ceriops deeandra, Rhizophora apiculata, Rhizophora lamarckii) completely inhibited the Human Immunodeficiency Virus (HIV) adsorption to the cells. Meanwhile, Kumar, Ammani, and Siddhardha (2011) had concluded that leaves extracts of Derris trifoliata and Brugiera gymnorrhiza were significantly active against some pathogenic bacteria and fungi. Sivaperumal, Ramasamy, Inbaneson, and

Ravikumar (2010) had successfully tested bioactive compounds from Exoecaria agallocha against several antibiotic resistant bacterial pathogens.