A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy in Biomedical Science

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IN-VITRO AND IN-VIVO EVALUATION OF

THERAPEUTIC PROPERTIES OF SKIN MUCUS FROM ASIAN SWAMP EEL (Monopterus albus)

BY

AYAH REBHI MOHAMMAD HILLES

A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy in Biomedical Science

Kulliyyah of Allied Health Sciences International Islamic University Malaysia

FEBRUARY 2019

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ABSTRACT

Swamp eels have been widely accepted as sources of food, especially among various Asian cultures. However, their potential values as novel sources of therapeutic agents have not been widely appreciated. Like most other tropical fishes and amphibians, the outer integumentary system of Monopterus albus is covered with mucus layers, which may act as mechanical and biochemical barrier for their skin. The biochemical components of these mucus layers may have certain compounds that may be medically beneficial to human. The current study was interested to screen selected biological activities of skin mucus from the tropical eel in-vitro and in-vivo. The first stage of the study was the screening of the eel skin mucus extracts for bioactive compounds using gas chromatography-mass spectrometry and liquid chromatography quadrupole-time-of-flight mass spectrometry. Several compounds, which had been previously identified to have different biological activities mainly cytotoxic, antioxidant and antimicrobial activities were discovered. The second stage of the study aimed to demonstrate and investigate the anti-oxidative, anti-cancer and antimicrobial activities in-vitro models. The antioxidant results revealed that methanolic extract showed higher activity than aqueous extract with higher phenolic and flavonoid contents as well as higher antioxidant assays which including DPPH radical scavenging and β-carotene bleaching. Preliminary cytotoxicity study was demonstrated against human lung carcinoma cell line (A549) using cell viability assay which revealed that methanolic extract is more potent than aqueous extract as IC50 values were 621±0.09 µg/mL and 845 ± 0.25 µg/mL respectively. It was then established that the methanolic extract was able to induce apoptosis in A549 cell line by the activation of caspase-3, 8 and 9. Further analyses to investigate the mode of cell death induction and cell cycle arrest pathways by flow cytometry analysis revealed that the methanolic extract was able to induce late apoptosis and arrested the cells in G0/G1 phase. Assessment of antimicrobial activities of the eel mucus extracts against several bacterial and fungal strains was conducted using diffusion method. Methanolic extract at 100 µL/well was found to inhibit the growth of Microsporum gypseum and Aspergillus niger. Significantly. Furthermore, the methanolic extract at 100 µL/disc significantly inhibited the growth of Staphylococcus aureus and Escherichia coli. Comprehensive evaluation of antimicrobial activities against selected oral pathogens showed that methanolic extract exhibited high activity against Enterococcus faecalis, Streptococcus mutans, Streptococcus pyogenes, Klebsiella pneumoniae, Pseudomonas aeruginosa, and Candida albicans. For in-vivo study, a topical gel delivery system used from eel skin mucus formulated gel to apply into the infected rat skins. Sprague Dawley rats were divided into normal, positive control, negative control, and treated groups. The infections were introduced to the rats by intracutaneous injections of pathogenic bacteria and fungi. The development of impetigo, tinea capitis and cutaneous candidiasis in the animal model was confirmed based on the clinical and histological observations. Following that, the infected rats were treated topically with the formulated gel from eel mucus extract. The histological analysis of the skin tissues which treated with the formulated gel was shown a complete recovery in the skin tissues at a similar rate as the control antibiotic groups. In conclusion, the present study revealed that eel skin mucus formulated gel efficient therapeutic candidate in treating skin infections which can be considered as a novel discovery as a natural alternative treatment for certain skin diseases.

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ثحبلا ةصلاخ

ايشلأا نم دحاو ةديدلجا ةيرحبلا ةيودلأا ريوطتو فاشتكا دعي ء

يثلث نم رثكأ تاطيلمحا يطغت .ارخؤم ةيرحبلا مولعلا لامج في يادتح رثكلأا

تنااويلحا دلج يطغت .ةيرحبلا تانئاكلا نم ةيودلأا فاشكتسا يرورضلا نمف ،لياتلباو ،ضرلأا حطس ،طاخلما نم ةقبطب ةيئامبرلاو ةيرحبلا

عقنتسلما نابعث نم دللجا طاخلم ةيجولويبلا ةطشنلأا ديدتح لىإ فدته ةساردلا هذه .مهدللج يكيناكيمو يئايميك زجاح ةباثبم لمعي يذلا يويسلآا (Monopterus albus) يه ةساردلا نم لىولأا ةلحرلما تناكو .يلحا مسلجا فيو برتخلما في

صحف ايجولويب ةطشنلا تابكرلما

و يلتكلا فيطلل نيوللا زاغلا مادختسبا لا

نيوللا لئاس -

يعبار - لما فايط ل ةفلتمخ ةطشنأ كلتتم انهأ ينبت تابكرلما ديدتح للاخ نم ،نايرطلا نمز

ةصاخ ب ةيمس ا لىإ ةساردلا نم ةيناثلا ةلحرلما فدته ، كلذل .تباوركيلما تاداضمو ةدسكلأا تاداضمو ،يالالخ في ةطشنلأا هذه صحف

لوناثيلما صلختسلم ةدسكلأا تاداضم نأ جئاتنلا تفشك .يئالما صلختسلما نم ىلعأ ةطشنأ لوناثيلما صلختسم رهظأ ،ماع لكشب .برتخلما ةميق لياجمإ غلب ثيح ،ايلاع اطاشن كلتم IC50

ليونيفلا ىوتحملل

mg(GAE)/g

±0.11 120.29 ،

و ديونوفلافلا ىوتمح لياجمإ غلب

260.28±0.19mg (QE)/g و ،

لا صحف حساكلا يرذلجا

0.1±0.08 mg/mL ضيبلما ينتوراك اتيب صحفو

23.2±0.44%

. ةئرلا ناطرسل ةيرشبلا يالالخا دض ةيوللخا ةيمسلل ةيلولأا ةساردلا تراشأ دق (A549)

،يالالخا ءاقب صحف مادختسبا

ةوق رثكأ لوناثيلما صلختسم نأ رهظأ يذلاو )

mL µg/

±0.09 621 IC50

( ترابتخا ءارجإ دعب يالالخا توم ةيلآ ديدتح تم ، كلذ دعب.

سابساك ليعفت قيرط نع جمبرلما يالالخا توم للاخ نم يالالخا توم نع لوؤسم لوناثيلما صلختسم نأ تبثو ،ةفلتمخ ةيئايميك 3

و 8 و

9 يالالخا لاقتعاو رخأتلما جمبرلما يالالخا توم ثحتسا لوناثيلما صلختسم . ةلحرم في

G0/G1 تباوركيملل ةداضلما صئاصلخا تيرجأ.

،رشنلا ةقيرط مادختسبا تناك ثيح

زيكترل ةيسبلجا ءاغيوبلا دض لوناثيلما صلختسم طيبثت ةقطنم 100 µL/well

ةميقب

25.72±0.75mm تناك رجينلا تايشاشر دض امنيب

11.21±0.59mm صلختسم طيبثت ةقطنم تناك ،هسفن تقولا فيو .

زيكترل لوناثيلما 100 µL/disc

ةيبهذلا ةيدوقنعلا تاروكلما دض ةميقب

9.91±0.06mm تناك ةينولوقلا دض امنيب

10.79±0.17mm رثكأ لوناثيلما صلختسم نبأ مفلا قيرط نع ضارملأا تاببسم دض تباوركيلما ةداضم ةطشنلأل لماش مييقت رهظأ .

ف ةيزابرلا ةيوعلما تاروكلما دض هتيلاعف تناك ثيح ةيلاع )

mL g/

±0.73μ 7.15 IC50

( ةرفاطلا ةيدقعلا ثم ،

±0.15 12.89 IC50

(

) mL g/

،μ ةحيقلما ةيدقعلا اهيلت )

mL g/

±0.91μ 9.75 IC50

( ةيوئرلا ةليسبلكلا ثم ، )

mL g/

±0.28μ 13.56 IC50

( ثم ،

ةيرانجزلا ةفئازلا )

mL g/

±0.43μ 23.44 IC50

( ضيبلل تاضيبلما دض ناك طاشن نىدأو )

mL g/

±0.5μ 379.91 IC50

.( تتم

ةسارد ماظنل يلحا مسلجا في جلاعلا

لما نارئفلا دولج ىلع قبطتل تمدختسا ثيح عقنتسلما نابعث دللج طاخلما ملاه نم مدختسي يعضو

لا لىإ ليواد غابرس نارئف ميسقت تم .ةباصلما مكحت

نع نارئفلا لىإ ىودعلا لاخدإ تم .ةلجاعلما تاعوملمجاو بيلسلا مكحتلاو ، بيايجلإاو يعيبطلا

دللجا تتح نقلحا قيرط با

ةضيبلما ةيدللجا ضارملأاو سأرلا ةفعس ،ءباوقلا نم ةيناويح جذانم ريوطت تم ،ضارملأل ةببسلما تيارطفلاو يايرتكبل

ةيريرسلا تاظحلالما نم ققحتلا للاخ نم يجيسنلا ليلحتلا حضويو .رضلمحا ملالهبا ايعضوم ةباصلما نارئفلا جلاع تم ،ةيجيسنلاو

لج دول

ملالهبا ةلجاعلما نارئفلا

، دللجا ةجسنأ في لماكلا شاعتنلاا في لالحا وه امك اماتم

ملالها نأ ةساردلا تفشك ،ماتلخا فيو .ةيويلحا تاداضلما

شرم عقنتسلما نابعث دلج طامخ نم رضلمحا ل ةلاعف تاجلاعل ح

ضعب جلاعك ديدج فاشتكا في رظنت تيلا ةيدللجا تبااهتللإ يعيبط

ليدب

دللجا ضارملأ .

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APPROVAL PAGE

The thesis of Ayah Rebhi Mohammad Hilles has been approved by the following:

_____________________________

Muhammad Nor Omar Supervisor

_____________________________

Mohd Arifin Kaderi Co-Supervisor

_____________________________

Syed Mahmood Co-Supervisor

_____________________________

Radiah Abdul Ghani Internal Examiner

_____________________________

Shaharum Shamsudin External Examiner

_____________________________

Nadeem Akhtar External Examiner

_____________________________

Zarina Zainuddin Chairman

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DECLARATION

I hereby declare that this dissertation 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.

Ayah Rebhi Mohammad Hilles

Signature ... Date ...

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T P

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

IN-VITRO AND IN-VIVO EVALUATION OF THERAPEUTIC PROPERTIES OF SKIN MUCUS FROM ASIAN SWAMP EEL

(Monopterus albus)

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 Commercialization policy.

Affirmed by Ayah Rebhi Mohammad Hilles

……..……….. ………..

Signature Date

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ACKNOWLEDGEMENTS

The real of infinite and grateful praises, thanks to ALLAH for all this gift thus this text would not have been possible without the help of ALLAH under whose blessing and guidance I have completed this thesis as a requirement for my PhD of Biomedical Sciences and may His peace and blessings be upon our beloved Prophet Mohammad.

Almighty ALLAH was the most merciful in providing me the opportunity to have worked with great people who have contributed in various ways to my research and thesis that deserve special mention.

First and foremost, special thanks go to my parents, my Father Rebhi Mohammad Hilles, my Mother Amal Saqer Hilles, who have been my backbone, source of motivation and courage, to keep me moving forward, they brought me up to reach this level. The preparation of this research was a challenging time in my life. I would not have succeeded in anything without my parents who made everything possible. I dedicate this research to them, without their support, this research would not have seen the light. I would like to extend heartfelt gratitude to my siblings; Basil, Nazik, Saeed, Masood and Yara, who have given me effective incentive and encouragement which kept me motivated, sane and on schedule to complete this research. Without their support and pep talks, this research and thesis would not have been possible. My deepest gratitude to all my all colleagues and friends for their support and encouragement during my PhD journey.

Secondly, it was not possible for this work to be real, in this form, without the experience and confident guidance from Prof. Dato’ Paduka Dr. Ridzwan Hashim, who has shown faith in me and my abilities as a student from day one. Without his assistance, this research would not have been possible. My highest appreciation also goes to Prof.

Dr. Muhammad Nor Omar who has given me support and encouragement. I would like to express my deepest appreciation to Dr. Mohd Arifin Kaderi, without his promptness in reading and commenting, this research would not have come to fruition, my deep respect and appreciation goes to him. I would like to thank Dr. Syed Mahmood for his continuous support, immense knowledge, motivation and guidance gave a real value to my research. I could not have imagined having a better advisor and mentor for my research such as him. Words cannot express how grateful I am to him and I will be grateful forever. My heartfelt gratitude goes to Dr. Ahmad Aidil Arafat Dzulkarnain, our deputy dean and Dr. Zarina Zainuddin (the deputy dean of CPS), the challenging we are facing as students become much easier with their help and support. My greatest appreciation flows to my Postgraduate Office especially sister Intan who is always help me and all the students, her smile enough to lighten up my whole day. I really thank her from deep of my heart. I want to thank Centre of Postgraduate Studies (CPS) for helping me and make my procedure smooth, especially brother Ihsan who has a great impact in helping me and all the students in our thesis, my highest appreciation goes to him.

Last but not least, I would like to express my deep and special thanks to IIUM for giving me this knowledge to contribute to others. I was so blessed to be a student in IIUM and I wish this piece of work will be beneficial to the Ummah and will encourage other fruitful work in the future.

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LIST OF TABLES

Table No. Page No.

3.1 List of chemicals 38

3.2 GC-MS parameters 40

3.3 The Gradient grade of solvents in the mobile phase using LC-QTOF-

MS 41

4.1 The bioactive compounds detected using GC-MS 63

4.2 The compounds detected in ESM aqueous and methanolic extracts

using LC-QTOF-MS 65

4.3 Total phenolic content of ESM aqueous and methanolic extracts 69 4.4 Total flavonoid content of ESM aqueous and methanolic extracts 70 4.5 DPPH free radical scavenging activity of ESM aqueous and

methanolic extract. *Significant difference at p < 0.05 (one-way

ANOVA) 71

4.6 IC50 of ESM aqueous and methanolic extracts 72

4.7 Caspase-3 activity after treatment of A549 cells treated with ESM methanolic and aqueous extracts along with positive control (Taxol), negative control (untreated cells) for 24, 48 and 72 hrs 76 4.8 Caspase 8 activity after treatment of A549 cells with ESM methanolic

and aqueous extracts along with positive control (Taxol) and negative

control (untreated cells) for 24, 48 and 72 hrs 77

4.9 Caspase 9 activity after treatment of A549 cells with ESM methanolic and aqueous extracts along with positive control (Taxol) and negative

control (untreated cells) for 24, 48 and 72 hrs 78

4.10 Apoptosis induced by ESM aqueous and methanolic extracts 80 4.11 Antibacterial activity of ESM extracts against E. coli 92 4.12 Antibacterial activity of ESM extracts against E. coli 92 4.13 Antibacterial activity of ESM extracts against S. aureus 93

4.14 Antifungal activity of ESM against M. gypseum 94

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4.15 Antifungal activity of ESM against A. niger using well diffusion method (mm). * Significant difference at p < 0.05 (one-way ANOVA) 95 4.16 Determination of IC50 of ESM extracts. Data expressed in μg/mL.

Mean ± SD (n = 3). * Significant difference at p < 0.001 (one-way

ANOVA) 96

4.17 Antibacterial activity of ESM extract against E. faecalis using percentage of inhibition method. * Significant difference at p < 0.05

(one-way ANOVA) 97

4.18 Antibacterial activity of ESM extract against S. mutans using percentage of inhibition method. * Significant difference at p < 0.05

(one-way ANOVA) 98

4.19 Antibacterial activity of ESM extract against S. pyogenes using Percentage of inhibition method. * Significant difference at p < 0.05

(one-way ANOVA) 99

4.20 Antibacterial activity of ESM extract against K. pneumoniae using percentage of inhibition assay. * Significant difference at p < 0.05 (one-

way ANOVA) 100

4.21 Antibacterial activity of ESM extract against P. aeruginosa using percentage of inhibition assay. * Significant difference at p < 0.05 (one-

way ANOVA) 101

4.22 Antifungal activity of ESM extract against C. albicans using Percentage of inhibition method. * Significant difference at p < 0.05

(one-way ANOVA) 102

4.23 The clinical and histological features observed in the infected rats 105 5.1 Volatile constitutes of Asian swamp eel skin mucus which detected in

other samples and their biological activities using GC-MS 169 5.2 The biological activities of the bioactive compounds detected in ESM

aqueous and methanolic extracts using LC-QTOF-MS 172

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LIST OF FIGURS

Figure No. Page No.

2.1 Asian swamp eel (Monopterus albus) shows eel eyes covered with a

thin layer of skin (Image captured by Ayah Hilles) 16

2.1 The classical model of cell cycle 23

3.1 Schematic diagram of the extract preparation process 39

3.2 Flow chart of research activities 59

3.3 Animal study overview 60

4.1 GC-MS Chromatogram of all the compounds detected in ESM

methanolic extract. 62

4.2 Chromatogram of all compounds detected in ESM aqueous extract

using LC-QTOF-MS 64

4.3 Chromatogram of all compounds detected in ESM methanolic

extract using LC-QTOF-MS 64

4.4 (A) Chromatogram of 2-Octylphenol in ESM methanolic extract at

0.42 min (B) Terrestroside F in ESM methanolic extract at 0.43 min 66 4.5 (A) Chromatogram of Ethyl-α-O-D-galactopyranoside in ESM

methanolic extract at 2.68 min. (B) Ethyl-α-O-D-galactopyranoside

in ESM aqueous extract at 2.69 min 66

4.6 (A) Chromatogram of isotope peak of 3-β-D-Glucopyranosylox- ybutanol-2 in ESM aqueous extract at 3.38 min. (B) Peonidin in ESM

methanolic extract at 5.60 min 66

4.7 (A) Chromatogram of isotope peak of Astragaloside VI in ESM methanolic extract at 10.38 min. (B) Eicosenoic acid in ESM

methanolic extract at 10.49 min 67

4.8 (A) Chromatogram of Benzyl benzoate in ESM methanolic extract at 10.49 min. (B) Momor-cerebrosid Ι in ESM aqueous extract at 10.50

min 67

4.9 (A) Chromatogram of 6′-O-Palmitoyl-sitosterol-3-O-β-D-glucoside in ESM methanolic extract at 10.55 min. (B) 3',4',5',5,7,8-Hexame-thoxy

flavone in ESM methanolic extract at 10.58 min 67

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4.10 (A) Chromatogram of Bufotalinin in ESM aqueous extract at 10.61 min. (B) Bufotalinin in ESM methanolic and aqueous extract at 10.60

min 68

4.11 A) Chromatogram of Progenin II in ESM methanolic and aqueous extracts at 10.83 min. (B) Salvianolic acid G in ESM methanolic

extract at 13.54 min 68

4.12 Total phenolic content of ESM aqueous and methanolic extracts (mg GAE/g). Values are expressed as gallic acid equivalents (GAE) mg/g

extract 69

4.13 Total flavonoids content of ESM aquous and methanolic extracts mg (QE)/g). Values are expressed as catechin equivalents mg (QE)/g 70 4.14 DPPH free radical scavenging activity of ESM aqueous and

methanolic extracts. Values are expressed as percentage (%) 71 4.15 The effects of ESM aqueous and methanolic extracts on β-carotene

oxidation by linoleate radical 72

4.16 Cell viability of ESM aqueous and methanolic extracts against A549.

Values are expressed as percentage (%) 73

4.17 Cell viability of ESM aqueous and methanolic extracts against 3T3-

L1. Values are expressed as percentage (%) 73

4.18 Cellular caspases shows the average caspase-3 activity of ESM aqueous and methanolic extracts against A549 cells after 24, 48 and 72 hrs of treatment, with respect to untreated cells (negative control)

and Taxol (positive control) 76

4.19 Cellular caspases shows the average caspase-3 activity of ESM aqueous and methanolic extracts against A549 cells after 24, 48 and 72 hrs of treatment, with respect to untreated cells (negative control)

4.20 Cellular caspases shows the fold change caspase-9 activity of ESM aqueous and methanolic extracts against A549 cells after 24, 48 and 72 hrs of treatment, with respect to untreated cells (negative control)

4.21 Apoptosis induced by ESM aqueous and methanolic extracts against A549 cells. Cells were treated with a concentration of IC50 for 72 hrs.

Taxol was used as a positive control and untreated cells was used as a

negative control 80

4.22 The scatter plot of annexin-V versus ratio of A549 cells-treated with

ESM methanolic extract 81

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ESM aqueous extract 82

Taxol 83

4.25 The scatter plot of annexin-V versus ratio of untreated A549 cells 84 4.26 Flow cytometric histogram analysis show the effects on cell cycle

arrest of A549 cells upon treatment of (A) ESM methanolic extract, (B) ESM aqueous extract, (C) Taxol and (D) untreated cells 86 4.27 Antibacterial activity of ESM aqueous and methanolic extracts against

E. coli using disc diffusion method 87

4.28 Antibacterial activity of ESM aqueous and methanolic extracts against

S. aureus using disc diffusion method 88

4.29 Percentage of growth inhibition for ESM aqueous and methanolic extracts against E. coli. Penicillin was used as a positive control 90 4.30 Percentage of growth inhibition for ESM aqueous and methanolic

extracts against S. aureus. Penicillin was used as a positive control 91 4.31 Survival rate of ESM aqueous and methanolic extracts against E. coli.

Penicillin was used as a positive control 92

4.32 Survival rate of ESM aqueous and methanolic extracts against S.

aureus. Penicillin was used as a positive control 93 4.33 Antifungal activity of ESM aqueous and methanolic extracts against

M. gypseum. Ketoconazole was used as a positive control 94 4.34 Antifungal activity of ESM aqueous and methanolic extracts against

A. niger. Ketoconazole was used as a positive control 95 4.35 Antibacterial activity of ESM aqueous and methanolic extracts against

E. faecalis compared with positive control (penicillin) 97 4.36 Antibacterial activity of ESM aqueous and methanolic extracts against

S. mutans compared with positive control (penicillin) 98 4.37 Antibacterial activity of ESM aqueous and methanolic extracts S.

pyogenes compared with positive control (penicillin) 99 4.38 Antibacterial activity of ESM aqueous and methanolic extracts K.

pneumoniae compared with positive control (penicillin) 100 4.39 Antibacterial activity of ESM aqueous and methanolic extracts against

P. aeruginosa compared with positive control (penicillin) 101

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4.40 Antifungal activity of ESM aqueous and methanolic extracts against

C. albicans compared with positive control (nystatin) 102

4.41 Rats infected with S. aureus 106

4.42 Rats infected with S. pyogenes 107

4.43 Rats infected with M. gypsum shows the black dots and hairless on

their skin 108

4.44 Rats infected with C. albicans shows the spread of rashes on the

injection sites of the skin 109

4.45 Histological features of skin tissue shows a cross section of normal untreated skin tissue (H&E, × 4). (B) shows a longitudinal section of

normal untreated skin tissue (H&E, × 4) 110

4.46 Histological features of the skin tissues infected with S. aureus. It shows a cross section of infected skin tissue with necrotic cell debris

and spongiform pustules contain numerous neutrophils. (H&E, × 4) 111 4.47 Histological features of the skin tissues infected with S. aureus. It

shows a longitudinal section of infected skin tissue. Small red arrows show the neutrophils numerus and small black arrows show the

macrophages. (H&E, × 4) 112

4.48 Histological features of the skin tissues infected with S. aureus. It shows a longitudinal section of infected skin tissue. Small red arrows show the neutrophils numerus and small black arrows show the

macrophages. (H&E, × 4) 113

4.49 Histological features of the skin tissues infected with S. aureus. It shows a cross section of subcorneal pustule containing numerous

neutrophils (H&E, × 20) 114

4.50 Histological features of the skin tissues infected with S. pyogenes. It shows a cross section of necrotic cell debris and spongiform pustules

contain numerous neutrophils. (H&E, × 4) 115

4.51 Histological features of the skin tissues infected with S. pyogenes. It shows a longitudinal section of acute Inflammatory cells and

neutrophils (H&E, × 4) 116

4.52 Histological features of the skin tissues infected with S. pyogenes. It shows a longitudinal section of localised infection at the injection site with neutrophils and macrophages Inflammation in the reticular

dermis (H&E, × 4) 117

4.53 Histological features of the skin tissues infected with S. pyogenes. It shows a longitudinal section of necrosis and intense inflammatory

cells (H&E, × 20) 118

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4.54 Histological features of the skin tissues infected with M. gypsum. (A) shows a longitudinal section of abscess wall and necrotic debris (H&E, × 4). (B) shows longitudinal section of abscess wall and

necrotic debris (H&E, × 40) 119

4.55 Histological features of the skin tissues infected with M. gypsum. (A) shows longitudinal section of abscess wall (H&E, × 4). (B) shows a

longitudinal section of abscess wall (H&E, × 20) 120 4.56 Histological features of the skin tissues infected with M. gypsum. It

shows a longitudinal section of neutrophils around hair follicle (H&E,

× 4) 121

4.57 Histological features of the skin tissues infected with M. gypsum. (A) shows a longitudinal section of abscess wall and necrotic debris (H&E, × 4). (B) shows longitudinal section of abscess wall and

necrotic debris (H&E, × 40) 122

4.58 Histological features of the skin tissues infected with C. albicans. It shows a longitudinal section of tinea capitis infection. Long black arrows represent giant cells, small black arrow represent macrophages, long red arrows represent granuloma formation and

small red arrows represent neutrophils (H&E, × 4) 123 4.59 Histological features of the skin tissues infected with C. albicans.

Black arrows show a longitudinal section of granulomas (H&E, × 4) 124 4.60 Histological features of the skin tissues infected with C. albicans. It

shows a longitudinal section of numerus inflammatory cells including neutrophils, macrophages and giant cells in the reticular dermis. Long black arrows represent the giant cells, short black arrows represent

neutrophils and red arrows represent macrophages (H&E, × 4) 125 4.61 Histological features of the skin tissues infected with C. albicans. It

shows a longitudinal section of numerus inflammatory cells including neutrophils, macrophages and giant cells in the reticular dermis. Long black arrows represent the giant cells, short black arrows represent

neutrophils and red arrows represent macrophages (H&E, × 4) 126 4.62 Histological features of the skin tissues infected with C. albicans. It

shows a longitudinal section of abscess formation (H&E, × 4) 127 4.63 (A) shows the skin of rat recovery after treatment with mupirocin. (B)

skin of rat recovery after treatment with ketoconazole 128 4.64 (A) shows the skin of rat recovery from bacterial infection after

treatment with ESM gel (B) shows the skin of rat recovery from fungal

infection after treatment with ESM gel 129

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4.65 Histological features of the skin tissues treated with antibiotic (A) shows a cross section of skin layers with normal appearance (H&E, × 4). (B) shows a longitudinal section of fibroblasts and fibrosis

formation (H&E, × 4) 130

4.66 Histological features of the skin tissues treated with mupirocin ointment against S. aureus. (A) shows a longitudinal section of infection recovery by collagen fibres formation with no sign of inflammation. (B) shows collagen fibres and normal hair follicles. (C) shows collagen fibres and normal sebaceous glands. (D) shows thick

collagen fibres and normal sebaceous glands (H&E, × 20). 131 4.67 Histological features of the skin tissues treated with mupirocin

ointment against S. pyogenes. (A, B, C, and D) show a longitudinal section of infection recovery by collagen fibres and normal hair

follicles (H&E, × 20) 132

4.68 Histological features of the skin tissues treated with ketoconazole ointment against M. gypsum. (A) shows thick collagen bundles and normal hair follicles. (B) shows collagen bundles (C) Intense of thin

collagen fibres. (D) (H&E, × 40) 133

4.69 Histological features of the skin tissues treated with ketoconazole ointment against C. albicans. It shows a longitudinal section of infection recovery by collagen fibres formation with no sign of

inflammation (H&E, × 4) 134

4.70 Histological features of the skin tissues treated with ESM gel (A) shows a cross section of skin layers with normal appearance (H&E, × 4). (B) black arrows show a longitudinal section of fibroblasts

formation (H&E, × 20) 135

4.71 Histological features of the skin tissues treated with ESM gel against S. aureus. It shows a longitudinal section of infection recovery by

collagen fibres formation with no sign of inflammation (H&E, × 40) 136 4.72 Histological features of the skin tissues treated with ESM gel against

S. pyogenes. (A and B) show a longitudinal section of infection recovery by collagen fibres formation with no sign of inflammation (H&E, × 20). (C and D) show a longitudinal section of infection

recovery by collagen (H&E, × 40) 137

4.73 Histological features of the skin tissues treated with ESM gel against M. gypsum. It shows a longitudinal section of infection recovery by

collagen fibres formation with no sign of inflammation (H&E, × 40) 138 4.74 Histological features of the skin tissues treated with ESM gel against

C. albicans. It shows a longitudinal section of infection recovery by

collagen fibres formation with no sign of inflammation (H&E, × 40) 139

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LIST OF ABBREVIATIONS

ESM SSTIs GC-MS

LC-QTOF-MS µg/mL

TPC TFC DPPH IC50

SD PBS MTT RPM NA NB PDA PDB MIC MBC TEA ANOVA H&E

Eel skin mucus

Skin and soft tissue infections

Gas Chromatography- Mass Spectrometery

Liquid chromatography quadrupole time flight mass spectrometry Microgram per millilitre

Total Phenol Content Total Flavonoids Content 2,2-diphenyl-1-picrylhydrazyl

Inhibition concentration (reduces the effect by 50%) Standard Deviation

Phosphate Buffered Saline

3-(4,5- dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide Revolutions per minute

Nutrient Agar Nutrient Broth

Potato Dextrose Agar Potato Dextrose Broth

Minimum Inhibitory Concentration Minimum Bactericidal Concentration Triethanolamine

One-way analysis of variance Haematoxylin and eosin stain

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LIST OF SYMBOLES

β g M S µg/mL mg GAE/g mg QE/g

°C

%

<

±

=

*

Beta Gram Mitosis Synthesis

Microgram per millilitre

Milligrams of gallic acid equivalent per gram of dry weight Milligrams of quercetin equivalent per gram of dry weight Degree Celsius

Percent Less than Plus-minus Equal to

Statistical significance denotation

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CHAPTER ONE INTRODUCTION

1.1 BACKGROUND OF THE STUDY

Development and discovery of new marine drugs consider one of the most challenging fields in the recent marine sciences area. The water covers more than two-thirds of the Earth’s surface and there are almost 90% of the world’s species found in the marine environment (Napolitano et al., 2009), therefore, it is necessary to explore new drugs from marine organisms. Finding new, anti-inflammatory agents with fewer side effects is highly needed. Therefore, many types of research have been proven the anti- inflammatory activity of marine organisms, overall the fish has long-chain omega-3 fatty acids which is potent anti-inflammatory (Wall et al., 2010).

Marine natural products have a novel antioxidant prototype (Takamastu et al., 2003). Fish and marine invertebrates stimulate the induction of antioxidant defense systems (Abele and Puntarulo, 2004). Marine plants play a critical role to fulfill the requirement of food and nutrition, as it has high dietary fibres, low energy density, non- starchy vegetables, and fruits, which might protect against some cancers (Marmot et al., 2007). The marine environment provides novel leads against fungal, parasitic, bacterial, and viral diseases. Many marine natural products have successfully advanced into the late stages of clinical trials (Donia and Hamann, 2003).

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Asian swamp eel (Monopterus albus) belongs to the family of Synbranchidae under Synbranchiformes order (Cheng et al., 2003). It is a freshwater fish which distributed widely in the East of India mainly across Greater Sunda Islands, Malay Peninsula and Indochinese Peninsula, it is also broadly distributed in the Southern areas of East Asia including, southeastern China, Western Japanese Archipelago, and Korean Peninsula (Banarescu, 1990). However, the eel is rarely found in the United States, as this species mainly distributed in Asia (Collins et al., 2002). It lives in muddy places, rice paddies, and slow-flowing currents areas. Asian swamp eel has a unique morphological elongated body which is similar to snake and covered with a thick layer of the mucus with no scales or fins. It can breathe air through the buccal mucosa (Chan and Phillips, 1967).

The skin of marine animals and amphibians are covered with a mucus layer, which acts as a biochemical and mechanical barrier for their skin. Several mucus sources have been isolated and studied for their biochemical and immunological functions, but the precise mechanism of action still not fully understood. Our study aimed to investigate the antimicrobial potential activity of Malaysian local swamp eel (Monopterus albus) skin mucus. Asian swamp eel mucus is secreted by the epidermal goblet cells in the epidermis which composed from inorganic salts, immunoglobulins, lipids and gel-forming macromolecules such as mucins, and other glycoproteins suspended in water (Bragadeeswaran and Thangaraj, 2011), which gives the mucus lubricating properties (Pearson and Brownlee, 2005).

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The mucus layer is continuously replaced which protect the eel from stable colonization by bacteria, parasites, and fungi (Villarroel et al., 2007). The functional properties of the mucus depend on its ability to form a gel on the epithelial surface (Martínez‐Antón et al., 2006), which produce antimicrobial molecules serve as the first line of a host’s defense against microbial invasion (Manivasagan et al., 2009). The mucopolysaccharides in the mucus have a high value for immunoprotection and defense mechanism (Watanabe et al., 2012; Dirks et., 2014). It has been proven that mucin has the potential of antimicrobial and noxious properties (Knouft et al., 2003) as it plays an important role to protect the eel from pathogens (Yan et al., 2010). Overall mucus layer on the surface of eel functions as a physical and biochemical barrier between the eel and its environment (Palaksha et al., 2008).

Human skin infections can be caused due to many factors, such as low socioeconomic status, poor skin health, low level of hygiene and lack of awareness (Goonmatee and Rajesh, 2013; Kingman, 2005; Balai, 2012; Puri and Puri, 2013). It has been reported in Bari and Sierra Leone that skin infection was 42 % in African population because of social and environmental factors. (Ul Bari, 2007). The infection in Tunisia has been demonstrated as 16.9% fungal infection and 11.9% eczema were associated with their climatic conditions (Souissi, et al., 2006). In Nigeria, the most common fungal skin infection is tinea versicolor which correlates with several factors such as humid environment, heavy sweating, malnutrition, and genetics. Furthermore, the treatment from this skin fungal infection is found to be quite expensive such as Clotrimazole and due to the poor socioeconomic status in the infected population thus the number of infected patients with skin infections are very high (Oladele, et al., 2010).

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It has also been reported that one-third of Mauritian population are infected with skin diseases with acne which is the most prevalent skin infection, followed by eczema, athlete foot and versicolor infection due to gender, age, personal hygiene, climatic conditions and level of awareness (Goonmatee and Rajesh, 2013). Therefore, the knowledge is an essential prevention factor to prevent skin infection (Schofield et al., 2009). The health knowledge in skin infections is necessary with the prevention, and it is important to seek primary health care to prevent complication and serious infection.

knowledge and education about skin infections can play a critical role in prevention and management of skin infections (White et al., 2013).

1.2 PROBLEM STATEMENT AND SIGNIFICANCE OF THE STUDY

In the recent decades, the interest in evaluating therapeutic effects of aquatic natural products has been increased, therefore researches exploration of new alternative treatment from natural sources such as eel that possess no/minimal toxic effect is highly needed. According to WHO, 80% of the developing countries in the world’s population rely on plant-derived medicines for the health care (Gurib-Fakim, 2006). Marine floras are widely unexplored as potent medicines for the dreadful human disease (Sithranga Boopathy and Kathiresan, 2011). It has been isolated more than 10,000 compounds from marine organisms and there are hundreds of new compounds are still being discovered every year. Around 300 patents were issued on bioactive marine natural products between 1969 and 1999 (Kathiresan et al., 2008). Discovery and development of new aquatic drugs remain one of the most challenging areas in recent marine and freshwater sciences.

A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy in Biomedical Science

IN-VITRO AND IN-VIVO EVALUATION OF

THERAPEUTIC PROPERTIES OF SKIN MUCUS FROM ASIAN SWAMP EEL (Monopterus albus)

BY

AYAH REBHI MOHAMMAD HILLES

A thesis submitted in fulfilment of the requirement for the degree of Doctor of Philosophy in Biomedical Science

Kulliyyah of Allied Health Sciences International Islamic University Malaysia

FEBRUARY 2019

ABSTRACT

ثحبلا ةصلاخ

APPROVAL PAGE

DECLARATION

T P

INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

IN-VITRO AND IN-VIVO EVALUATION OF THERAPEUTIC PROPERTIES OF SKIN MUCUS FROM ASIAN SWAMP EEL

(Monopterus albus)

ACKNOWLEDGEMENTS

TABLE OF CONTENTS

LIST OF TABLES

LIST OF FIGURS

LIST OF ABBREVIATIONS

LIST OF SYMBOLES

CHAPTER ONE INTRODUCTION