MEDICINAL PLANTS

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SCREENING OF ANTIBACTERIAL ACTIVITIES AND PEPTIDES FROM SELECTED MALAYSIAN

MEDICINAL PLANTS

BY

ASMA BENAFRI

A thesis submitted in fulfilment of the requirement for the degree of Master of Science (Bio-Medical)

Kulliyyah of Science

International Islamic University Malaysia

JUNE 2009

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ABSTRACT

The Malaysian medicinal plants are used in the society for traditional medicine. They have shown a good effect to cure some diseases. Nowadays, more researches are focusing on natural products such as plants to find new antimicrobial agents, to overcome the problem of bacterial resistance. Hence, the aim of this study is to investigate for antibacterial activity of different extracts obtained from selected medicinal plants and to analyze the presence of antibacterial peptides in the active extracts. Four promising plants, Eurycoma longifolia, Labisia pumila (var. alata), Annona muricata and Annona squamosa have been selected based on their ethnomedicinal use to conduct this research. A broad range of methods and protocols were applied through experimental work. Leaves stems and roots of the plants chosen were screened for antibacterial activity using several solvents and buffers, with different concentrations ranging from 10 w/v to 100 w/v. The antibacterial activity was evaluated based on the diameter of inhibition zone by using agar well diffusion assay. Most of the extracts of selected plants prepared in ethanol, methanol and acetone exhibited antibacterial activity against both groups of bacteria Gram positive and Gram negative. Whereas only three aqueous extracts, E. longifolia leaves, Labisia pumila leaves and stems were active against B.subtilis, P. aeruginosa and M. luteus.

The range of diameter of inhibition zone of the active extracts screened is from 8mm to 25mm. All the extracts were concentrated and re-suspended in phosphate buffer, then tested again for antibacterial activity. Only, the active aqueous extracts were fractionated by size exclusion chromatography using Sephadex G50. The eluted fractions were tested for antibacterial activity and then analyzed by their loading in 15% SDS -PAGE and stained by silver nitrate to visualize the quality of the peptides purified. The molecular weight of purified antibacterial peptide in E. longifolia and L.

pumila aqueous leaves extract was estimated to be approximately 10.6 KDa and 9.9 KDa respectively. In conclusions, Eurycoma longifolia and Labisia pumila may contain antibacterial peptides which will be more purified, tested again for antibacterial activity, sequenced and commercialized in the future.

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ﺚﺤﺒﻟﺍ ﺺﺨﻠﻣ

ﰲ ﺍﲑﺜﻛ ﺖﻠﻤﻌﺘﺳﺍ ﺚﻴﺣ ﺎﻳﺰﻴﻟﺎﲟ ﻱﺪﻴﻠﻘﺘﻟﺍ ﺐﻄﻟﺍﺭﻮﻄﺗ ﰲ ﺎﻴﺳﺎﺳﺃ ﺍﺭﻭﺩ ﺏﺎﺸﻋﻷﺍ ﺖﺒﻌﻟ ﺪﻘﻟ ﻊﻤﺘﺍ . ﺮﺿﺎﳊﺍ ﻲﻔﻓ ،ﺽﺍﺮﻣﻷﺍ ﺾﻌﺑ ﻰﻠﻋ ﺍﺪﻴﺟ ﻻﻮﻌﻔﻣ ﺏﺎﺸﻋﻷﺍ ﻩﺬﻫ ﺕﺮﻬﻇﺃ ﺚﻴﺣ

ﺓﺪﻳﺪﺟ ﺔﻳﲑﺘﻜﺑ ﺕﺍﺩﺎﻀﻣ ﺩﺎﳚﻹ ﺏﺎﺸﻋﻷﺍ ﻞﺜﻣ ﺔﻴﻌﻴﺒﻄﻟﺍ ﺩﺭﺍﻮﳌﺍ ﻰﻠﻋ ﺙﺎﲝﻷﺍ ﻦﻣ ﲑﺜﻛ ﺪﻤﺘﻌﺗ ﺘﻟﺍ ﻞﺟﺃ ﻦﻣ ﺓﺩﺎﻀﳌﺍ ﺔﻳﻭﺩﻸﻟ ﺎﻳﲑﺘﻜﺒﻟﺍ ﺔﻣﻭﺎﻘﻣ ﺔﻠﻜﺸﻣ ﻰﻠﻋ ﺐﻠﻐ

. ﻩﺬﻫ ﻑﺪ ،ﺐﺒﺴﻟﺍ ﺍﺬﳍ

ﻯﺮﺧﻷﺍ ﺕﺍﺩﺎﻀﳌﺍ ﻦﻋ ﻞﻳﺪﺒﻛ ﺔﻳﺪﻴﺘﺒﻴﺑ ﺔﻌﻴﺒﻃ ﻱﺫ ﻱﲑﺘﻜﺑ ﺩﺎﻀﻣ ﻦﻋ ﺚﺤﺒﻠﻟ ﺔﺳﺍﺭﺪﻟﺍ ﺔﻓﻭﺮﻌﳌﺍ . ﻱﺪﻴﻠﻘﺗ ﱯﻃ ﻝﺎﻤﻌﺘﺳﺍ ﺕﺍﺫ ﺕﺎﺗﺎﺒﻧ ﻊﺑﺭﺃ ﺕﲑﺘﺧﺍ ﺪﻘﻓ ﺔﺳﺍﺭﺪﻟﺍ ﻑﺪﻫ ﻖﻴﻘﺤﺘﻟ

ﻲﻫﻭ : ،ﻼﻣﻮﺑ ﺎﺴﻴﺑﻻ ،ﺎﻴﻟﻮﻔﻴﳒﻮﻟ ﺎﻣﻮﻜﻳﺭﻭﺃ ﺍﺯﻮﻣﺍﻮﻜﺳ ﺎﻧﻮﻧﺃ ﻭ ،ﺎﺗﺎﻜﻳﺭﻮﻣ ﺎﻧﻮﻧﺃ

. ﺖﻠﻤﻌﺘﺳﺍ

ﺕﺍﺩﺎﻀﻣ ﺩﻮﺟﻭ ﻦﻣ ﺪﻛﺄﺘﻠﻟ ﻭ ﻒﺸﻜﻠﻟ ﺔﻔﻠﺘﳐ ﻕﺮﻃ ﺕﺍﺫ ﺕﺍﺭﺎﺒﺘﺧﻹﺍ ﻦﻣ ﺪﻳﺪﻌﻟﺍ ﺔﻳﲑﺘﻜﺑ

ﺱﺎﻴﻗ ﻰﻠﻋ ﺩﺎﻤﺘﻋﻹﺎﺑ ﻚﻟﺫﻭ ﻥﺎﺼﻏﻷﺍ ﻭ ﻕﺍﺭﻭﻷﺍ ﻭ ﺭﻭﺬﳉﺎﻛ ﺕﺎﺗﺎﺒﻨﻟﺍ ﻦﻣ ﺔﻔﻠﺘﳐ ﻖﻃﺎﻨﻣ ﰲ ﻮﻨﺘﻣ ﻞﻴﻟﺎﳏ ﰲ ﺕﺎﺼﻠﺨﺘﺴﳌﺍ ﺕﺮﻀﺣ ﺚﻴﺣ ﻂﻴﺒﺜﺘﻟﺍ ﺔﻘﻄﻨﻣ ﺓﺮﺋﺍﺩ ﺮﻄﻗ ﺕﺍﺰﻴﻛﺮﺗ ﺕﺍﺫ ﺔﻋ

ﲔﺑ ﺡﻭﺍﺮﺘﺗ ﺔﻔﻠﺘﳐ 10

ﻎﻣ / ﻭ ﻞﻣ 100 ﻎﻣ / ﻞﻴﻟﺎﳏ ﰲ ﺓﺮﻀﶈﺍ ﺕﺎﺼﻠﺨﺘﺴﳌﺍ ﻩﺬﻫ ﻢﻈﻌﻣ ،ﻞﻣ

ﺕﺎﺼﻠﺨﺘﺴﻤﻠﻟ ﺔﺒﺴﻨﻟﺎﺑ ﻦﻜﻟ ،ﺔﻠﻤﻌﺘﺴﳌﺍ ﺎﻳﲑﺘﻜﺒﻟﺍ ﻉﺍﻮﻧﺃ ﻞﻛ ﺪﺿ ﺔﻴﻟﺎﻌﻓ ﺕﺮﻬﻇﺃ ﺪﻗ ﺔﻳﻮﻀﻋ ﺔﻴﺋﺎﳌﺍ ﻞﻴﻟﺎﶈﺍ ﰲ ﺓﺮﻀﶈﺍ ,

ﻉﺍﻮﻧﺃ ﺾﻌﺑ ﻰﻠﻋ ﺔﻴﻟﺎﻌﻓ ﻂﻘﻓ ﺕﺎﺼﻠﺨﺘﺴﻣ ﺔﺛﻼﺛ ﺕﺮﻬﻇﺃ ﺪﻘﻓ

ﺘﻜﺒﻟﺍ ﱄﺎﺘﻟﺎﻛ ﻲﻫ ﻭ ﺎﻳﲑ :

ﻕﺍﺭﻭﺃ ﻦﻣ ﺺﻠﺨﺘﺴﻣ ،ﺎﻴﻟﻮﻔﻴﳒﻮﻟ ﺎﻣﻮﻜﻳﺭﻭﺃ ﻕﺍﺭﻭﺃ ﻦﻣ ﺺﻠﺨﺘﺴﻣ

ﻼﻣﻮﺑ ﺎﻴﺴﺑﻻ ﻕﺎﺳﻭ .

ﻭ ﻯﺮﺧﺃ ﺏﺭﺎﲡ ﺖﻳﺮﺟﺃ ﺪﻘﻓ ، ﺕﺎﺼﻠﺨﺘﺴﳌﺍ ﺔﻴﻟﺎﻌﻓ ﺭﺎﺒﺘﺧﺍ ﺪﻌﺑ

ﻭ ﻞﻣﺎﻌﻟﺍ ﺔﻴﻋﻮﻧ ﺰﻴﻴﲤ ﻭ ﺔﻴﻘﻨﺘﻟ ﺖﻣﺪﺨﺘﺳﺍ ﺚﻴﺣ ﺲﻳﺰﻳﺭﻮﻓﻭﺮﺘﻜﻟﺇ ﻭ ﺎﻴﻓﺍﺮﻏﻮﺘﻣﻭﺮﻛ ﻲﻫ ﺍ ﺩﺎﻀﳌﺍ ﺔﻴﻟﺎﻌﻓ ﻰﻠﻋ ﻝﻭﺆﺴﳌﺍ ﺐﻛﺮﳌﺍ ﻱﲑﺘﻜﺒﻟ

. ﺖﻠﻤﻌﺘﺳﺍ ﺪﻘﻓ ﺚﺤﺒﻟﺍ ﺔﻠﺻﺍﻮﳌﻭ

ﻂﻘﻓ ﺔﻴﺋﺎﳌﺍ ﻞﻴﻟﺎﶈﺍ ﰲ ﺓﺮﻀﶈﺍ ﺕﺎﺼﻠﺨﺘﺴﳌﺍ .

ﻥﺃ ﺕﺮﻬﻇﺃ ﺪﻗ ﺏﺭﺎﺠﺘﻟﺍ ﻩﺬﻫ ﺞﺋﺎﺘﻧ ﻥﺇ

ﺩﺎﻀﳌﺍ ﺔﻴﻟﺎﻌﻓ ﻦﻋ ﻝﻭﺆﺴﳌﺍ ﻞﻣﺎﻌﻟﺍ ﻰﻠﻋ ﻱﻮﺘﲢ ﺔﻴﺋﺎﳌﺍ ﻞﻴﻟﺎﶈﺍ ﰲ ﺓﺮﻀﶈﺍ ﺔﻟﺎﻌﻔﻟﺍ ﺕﺎﺼﻠﺨﺘﺴﳌﺍ ﺔﻳﺪﻴﺘﺒﻴﺑ ﺔﻌﻴﺒﻃ ﻭﺫ ﻪﻧﺃ ﻒﺸﺘﻛﺍ ﻱﺬﻟﺍ ﻭ ﻱﲑﺘﻜﺒﻟﺍ .

ﺎﳋﺍ ﰲ ﻭ ﺙﺎﲝﺃ ﺀﺍﺮﺟﻹ ﺕﺎﻴﺻﻮﺗ ،ﺔﲤ

ﻢﻴﻤﻌﺗ ﻞﺟﺃ ﻦﻣ ﻞﻤﻌﻟﺍ ﺔﻠﺻﺍﻮﳌ ﻭ ﺎﻬﻴﻠﻋ ﻞﺼﺤﺘﳌﺍ ﺞﺋﺎﺘﻨﻟﺍ ﻦﻣ ﻖﻘﺤﺘﻟﺍ ﻞﺟﺃ ﻦﻣ ﻯﺮﺧﺃ

ﺔﻴﻧﺎﺴﻧﻹﺍ ﺔﻣﺪﳋ ﻑﺎﺸﺘﻛﻹﺍ ﺍﺬﻫ ﻡﺍﺪﺨﺘﺳﺍ

.

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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.

……….

Abd-ElAziem Farouk Gad 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.

………..

Syed Zahir Idid Co-supervisor

………

Ridzwan Hashim Internal Examiner

……….

Norihan Mohd. Saleh External Examiner

This thesis was submitted to the Department of Biomedical Science and is accepted as fulfilment of the requirement for the degree of Master of Science.

………

Nurulwahida Saad Head, Department of Biomedical Science

This thesis was submitted to the Kulliyyah of Science and is accepted as fulfilment of the requirement for the degree of Master of Science.

……….

Ridzwan Hashim

Dean, Kulliyyah of Science

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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.

Asma Benafri

Signature ……… Date ………..

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INTERNATIONAL ISLAMIC UNIVERSITY MALAYSIA

DECLARATION OF COPYRIGHT AND AFFIRMATION OF FAIR USE OF UNPUBLISHED RESEARCH

THE SCREENING OF ANTIBACTERIAL COMPOUNDS FROM MALAYSIAN MEDICINAL PLANTS: IDENTIFICATION OF

ANTIBACTERIAL PEPTIDES.

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 Asma Benafri.

……… ………

Signature Date

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I dedicate this work to, my beloved husband Djamel Dilmi for his patience, endurance and support through out my study, to my lovely son Mouhamed Is`haq and to my mother Habiba Benrekia, my sisters and brothers and to my father and mother in law, brothers and sisters in law. I would also like to thank my uncle Ali, his wife and all his family Also a special thank goes to my sister Hanane who helped me a lot and to my best friend Leila, all my friends in the lab and friends in Malaysia.

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ACKNOWLEDGEMENTS

Thanks to Allah for his blessings and guidance. I would like to thank my supervisor Assoc. Prof. Dr. Abd-ElAziem Farouk Gad and my co-supervisor Assoc. Prof. Dr Zahir Idid, and Dr Noorbatcha and Dr Azzedin Messikh for their help and advice. I would like also to thank my internal examiner Prof. Dr. Ridzwan Hashim, Dean of KOS, my external examiner Dr. Norihan Mohd. Saleh and also Assoc. Prof. Dr Othman Abd Samah, Deputy Dean of Post graduate Studies and Research. My special thanks and appreciation to Center of Post graduate Studies, IIUM for awarding a scholarship and the full financial support to finish my study in Malaysia successfully.

Finally, I wish to extend my gratitude to IIUM family especially the staff of both Kulliyyah of Science and Kulliyyah of Engineering and every body who helped me from far or near.

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...

……..………..

2.2 Plant material ………..………...

2.3 Test organisms………..………..

2.4 Preparation of extracts……….

2.5 Assay for antibacterial activity……….

2.6 Antibacterial activity of different concentrations of E. longifolia……..

2.7 The growth of Staphylococcus aureus in the presence of E. longifolia….

2.8 The growth of Micrococcus luteus in the presence of Labisia……..…….

2.10 Purification (size exclusion chromatography)………..

2.11 Antibacterial assay of fractions………

2.12 Optical density of fractions collected………..

2.13 SDS – polyacrylamide gel Electrophoresis……….

2.13.1 Preparation of SDS-PAGE gels………

2.13.2 Preparation of samples and running the gel……….

2.13.3 Gel staining and destining using coomassie brilliant blue R-250.

2.13.4 Gel staining using silver nitrate………

2.14 Measuring protein concentration by colorimetric………..

CHAPTER 3: RESULTS………...

3.1 Results ……….….

CHAPTER 4: DISCUSSION………..

4.1 Evaluation of the antibacterial activity ………..……….

4.2 Purification (sec) and SDS-PAGE analysis ………

4.3 Protein concentration……….

CHAPTER 5: CONCLUSION………...

BIBLIOGRAPHY……….

APPENDIX I ………...

APPENDIX II ………....

APPENDIX III …………..………....

31 31 31 31 34 34 35 35 35 36 36 37 39 39 39 39 40 41 42 42

73 73 82 86 88 92 99 100 105

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LIST OF FIGURES

Figure No. Page No.

1.1 Schematic of an SEC separation showing the separation of

low and high molecular polymers 6

1.2 3.1

Model for the mechanism of action of cationic antimicrobial peptides

Inhibition zone caused by root extract of Labisia pumila

20 48 3.2

3.3 3.4

Inhibition zone caused by Extracts From leaves and stem of Labisia pumila

Inhibition zone caused by extract from Annona muricata and Annona squamosa

Inhibition zone caused by extract from leave and stem of Eurycoma longifolia

49 50 51 3.5

3.6

3.7

3.8

3.9 3.10 3.11

Graph of diameter of inhibition zone caused by different concentrations of E. longifolia extracts in acetone on B.

subtilis (A: Acetone)

Graph of diameter of inhibition zone caused by different concentrations of E. longifolia extracts in acetone on S.

marcescens (A: Acetone)

Graph of diameter of inhibition zone caused by different concentrations of L. pumila extracts in acetone on B.

subtilis (A: Acetone)

Graph of diameter of inhibition zone caused by different concentrations of L. pumila extracts in acetone on S marcescens . (A: Acetone)

Effect of the phosphate buffer leaves extract of E. longifolia on the growth of Staphylococcus aureus

Effect of the phosphate buffer stem extract of L. pumila on the growth of M.luteus

Growth of M. luteus in the presence of Labisia pumila stem extract in phosphate buffer

52

53

54

55 56 57 58 3.12

3.13

3.14

3.15

Sephadex^TMG-50 chromatographic separation of antibacterial peptides from Eurycoma longifolia leaves extract. Buffer, 0.1M of phosphate (PH7)

Sephadex^TMG-50 chromatographic separation of antibacterial peptides from Labisia pumila leaves extract.

Buffer, 0.1M of phosphate (PH7)

15% SDS-PAGE of peptide extracted from leaves of Eurycoma longifolia for the active fractions eluted from size exclusion chromatography. Stained with silver nitrate (M:

protein marker)

15% SDS-PAGE of peptide extracted from leaves of Labisia pumila for the active fractions eluted from size exclusion chromatography. Stained with silver nitrate (M: protein marker)

59

60

61

62

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xii 3.16

3.17

3.18

3.19

15% SDS-PAGE of the leaves aqueous extract from

E .longifolia and some non active fractions eluted from size exclusion chromatography. Stained with silver nitrate (E:

crude extract; M: protein marker, 70 to 146: non active fractions)

15% SDS-PAGE of the leaves aqueous extract from L.

pumila and some of active fractions of stem extract eluted from size exclusion chromatography. Stained with silver nitrate (1: crude extract of leaves; 2: crude extract of stem M: protein marker, 69, 77, 83 active fractions of Stem extract).

Determination of molecular weight of target peptide extracted from E. lomgifolia leaves by using graph of log molecular weight of standard proteins on SDS-PAGE versus their distance traveled on the gel

Standard curve of different concentrations of Serum Bovine Albumine (BSA)

63

64

65 66

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Table No.

1.1

LIST OF TABLES

Common protein stains used for detection after electrophoresis (Stewart and Ebel, 2000)

Page No.

7 1.2

1.3 1.4 1.5 2.1 3.1 3.2 3.3 3.4 3.5 3.6 3.7

3.8

3.9 3.10 3.11 3.12

Prevalence of MRSA strains resistant to antibiotics in Central Europe. (Andre, 2005)

Antimicrobial peptides from different sources (Andre, 2005) Insect innate immune system: in vitro activity (Andre, 2005) Plant innate immune system: in vitro activity (Andre, 2005) Ethno-botanical data on medicinal plants used in this study Inhibition zone in mm indicating antibacterial activity of extracts from Annona squamosa and Annona muricata

Inhibition zone in mm indicating antibacterial activity of extracts from of E.longifolia

Inhibition zone in mm indicating antibacterial activity of extracts from of Labisia pumila

Inhibition zone in mm indicating antibacterial activity of control antibiotics

Inhibition zone in mm indicating antibacterial activity of solvents

Protein concentration of active extracts and fractions Two sample T test for antibacerial activity of two different concentrations of of Eurycoma longifolia acetone extracts on B.subtilis

Two sample T test for Antibacterial activity of of two different concentrations of Eurycoma longifolia acetone extracts on S marcescens

Two sample T test for antibacterial avtivity of of two different concentrations of L.pumila acetone extracts on B. subtilis Two sample T test for Antibacterial avtivity of two different concentrations of L.pumila acetone extracts L. pumila on S.

marcescens

Two sample T test for the growth of M. luteus in the presence of aqueous steme extract of L.pumila

Two sample T test for the growth of S. aureus in the presence of aqueous leaves extract of E. longifolia

9 15 16 18 33 43 45 46 47 47 67 68

69

69 70

71 72

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LIST OF ABBREVIATIONS

g gram

mg milligram

µg microgram

ml milliliter

µl micro liter

mm millimeter

KDa kilo Dalton

Rpm revolutions per minute SDS Sodium Deodecyl Sulfate SEC Size Exclusion Chromatography PAGE Poly Acrylamide Gel Electrophoresis W/v weight/volume

OD Optical Density

AMPs Antimicrobial peptides

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1

CHAPTER ONE INTRODUCTION

1.1 INTRODUCTION

Plants have long provided mankind with herbal remedies for many infectious diseases from minor infections to dysentery, skin diseases, asthma and malaria by using traditional methods. They continue to play a major role in primary health care as therapeutic remedies in developing countries. The search for biologically active extracts based on traditionally used plants is relevant due to the appearance of microbial resistance of many antibiotics and the occurrence of fatal opportunistic infections (Tshikalange, Meyer, and Hussein, 2005). Plants do not possess an immune system, such as found in higher vertebrates to protect themselves against pathogens.

Their resistance is mainly based on a dynamic defense system composed of antimicrobial agents (Cammue, De Bolle, Schoofs, Terras, Thevissen, Osborn, Rees, and Broekaert, 1994).

1.2 ADVANTAGES OF USING PLANTS AS A SOURCE OF ANTIBACTERIAL PEPTIDES

Plant kingdom is chosen for antibacterial peptides search because of many reasons.

First, plants have been used for many centuries and tested by millions of people. Also, it was reported that with the exception of thionin, antimicrobial peptides (AMPs) from plants are specific to fungi, yeast and bacteria; they are not toxic to human cells (Xio, 1994). So, the health risk by using medicinal plants is minimized (Xiao, 1994).

Second, plants can be harvested easily and the research can be conducted smoothly.

Also, the plant system is less complicated than other bio-sources like animals, so the

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purification and extraction techniques for the antibacterial peptides are less complicated. Moreover, plants are considered as an excellent system because of their protein expression potential and minimal production costs. Third, AMPs can significantly contribute to the development of plant crops with enhanced resistance to pathogens. Finally, the current research focuses on medicinal plants because they play an important role in the country’s economy (Shaharuddin, Abd Rahman & Zahari, 2004).

For these advantages, four promising medicinal plants (E. longifolia, L. pumila, A. muricata, and A. squamosa) that are used traditionally by local inhabitants of Malaysia have been chosen as a source for research into antibacterial agents.

1.3 PROBLEM STATEMENT

The past three decades have seen a dramatic increase in microbial resistance to antimicrobial agents that has created an immense clinical problem in the treatment of infectious disease (Prashanth, Asha, and Amit, 2001). There is not only the loss of effectiveness of antibiotics against multi-drug resistance bacteria but also the problem of the receding budget for treatment of infectious diseases (Karaman, Sahin, Gulluce, Ogutcu, Sengul, and Adiguzel, 2003).

Due to the emergence of drug resistance in human pathogens, new prototype antimicrobial agents are needed to address this situation. This prompted the evaluation of plants as a source of potential chemotherapeutic agents with antimicrobial activity based on their medical uses.

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3 1.4 AIM OF STUDY

Medicinal plants (E. longifolia, L. pumil, A. muricata and A. squamosa) were evaluated for potential antibacterial activity, in order to:

1. Screen for the presence of antibacterial activities in different plant extracts obtained from different parts of selected medicinal plants.

2. Analyse the presence of antibacterial peptides obtained from selected plant extracts

1.5 SCOPES AND LIMITATION OF STUDY

This study focuses on the screening for antibacterial peptides from the Malaysian medicinal plants, Annona muricata, Annona squamosa, Eurycoma longifolia and Labisia pumila. These species are well known, especially Labisia pumila and Eurycoma longifolia used by Malaysian herbal manufacturers to produce a wide variety of health related products. This study is limited to the characterization and identification of active antibacterial peptides from the medicinal plants.

1.5.1 Screening for antibacterial agents from medicinal plants

Plants contain numerous biologically active compounds, many of which have been shown to have antimicrobial properties. Plant-derived medicines have been part of traditional healthcare in most parts of the world for thousands of years and there is increasing interest in plants as sources of agents to fight microbial diseases (Chariandy, Seaforth, Phelps, Pollard and Khambay, 1999). Given the alarming incidence of antibiotic resistance in bacteria of medical importance (Monroe and Polk, 2000), there is a constant need for new and effective therapeutic agents.

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Numerous plants species have been utilized as traditional medicines in Malaysia. However, some of these have been investigated for their antimicrobial properties. Hence, it is necessary to establish the scientific basis for the therapeutic action of traditional medicinal plants as these may serve as the source for the development of more effective drug.

The current research is about screening for antibacterial compounds or peptides from four species of Malaysian medicinal plants. The Malaysian forests represent the richest of the region’s tropical forest. Many writers and scientists have amply cited its potential as a vast pharmaceutical storehouse. The diverse flora it holds offer great abundance of complex biocompounds such as alkaloids, antibiotics, enzymes and hormones that can potentially serve the need of modern medicine. There are four plant species used in this study Annona muricata and Annona squamosa (Annonaceae), Labisia pumila (Myrsinaceae) and Eurycoma longifolia (Simarubaceae) are used as traditional medicine in South East Asia including Malaysia.

Annonaceae is a large floral family comprising 120 genera and more than 2000 species including Annona squamosa and Annona muricata. This family of plant is well known and widely used as medicinal plants amongst the local population. They are generally used for treatment of asthma, cough, fever, intoxication, rheumatism, diarrhea, tumors, toothache, ulcers and wounds.

Labisia pumila (Kacip Fatimah) is one of the most popular and potent ingredients in traditional herbal "jamu" for afterbirth care. A decoction of the plant is used to expedite labor and as a protective medicine after childbirth. It is also used to treat gonorrhea, dysmenorrhoea, rheumatism, dysentery, and flatulence.

Eurycoma longifolia (Tongkat Ali) is a small tree that has been as popular as a traditional aphrodisiac in Malaysia that the over harvesting has placed it in danger of

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being extinct and is now on Malaysia’s “protected plant list”. It can be found in South East Asian rainforest areas such as Indonesia, Thailand, Laos, Vietnam and Cambodia. For centuries it has been used as an aphrodisiac and as relief for a variety of ailments from post partum to antimalarial, antiulcer, antipyretic, intestinal worms as well as malarial parasites (Kuo, Damu, Lee, and Wu, 2004).

1.5.2

Identification and characterization of active antibacterial peptide

Due to the worldwide problem of increasing economic losses and health problems caused by fungi and bacteria due to the spreading of microbial resistance, several studies have been focused on the screening of novel plant peptides that possesses antimicrobial properties. These peptides are characterized by a low molecular weight (<10 KDa) and a positive charge (Pelegrini, Noronha, Muniz, Vasconcelos, Chiarello, Oliveira, and Franco, 2006).

In the current research, four plant species were screened for antibacterial peptides. Only two samples, which are E. longifolia, leave extract and L. pumila stem extract showed activity in phosphate buffer thus the presence of antibacterial peptides is highly suspected. In this step of the research, size exclusion chromatography and SDS electrophoresis techniques were used for the purification and characterization of antibacterial peptides that may be present in both samples.

Because of the complexicity of proteins, no single analytical method can detect all possible chemical, physical and immunological changes in the proteins. Several analytical techniques (electrophoresis, spectroscopy, chromatography, thermal analysis, immunoassays and bioassays) may be required to characterize a protein completely (Ajay, 2006). In the current study, SEC as shown in Figure1.2 and SDS- PAGE were applied.

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Figure 1.1: Schematic of an SEC separation showing the separation of low and high molecular weight polymers (Sadao and Howard, 1999).

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7 Table 1.1

Common protein stains used for detection after electrophoresis (Stewart and Ebel, 2000)

Stain Advantages Disadvantages

India ink Cheap, sensitive

Enzyme detection often inhibited

Amido black cheap Low sensitivity

Ponceau S

Cheap, compatible with antigen

detection systems Low sensitivity Commassie Brilliant

Blue R Sensitive

High background due to

non specific binding

Silver stain -several versions

very sensitive ~100 fold more sensitive than Coommassie Brilliant Blue stains

Very high background due to

nonspecific binding enzyme detection often inhibited

1.6 SIGNIFICANCE OF STUDY

The findings of this study will contribute to find a new, inexpensive and alternative antibacterial peptide from Malaysian medicinal plants. Moreover, it is hoped that this research will help the medicinal plant research and development to gain an insight into the immense potential of these bio-resources.

1.7 LITERATURE REVIEW 1.7.1 Antimicrobial agents

Antibiotics are unique therapeutic agents in several respects. An antibiotic is a derivative produced by the metabolisms of microorganisms that possess antibacterial activity at low concentration .The use of antimicrobial agents is directed towards invading organisms rather than creating alteration in host physiology.

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From 1945, research in the field in antibacterial chemotherapy therapy followed two directions (i) synthetic antibacterial agents not found in nature and (ii) antibiotics extracted from the fermentation of molds such Actinomycetes and other bacteria genera.

In 1929, Fleming found a very active substance known as penicillin which was capable of inhibiting the growth of Staphylococcus aureus and Streptococcus pyogenes and that was also particularly devoid of toxicity. The penicillin was tested on patient for the first time in 1941. After this, all major families of antibiotics were discovered, cephems, aminoglycosides, tetracyclines, macrolides, peptide antibiotics, chloramphenicol, ansamycins and linsamides as well as synthetic antibacterial agents (Andre, 2005).

1.7.2 Epidemiology of resistance

Recent studies have documented that there is an epidemic of bacterial resistance that is due to the overuse and misuse of particular antibiotics. Shortly after the introduction of penicillin in the 1940s, resistance developed in Staphylococcus aureus. Nowadays, resistance includes potent antibacterial agents which are used as a last resort, including methicillin and vancomycin. Approximately 30% of hospital strains of enterococci are currently vancomycin-resistant and nearly half of the infections due to Staphylococcus aureus are methicillin-resistant. Table1.1 shows the percentage of MRSA isolate in central Europe (Andre, 2005). Given this situation, there has been an urgent need to develop new bactericidal agents which target resistant Gram-positive pathogens (Straus and Hancock, 2006). As much as antibiotics have been developed and used, resistance has emerged in many others species. At the present time 80% of strains are resistant to penicillin G.

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Studies performed in hospitals have suggested a relationship between the indiscriminate use of antimicrobial drugs and resistance of microorganisms to many antibiotics and created immense clinical problems in the treatment of infectious diseases (Monroe and Polk, 2000).

The drug-resistant bacteria have further complicated the treatment of infectious diseases in immunocompromised and AIDS patients especially in the case of nosocomial infections. There is not only the loss of effective antibiotics against multi- drug resistant bacteria, but also the global problem for the loss of budget for infectious diseases treatment. In the emergence of drug resistance in human pathogenic organisms, there is a need to develop alternative antimicrobial drugs for the treatment of infectious diseases (Lister, 2006). One approach is to screen new, inexpensive and effective drugs from other sources, including plants, for possible antimicrobial properties (Prashanth et al, 2001).

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10 Table 1.2

Prevalence of MRSA strains resistant to antibiotics in Central Europe (Andre, 2005)

Country

Prevalence of strains resistatnt to antibiotic (%)

Methicillin Ciprofloxacin ErythromycinA Co-trimoxazole Rifampycin

Austria 20.6 83.9 51.6 63.4 22.6

Germany 5.3 91.7 41.7 54.2 20.8

Italy 30.4 89.8 43.8 54 52.6

Czech

Republic 4.3 47.1 94.4 17.6 5.9

Hungary 8.6 33.3 85.2 70.4 7.4

Poland 19.7 26.3 36.8 42.1 5.2

1.7.3

Traditional medicinal plants as alternative medicine

The world health organization estimates that 80% of people living in developing countries almost exclusively use traditional medicine. This means that in the order of 3300 million people use medicinal plants on a regular basis. Medicinal plants used in traditional medicine should therefore be studied for safety and efficacy (Eloff, 1998).

Medicinal components from plants also play an important role for instance in conventional western medicine. In 1984, at least 25% of the prescription drugs issued in the USA and Canada were derived from plant, 119 secondary plant metabolites that are used globally as drug. It has been estimated that 14-28% of higher plant species are used medicinally, that only 15% of all angiosperms have been investigated chemically and that 74% of pharmacologically-active plant derived component were discovered after following up of ethno-medicinal use of the plant (Eloff, 1998). South

MEDICINAL PLANTS

SCREENING OF ANTIBACTERIAL ACTIVITIES AND PEPTIDES FROM SELECTED MALAYSIAN