universiti putra malaysia phytochemical …psasir.upm.edu.my/39172/7/fp 2013 17 ir.pdfuniversiti...

UNIVERSITI PUTRA MALAYSIA

PHYTOCHEMICAL COMPOUND DETERMINATION OF Entada rheedei SPRENG AND ITS ANTIFUNGAL ACTIVITY ON SELECTED SOILBORNE FUNGAL

PHYTOPATHOGENS

NENI KARTINI BT CHE MOHD RAMLI

FP 2013 17

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PHYTOCHEMICAL COMPOUND

DETERMINATION OF Entada rheedei SPRENG

AND ITS ANTIFUNGAL ACTIVITY ON

SELECTED SOILBORNE FUNGAL

PHYTOPATHOGENS


DOCTOR OF PHILOSOPHY UNIVERSITI PUTRA MALAYSIA

2013

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PHYTOCHEMICAL COMPOUND DETERMINATION OF Entada rheedei

SPRENG AND ITS ANTIUNGAL ACTIVITY ON SELECTED SOILBORNE

FUNGAL PHYTOPATHOGENS

By


Thesis Submitted to the School of Graduate Studies,

Universiti Putra Malaysia, in Fulfillment of the

Requirements for the Degree of Doctor of Philosophy

May 2013

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COPYRIGHT

All materials contained within the thesis, including without limitation text, logos, icons, photographs and all other artwork, is copyright material of Universiti Putra Malaysia unless otherwise stated. Use may be made of any material contained within the thesis for non-commercial purposes from the copyright holder. Commercial use of material may only be made with the express, prior, written permission of Universiti Putra Malaysia.

Copyright © Universiti Putra Malaysia

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DEDICATION

I dedicated this precious effort, the fruit of my thoughts and study to my affectionate

mother, brothers and sisters, husband Samsuri and children Nurul Shuhada,

Muhammad Shahril Najmi, Muhammad Shahril Nazim, Muhammad Syamil Nabil

and Muhammad Syamim Naqib who inspired me to the higher destiny of life.

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Abstract of thesis presented to the Senate of Universiti Putra Malaysia in fulfillment

of the requirement for the degree of Doctor of Philosophy

PHYTOCHEMICAL COMPOUND DETERMINATION OF Entada

rheedei SPRENG AND ITS ANTIFUNGAL ACTIVITY ON SELECTED

SOILBORNE FUNGAL PHYTOPATHOGENS

By

NENI KARTINI CHE MOHD RAMLI

May 2013

Chairman : Associate Professor Kamaruzaman Sijam, PhD

Faculty : Agriculture

This study presents the potential use of natural products from Entada rheedei Spreng.

as biofungicide and their antifungal activity against five common soilborne fungal

pathogens. The dried leaves, stems and seeds of E. rheedei were extracted by

sequential extraction using petroleum ether, chloroform, methanol and water. Among

the four solvents used, methanol (MeOH) was proven to be the best extracting

solvent by giving the highest quantity of crude extract yielding 7.74g (3.87%) of

stems crude extract, 1.861g (0.93%) of leaves crude extract and 26.6g (13.3%) of

seed crude extract. The gained crude extracts were then screened via in vitro assays

for their antifungal activity, using well diffusion method, against five selected

soilborne fungal pathogens Rhizoctonia solani, Fusarium commune, Fusarium

oxysporum f.sp. cubense (FOC), Pythium ultimum and Phytophthora palmivora. The

petroleum ether and chloroform extracts of stems, seeds and leaves of E. rheedei

failed to inhibit the growth of all tested soilborne fungal phytopathogens. The best

inhibitory effect was elicited by the stems and seeds crude methanol extracts against

Fusarium commune (51.4% and 50% radial growth inhibition respectively), while

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crude water and methanol extracts of seed against FOC showed the least significant

effect (15% radial growth inhibition). Nevertheless, seed crude methanol and water

extracts gave an impressive inhibitory on radial growth of F. commune with 50% and

36.7% inhibition respectively. Moreover, stem crude methanol and water extracts

also showed moderately significant inhibitory against FOC (45% and 42.5%

inhibitory respectively). The results also showed that all the stem and seed methanol

and water crude extracts failed to inhibit the growth of R. solani, P. ultimum and P.

palmivora. The Effective Inhibitory Concentration (EIC) of stem and seed crude

methanol and water were determined at 100mg/mL. The phytochemical analysis of

the active crude extracts revealed the presence of tannins, saponins, glycosides,

triterpenoid, and alkaloid which only present in seed. Light and scanning electron

microscopic observation on the effect of active crude extracts on spore germination

and hyphal growth of F. commune and FOC illustrated that the spore and hyphae

were collapsed and lysed; cell wall were degraded and shrank; while the spore

(macroconidia) failed to germinate. The active crude extracts were further purified

through Vacuum Liquid Chromatography (VLC) fractionation and resulted in the

total of 22 fractions with 11 fractions for each stem and seed methanolic extracts.

The obtained fractions were then screened for the antifungal activity against the

selected soilborne fungal pathogens. Results showed some improvement on

antifungal activity of the stem and seed methanolic fractions. The fraction eluted

with chloroform: methanol in the ratio of 6:5 (F5) and 5:5 (F6) of stem methanol

proved to be the most active fractions against F. commune (56.8% and 45.9% growth

inhibition) and FOC (48.7% and 43.6% growth inhibition). All fractions of stem and

seed methanolic extracts showed some improvement in their antifungal activity

against R. solani and P. palmivora, which were not shown previously by any of their

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parent crude extracts. The TLC phytochemical analysis showed the presence of

saponin and tannin in the fractions F4, F5, F6 and F7 of stem and seed methanolic

extracts. The determination of major active compound in the active fractions via

High Performance Liquid Chromatography (HPLC) analysis, to confirm the major

constituent contained in the active fractions, showed that the active fractions (F4-F7)

of stem and seed methanolic extracts of E. rheedei contain saponin as major active

compound.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Doktor Falsafah.

PENENTUAN SEBATIAN KIMIA TUMBUHAN Entada rheedei SPRENG

DAN AKTIVITI ANTIKULATNYA KE ATAS PATOGEN TUMBUHAN

KULAT BAWAAN TANAH TERPILIH

Oleh

NENI KARTINI CHE MOHD RAMLI

Mei 2013

Pengerusi : Prof Madya Kamaruzaman Sijam, PhD

Fakulti : Pertanian

Kajian menunjukkan potensi kegunaan hasilan semulajadi daripada pokok Entada

rheedei Spreng sebagai racun kulat biologi dan aktiviti antikulatnya terhadap lima

jenis patogen kulat bawaan tanah. Daun, batang dan biji kering Entada rheedei

diekstrak melalui pengekstrakan urutan menggunakan empat jenis larutan petroleum

ether, kloroform, methanol dan air. Methanol (MeOH) telah terbukti sebagai pelarut

pengekstrak terbaik dengan memberikan kuantiti ekstrak mentah terbanyak dengan

menghasilkan 7.74g (3.87%) ekstrak mentah batang, 1.861g (0.93%) ekstrak mentah

daun dan 26.6g (13.3%) ekstrak mentah biji. Ekstrak-ekstrak mentah yang kedapatan

di saringkan untuk aktiviti kulatnya secara in vitro dengan kaedah pembauran lubang

terhadap lima patogen kulat bawaan tanah terpilih Rhizoctonia solani, Fusarium

commune, Fusarium oxysporum f.sp. cubense (FOC), Pythium ultimum dan

Phytophthora palmivora. Ekstrak petroleum ether dan kloroform extracts batang, biji

dan daun gagal merencat pertumbuhan semua kulat pathogen tumbuhan yang diuji.

Perencatan terbaik ditunjukkan oleh ekstrak mentah methanol batang dan buah

terhadap Fusarium commune (masing-masing dengan 51.4% dan 50% perencatan

jejari pertumbuhan), Sementara ekstrak air dan methanol biji memberikan kesan

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yang sedikit terhadap FOC (15% perencatan jejari pertumbuhan). Namun, ekstrak

mentah methanol dan air biji memberikan perencatan jejari pertumbuhan yang

menggalakan terhadap F. commune dengan 50% dan 36.7% perencatan masing-

masing. Seterusnya, ekstrak mentah methanol dan air batang juga menunjukkan

perencatan yang sederhana terhadap FOC (45% dan 42.5% perencatan masing-

masing). Keputusan juga menunjukkan semua ekstrak mentah methanol dan air

batang dan biji gagal untuk merencat R. solani, P. ultimum dan P. palmivora.

Kepekatan Perencatan Berkesan (KPB) ekstrak mentah methanol dan air batang dan

biji telah ditentukan pada 100mg/mL. Analisa fitokimia ke atas ekstrak aktif E.

rheedei mendapati kehadiran tannins, saponin, glycosides, triterpenoid, dan alkaloid

yang hanya terdapat dalam biji. Cerapan mikroskopik melalui mikroskop cahaya dan

elektron terhadap kesan ekstrak mentah aktif terhadap percambahan spora dan

pertumbuhan hifa kulat F. commune dan FOC memperlihatkan spora dan hifa kulat

meruntuh dan pecah; dinding sel hancur dan mengecut; sementara spora

(makrokonidia) gagal untuk bercambah. Ekstrak mentah yang aktif seterusnya di

tulinkan melalui fraksinasi Kromatografi Cairan Hampagas (KCH) dan telah

menghasilkan sejumlah 22 fraksi dengan 11 fraksi untuk setiap ekstrak mentah

methanol batang dan biji. Fraksi yang kedapatan seterusnya disaringkan untuk

aktiviti antikulatnya terhadap patogen kulat bawaan tanah terpilih. Keputusan telah

menunjukkan peningkatan terhadap aktiviti antikulat fraksi methanol batang dan biji.

Fraksi batang methanol yang di alirkan dengan pelarut kloroform: methanol dalam

nisbah 6:5 (F5) dan 5:5 (F6) terbukti menjadi fraksi yang teraktif terhadap F.

commune (56.8% dan 45.9% perencatan pertumbuhan) dan FOC (48.7% and 43.6%

perencatan pertumbuhan). Semua fraksi ekstrak methanol batang dan biji

menunjukkan peningkatan dalam aktiviti antikulatnya terhadap R. solani dan P.

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palmivora, yang mana sebelumnya tidak ditunjukkan oleh ekstrak mentah induk

mereka. Analisa fitokimia TLC menunjukkan kehadiran saponin dan tannin di dalam

fraksi-fraksi F4, F5, F6 dan F7 ekstrak methanol batang dan biji. Penentuan sebatian

utama yang aktif didalam fraksi yang aktif melalui analisa Kromatografi Cairan

Prestasi Tinggi, bagi mempastikan kandungan utama dalam fraksi aktif telah

menunjukkan fraksi-fraksi aktif (F4-F7) ekstrak methanol batang dan biji

mengandungi saponin sebagai sebatian aktif utama.

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ACKNOWLEDGMENTS

All praises and thanks are to Allah SWT, the magnificent and merciful.

I avail myself of this opportunity to record my sincerest thanks and appreciation to

Associate Professor Dr. Kamaruzaman Sijam, Chairman of supervisory committee

and also to my supervisory committee members, Professor Dr. Dzolkifli Omar, and

Professor Dr. Rita Muhammad, for their dedicated efforts, constructive comments,

support, invaluable advice, intellectual guidance and encouragement in conducting

my research and in the preparation of this thesis.

I am exceedingly grateful to the Malaysian Government for providing financial

support through FRGS GRANT (No 011000070005) and also to the Universiti

Teknologi MARA Pahang for allow me to study for Doctor of Philosophy

programme in the Universiti Putra Malaysia.

My special thanks to all my friends and staffs in Chemistry Laboratory, UiTM

Pahang and Plant Pathology, Microbiology and Nematology Laboratories, Faculty of

Agriculture, UPM for their support, courage and assistance in doing my research.

Finally, I also take this opportunity to express my deepest gratitude to my Mother in

peace, brothers, sister, husband Samsuri, daughter Nurul Shuhada, sons Najmi,

Nazim, Nabil and Naqib. I thank them for all their love, support and encouragement

throughout my study in the Universiti Putra Malaysia and my whole life.

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APPROVAL

I certify that a Thesis Examination Committee has met on 31st May 2013 to conduct

the final examination of Neni Kartini Che Mohd Ramli on her thesis entitled

“Phytochemical compounds determination of Entada rheedei Spreng and its

antifungal activity against selected soilborne fungal phytopathogens” in accordance

with the Universities and University College Act 1971 and the Constitution of the

Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The committee

recommends that the student be awarded the Doctor of Philosophy.

Members of the Thesis Examination Committee were as follows:

Lau Wei Hong, PhD

Department of Crop Protection,

Faculty of Agriculture,

Universiti Putra Malaysia

(Chairman)

Zainal Abidin Mior Ahmad, PhD

Associate Professor,




(Internal Examiner)

Sariah Meon, PhD

Professor,




(Internal Examiner)

Jamal Ragheb Said Qasem, PhD

Professor,

Department of Plant Protection,

Faculty of agriculture,

University of Jordan

(External Examiner)

__________________________

SEOW HENG FONG, PhD

Professor and Deputy Dean

School of Graduate Studies


Date :

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This thesis was submitted to the Senate of Universiti Putra Malaysia and has been

accepted as fulfillment of the requirement for the degree of Doctor of Philosophy.

The members of the Supervisory Committee were as follows

Kamaruzaman Sijam, PhD

Associate Professor

Faculty of Agriculture


(Chairman)

Dzolkhifli Omar, PhD

Professor



(Member)

Rita Muhamad, PhD

Professor



(Member)

Siti Zaiton Mad So’ad, Phd

Assistant Professor

Kuliyyah of Pharmacy

International Islamic University Kuantan, Pahang

(Member)

___________________________

BUJANG BIN KIM HUAT, PhD

Professor and Dean School of Graduate Studies


Date:

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DECLARATION

Declaration by graduate student

I hereby confirm that:

this thesis is my original work;

quotations, illustrations and citations have been duly referenced;

this thesis has not been submitted previously or concurrently for any other degree

at any other institutions;

intellectual property from the thesis and copyright of thesis are fully-owned by

Universiti Putra Malaysia, as according to the Universiti Putra Malaysia

(Research) Rules 2012;

written permission must be obtained from supervisor and the office of Deputy

Vice-Chancellor (Research and Innovation) before thesis is published (in the form

of written, printed or in electronic form) including books, journals, modules,

proceedings, popular writings, seminar papers, manuscripts, posters, reports,

lecture notes, learning modules or any other materials as stated in the Universiti

Putra Malaysia (Research) Rules 2012;

there is no plagiarism or data falsification/fabrication in the thesis, and scholarly

integrity is upheld as according to the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) and the Universiti Putra Malaysia

(Research) Rules 2012. The thesis has undergone plagiarism detection software.

Signature: _______________________ Date: 31 May 2013

Name and Matric No.: Neni Kartini Che Mohd Ramli (GS19109)

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL ix

DECLARATION xi

LIST OF TABLES xv

LIST OF FIGURES xvi

LIST OF ABBREVATION xviii

CHAPTER

1 GENERAL INTRODUCTION 1

1.1 Background 1

1.2 The objectives of the study 4

2 LITERATURE RIVIEW 5

2.1 Entada rheedei plant 5

2.2 Soilborne diseases and their managements 7

2.3 Implication of chemical control method used in agriculture 14

2.4 Potential used of plant products as bio-fungicide 18

2.5 Phytochemicals constituents in Entada and their biological

activity

22

2.6 Mechanism of antifungal activity of plant extracts 26

3 ANTIFUNGAL PROPERTIES OF Entada rheedei CRUDE

EXTRACTS AGAINST SELECTED SOILBORNE FUNGAL

PHYTOPATHOGENS

31

3.1 Introduction 31

3.2 Materials and Methods 34

3.2.1 Plant Material 34

3.2.2 Extraction Process 34

3.2.2.1 Water extraction 34

3.2.2.2 Solvent Extraction 35

3.2.3 Assay for antifungal activity 36

3.2.3.1 Preparation of fungal pathogens 36

3.2.3.2 Preparation of the tested extracts 37

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3.2.3.3 Antifungal activity bioassay 38

3.2.4 Determination of effective inhibitory concentration

(EIC) in vitro 39

3.2.5 Effect of active crude extracts on spore germination 41

3.2.6 Microscopic observation on the effects of active

crude extracts on the hyphal growth 43

3.2.6.1 Preparation of the fungal culture stub 43

3.2.6.2 Scanning electron microscopic observation 44

3.2.7 Statistical analysis 44

3.3 Results and Discussion 45

3.3.1 Extraction Results 45

3.3.2 Antifungal activity of E. rheedei crude extracts 49

3.3.2.1 Effect of water extracts on the growth of

soilborne fungal growth 49

3.3.2.2 Effect of solvents extracts on the growth of

soilborne fungal pathogens 52

3.3.3 Determination of effective inhibitory concentration

(EIC) 59

3.2.4 Spore germination assay 60

3.2.5 Microscopic observation of the effects of active

extracts on hyphal and spore of FOC and F.

commune growth

64

3.4 Conclusions 69

4 DETERMINATION OF THE PHYTOCHEMICAL

COMPOSITION IN THE ACTIVE CRUDE EXTRACTS OF

Entada rheedeiI SPRENG

71

4.1 Introduction 71


4.2.1 Alkaloids (Meyer’s reagent test) 73

4.2.2 Flavonoids (Shinoda test) 73

4.2.3 Glycosides (reducing sugar) 73

4.2.4 Saponin (Froth test) 73

4.2.5 Triterpenoid/Steroid (Liebermann-Burchard Test) 74

4.2.6 Tannins 74

4.3 Results and Discussion 75

4.4 Conclusions 82

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5 FRACTIONATION OF THE ACTIVE CRUDE EXTRACTS AND

SCREENING FOR ANTIFUNGAL ACTIVITY AGAINST

SELECTED SOILBORNE FUNGAL PHYTOPATHOGENS

83

5.1 Introduction 83


5.2.1 Fractionation of active crude extracts 84

5.2.1.1 Admixture 85

5.2.1.2 Column packing 85

5.2.2 Assay for antifungal activity 88

5.2.3 Identification of bioactive compounds in active fractions 90

5.2.3.1 Thin Layer Chromatography phytochemical

analysis 90

5.2.3.2 HPLC analysis of E. rheedei active fractions 91

5.3 Results and discussion 93

5.3.1 Fractionation of active crude extracts 93

5.3.2 Antifungal activity assay 100

5.3.3 Identification of bioactive compounds in active fractions 110

5.3.3.1 Thin Layer chromatography phytochemical

analysis 110

5.3.3.2 HPLC analysis of E. rheedei active fractions 117

5.4 Conclusions 124

6 GENERAL CONCLUSION AND RECOMMENDATIONS FOR

FUTURE RESEARCH 126

6.1 Summary 126

6.2 General conclusions 129

6.3 Recommendations for future research 131

REFERENCES 132

APPENDICES 173

BIODATA OF STUDENT 177

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