BIOTRANSFORMATION OF ARTONIN E BY LOCALLY ISOLATED

MICROORGANISMS

LAI YEW SENG

A thesis submitted in fulfillment of the

requirements for the award of the degree of

Master of Science (Bioscience)

Faculty of Biosciences and Medical Engineering

Universiti Teknologi Malaysia

March 2013

v

A special dedication for my family, supervisors, lecturers and friends:-

vi

ACKNOWLEDGEMENTS

In preparing this thesis, I came in contact with many people, researchers and

academicians. They had contributed towards my further understanding and

developed innovative thoughts on the research itself. In particular, I would like to

express my deepest gratitude towards my supervisor, Dr. Shaza Eva binti Mohamad

for her care, concern and guidance throughout the project. I am also very thankful to

my co-supervisor, Dr Shajarahtunnur binti Jamil for her encouragement, guidance

and advices.

I would like to express my sincere appreciation to all my lecturers, who have

had given me advices throughout my study in UTM. I am also grateful to MOSTI

(VOT 79292) for financial support, Faculty Bioscience and Bioengineering, UTM for

giving me the chance to study in this field and Research Management Center for

managing the distribution of funds for this project.

Special appreciation goes to all the laboratory assistants for their kindness

and assistance during the project. Thanks to all my friends for their cooperation

during the progress of this study. A meaningful gratitude dedicated to my beloved

family for their understandings, support and inspirations throughout the project.

vii

ABSTRACT

Biotransformation is a process that involves modification of the structure of a

chemical compound by microbial activities. It has advantages over chemical

synthesis as it is able to be operated at near room temperature, with non-extreme pH

as well as it is a highly stereospecific reaction. The purpose of this research are to

study the ability of selected microbes to transform artonin E, a flavonoid extracted

from the bark of local plant, Artocarpus tesymanii and identify potential

transformation products. 17 types of microbes were isolated from various sources,

however 3 types of bacteria and 2 types of fungi were able to grow in the presence of

artonin E. They were identified by 16S rRNA and Internal Transcrib Spacer Region

for bacteria and fungi respectively. These microbes were Bacillus cereus, Bacillus

subtilis, Bacillus pumillus, Aspergillus fumigatus and Aspergillus aculeatus. The

transformation products were analysed by High Performance Liquid

Chromatography (HPLC) and Thin Layer Chromatography (TLC). There was a

significant peak which corresponded to the biotransformation product as shown in

the HPLC chromatogram at retention time of approximately 3.9 minutes. The

percentage area of the peak was shown to increase from 6.89% at day 0 to 11.80% at

day 1 followed by decreasing of the percentage area of the remaining artonin E

concentration in the reaction medium. This was further confirmed by TLC where a

compound termed as P1 (transformation product) emerged at the Rf value of 0.37.

Artonin E showed a moderate antioxidant property with IC50 value of 81.61µg/mL

while the biotransformation product showed very weak antioxidant property with

IC50 more than 200 µg/mL. Sample was sent for structure elucidation using Nuclear

Magnetic Resonance Spectroscopy (NMR) but due to limited concentration of

sample produced in the transformation, the structure could not be fully analyzed.

viii

ABSTRAK

Biotransformasi adalah proses yang melibatkan pengubahsuaian sebatian

kimia oleh aktiviti mikrob. Ia mempunyai kelebihan berbanding sintesis kimia biasa

kerana ianya dapat beroperasi pada suhu bilik dan pH yang tidak melampau dan

tindakbalas yang dilakukan juga adalah sangat stereospesifik. Tujuan kajian ini ialah

untuk mengkaji keupayaan mikrob terpilih untuk mengubah artonin E, flavonoid

yang diekstrak daripada kulit tumbuhan tempatan iaitu Artocarpus tesymanii dan

juga untuk mengenalpasti produk transformasi. 17 mikrob yang dipencilkan dari

pelbagai sumber , tetapi hanya 3 jenis bakteria dan 2 jenis fungus telah dipilih di

mana mikrob ini mempunyai kebolehan hidup dengan kehadiran artonin E serta

telah dikenal pasti melalui analisis 16S rRNA dan Internal Transcrib Spacer. Mikrob

ini adalah Bacillus cereus, Bacillus subtilis, Bacillus pumillus, Aspergillus fumigatus

and Aspergillus aculeatus. Produk transformasi dianalisa menggunakan

Kromatografi Cecair Prestasi Tinggi (HPLC) dan Lapisan Kromatografi Nipis (TLC).

Satu puncak ketara yang dipercayai produk biotransformasi ditunjukkan pada

kromatogram HPLC pada masa kira-kira 3.9 minit. Terdapat peningkatan peratusan

keluasan dari 6.89% pada hari 0 ke 11.80% pada hari pertama dan diikuti dengan

penurunan peratusan keluasan baki kepekatan artonin E. Ini telah dibuktikan juga

oleh TLC di mana satu sebatian yang dinamakan P1 muncul pada nilai Rf

0.37. Artonin E menunjukkan ciri-ciri antioksidan yang sederhana di mana nilai IC50

81.61μg/mL, manakala produk biotransformasi menunjukkan ciri antioksidan yang

sangat lemah berbanding artonin E dengan nilai IC50 adalah 200 μg/mL. Sampel

telah dihantar untuk pengenalpastian struktur dengan menggunakan Spektroskopi

Resonan Magnetik Nuklear (RMN) tetapi disebabkan kepekatan sampel yang

dihasilkan dalam transformasi adalah terhad, struktur tidak dapat dianalisa

sepenuhnya.

ix

TABLE OF CONTENT

CHAPTER TITLE PAGE

TITLE

SUPERVISOR ENDORSEMENT ii

DECLARATION iv

DEDICATION v

ACKNOWLEDGEMENTS vi

ABSTRACT vii

ABSTRAK viii

TABLE OF CONTENTS ix

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATION xvi

1 INTRODUCTION

1.1 General Introduction 1

1.2 Problem Statement 2

1.3 Scope of Research 3

1.4 Research Objective 4

2 LITERATURE REVIEW

2.1 Transformation of flavonoid by Microorganisms 5

x

2.2 Flavonoid 6

2.3 Artonin E 10

2.4 Major Reaction Involved in Biotransformation of

Flavonoid

10

2.5 Transformation of Flavonoid by Recombinant Strain 11

2.6 16S rRNA Analysis for Bacterial Identification 13

2.7 Polymerase Chain Reaction 15

3 MATERIALS AND METHODS

3.1 Isolation and Screening of the Potential

Microorganism

17

3.1.1 Preparation of Growing Medium 18

3.1.2 Harvesting the Bacteria 18

3.1.3 Harvesting of the Fungi 19

3.2 Identification of the Potential Microorganism 19

3.2.1 Gram Staining 19

3.2.2 16S rRNA Analysis 20

3.2.2.1 Polymerase Chain Reaction for

Bacteria

21

3.2.3 Internal Transcrib Spacer for Identification of

Fungi

22

3.2.3.1 Polymerase Chain Reaction for Fungi

by Internal Transcribed Spacer

22

3.3 Biotransformation of Artonin E 23

3.3.1 Preparation of the Artonin E Stock Solution 24

3.3.2 Optimization of Artonin E Biotransformation 24

3.3.2.1 Preparation of Buffer for

Optimization

of Artonin E Biotransformation

24

3.4 Identification of Artonin E Biotransformation 25

3.4.1 High Performance Liquid Chromatography

(HPLC)

25

3.4.2 Thin Layer Chromatography (TLC) 27

xi

3.4.3 Extraction of Unknown Product From Thin

Layer

Chromatography Plate

28

3.4.4 Free Radical Scavenging Activity (DPPH)

Assay

28

4 RESULTS AND DISCUSSION

4.1 Isolation and Screening of Potential Microorganism 30

4.2 Identification of Potential Microorganism 31

4.3 pH and Composition of Mobile Phase 36

4.4 Biotransformation of Artonin E by Bacteria 36

4.5 Biotransformation of Artonin E by Fungus 37

4.5.1 Biotransformation of Artonin E by Aspergillus

fumigatus

38

4.5.2 Biotransformation of Artonin E by Aspergillus

aculeatuss

39

4.6 Identification of Artonin E Biotransformation 40

4.6.1 Analysis of the Biotransformation Product by

HPLC

42

4.6.2 Analysis of the Biotransformation Product by

TLC

46

4.6.3 Free Radical Scavenging Activity (DPPH)

Assay

48

4.6.4 Nuclear Magnetic Resonance Spectroscopy 51

5 CONCLUSION AND FUTURE WORKS 54

REFERENCES 59

APPENDICES 1-5 63-70

xii

LIST OF TABLES

TABLE NO. TITLE PAGE

3.1 Sequences for the Respective Universal Primer 21

3.2 PCR Cycle Profile 21

3.3 PCR Cycle Profile for ITS 23

4.1 Source of Microorganisms 35

4.2 Transformation Products of Artonin E by Bacillus

subtilis Analyzed by HPLC

37

4.3 Transformation Products of Artonin E by Bacillus

pumillus Analyzed by HPLC

37

4.4 Transformation Products of Artonin E by Aspergillus

fumigatus Analyzed by HPLC

39

4.5 Transformation Products of Artonin E by Aspergillus

aculeatus Analyzed by HPLC

40

4.6 The Rf value of Artonin E, control and its

transformation product

48

xiii

LIST OF FIGURES

FIGURE NO. TITLE PAGE

2.1 2-phenylchromen-4-one (flavone), (A,B)

aromatic ring; C) oxygen-containing

heterocyclic ring

7

2.2 6 major subgroups of flavonoid: (a) chalcone;

(b) flavone; (c) flavonol; (d) flavanone; (e)

catechin; (f) isoflavone and (g) isoflavanone

adapted from Alan Crozier et al., 2009

9

2.3 The structure of artonin E 10

2.4 Biotransformation of flavonoid by

recombinant strains (Chemler et al. 2009)

13

4.1 Agarose gel quantification for 16S rRNA

analysis with the 1kb DNA ladder as the

marker

33

4.2 Agarose gel quantification for Internal

Transcrib Spacer Region analysis with the

100bp DNA ladder as the marker

33

4.3 Gram staining (a) Bacillus cereus; (b) Bacillus

subtilis; (c) Bacillus pumillus

34

4.4 (a) Aspergillus fumigatus from the stem of

Artocarpus tesymanii in PDA plate; (b)

Aspergillus aculeatus from the stem of

Artocarpus heterophyllus in PDA plate

35

4.5 Chromatogram for artonin E that extracted

from the bark Artocarpus tesymanii. artonin E

was detected at retention time of 3.320

38

xiv

minutes

4.6 1.5mM artonin E 41

4.7 After addition of phosphate buffer, pH 7. (a)

Control,C1: Phosphate buffer pH 7 and

1.5mM artonin E; (b) Biotransformation

medium after addition of Phosphate buffer pH

7

41

4.8 (a) Control,C1: Phosphate buffer pH 7 and

1.5mM artonin E;(b)complete

Biotransformation medium: Phosphate buffer

pH 7, 1.5mM artonin E and fungi

42

4.9 Chromatogram for artonin E that extracted

from the bark Artocarpus tesymanii. Artonin E

was detected at retention time of 3.320

minutes

43

4.10 Chromatogram of artonin E and Controls. 1.5

mM Artonin E; Phosphate buffer pH 7 and

artonin E; Phosphate buffer pH 7 and Fungi;

Phosphate buffer pH 7, Fungi and Methanol;

Phosphate buffer pH 7, and Methanol

44

4.11 Chromatogram for the Sample by Day. Initial

Day; Day 1; Day 2; Day 3

45

4.12 The relationship between the percentage area

of 1.5mM artonin E and the percentage area of

biotransformation product done by Aspergillus

aculeatus. Remaining 1.5mM artonin E;

Transformed Product

45

4.13 Chromatogram of TLC for detection of the

suspected product done by Aspergillus

aculeatus. (a) 1.5mM Artonin E; (b) C1:

Phosphate buffer pH 7 and artonin E; (c) C2:

Phosphate buffer pH 7 and Methanol; (d) C3:

Phosphate buffer pH 7 and fungi; (e) C4:

47

xv

Phosphate buffer pH 7 ,fungi and Methanol;

(f) Initial Day; (g ) Day 1; (h) Day 2; (i) Day

3; P1, Potential Transformed Product

4.14 The percentage scavenging activity of artonin

E at different concentration

49

4.15 Standard curve of scavenging ability for

artonin E

49

4.16 The percentage scavenging activity between

1.5mM artonin E and transformed product,

P1

50

4.17 The relationship of percentage scavenging

activity. 1.5 mM artonin E; Product, P1;

Reaction mixture, day 1; Reaction mixture,

day 2

51

4.18 1H NMR spectrum for artonin E 52

4.19 1H NMR spectrum for sample extracted from

TLC plate

53

xvi

LIST OF ABBREVIATION

ACN-----Acetonitril

bp ------- base pair

DNA ---- Deoxyribonucleic acid

DPPH--- 1,1-diphenyl-2-picrylhydrazyl

NMR-----Nuclear Magnetic Resonance

EDTA -- Ethylene-diamine tetraacetate

EtBr ---- Ethidium bromide

HPLC -- High performance liquid chromatography

ITS----- Internal Transcribed Spacer

OD ---- Optical density

PCR --- Polymerase chain reaction

PDA--- Potato Dextrose Agar

ppm-- - Part per million

Rf-------Value of Thin Layer Chromatography

rpm --- Rotation per minute

TLC---Thin Layer Chromatography

Tm --- Melting temperature

UV --- Ultraviolet light

mM ---- Millimolar

mg ----- Milligram

mL -------Millilitre

nm ------Nanometer

μg ------Microgram

μL ------Microlitre

μM-----Micromolar

CHAPTER 1

INTRODUCTION

1.1 General Introduction

Intensive research on natural products has been carried out in great depth due to

its therapeutic properties. In order to enhance its pharmacological activities, these

natural products were studied in details by looking at potential functional groups that

could be manipulated to improve its efficacy (Havsteen, 1983). Flavonoid, a

polyphenolic compound that exists in the plant has been given particular attention.

It has been reported to possess bactericidal action (Havsteen, 1983) and also the

ability to inhibit various enzymes such as glutathione reductase (Elliot et al., 1992),

mitochondria succinoxidase (Hodnick et al., 1986), cyclooxygenase and lipoxygenase

(Laughton et al., 1989) and to act as antioxidants due to its ability to chelate iron (Ueno

et al., 1984). There are a lot of previous studies showing the transformation of different

sources of flavonoid with different types of microbes and enzymes (Robak and

Gryglewski, 1988; Afanas’Ev et al., 1989; Jovanovic et al., 1994).

Biotransformation or biocatalysis specifically involves the chemical modification

or the modification made by a microorganism on the structure of the compound.

Biocatalysis is more favorable compared to chemical synthesis due to the fact that the

microbial transformation can be operated in non-extreme pH condition, near room

2

temperature not forgetting its high stereospecificity. In addition, biotransformation is

the useful tools for the production of medicinal chemicals from natural products

(Demetzos et.al.,1997).

1.2 Problem Statement

Previous studies has shown that flavonoid possesses diverse physiological

and pharmacological activities such as astrogenic, antilipoperoxidant, antitumor,

antiplatelet, antiviral, antifungal, antibacterial, antihemolytic, anti-ischemic,

antiallergic and anti-inflammatory.

Artonin E is a type of isoprenyl flavoid extracted from the bark of Artocarpus

teysmanii and it was previously shown to have antimicrobial activity against

Escherichia coli and Bacillus subtilis (Jagtap and Bapat, 2010). In addition, the

artonin E was proven to give some potential transformation products as shown by

Tang (2007).

Microbial biotransformation is one of the best choices to produce medicinal

chemicals from the natural products (Demetzos et al., 1997). It can stimulate the

production of biomass and microbial genetic systems that are generally well

understood. In addition, it may mimic mammalian catabolism, and possibly allow the

production of useful intermediates or metabolites in sufficiently large quantities to

enable identification and usage in drug toxicity studies (Rathbone et al., 2002).

However, chemical synthesis which often produces many chemical groups

such as hydroxyl groups, sulphate groups and others tend to be too randomly

allocated (Barron D., et. al., 1988) and may be difficult to purify. Furthermore, it is

also likely to generate toxic waste products (Seeger et al., 2003).

In this study, locally isolated microorganisms will be used to screen their

3

ability to transform the artonin E. The potential biotransformation product will be

analysed by HPLC and TLC followed by investigation of their scavenging ability

using the free radical scavenging activity (DPPH) assay.

1.3 Scope of Research

In this study, the microorganism will be isolated from different parts of

several Artocarpus sp. such as the bark, the skin of the fruit and also the soil around

the Artocarpus tree. These parts of the tree and surroundings will be selected as

microorganisms from these sources could have the ability to grow in the presence of

artonin E, by utilizing this compound as their natural carbon sources. Artonin E was

provided by Dr. Shajarahtunnur Jamil, Department of Chemistry, Faculty of Science,

UTM where the artonin E was isolated from the bark of Artocarpus tesymanii.

The isolated microorganisms that can grow in the presence of the artonin E

will be characterized by gram staining. They will be further identified by 16S rRNA

analysis and Internal Transcribed Spacer Region for fungi identification. The

transformation of the artonin E will be carried out using locally isolated

microorganisms. The transformed products will be subjected to Thin Layer

Chromatography (TLC), High Performance Liquid Chromatography (HPLC). The

structure of the biotransformation products will be elucidated by NMR spectroscopy.

Antioxidant assay will be performed to evaluate the antioxidant property of the

bitransformation products.

4

1.4 Research Objective

The objectives of this study are stated as below:

1. To screen microorganisms from different sources that have the ability to

transform artonin E from Artocarpus sp.

2. To identify the potential microorganisms using gram staining and further

analyzed by 16S rRNA analaysis and Internal Transcribed Spacer Region

for fungi identification.

3. To optimize the biotransformation reaction and identify the

transformation products using Thin Layer Chromatography (TLC) and

High Performance Liquid Chromatography (HPLC).

4. To identify the biotransformed products by Nuclear Magnetic Resonance

(NMR) Spectroscopy and evaluate their antioxidant property using free

radical scavenging (DPPH) assay.

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