universiti putra malaysia comparative analysis of different grades of gaharu using nmr...

UNIVERSITI PUTRA MALAYSIA

COMPARATIVE ANALYSIS OF DIFFERENT GRADES OF GAHARU USING NMR- AND GCMS- BASED METABOLOMICS APPROACH

SITI NAZIRAH ISMAIL

IB 2015 39

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COMPARATIVE ANALYSIS OF DIFFERENT GRADES OF GAHARU USING

NMR- AND GCMS- BASED METABOLOMICS APPROACH

By

SITI NAZIRAH ISMAIL

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia,

in Fulfilment of the Requirements for the

Degree of Master of Science

June, 2015

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

of the requirement for the degree of Master of Science

COMPARATIVE ANALYSIS OF DIFFERENT GRADES OF GAHARU USING

NMR- AND GCMS- BASED METABOLOMICS APPROACH

By

SITI NAZIRAH ISMAIL

June 2015

Chairman: Professor Khozirah Shaari, PhD

Faculty: Institute of Bioscience

Gaharu, also referred to as agarwood, kalambak, aloeswood or eaglewood, is the

resinous heartwood of infected Aquilaria sp. where infection may occur due to the

attack by fungi, insects or other external injuries. In gaharu trade, gaharu wood is

graded by experts using organoleptic methods because there is no other scientific

method to determine the grades. Normally they are graded according to observable

traits such as odor upon burning, color and wood density which reflects the resin

content of the woods. Basically price of gaharu follow the grades with highest grade

being the most expensive. The aim of this study was to carry out a comparative

analysis of the different grades of gaharu by making use of their multicomponent

metabolite profiles in order to ascertain whether they can be used to differentiate

between one grade to another. To achieve this aim, 1H Nuclear Magnetic Resonance

(1H NMR) and Gas Chromatography Mass Spectrometry Spectrometry (GCMS) combined with multivariate data analysis were used to analyze the variation between

the metabolite profiles of the different grades of gaharu samples.

Gaharu woods finely milled into fine powder, then extracted by using distilled

methanol and dried using Genevac. The gaharu crudes were proceed for 1H NMR

analysis, by dilution with deuterated methanol. Visual inspection on 1H-NMR gaharu

spectra shows that the grades have similar profile but different concentration of various

constituents. Major constituents identified are aromatics (δ6.0-7.5), sugar and

glycosides (δ2.5-5.0), fatty acids/aliphatics (δ0.5-2.0) and aldehydic compound (δ9.32).

However, since the difference among the grades is not clear from visual analysis, the analysis were carried out using multivariate analysis. Principal Component Analysis

(PCA) model overview of 4 batches of all grades of gaharu does not provide useful

information, thus further analysis was carried out on consistency of individual grade for

each batch of gaharu to see which batch and grades are suitable as class model.

According to their chemical constituents, the grades reclassified into 5 new groups (A,

B, C, D and E). Group A more significant in sugar constituents (4.24-4.28 ppm) and

possibly terpenoids (1.12, 1.16, 1.32, and 2.04 ppm), while group C and E are different

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from the rest due to higher aliphatics (3.0, 4.0, 4.52 and 7.2 ppm). On the other hand,

group B and D different from other grades by possibly terpenoidal constituents as well

(0.80, 0.84, 0.88, 1.24, and 1.28 ppm).

In order to identify the chemical markers that contributed to the differentiation of the

gaharu, NMR experiments, comprising 1D and 2D as well as J-resolve, were carried

out on selected fractions of the gaharu extract. The highest grade gaharu was selected

for the fractionation via solid phase extraction (SPE). An ethylphenylchromone type

compound was identified as on of the major constituents in the 50% SPE fractions of gaharu grade 1. Further analysis by Liquid Chromatography-Mass Spectrometry

(LCMS) resulted in the identification of 6-hydroxyethylphenylchromone and

dihydroxy-2-[(2-phenyl)-ethyl]chromones, two fatty acids which were (9Z,12Z)-15,16-

dihydroxyoctadeca-9,12-octadecadienoic acid and (11Z,14Z)-17,18-dihydroxyicosa-

11,14-dienoic acid as well as sesquiterpenes which were aquilarone B, aquilarone D or

E, and aquilarone F.

Gaharu smoke was captured on the glass fiber filter using a simple lab setup

comprising an incense burner, connected to a glass tube containing the glass fiber. To

ensure absorption of smoke, the setup was linked to a vacuum line. The fiber filter containing smoke were directly analyzed by headspace GCMS analysis. Volatile

constituents detected in the gaharu smoke included aldehydes (benzaldehyde and

cuminaldehyde) and aromatics (toluene, 2-methylanisole, p-cresol, creosol, anisole, o-

cresol and acetophenone). Other constituents detected were furfuryl alcohol and

benzylideneacetone. Orthogonal Partial Least Square-Discriminant Analysis (OPLS-

DA) of the gaharu smoke profiles can be differentiated into a terpene rich and a

chromone rich groups. The terpene rich group consisted of the higher grades gaharu

samples, while the chromone rich group consisted of the lower grade samples.

Further treatment of the variables using Variable Importance in Projection (VIP) for

independent variables resulted in the identification of p-cresol (m/z 107.05), 2-

methylanisole (m/z 122.10), toluene (m/z 91.05) and p-anisaldehyde (m/z 135.10), as

some of the discriminating chemical markers present in the high grade gaharu smoke. Meanwhile furfuryl alcohol (m/z 98.05) and 5-methylfurfural (m/z 110.05) were some

of the markers present in lower grades. On the overall, this study has shown that

spectrometry-based metabolomics could offer a better approach in grading of gaharu

qualities.

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk Ijazah Master Sains

ANALISIS PERBEZAAN ANTARA GRED GAHARU MENGGUNAKAN

PENDEKATAN METABOLOMIK BERASASKAN RESONAN MAGNETIK

NUKLEUS DAN KROMATOGRAFI GAS-SPEKTROSKOPI JISIM

Oleh

SITI NAZIRAH ISMAIL

Jun 2015

Pengerusi: Profesor Khozirah Shaari, PhD

Fakulti: Institut Biosains

Gaharu,yang turut dikenali sebagai agarwood, kalambak, aloeswood atau eaglewood

adalah bahagian berdamar pada kayu teras bagi kayu dari spesis Aquilaria yang

dijangkiti oleh jangkitan yang mungkin berpunca oleh serangan fungus, serangga atau

kecederaan luaran. Dalam perdagangan gaharu, kayu gaharu digredkan oleh pakar

menggunakan cara organoleptik kerana tiada cara saintifik untuk menentukan gred

gaharu. Kebiasaannya, gaharu digredkan berdasarkan ciri-ciri yang dapat dilihat seperti

bau ketika pembakaran, warna dan densiti kayu yang memaparkan kandungan damar

pada kayu. Pada asasnya, harga gaharu adalah mengikut gred dimana gred tertinggi

adalah yang paling mahal. Matlamat penyelidikan ini adalah untuk menganalisis

perbezaan antara gred gaharu dengan menggunapakai profil komponen pelbagai

metabolit dalam memastikan samada ia boleh digunakan untuk memebezakan antara gred. Untuk mencapai objektif tersebut, 1H Nuclear Resonan Magnetik Nukleus Proton

(1H NMR) dan Spektrometri Jisim Kromatografi Gas (GCMS) digabungkan dengan

analisis data multivariat digunakan untuk menganalisis variasi antara profil metabolit

bagi gred gaharu yang berbeza.

Kayu gaharu dikisar menjadi serbuk halus, kemudian diekstrak menggunakan metanol

dan dikeringkan menggunakan Genevac. Kemudian, pati gaharu digunakan untuk

analisis 1H NMR dengan mencairkannya bersama metanol terdeuterad. Analisis secara

visual menunjukkan bahawa kesemua gred mempunyai profil yang sama tetapi berbeza

dari segi kepekatan pelbagai komponen. Komponen utama yang dikenalpasti adalah aromatik (δ6.0-7.5), gula dan glikosida (δ2.5-5.0), asid lemak/alifatik (δ0.5-2.0) dan

sebatian aldehida (δ9.32). Walaubagaimanapun, memandangkan beza antara gred tidak

jelas melalui analisis secara visual, analisis diteruskan menggunakan analisis data

multivariat. Model gambaran keseluruhan bagi analisis komponen utama (PCA) untuk

keempat-empat kumpulan bagi kesemua gred gaharu tidak memberikan maklumat

penuh, jadi analisis multivariat dijalankan untuk melihat kekonsistenan bagi setiap gred

bagi memilih gred yang sesuai digunakan sebagai model kelas. Berdasarkan komponen

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kimia, gred-gred tersebut diklasifikasi semula kepada 5 kumpulan (A, B, C, D and E).

Kumpulan-kumpulan tersebut mempunyai metabolit berbeza dan taburan metabolit

yang berbeza. Kumpulan A lebih signifikan dalam kandungan gula (4.24-4.28 ppm)

dan mungkin terpenoid (1.12, 1.16, 1.32, and 2.04 ppm), manakala kumpulan C dan E

berbeza dari kumpulan lain disebabkan oleh kandungan alifatik (3.0, 4.0, 4.52 and 7.2

ppm). Sementara itu kumpulan B dan D berbeza dari kumpulan lain mungkin

disebabkan oleh terpenoid juga (0.80, 0.84, 0.88, 1.24, and 1.28 ppm).

Analisis fitokimia oleh 1D NMR, 2D NMR dan J-resolve membenarkan pengenalpastian sebatian jenis etilfenilkromon sebagai komponen utama dalam gaharu

gred 1. Analisis selanjutnya oleh Spektrometri Jisim-Kromatografi Cecair (LCMS)

menunjukkan pengesanan 6-hidroksietilfenilkromon dan dihidroksi-2-[(2-fenil)-

etil]kromon, serta dua asid lemak iaitu asid (9Z,12Z)-15,16-dihidroksioktadeka-9,12-

oktadekadienoik dan asid (11Z,14Z)-17,18-dihidroksiikosa-11,14-dienoik dan

seskuiterpen iaitu akuilaron F, akuilaron D atau E, dan akuilaron B.

Asap gaharu dikumpul dalam penapis fiber kaca menggunakan persediaan makmal

secara ringkas, kemudian disambungkan ke tiub kaca. Untuk memastikan penyerapan

asap, ia disambungkan ke vakum. Penapis fiber kaca dianalisis terus menggunakan analisis headspace GCMS. Komponen meruap yang dikesan dalam asap gaharu

adalah aldehida, (benzaldehida dan kuminaldehida) dan aromatik (toluena, 2-

metilylanisol, p-kresol, kreosol, anisol, o-kresol and asetofenon). Komponen lain yang

dikesan dalam asap gaharu adalah alkohol furfuril dan benzilidinaaseton.

Analisis Diskriminan-Ortogon Persegi Separa Kurangnya (OPLS-DA) pada profil asap

boleh dibahagikan kepada sampel kaya terpen dan sampel kaya kromon. Kumpulan

kaya terpen mempunyai gred gaharu yang tinggi, manakala kumpulan kaya kromon

memiliki sampel gred rendah Seterusnya, analisis variabel diteruskan menggunakan

Pembolehubah Penting dalam Unjuran (VIP) kepada p-kresol (m/z 107.05), 2-

metilanisol (m/z 122.1), toluena (m/z 91.05)and p-anisaldehida (m/z 135.1) sebagai sebahagian daripada penanda pembezaan bahan kimia yang wujud dalam gaharu gred

tinggi sementara itu alkohol furfuril (m/z 98.05) and 5-metilfurfural (m/z 110.05)

adalah beberapa penanda yang wujud dalam gred rendah. Secara keseluruhannya,

kajian ini menunjukkan bahawa metabolimik berdasarkan-spektrometri mampu

menawarkan pendekatan yang lebih baik dalam penggredan kualiti gaharu.

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ACKNOWLEDGEMENT

"In the name of Allah S.W.T.,

All gratification is referred to Allah S.W.T."

Alhamdulillah, with His blessing, I have successfully completed this study. My deepest

appreciation and thanks to everyone that helped me through this project. Without their

support and help, this thesis would not have been completed.

First of all, I want to express my deepest gratitude to my supervisor, Prof. Dr. Khozirah

Shaari for her advice, guidance, concern, and support throughout completing this study.

My appreciation is also extended to my lecturer, Dr. Alfi Khatib for his valuable guide

on multivariate analysis, helpful advice and suggestions.

My sincerest thanks are also extended to Dr. Faridah Abas, Dr. Maulidiani, Dr. Intan

Safinar Ismail, all of my friends and staff of Laboratory of Natural Product, Institute

Bioscience, for their camaraderie and kind help throughout our working together at the

Laboratory of Natural Products.

Last but not least, my sincerest thanks to my family for their support, patience, love

and constant prayers. Thank you

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I certify that a Thesis Examination Committee has met on 25 June 2015 to conduct the

final examination of Siti Nazirah binti Ismail on her thesis entitled "Comparative

Analysis Of Different Grades Of Gaharu Using Nmr- And Gcms- Based Metabolomics

Approach" in accordance with the Universities and University Colleges 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 Master of Science.

Members of the Thesis Examination Committee were as follows:

Gwendoline Ee Cheng Lian, PhD Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Faridah Abas, PhD

Associate Professor

Faculty of Food Science Technology


(Internal Examiner)

Jalifah Latip , PhD

Associate Professor.

Faculty of Science and Technology

Universiti Kebangsaan Malaysia

(External Examiner)

ZULKARNAIN ZAINAL, PhD Professor and Deputy Dean

School of Graduate Studies


Date: 22 September 2015

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

accepted as fulfilment of the requirement for the degree of Master of Science. The

members of the Supervisory Committee were as follows:

Khozirah Shaari, PhD

Professor

Faculty of Science

University Putra Malaysia (Chairman)

Intan Safinar Ismail, PhD

Associate Professor

Faculty of Science


(Member)

BUJANG KIM HUAT, PhD

Professor and Dean

School of Graduate Studies


Date:

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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: __________________

Name and Matric No.: _____________________________________

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Declaration by Members of Supervisory Committee

This is to confirm that:

the research conducted and the writing of this thesis was under our supervision; supervision responsibilities as stated in the Universiti Putra Malaysia (Graduate

Studies) Rules 2003 (Revision 2012-2013) are adhered to.

Signature: Signature:

Name of Chairman of Supervisory

Name of Member of Supervisory

Committee: Prof. Dr. Khozirah

Shaari

Committee: Prof. Madya Dr.

Intan Safinar

Ismail

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TABLE OF CONTENTS

Page

ABSTRACT i

ABSTRAK iii

ACKNOWLEDGEMENTS v

APPROVAL vi

DECLARATION viii

LIST OF TABLES xii

LIST OF FIGURES xiii

LIST OF ABBREVIATIONS xvii

CHAPTER

1 GENERAL INTRODUCTION 1.1 Background

1.2 Problem Statement

1

1

2

2 LITERATURE REVIEW

2.1 Gaharu and gaharu producing species 4

2.1.1 Trade and uses of gaharu 5

2.1.2 Gaharu formation 7

2.1.3 Grading the quality of gaharu 10

2.2 Chemical properties 13

2.2.1 Sesquiterpenes and chromones in gaharu

oil and extracts

14

2.2.2 Simple volatile aromatic compounds from

gaharu smoke

16

2.3 Gas Chromatography Mass Spectrometry (GCMS) and

analysis of plant volatiles

21

2.4 Metabolomics as a tool in research

22

3 MATERIALS AND METHOD

3.1 Gaharu samples 25 3.2 Chemicals and reagents 28

3.3 General instrumentation and equipments 28

3.4 Methodology 28

3.4.1 Preparation of gaharu extracts 28 3.5 NMR measurements 30

3.5.1 NMR sample preparation 30

3.5.2 Data acquisition 31

3.5.3 Data preprocessing 31

3.5.4 Multivariate data analysis 32

3.6 Identification of differentiating chemical markers 32

3.6.1 Solid phase extraction (SPE) 32

3.6.2 2D NMR analysis 32

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3.6.3 LCMS/MS 32

3.7 Analysis of gaharu smoke by GCMS 32

3.7.1 Sample preparation 32

3.7.2 Headspace GCMS analysis 34

3.7.3 Data preprocessing 34

3.7.4 Multivariate data analysis

34

4

RESULTS AND DISCUSSION

4.1 NMR-based discriminative analysis of different grades

of gaharu

35

4.1.1 Visual analysis of 1H NMR spectra 35

4.1.2 Multivariate data analysis of 1H NMR

spectra of various grades of gaharu

4.1.3 Reclassification of gaharu grades

38

44

4.2 Identification of differentiating chemical markers 48

4.2.1 Solid phase extraction (SPE) 48 4.2.2 Identification of ethylphenylchromone type

of compound in gaharu grade 1 extract

49

4.2.3 Identification of chromones, fatty acids and

sesquiterpenes in gaharu grade 1 extract by

LCMS

56

4.3 GCMS-based discriminative analysis of smoke from

different grades of gaharu

62

4.3.1 Visual analysis of headspace GCMS

chromatogram

62

4.3.2 GCMS-based multivariate data analysis of

gaharu smoke

74

5 CONCLUSION

76

REFERENCES 78

APPENDICES 84

BIODATA OF STUDENT 92

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LIST OF TABLES

Table Page

2.1 Grades and market prices for gaharu 5

2.2 Simple volatile compounds found in gaharu 16

2.3 Classification of metabolomic 22

4.1 Reclassification of gaharu 44

4.2 Tentative compounds 47

4.3 Selected 1H and 13C assignments for the ethylphenylchromone

in 50% SPE fraction of gaharu extract (grade 1)

55

4.4 Volatiles and simple aromatic compounds identified in gaharu

smoke grade1

64


smoke grade 2

65


smoke grade 3

66


smoke grade 4

67


smoke grade 5

68


smoke grade 6

69


smoke grade 7

70


smoke grade 8

71

4.12 Summary of compounds detected in smoke of gaharu grade 1-8

73

4.13 Major chemical compounds in higher grade of gaharu

75

4.14 Major chemical compounds in lower grade of gaharu 75

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LIST OF FIGURES

Figures Page

2.1 A. malaccensis seed 4

2.2 A. malaccensis flower 4

2.3 Trees deliberately wounded 8

2.4 Drilling and inoculation of fungal inoculum 8

2.5 Resin of agarwood embedded in heartwood 9

2.6 Gaharu formation in wood sample from wounded stem of

Aquilaria malaccensis

9

2.7 Wood block containing gaharu 10

2.8 (a) The most expensive grade of gaharu, Super King, (b)

second most expensive grade, Super grade, (c)(d) the first

two grades in the grade A1-A10 range, (e) grade B1 (f)

grade C1

12

2.9 Chromones in gaharu extract 16

2.10 Flowchart showing the analytical flow for a plant

metabolomics study

23

3.1 Gaharu samples for batch 2 26

3.2 Gaharu samples for batch 3 27

3.3 Gaharu samples for batch 4

28

3.4 Preparation of gaharu extract

29

3.5 Left: mortar pestel, right; portable grinder

30

3.6 Left: Ultrasonic bath (LC 60H), right: MiVac (Genevac)

30

3.7 Sample preparation for NMR analysis 31

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3.8 Gaharu smoke sample preparation scheme 33

3.9 Setup for gaharu smoke sample preparation 33

4.1 Figure 4.1. 1H NMR spectra of the gaharu extract of Aquilaria malaccensis from δ 0.5-10.00 ppm. (a) grade 1,

(b) grade 2, (c) grade 3, (d) grade 4, (e) grade 5, (f) grade 6,

(g) grade 7 and (h) grade 8

36

4.2 The expanded NMR spectra of the aldehydic region of the

gaharu extract for each grade

37

4.3 The expanded NMR spectra of the sugar region of the

gaharu extract for each grade

37

4.4 Figure 4.4. PCA-X score plot model build from the 1H NMR

data sets of grade 1 to grade 8 for all 4 batches of gaharu samples. Color codes refer to each batch

38

4.5 PCA-X score plot by grade (a-h). Color code refer to each

batch

43

4.6 Summary of fit for PLS-DA model for reclassified gaharu

group A to E

44

4.7 Permutation test of PLS-DA class model for the reclassified

gaharu group A to E

45

4.8 PLS-DA score plot of reclassified gaharu grades (Group A

to E) for batch 3 and 4. Color codes refer to the reclassified

grades

46

4.9 Loading plot for the PLS-DA score plot of reclassified

gaharu group (A to E) in batch 3 and 4.

47

4.10 1H NMR profile of SPE fractions of gaharu grade 1 extract

49

4.11 Selected HMBC correlations for 6-

hydroxyethylphenylchromone

50

4.12 1H NMR spectrum of gaharu grade 1 50% MeOH fraction

50

4.13 13C NMR spectrum of gaharu grade 1 50% MeOH fraction

50

4.14 Two-dimensional 1H-1H J-resolve of gaharu grade 1 50%

MeOH

51

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4.15 HSQC spectrum of gaharu grade 1 50% MeOH fraction

(6.0-7.5 ppm)

52

4.16 HSQC spectrum of gaharu grade 1 50% MeOH fraction

(2.5-4.9 ppm)

53

4.17 HMBC spectrum of gaharu grade 1 50% MeOH fraction

(5.8-7.6 ppm)

54

4.18 HMBC spectrum of gaharu grade 1 50% MeOH fraction (2.5-5.0 ppm)

55

4.19 LCMS spectrum of gaharu grade 1 80% MeOH fraction (RT

– 7.22 min)

56


– 5.90 min)

57


– 6.81 min)

57


– 8.55 min)

58


– 9.47 min)

58

4.24 Fatty acids in gaharu grade 1 extract. a) (9Z,12Z)-15,16-

dihydroxyoctadeca-9,12-dienoic acid b) (11Z,14Z)-17,18-

dihydroxyicosa-11,14-dienoic acid

59


– 6.90 min)

59


– 4.13a min)

60


– 4.13b min)

60


– 4.74 min)

61

4.29 Aquilarone F 61

4.30 Aquilarone D 61

4.31 Aquilarone E 61

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4.32 Aquilarone B 61


– 5.37 min)

62

4.34 TIC for grade 1 64








4.42 OPLS-DA score scatter plot of the smoke of gaharu

reclassified grades (group A to E) of batch 3 and 4.

74

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LIST OF ABBREVIATIONS

δ - Chemical shift in ppm

ppm - Parts per million

°C - Degree in Celsius

µg - Microgram

µL - Microliter

µm - Micrometer

1H - Proton NMR

HMBC - Heteronuclear multiple bond correlation

HSQC - Heteronuclear single quantum coherence

Hz - Hertz

MHz - Megahertz

Psi - Pounds per square inch

J - Coupling constant in hertz

m - Multiplet

kg - Kilogram

g - Gram

mm - Milimiter

m - Meter

mL - Mililiter

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mg - Miligram

min - Minute

MTIB - Malaysian Timber Industry Board

NMR - Nuclear Magnetic Resonance

PCA - Principal component analysis

FID - Free induction decay

PC - Principle component

s - Singlet

SIMCA-P - Soft Independent Modeling Class Analogy-P

PLS-DA - Partial Least Square-Discriminant Analysis

VIP - Variable Importance in Projection for Independent Variables

RT - Retention time

OPLS-DA - Orthogonal Partial Least Square-Discriminant Analysis

CITES - Convention on International Trade in Endangered Species of

Wild Fauna and Flora

TRAFFIC - The Wildlife Trade Monitoring Network

Eds - Editors

IUCN - World Conservation Union

SPE - Solid phase extraction

UHPLC - Ultra High Performance Liquid Chromatography

ANOVA - Analysis of Variance

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w/w - Weight over weight

GCMS - Gas Chromatography Mass Spectrometry

NIST - National Institute of Standards and Technology

FFNSC - Flavour and Fragrance Natural and Synthetic Compounds

kV - Kilovolt

rpm - Revolutions per minute

TMS - Tetramethylsilane

FTIR - Fourier transform infrared spectrometer

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CHAPTER 1

GENERAL INTRODUCTION

1.1 Background

Gaharu is the resinous heartwood of infected Aquilara species, traded for perfumery,

aesthetic and religious purposes. Depending on the country that it is traded in, gaharu

is also known as eaglewood, aloeswood, agarwood, kalamabak, jin-koh (Japanese),

oudh (Arabic), tram huong (Vietnamese), chen xiang (Chinese) (Persoon, 2008). It is

also well known as 'gold of the forest' due to its high price which varies

indiscriminately, very much dependent on demand and availability. The supply of this

valuable material is slowly, but certainly, depleting due to over collection from its

natural stand (Persoon, 2008).

Gaharu producing trees grow typically in a mixed forest habitat and usually grow up to

30 m tall with 1 m maximum diameter at breast height (Persoon, 2008). Among the

gaharu-producing species, Aquilaria malaccensis grows up to 75 m above sea level,

thus it is easier to harvest the resinous wood in comparison to Aquilaria hirta.

However, Aquilaria hirta which grows 300 - 600 meters above sea level, produces

better grade of gaharu which are usually very hard in texture and dark in color

indicating its high quality.

Gaharu are usually obtained from Orang Asli communities, via middleman or traders

and resold at higher price. Different countries import different types of gaharu

depending on the final intended use or application (Persoon, 2007). For instance,

Japan buys gaharu in the form of wood blocks to make religious sculptures and other

items of worship and aesthetic purposes, while countries in the Middle East prefer

wood chunks, for burning as incense in aromatherapy, and powdered agarwood, for

hydrodistillation and perfumery (Chakrabarty et al., 1994).

Recently, there has been an increase in products on the market which incorporate

gaharu as one of their main active ingredients, for example gaharu tea, gaharu coffee,

skincare and cosmeticeutical products. These development has more or less

contributed significantly in the increase in demand for gaharu (Persoon, 2008). In order

to address the supply and demand issue, plantations of A. malaccensis and several

other gaharu producing species were established and artificially induced to produce

gaharu (MTIB, 2010).

Gaharu is largely believed to be the product of fungal infection of the producing tree,

sustained through injury on trunks, branches and roots of the life tree. Artificial

induction of gaharu has been the subject of research carried out in various countries

including Thailand and Malaysia. Researchers and planters have tried wounding

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plantation trees and mimicking fungal infection using fungal or chemical based

inoculants (Persoon, 2008; MTIB, 2010). Although some gaharu was successfully

induced in the planted trees, the amount of gaharu produced was low and of undefined

quality in comparison to gaharu produced in natural stands. In some of the artificial

induction trials, some trees even died during the wounding and inoculation processes if

it is not done carefully (Turjaman, 2011). Theoretically, trees that grow in a natural

forest environment are expected to be more profilic and efficient producer of gaharu

since these species have to compete for nutrients and combat a whole range of external

predators and diseases. It is still unclear how much more efficient the plantation

species will be in comparison to the wild species. The whole process of gaharu

formation is very slow and thus, the returns promised by artificial production of gaharu

may take many years to realize.

The main chemical components of gaharu such as phenolic compounds and

sesquiterpenes viz. agarofuran, jinkohol, jinkoh-eremol and eudesmol, are believed to

play a very important role in plant defense (disease resistance) (Marinova et al., 2005).

The presence of these chemical components are therefore related to gaharu production

since an attack by pathogens or in response to changes in environmental condition or

external perturbation will affect their biosynthetic production. Usually, inoculated

planted trees are expected to only produce better quality of gaharu after 5 years

(Turjaman, 2011). As shown by the plantation and induction trials, often times the

quality of gaharu produced is very low (commonly grade C) and only suitable for oil

extraction. At most, the amount of gaharu that can be produced by a tree is

approximately 2 kg per tree. Thus, in terms of monetary returns, the most that a planter

can obtain is an estimated RM4000.00 per tree (MTIB, 2010).

In trade, the quality of gaharu depends largely on the traders and buyers, who decide

which grade is the best grade. The grader grades according to several criteria of the

wood that he has collected at a time. For example, if the grader was given a kilogram of

gaharu wood, he will classify all the wood pieces according to shape, color, density and

odor (usually upon burning) (MTIB, 2010). It is easiest to determine the best grade but

it is rather difficult to differentiate between the rest of the grades. The Malaysia Timber

Industry Board (MTIB) grades gaharu as Super King, Double Super, Super grade A

series (A1 to A10), Grade B series (B1 to B5), Grade C series (C1 to C10) and lastly,

the oil grade gaharu.

1.2 Problem statement

The classification of gaharu grades differ according to the product in trade and in

which country the trade is taking place. The type and number of gaharu grades may

vary widely between the countries. Different species have been shown to contain

different chemical components (Ishihara et al,. 1991 & 1993), but this may not

necessarily be the criteria that is important to traders and consumers who largely selects

the country of origin and wood quality as criteria for grading the gaharu products. The

scenario as a whole paints a very confusing system of grading which seems to rely

heavily on subjective preferences and thus making it very difficult to clearly delineate

between the various grades. Currently, there is no proper scientific method of

determining the grades of gaharu, and more research are needed in finding feasible

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methods for grading gaharu. To date, some methods developed based on electronic

measurements have met with some success, but more confirmatory data is needed to

assess whether the classification or grading is accurate and reproducible. A rapid and

practical method to differentiate between various grades of gaharu is needed. On this

basis, the current study was undertaken to address the following objectives:

1. To differentiate various grades of gaharu by applying 1H NMR-based

metabolomics on gaharu extracts.

2. To identify chemical markers responsible for the differentiation of gaharu

grades.

3. To differentiate various grades of gaharu by applying GCMS-based

metabolomics on gaharu smoke.

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UNIVERSITI PUTRA MALAYSIA

PENGESAHAN STATUS UNTUK TESIS/LAPORAN PROJEK DAN HAKCIPTA

SESI AKADEMIK : ________________

TAJUK TESIS/LAPORAN PROJEK :

____________________________________________________________________________

____________________________________________________________________________

____________________________________________________________________________

NAMA PELAJAR : ______________________________________________

Saya mengaku bahawa hakcipta dan harta intelek tesis/laporan projek ini adalah milik Universiti Putra Malaysia dan bersetuju disimpan di Perpustakaan UPM dengan syarat-syarat berikut :

1. Tesis/laporan projek adalah hak milik Universiti Putra Malaysia.

2. Perpustakaan Universiti Putra Malaysia mempunyai hak untuk membuat salinan untuk tujuan akademik sahaja.

3. Perpustakaan Universiti Putra Malaysia dibenarkan untuk membuat salinan tesis/laporan projek ini sebagai bahan pertukaran Institusi Pengajian Tinggi.

Tesis/laporan projek ini diklasifikasi sebagai :

*sila tandakan (√ )

SULIT (mengandungi maklumat di bawah Akta Rahsia Rasmi 1972)

TERHAD (mengandungi maklumat yang dihadkan edaran kepada umum oleh organisasi/institusi di mana penyelidikan telah dijalankan)

AKSES TERBUKA Saya bersetuju tesis/laporan projek ini dibenarkan diakses oleh umum dalam bentuk bercetak atau atas talian.

Tesis ini akan dibuat permohonan :

PATEN Embargo ____________ hingga ___________ (tarikh) (tarikh)

Pengesahan oleh: ____________________ ______________________ (Tandatangan Pelajar) (Tandatangan Pengerusi Jawatankuasa Penyeliaan) No Kad Pengenalan / No Pasport.: Nama: Tarikh : Tarikh : [Nota : Sekiranya tesis/laporan projek ini SULIT atau TERHAD, sila sertakan surat dari organisasi/institusi

tersebut yang dinyatakan tempoh masa dan sebab bahan adalah sulit atau terhad.]

CHAPTER 2LITERATURE REVIEW2.1 Gaharu and gaharu-producing species2.1.1 Trade and uses of gaharuFigure 2.9: Chromones in gaharu extract2.2.2 Simple volatile aromatic compounds from gaharu smokeBurning gaharu will produce smoke which consists of mostly volatile products of pyrolysis. The gaharu smoke has been reported to be impart energizing and spiritually calming effect, as well as a warm, long lasting perfume (Naef, 2011). A GCMS analys...Thirty seven simple volatile aromatic compounds have been detected in gaharu smoke as shown in Table 2.2. (Naef, 2011). Benzylacetone (23) is a common and major constituent associated with the resin (Qi et al., 1998). Benzylacetone and various chrom...2.4 Metabolomics as a tool in researchCHAPTER 2REFERENCES

universiti putra malaysia comparative analysis of different grades of gaharu using nmr...

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