UNIVERSITI PUTRA MALAYSIA

SEARCH FOR NEW ANTIOXIDANTS AND OTHER RELATED BIOACTIVE COMPOUNDS FROM ZINGIBERACEOUS SPECIES

HABSAH MOHAMAD

FSAS 2002 55

SEARCH FOR NEW ANTIOXIDANTS AND OTHER RELATED BIOACTIVE COMPOUNDS FROM ZINGIBERACEOUS SPECIES

By

HABSAH MOHAMAD

Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirement for the Degree of Doctor of

Philosophy

November 2002

Abstract of the thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of the requirements for the degree of Doctor of Philosophy

SEARCH FOR NEW ANTIOXIDANTS AND OTHER RELATED BIOACTIVE COMPOUNDS FROM ZINGIBERACEOUS SPECIES

By

HABSAH MOHAMAD

November 2002

Chairman: Prof. Dr. Md. Nordin Hj . Laj is

Faculty: Science and Environmental Studies

Thirty-one species from Zingiberacea family were screened for their

antioxidant (FTC method), antimicrobial (disc diffusion method), and

antitumour promoting (EBV EA assay method) activities. Three species,

Alpinia zerumbet, Alpinia raJfle3iana and Etlingera elatiar, were selected for

further study, based on their t�romising preliminary biological activities.

Five known compounds were isolatpd hom the rhizomes of Alpinia

zerumbet, namely S,6-dehydrokawain, flavokawin B, l,7-diphenyl-S-

hydroxy-6-heptene-3-one, (-)-pinocembrin, and a mixture of stigmasterol

and p-sitosterol. From the fruits of Alpinia raffiesiana, seven compounds

were isolated, namely 5,6-dehydrokawain: flavokawin B, l,7-diphenyl-5-

hydroxy-6-heptene-3-one, (-)-pinocembrin, cardamonin, (-)-pinostrobin,

and 2',3',4',6' -tetrahydroxychaIcone. This is the first report on the isolation

of 2',3',4',6' -tetrahydroxychalcone from Alpinia. From the rhizomes of

I I

Etlingera eZrztior, 11 compounds were isolated, namely stigmast-4-en-3-one,

stigmast-4-ene-3,6-dione, stigmast-4-en-6,B-ol-3-one, stigmast-4-en-6a-ol-3-

one, a mixture of stigmasterol and �-sitosterol, 5a,8a-epidioxyergosta-6,22-

dien-3,B-ol, 16-hydroxylabda-8(17),II,13-trien-16,15-olide, tetracosanoic

acid, 1-(4-hydroxy -3-methoxyp hen y 1 )-7-(4-h Y droxyp hen y 1 )-1,6-heptadiene-

3,5-dione or demethoxycurcumin 1,7-bis(4-hydroxyphenyl)-2,4,6-

heptatrienone, 1,7 -bis( 4-hydroxyphenyl)-I,4,6-heptatrien-3-one. 16-

Hydroxylabda-8(17),11,13-trien-16,15-olide and 1,7 -bis( 4-hydroxyphenyl)-

2,4,6-heptatrienone were identified as new compounds. The structure of

these compounds were established based on spectral data and comparison

with literature data.

The three diarylheptanoids, demethoxycu rcumin, 1,7 -bis( 4-

hydroxyphenyl)-2,4,6-heptatrienone, 1,7 -bis( 4-hydroxyphenyl)-I,4,6-

heptatrien-3-one, were found to have high antioxidant activity. 5,6-

Dehydrokawain and (-)-pinocembrin showed weak antioxidant activity.

Flavokawin B was found to be cytotoxic to a number of cell lines including

MCF-7 and T-47D (Human, mammary carcinoma, positive estrogen

receptor) . Cardarnonin showed cytotoxic activity against CEM-SS cell line .

5,6-Dehydrokawain, 1,7 -di P hen y 1-5-hydroxy -6-heptene-3-one, (-)-

pinocernbrin, stigmast-4-en-3-one, stigmast -4-ene-3,6-dione, stigmast -4-en-

6�-01-3-one, and tetracosanoic acid showed antitumour promoting activity.

III

Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi keperluan ijazah Doktor Falsafah

PENCARIAN SEBATIAN ANTIOKSIDAN BARD DAN SEBATIAN BIOAKTIF YANG BERKAITAN DARIPADA SPESIES ZINGIBERACEA

Oleh

HABSAH MOHAMAD

November 2002

Pengerusi: Prof. Dr. Md. Nordin Hj. Lajis

Fakulti: Sains dan Pengajian Alam Sekitar

Tiga puluh satu spesies daripada keluarga Zingiberaceae telah dikaji untuk

menentukan aktiviti antioksidan (kaedah FTC), aktiviti antimikrobial

(kaedah pembauran cakera), dan aktiviti anti-penggalakan tumor (kaedah

EBV EA). Tiga spesies, iaitu Alpinia zerumbet, Alpinia rafflesiana dan

Etlingera elatiol� telah dipilih untuk kajian lebih lanjut, berdasarkan aktiviti

biologi yang tinggi daripada ekstrak mentah spesies ini. Lima sebatian

yang diketahui telah dipencilkan daripada rizom Alpinia zerumbet, , iaitu

5,6-dehidrokawain, flavokawin B, 1,7-difenil-5-hidroksi-6-hepten-3-on, (-)-

pinosembrin, dan campuran �-sitosterol dan stigmasterol . Tujuh sebatian

telah dipencilkan daripada buah Alpinia rafflesiana, iaitu 5,6-

dehidrokawain, flavokawin B, 1,7-difenil-5-hidroksi-6-hepten-3-on, (-)-

pinosembrin, kardamonin, (-)-pinostrobin dan 2' ,3' ,4' ,6' -

tetrahidroksikalkon. Ini merupakan laporan yang pertama mengenai

pemencilan 2' ,3',4' ,6' -tetrahidroksikalkon daripada Alpinia. Sebelas

lV

sebatian telah diasingkan daripada rizom Etlingera elatior" iaitu stigmast-4-

en-3-on, stigmast-4-en-3,6-dion, stigmast-4-en-6a-ol-3-on, stigmast-4-en-6,B­

ol-3-on, campuran ,B-sitosterol dan stigmasterol, 5a,8a-epidioksiergosta-

6,22-dien-3,B-ol,

tetrakosanoik,

16-hidroksilabda-8(17),ll,13-trien-16,15-olida, asid

1-( 4-hidroksi-3-metoksifenil)-7 -( 4-hidroksifenil)-l,6-

heptadien-3,5-dion, 1,7 -bis( 4-hidroksifenil)-2,4,6-heptatrienon dan 1,7-

bis( 4-hidro ks ife nil )-1,4,6-hepta trien-3-on. 5ebatian 16-hidroksilabda-

8(17),11,13-trien-16,15-olida dan l,7-bis(4-hidroksifenil)-2,4,6-heptatrienon

merupakan sebatian baru. 5truktur sebatian-sebatian ini dikenalpasti

berdasarkan data spektroskopi dan perbandingan dengan data literatur.

Ketiga-tiga sebatian diarilheptanoid, iaitu demetoksikurkumin, l,7-bis(4-

hidroksifenil)-2,4,6-hepta trienon dan 1,7 -bis( 4-hidroksifenil)-1,4,6-

heptatrien-3-on, menunjukkan aktiviti antioksidaan yang tinggi. 5,6-

Dehidrokawain dan (-)-pinosembrin menunjukkan aktiviti antioksidaan

yang rendah. Flavokawin B didapati sitotoksik terhadap beberapa talian

sel termasuk MCF-7 dan T-47D (sel karsinoma mamari manusia, estrogen

reseptor positif) . Kardamonin didapati sitotoksik terhadap talian sel CEM-

55 . 5ebatian 5,6-dehidrokawain, 1,7 -difenil-5-hidroksi-6-hepten-3-on, (-)­

pinosembrin, stigmast-4-en-3-on, stigmast-4-en-3,6-dion, stigmast-4-en-6a­

ol-3-on, stigmast-4-en-6,B-ol-3-on dan asid tetrakosanoik menunjukkan

aktiviti anti-penggalakan tumor.

v

ACKNOWLEDGEMENTS

In the name of Allah the most Gracious and the most Merciful.

My sincere gratitude goes to a number of people, without whose support

this thesis would not have been possible. In particular, I would like to

extend my heartfelt gratitude to

My supervisor:

Prof. Dr. Md. Nordin Hj .Lajis

My co-supervisors:

Prof. Dr. Abdul Manaf Ali

Assoc. Prof. Dr. Mohd AspoUah Hj. Sukari

Assoc. Prof. Dr. Taufiq Yap Yun Hin

Others:

Kolej Universiti Sains dan Teknologi Malaysia, Terengganu.

Prof. Dr. Nobuji Nakatani, Dr. Hiroe Kikuzaki, En. Ahmad Abdul Rahman,

Assoc. Prof. Dr. Khozirah Shaari, En. Nasir Tsafe Umar, En. Abdul Ghafar

Colleagues:

Amran, Mukram, Lim Yang Mooi, Yih Yih, Faridah, Pn. Rohaya, Samsul

and Sharin.

VI

I certify that an Examination Committee met on 26th November 2002 to conduct the final examination of Habsah Mohamad on her Doctor of Philosophy thesis entitled "Search for New Antioxidants and Other Related Compounds from Zingiberaceous Species" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia (Higher Degree) Regulations 1981 . The Committee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:

FAUJAN HJ. AHMAD, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Chairman)

MOHO NORDIN HJ. LAJIS, Ph.D. Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)

MOHO ASPOLLAH HJ. SUKARI, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)

ABDUL MANAF ALI, Ph.D.

Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

T AVFIQ YAP YVN HIN, Ph.D., CChem., MRSC

Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)

NOBVII NAKATANI, Ph.D. Professor Food Chemistry, Graduate School of Human Life Science Osaka City University (Independent Examiner)

S Professor/Deputy Dean School of Graduate Studies Universiti Putra Malaysia

Date:3 1 DEC 2002.

VII

The thesis submitted to the Senate of Universiti Putra Malaysia has been accepted as fulfilment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows:

MOHD NORDIN HI. LAIIS, Ph.D.

Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Chairman)

MOHD ASPOLLAH HI. SUKARI, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)

ABDUL MANAF ALI, Ph.D. Professor Faculty of Food Science and Biotechnology Universiti Putra Malaysia (Member)

TAUFIQ YAP YUN HIN, Ph.D., CChem., MRSC Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)

AINI IDERIS, Ph.D. Professor / Dean School of Graduate Studies Universiti Putra Malaysia

Date: 1 3 FEB 2003

Vlll

DECLARATION

I hereby declare that the thesis is based on my original work except for quotations and citations, which have been duly acknowledged. I also declare that it has not been previously or concurrently submitted for any degree at UPM or other institutions.

HABSAH MOHAMAD

Date: 31st December 2002

IX

TABLE OF CONTENTS

ABSTRACT Page 11

ABSTRAK ACKNOWLEDGEMENTS APPROVAL

IV vi V11

DECLARA nON IX T ABLE OF CONTENTS LIST OF TABLES

X Xlll XV XXI

LIST OF FIGURES LIST OF ABBREVIA nONS

CHAPTER

1 INTRODUCTION 1

2 LITERATURE REVIEW Antioxidants in Disease Prevention 7

What Are Antioxidants and Free Radicals? 7 Damage Caused by Free Radicals to Lipids, Proteins and 10 DNA Mechanism of Lipid Oxidation and Antioxidant Defenses 11 Natural Food Sources and Dietary Antioxidants in the 16 Prevention of Specific Cancers Cancer Development and Mechanism of Prevention by 19 Antioxidants

Zingiberacea Family 21 The Ginger Family (Zingiberaceae) 21 Use and Commercial Importance of Zingiberaceae 24 Phytochemical Study on Zingiberaceae 25 Bioactive Compounds from Zingiberacea Family 26

Overview on Alpznza 34 Botany and Geographic Distribution 34 Economic and Ethnopharmacognostic Use of Alpznza 36 Phytochemical Work on Alpznza 37

A. zerumbet (Pers.) B. L. Burtt & R. M. Smith ( A. nlltans Rose.) 77 Botanical Aspect of A. zerumbet 77 Uses of A. zerumbet 79 Phytochemical and Biological Activity Study of A. zerumbet 79

A. raffles zana Wall. Ex. Bak. 81 Botanical Aspect of A. raffles zana 81 Phytochemical and Biological Activity Study of A. raffle szana 83

x

Overview on Etlingera: The 'Nicolaia' Group Etlingera Elahor (Jack) R.M. Smitiz

Botany and Geographic Distribution Uses, Medicinal Value, Biological Activity and Phytochemical Study of Etlingera elatior

Biosynthesis Biosynthesis of Terpenoids Biosynthesis of Flavonoids, Styrylpyrones and Diarylheptanoids

3 METHODOLOGY

83 84 84 86

86 87 91

General Instrumentation 95 Chromatographic Methods 95 Solvents 97 Isolation of Compounds from Alpinia zerumbet 97

Plant Material 97 Extraction and Isolation 97

Isolation of Constituents from Alpinia rafflesiana 99 Plant Material 99 Extraction and Fractionation of MeOH extract 99 Isolation of ARl, AR2, AR3, AR6, AR7, AR9 and AR5 102

Isolation of Constituents from Etlingera elatior 106 Plant Material 106 Fractionation of CHCb Extract 106 Isolation of N4, N5 and N6 107 Fractionation of Hexane Extract 110 Isolation of N1, N8, N9, NIO and N12 110 Fractionation of Ethyl Acetate Extract 111 Isolation of NSH2 114 Isolation of NSH4 and NSH5 114

Bioassay Procedures 129 Antioxidant Activity 129 Antimicrobial Assay 132 Antitumour Promoting Activity 133 Cytotoxicity Assay 134

4 COMPOUNDS ISOLATED FROM ALPINIA ZERUMBET, ALPINIA RAFFLESIANA AND ETLINGERA ELA TIOR Structural Elucidation of the Compounds Alpinia zerumbet

Isolation of the Compounds from Alpinia zerumbet Characterisation of the Compounds Isolated from A. zerumbet

138 140 140 141

Xl

Alpinia Rafflesiana 169 Isolation of the Compounds from the Fruits of A. raffiesiana 169 Characterisation of the Compounds Isolated from 170 A. raffle siana

Etlingera elatior 187 Isolation of the Compounds from E. elatior 187 Characterisation of the Compounds Isolated from E. elatior 189

Biogenesis in Etlingera elatior 262 Relationship between the Species 265

5 BIOLOGICAL ACTIVITY STUDIES Biological Activity of Zingiberaceae Plant Extracts 267

Screening for Antioxidant Activity 267 Screening for Antimicrobial Activity 278 Screening for Anti-tumour Promoting Activity 280

Biological Activities of Alpinia zerumbet 284 Antioxidant Activity of Fractions from CHCL3 Extract 284 of Alpinia zerumbet Antioxidant and DPPH Free Radical Scavenging Activities of 286 Pure Compounds Isolated from Alpinia zerumbet Antitumour Promoting Activity of Pure Compounds Isolated 289 from Alpinia Zerunzbet Cytotoxic Activity of Pure Compounds Isolated from Alpinia 290 zerumbet

Biological Activities of Etlingera elatior 293 Antioxidant Activity of Fractions From Ethyl Acetate 293 Extracts of Etlingera elatior Antioxidant Activity of Diarylheptanoids from Ethyl Acetate 294 Extract of Etlingera elatior Antitumour Promoting Activity of Etlingera elatior 298 Cytotoxic Activity of Extracts from Etlingera elatior 301

Biological Activities of Alpinia raffiesiana 302 DPPH Free Radical Scavenging Activity 303 Antitumour Promoting Activity of Extracts and Fractions 305 from Alpinia rafflesiana Cytotoxic Activity of Extracts, Fractions and Cardamonin 306 from Alpinia rafflesiana

6 CONCLUSION 309 BIBLIOGRAPHY 317 BIODATA OF THE AUTHOR 333

XII

LIST OF TABLES

Table Page

2.1 Classification of antioxidants 9 2.2 Free radicals and their effects 10 2.3 Natural food sources of antioxidants 18 2.4 Order Zingiberales 22 2.5 Tribes and genera of Zingiberaceae family 23 2.6 Alpinia species from Peninsular Malaysia and Singapore 35 2.7 Chemical variations in the Alpinia species from Malaysia 43 2.8 Compound isolated from A. blepharocalyx 45 2.9 Compounds isolated from A. katsumadai 47 2.10 Compounds isolated from A. officinarum Hance 48 2.11 Compounds isolated from Alpinia oxyphylla 49 2.12 Compounds isolated from Renealmia alpinia Rottb. 50 2.13 Compounds isolated from Alpinia formosana 51 2.14 Compounds isolated from Alpinia speciosa 52 2.15 Compounds isolated from Alpinia japonica 53 2.16 Compounds isolated from Alpinia chinensis 54 2.17 Compounds isolated from A. calcarata 56 2.18 Compounds isolated from A. J1abellata 57 4.1 The assignment of protons and carbons of 5,6- 143

dehydrokawain (AN1) 4.2 The assignment of protons and carbons of flavokawin B (AN3) 150 4.3 The assignment of protons and carbons of 1,7-Diphenyl-5- 156

hydroxy-6-heptene-3-one (AN4) 4.4 Assignment of protons and carbons of (-)-pinocembrin (AN5) 162 4.5 Assignment of carbons for stigmasterol and p-sitosterol 168 4.6 Assingment of protons and carbons of cardamonin (AR3) 171 4.7 Assignment of protons and carbons of 5-hydroxy-7- 176

methoxyflavanone ((-)-pinostrobin) (AR9) 4.8 Assingment of protons and carbons for 2',3',4',6'- 183

tetrahydroxychalcone 4.9 Protons assignment of N1, N12, N4 and N6 208 4.10 Carbons assignment of N1, N12, N4 and N6 209 4.11 Assignments of protons and carbon of N5 216 4.12 Assignments of carbons of 16-hydroxylabda-8(17),l1,13- 225

trien-6,15-olide (N9) 4.13 Assignment of protons of 16-hydroxylabda-8(17),l1,13-trien- 226

16,15-olide (N9)

xiii

4.14 Assignments of protons and carbons of 1-(4-hydroxy 3- 237 methoxyphenyl)-7-(4-hydroxyphenyl)-l,6-heptadiene-3,5-dione or demethoxycurcumin (NSH2)

4.15 Assignment of carbons and protons of 245 l,7-bis(4-hydroxyphenyl)-2A,6-heptatrienone (NSH4)

4.16 Assingment of protons and carbons of l,7-bis(4- 255 hydroxyphenyl)-A,6-heptatrien-3-one (NSH5)

5.1 Minimum inhibitory dose (�lg/ disc) for antimicrobial activity 279 of Zingiberaceae extracts

5.2 Antitumour promoting activity of Zingiberaceae species 282 5.3 DPPH free radical scavenging activity of compounds isolated 289

from Alpinia zerumbet. 5.4 Antitumour promoting activity screening of compounds 290

isolated from Alpinia zerumbet 5.5 Cytotoxic activity of flavokawain B 292 5.6 Antitumour promoting activity of crude extracts of Etlingera 298

elatior 5.7 Antitumour promoting activity of compounds isolated from 299

Etlingera elatior 5.8 Cytotoxic activity of Etlingera elatior extracts 302 5.9 Antitumour promoting activity of the fractions from MeOH 306

extract of Alpinia rafflesiana 5.10 Cytotoxic effect of crude MeOH extracts, fractions and 308

cardamonin from Alpinia rafflesiana against CEM -SS cell line

XIV

Figure

1 .1 2.1 2.2 2.3 2.4 2.5 2.6 2.7 2.8

2.9

2.10 2.11 2.12 2.13 2.14 2.15 2.16 2.17 2.18 2.19 2.20 2.21 2.22 2.23 2.24 2.25 2.26 2.27 2.28 2.29 2.30 2.31 2.32 2.33 2.34 2.35 2.36

3.1

LIST OF FIGURES

Strategy for plant-derived drug discovery Steps in lipid autoxidation Inhibition of lipid oxidation by antioxidant (AH) Mechanism of chain-breaking inhibition by BHT 102 quenching mechanism of oc- tocopherol 102 quenching mechanism of oc- tocopherol Metal - chelating mechanism for flavones Mechanism of carcinogenesis Antioxidant gingerol related compounds and diarylheptanoids of ginger Antioxidant and anti-inflammatory compounds from Zingiber cassumunar Curcuminoids from Curcuma domestica Antioxidant compounds from Curcuma xanthorhiza Antioxidant compounds from Alpinia speciosa Plant of Alpinia zerumbet Inflorescence of Alpinia zerumbet Flower of Alpinia zerumbet Ripe Fruits of Alpinia zerumbet Alpinia rafflesiana foliage Inflorescence of Alpinia mfflesiana Unripe fruits of Alpinia. mfflesiana Flower of Alpinia mfflesiana Flower of Alpinia mfflesiana Flower shoots and rhizome of Etlingem elatior Fruits of Etlingem elatior Flower of Etlingem elatior (pink) Flower of Etlingem elatior (white) Flower of Etlingem elatior (red) Biosynthetic relationship in the terpenoids series Squalene as origin of triterpenes and steroids Elimination of the methyl group at the C-14 position Elimination of the methyl group at the C-4 position Modifications of the C-7 side chain of phytosterols Postulated biogenesis among several labdane diterpenes Mainstreams of secondary metabolism Biosynthesis of flavonoids Biosynthesis of styrylpyrone Biosynthesis of diarylheptanoids and gingerol related compound Isolation of 5,6-dehydrokawain (AN1)

Page

5 12 13 14 15 15 16 20 28

29

30 31 31 78 78 78 78 82 82 82 82 82 85 85 85 85 85 87 89 90 90 90 91 92 93 93 94

100

xv

3.2 Isolation of AN2, AN3,AN4 and AN5 101 3.3 Isolation of AR1,AR2, AR3, AR6, AR7, AR5 and AR9 105 3.4 Fractionation of CHCb extract of Etlingera elatior 108 3.5 Isolation of N4, N5 and N6 109 3.6 Fractionation of hexane extract and isolation of Nl0, Nl, 112

N8, N12, N9 and N4 3.7 Fractionation of ethyl acetate extract Etlingera elatior 113 3.8 Isolation of NSH2 115 3.9 Isolation of NSH4 and NSH5 116 4.1 Strategy of structural elucidation 139 4.2 Structure of 5,6-dehydrokawain (AN1) 143 4.3 Mass spectrum of 5,6-dehydrokawain (AN1) 144 4.4 IR spectrum of 5,6-dehydrokawain (AN1) 144 4.5 IH NMR spectrum of 5,6-dehydrokawain (AN1) 145 4.6 13C NMR spectrum of 5,6-dehydrokawain (AN1) 146 4.7 HMBC spectrum of 5,6-dehydrokawain (AN1) 147 4.8 Structure of flavokawin B (AN3) 149 4 .9 IR spectrum of flavokawin B (AN3) 151 4.10 Mass spectrum of flavokawin B (AN3) 151 4 .11 IH NMR spectrum of flavokawin B (AN3 152 4.12 13C NMR spectrum of flavokawin B (AN3) 153 4.13 Structure of 1,7-Di phen y 1-5-hydroxy -6-heptene-3-one 155

(AN4) 4.14 IR spectrum of 1,7-diphenyl-5-hydroxy-6-heptene-3-one 157

(AN4) 4.15 ElMS spectrum of 1,7-diphenyl-5-hydroxy-6-heptene-3- 157

one (AN4) 4.16 IH NMR spectrum of 1,7-diphenyl-5-hydroxy-6-heptene- 158

3-one (AN4) 4.17 13C NMR spectrum of 1,7-diphenyl-5-hydroxy-6-heptene- 159

3-one (AN4) 4.18 Structure of (-)-pinocembrin (AN5) 161 4.19 ElMS spectrum of (-)-pinocembrin (AN5) 163 4.20 Selected HMBC correlations in (-)-pinocembrin (AN5) 163 4.21 IH NMR spectrum of (-)-pinocembrin (AN5) 164 4.22 13C NMR spectrum of (-)-pinocembrin (AN5) 165 4.23 CD and UV spectrum of (-)-pinocembrin (AN5) 166 4.24 Structure of p-sitosterol and sitosterol 167 4.25 Cardamonin 171 4.26 IR spectrum of cardamonin (AR3) 172 4.27 ElMS spectrum of cardamonin (AR3) 172 4.28 IH NMR spectrum of cardamonin (AR3) 173 4.29 BC NMR spectrum of cardamonin (AR3) 174 4.30 5-hydroxy-7-methoxyflavanone ((-)-pinostrobin) (AR9) 176

XVI

4.31 IR spectrum of 5-hydroxy-7-methoxyflavanone 177 ((-)-pinostrobin) (AR9)

4.32 ElMS spectrum of 5-hydroxy-7-methoxyflavanone 177 ((-)-pinostrobin) (AR9)

4 .33 lH NMR spectrum of 5-hydroxy-7-methoxyflavanone 178 ((-)-pinostrobin) (AR9)

4.:H BC NMR spectrum of 5-hydroxy-7-methoxyflavanone « -)- 179 pinostrobin) (AR9)

4.35 2' ,3',4' ,6' -Tetrahydoxychalcone 181 4.36 HMBC correlation in the molecule of 2',3',4',6'- 182

tetrahydoxychalcone 4.37 1 H NMR spectrum of 2',3',4',6' -tetrahydoxychalcone 184

(AR5) 4.38 13C NMR spectrum of 2',3',4',6' -tetrahydoxychalcone 185

(AR5) 4.39 ElMS spectrum 2',3',4',6' -tetrahydoxychalcone (AR5) 186 4.40 Stigmast-4-en-3-one (N1) 190 4.41 lH-NMR spectrum of stigmast -4-en-3-one (N1) 191 4.42 13C-NMR spectrum of stigmast-4-en-3-one (N1) 192 4.43 IR spectrum of stigmast-4-en-3-one (N1) 193 4.44 ElMS spectrum of stigmast-4-en-3-one (N1) 193 4.45 lH-NMR spectrum (expanded) of stigmast -4-en-3-one 194

(N1) 4 .46 Stigmast -4-ene-3 ,6-dione (N12) 196 4.47 IR spectrum of stigmast-4-ene-3,6-dione (N12) 196 4.48 lH NMR spectrum of stigmast-4-ene-3,6-dione (N12) 197 4 .49 lH NMR spectrum of stigmast-4-ene-3,6-dione (N12) 198 4.50 13C NMR spectrum of stigmast-4-ene-3,6-dione (N12) 199 4.51 HMBC spectrum of stigmast-4-ene-3,6-dione (N12) 200 4.52 Stigmast-4-en-6�-ol-3-one (N4) 202 4.53 lH-NMR spectrum of stigmast-4-en-6�-ol-3-one (N4) 203 4.54 13C-NMR spectrum of stigmast-4-en-6�-ol-3-one (N4) 204 4.55 ElMS spectrum of stigmast-4-en-6�-ol-3-one (N4) 205 4.56 IR spectrum of stigmast-4-en-6�-ol-3-one (N4) 205 4.57 lH-NMR spectrum (expanded) of stigmast-4-en-6�-ol-3- 206

one (N4) 4.58 Stigmast-4-en-6cx-ol-3-one (N6) 207 4.59 lH NMR spectrum of mixture of stigmast-4-en-6cx-ol-3- 210

one (N6) and N5 4.60 lH NMR spectrum (expanded) of mixture of stigmast-4- 211

en-6cx-ol-3-one (N6) and N5 4.61 13C NMR spectrum (expanded) of mixture of stigmast-4- 212

en-6cx-ol-3-one (N6) and N5

XVll

4.62

4.63 4.64

4.6S

4.66

4.67

4.68

4.69

4.70 4.71

4.72

4.73

4.74

4.7S

4.76 4.77 4.78 4.79 4.80 4.81

4.82

4.83

4.84

13C NMR spectrum of mixture of stigmast-4-en-6a-ol-3-one (N6) and NS Sa,8a-Epidioxyergosta-6,22-dien-3p-ol ElMS spectrum of Sa,8a-epidioxyergosta-6,22-dien-3p-ol (NS) 13C-NMR spectrum of Sa,8a-epidioxyergosta-6,22-dien-3p-ol (NS) IH-NMR spectrum of Sa,8a-epidioxyergosta-6,22-dien-3p-ol (NS) IH-NMR spectrum (expanded) of Sa,8a-epidioxyergosta-6,22-dien-3�-ol (NS) HMBC spectrum of Sa,8a-epidioxyergosta-6,22-dien-3p-ol (NS) The characteristic mass spectral fragmentation of decalin nucleus of labdane-type diterpene 16-Hydroxy-8(17),11,13-labdatrien-16,1S-olide (N9) Selected HMBC, IH-IH COSY and NOESY correlation of 16-hydroxy-8(17),11,13-labdatrien-16,1S-olide (N9) ElMS spectrum of of 16-hydroxy-8(17),11,13-labdatrien-16,1S-olide (N9) IR spectrum of of 16-hydroxy-8(17),ll,13-labdatrien-16,1S-olide (N9) 13C-NMR spectrum of 16-hydroxy-8(17),ll,13-labdatrien-16,1S-olide (N9) IH-NMR spectrum of 16-hydroxylabda-8(17),ll,13-trien-16,1S-olide (N9) ElMS spectrum of tetracosanoic acid (N10) IR spectrum of tetracosanoic acid (N10) 13C NMR spectrum of tetracosanoic acid (N10) 1 H NMR spectrum of tetracosanoic acid (N10) p-diketone in its enol form 1-(4-hydroxy - 3-methoxyp hen y 1 )-7 -( 4-hydroxyp hen y 1 )-1,6-heptadiene-3,S-dione or demethoxycurcumin (NSH2) IH NMR spectrum of 1-(4-hydroxy- 3-methoxyphenyl)-7-(4-hydroxyphenyl)-1,6-heptadiene-3,S-dione or demethoxycurcumin (NSH2) IH-IH COSY correlations observed in 1-(4-hydroxy- 3-methoxyphenyl)-7-(4-hydroxyphenyl)-1,6-heptadiene-3,S-dione or demethoxycurcumin (NSH2) NOESY correlations observed in 1-(4-hydroxy 3-methoxyphenyl)-7-(4-hydroxyphenyl)-1,6-heptadiene-3,S-dione or demethoxycurcumin (NSH2)

213

21S 216

217

218

219

220

224

224 22S

227

227

228

229

231 231 232 233 234 236

238

239

240

XVlll

4.85 ElMS spectrum of 1-(4-hydroxy- 3-methoxyphenyl)-7- 241 (4-hydroxyphenyl)-l,6-heptadiene-3,5-dione or demethoxycurcumin (NSH2)

4.86 Selected HMBC and HMQC correlations observed in 241 1-(4-hydroxy- 3-methoxyphenyl)-7-(4-hydroxyphenyl)-1,6-heptadiene-3,5-dione or demethoxycurcumin (NSH2)

4.87 IH-IH COSY and HMBC correlations of 2,4,6- 243 heptatrienone moeity

4.88 Structure and fragmentation in ElMS spectrum of 1,7- 244 bis(4-hydroxyphenyl)-2,4,6-heptatrienone (Nsh4)

4.89 HMBC correlations of l,7-bis(4-hydroxyphenyl)-2,4,6- 244 hepta trienone

4.90 ElMS spectrum of 1,7-bis(4-hydroxyphenyl)-2,4,6- 246 heptatrienone (NSH4)

4.91 13C NMR spectrum of l,7-bis(4-hydroxyphenyl)-2,4,6- 247 heptatrienone (NSH4)

4.92 IH NMR spectrum of l,7-bis(4-hydroxyphenyl)-2,4,6- 248 heptatrienone (NSH4)

4.93 IH NMR spectrum (expanded) of l,7-bis(4- 249 hydroxyphenyl)-2,4,6- heptatrienone (NSH4)

4.94 IH-IH CG8Y correlation spectrum of 250 1,7-bis( 4-hy droxypheny 1)-2,4,6-hepta trienone (NSH 4)

4.95 HMBC spectrum of l,7-bis(4-hydroxyphenyl)-2,4,6- 251 heptatrienone (NSH4)

4.96 HMQC spectrum of l,7-bis(4-hydroxyphenyl)-2,4,6- 252 heptatrienone (NSH4)

4.97 Structure and HMBC correlations of l,7-bis 254 (4- hydroxyphenyl)-1,4,6-heptatrien-3-one (NSH5)

4.98 ElMS spectrum of 1,7-bis(4- hydroxyphenyl)-,4,6- 256 heptatrien-3-one (NSH5)

4.99 IH NMR spectrum of l,7-bis(4- hydroxyphenyl)-,4,6- 257 heptatrien-3-one (NSH5)

4.100 IH NMR spectrum (expanded) of l,7-bis(4- 258 h ydroxyp hen y 1)-,4,6-hepta trien-3-one (NSH5)

4.101 IH-IH COSY spectrum of l,7-bis(4- hydroxyphenyl)-,4,6- 259 heptatrien-3-one (NSH5)

4.102 HMBC and 13C NMR spectrums of l,7-bis(4- 260 hy droxyp heny 1)-,4,6-hepta trien -3-one (NSH5)

4.103 HMQC spectrum of l,7-bis(4-hydroxyphenyl)-2,4,6- 261 heptatrien-3-one (NSH5)

4.104 Biosynthesis of Nl, N4 and N12 from �-sitosterol 262 4.105 Biosynthesis of N5 from ergosterol 263

XIX

4.106 Biosynthesis of 16-hydroxylabda-8(17),11,13-trien-16,15- 263 olide

4.107 Biosynthesis of NSH2, NSH5 and NSH4 264 5.1 Antioxidant activity of Alpinia methanol extracts 271

(FTC method) 5.2 Antioxidant activity of Alpinia dichloromethane extracts 272

(FTC method) 5.3 Antioxidant activity of Costus extracts (FTC method) 273 5.4 Antioxidant activity of Zil1giber extracts (FTC method) 274 5.5 Antioxidant activity of Etlingera (Nicolaia group) 275

Extracts (FTC method) 5.6 Antioxidant activity of Curcuma and Alpinia chonchigera 276

Extracts (FTC method) 5.7 Antioxidant activity of Amomum and Etlingera extracts 277

(FTC method) 5.8 Antioxidant activity of the fraction from CHCL3 extract of 285

Alpinia zerumbet 5.9 Antioxidant activity of the compounds isolated from 287

Alpinia zerumbet 5.10 Antioxidant activity of fractions K (Fr .K), G (Fr.G) and I 288

(FrJ) from ethyl acetate extracts of Etlingera elatior 5.11 Antioxidative diarylheptanoids from Etlingera elatior 294 5.12 Antioxidant activity of demethoxycurcumin (NSH2), 295

1, 7-bi s( 4-h ydroxyp hen y 1)-2A,6-hepta trienone (NSH 4) and 1,7 -bis( 4-hydroxypheny 1)-lA,6-hepta trien-3-one (NSH5) (FTC method)

5.13 Structure of potential anti tumour promoting compound 296 isolated from Etlingera elatior

5.14 Structure of potential antitumour promoting compounds 300 isolated from Etlingera elatior

5.15 Scavenging activities of 2',4' -dihydroxy-6' - 304 methoxychalcone (cardamonin) (1), (-)-pinostrobin (2) and 2' ,3' A' ,6' -tetrahydroxychalcone (3) on DPPH radical compared to those of quercetin and tocotrienol

6.1 Compound isolated from Alpinin zerumbet and Alpinia 314 rafflesiana

6.2 Compounds isolated from Etlingera elatior 315 6.2 Compounds isolated from Etlingera elatior 316

xx

[a]D 8 br COSY BC CHCb CH2Cb d dd DPPH EA EBV ElMS EtOAc FGHMBC

FGHMQC -

IH HPLC IC id IR J Lit. m M MeOH MID Mol. wt. mp MS NMR NOE P s sh t TMS TPA UV

LIST OF ABBREVIATIONS

specific rotation at sodium D-line chemical shift in ppm broad Correlated Spectroscopy carbon-13 chloroform dichloromethane doublet doublet of doublets diphenyl-p-picrylhydrazyl early antigen Eipstein-Barr virus Electron impact mass spectroscopy ethyl acetate Field Gradient Heteronuclear Bond Connectivity (by 2D Multiple Quantum NMR) Field Gradient IH-Detected Heteronuclear Multiple Quantum Coherence (via Direct Coupling) proton High performance liquid chromatography inhibition concentration internal diameter infrared Coupling constant in Hz literature multiplet molar methanol minimum inhibition dose molecular weight melting point mass spectrumj mass spectrometry nuclear magnetic resonance Nuclear Overhauser Enhancement para singlet shoulder triplet tetramethylsilane 12-0-tetradecanoylphorbol-13-acetate ultraviolet

xxi

CHAPTER 1

INTRODUCTION

Antioxidant is a chemical substance extremely useful to humans. It helps

us to ward off many kinds of diseases related to lungs, kidneys, heart,

cardiovascular system, muscle and brain, and it helps to retard the aging

process. Antioxidant has the ability to prevent or delay the formation of

free radicals and lipid peroxidation in the human bodies, two main

causes of human diseases and aging.

Antioxidants can be obtained from synthesis and natural sources.

Antioxidants from natural sources include a wide variety of cODlpounds

from a wide range of classes, including plant-based antioxidants; amino

acids, peptides and protein hydrolyzates; phytates; phospholipids; and

vitamin and enzymes (Shahidi, 1997). Among the plants that have been

known to provide antioxidant compounds are those belonging to

Zingiberaceae, or the ginger family . Most members of the Zingiberaceae

family are recognizable by the characteristic aromatic leaves and fleshy

rhizomes when both of them are crushed, and also by the elliptic to

elliptic-oblong leaves arranged in two ranks on the leaf-shoot. The plants

vary in height and size. The Zingiberaceae family comprises about 1200

species, with 1000 occuring in tropical Asia. The richest area, with 24

genera and about 600 species, is the Malesian region, which includes

Malaysia, Indonesia, Brunei, Singapore, the Philippines and Papua New

Guinea. The Peninsular Malaysia itself is estimated to have about 18

genera with more than 160 species (Larsen, 1999) .

Through the ages, plants from Zingiberaceae family have been frequently

used as raw materials in traditional medicines and spices (Perry, 1980) .

Species such as Alpinia oxyplzylla, Curcuma domestica, Curcuma

xanthorrhiza, Zingiber officinale, Zingiber cassumunar and others have been

documented as extremely helpful to the elders (Itokawa et al., 1981a;

Burkill, 1966; Tilaar et al., 1991; Ibrahim and Rahman, 1988; Ammon et al.,

1992; and Shiobara et al., 1985). To date, considerable studies have

shown that Zingiberaceae species did display antioxidant property. For

instance, gingerol and diarylheptanoids related compounds isolated

from Zingiber officinale Rosc. have been observed to have antioxidant

activity (Kikuzaki and Nakatani, 1993) . In addition, Cassumunins A, B,

and C from Zingiber cassumunar were claimed to dislay both anti­

inflammatory and antioxidant activity (Masuda and Jitoe, 1994). Zingiber

is not the only genus in which antioxidant and related activities are

inherent. The genus of Curcuma and Alpinia are also potent candidates to

supply us with natural antioxidant and related activity compounds.

Curcumin isolated from Curcuma domestica has been reported to display

antioxidant activity (Toda et al., 1985) . Curcuminoids from Curcuma

2

xantorrlziza were also declared to have promising antioxidant activity

(Masuda et al., 1992) . In addition, l' -acetoxychavicol acetate and 1'­

acetoxyeugenol acetate isolated from Alpinia galanga were found to have

anti tumour activity (Itokawa et al., 1987) .

Because of the large number of species in the Zingiberaceae family, many

of them are still left unstudied, as evidenced by unavailability of such

reports. There is a huge opportunity for researchers to discover new

antioxidant compounds from these unexplored species. Furthermore, if

the plants had been selected for study, only certain parts of the plants

were the focus of the research. For example, for Alpinia zerumbet, only the

leaves (Mpalantinos et al., 1998) and seeds (Hong et al., 1996) have been

studied thus far. In addition, only the rhizomes of Alpinia rafflesiana that

have been studied (Sirat et al., 1996), while for the case of Etlingera elatior,

only the essential oil of flower shoots that has been studied (Wong et al.,

1993.

These previous successful studies, and the easy availability of a collection

of the unexplored Zingiberaceae species at the Laboratory of

Phytomedicine, Institute of Bioscience, Universiti Putra Malaysia and

throughout the country, became the impetus for us to continue searching

3