UNIVERSITI PUTRA MALAYSIA

ISOLATION OF KAEMPFERIA ANGUSTIFOLIA ROSC., ALPINIA CONCHIGERA GRIFF. AND CURCUMA MANGGA VAL. AND VAN ZIJP.

PHYTOCHEMICALS AND THEIR BIOACTIVITIES

TANG SOOK WAH

FS 2011 84

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ISOLATION OF KAEMPFERIA ANGUSTIFOLIA ROSC., ALPINIA

CONCHIGERA GRIFF. AND CURCUMA MANGGA VAL. AND VAN ZIJP.

PHYTOCHEMICALS AND THEIR BIOACTIVITIES

By

TANG SOOK WAH

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

in Fulfilment of the Requirements for the Degree of Doctor of Philosophy

April 2011

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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

ISOLATION OF KAEMPFERIA ANGUSTIFOLIA ROSC.,

ALPINIA CONCHIGERA GRIFF. AND CURCUMA MANGGA VAL. AND

VAN ZIJP. PHYTOCHEMICALS AND THEIR BIOACTIVITIES

By

TANG SOOK WAH

April 2011

Chairman: Professor Mohd. Aspollah Hj. Sukari, PhD

Faculty: Science

Zingiberaceae is one of the most important herbaceous families found in tropical

forests. In this research, Kaempferia angustifolia Rosc. (Kunci pepet), Alpinia

conchigera Griff. (lengkuas ranting) and Curcuma mangga Val. & Van Zijp. (mango

turmeric) were selected for phytochemical and bioactivity studies. Several classes of

chemical constituents have been isolated and characterized spectroscopically. This

included cyclohexane epoxides, chalcones, terpenes, phenylpropanoids and

diarylheptanoids.

From Kaempferia angustifolia, crotepoxide (29), boesenboxide (30), 2'-hydroxy-

4,4',6'-trimethoxychalcone (31), zeylenol (28), 6-methylzeylenol (32), abiet-8(14)-

enepenta-6,7,9,11,13-ol (kaempfolienol) (84), (24S)-24-methyl-lanosta-9(11),25-

dien-3β-ol (80), pipoxide (25) were isolated besides β-sitosterol (82) and its

glycoside (83). Similarly, β-sitosterol-3-O-β-D-glucopyranoside (83) was also

obtained from the rhizomes of Alpinia conchigera along with p-

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hydroxycinnamaldehyde (55), 1’-acetoxychavicol acetate (33) and trans-p-coumaryl

diacetate (53). Furthermore, β-sitosterol (82), curcumin (59), demethoxycurcumin

(60) and 12,17-epoxy-3β,17-dihydroxylabda-13-en-16,15-olide (curcumangganol)

(94) were yielded from the Curcuma mangga extract. The structure of zeylenol (28)

was modified by using chemical reactions to give zeylenol diacetate (89), zeylenol

triacetate (90) and zeylenol epoxide (91) derivatives. p-Hydroxycinnamaldehyde (55)

was also derivatized to p-methoxycinnamaldehyde (92) and p-

acetoxycinnamaldehyde (93).

5S,6S,7S,9S,10S,11R,13S-Abiet-8(14)-enepenta-6,7,9,11,13-ol (Kaempfolienol) (84)

and epimeric 12,17-epoxy-3β,17-dihydroxylabda-13-en-16,15-olide

(curcumangganol I and II) (94) were isolated as new compounds. The stereostructure

of compound 84 was determined on the basis of its single crystal X-ray

crystallography. Compounds 80, 83 and (+)-25 were firstly described from the

respective species. Structures of all the isolated phytochemicals and structurally-

modified compounds were elucidated by using various spectroscopic methods

[infrared (IR), mass spectrometry (MS) and nuclear magnetic resonance (NMR)] and

by comparison with the previous literature.

Crude extracts, isolated constituents and derivatives were subjected to cytotoxic

screening against four cancer cell lines (HL-60, HT-29, MCF-7 and HeLa) as well as

antimicrobial testing. Crude extracts of Kaempferia angustifolia were not active in

the cytotoxic assays, with IC50 values more than 30 µg/mL. Isolated phytochemicals

from this species exhibited different degrees of inhibitions against the cancer cell

lines tested, with 2’-hydroxy-4,4’,6’-trimethoxychalcone (31) and (24S)-24-methyl-

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lanosta-9(11),25-dien-3β-ol (80) demonstrating the most prominent cytotoxic

activities (IC50 < 8 µg/mL) against certain cell lines. As for Alpinia conchigera, non-

polar and semi-polar extracts showed strong cytotoxic properties against human

promyelocytic leukaemia (HL-60), human breast cancer (MCF-7) and human

cervical cancer (HeLa) cell lines. 1’-Acetoxychavicol acetate (33) and p-

hydroxycinnamaldehyde (55) displayed a broad spectrum of anticancer effects

against the tested cell lines. In addition, the chloroform extract of Curcuma mangga

and isolated compounds 59, 60 and 94 also exhibited potent cytotoxic activities in

the study.

In antimicrobial screening test, extracts and constituents of Kaempferia angustifolia

were not active. On the other hand, the crude hexane, chloroform and ethyl acetate

extracts of Alpinia conchigera were strongly active against Salmonella choleraesuis

in the antimicrobial assay. In antifungal screening test, hexane and ethyl acetate

extracts of Alpinia conchigera showed slight inhibition on Saccharomyces cerevisiae.

4- Hydroxycinnamaldehyde (55) showed a remarkable activity in the antimicrobial

testing with a diameter of inhibition zone > 20 mm. The chloroform-soluble fraction

of Curcuma mangga and demethoxycurcumin (60) showed negative results in the

antimicrobial and antifungal screenings but curcumangganol (94) from the extract

demonstrated a moderate inhibitory activity towards Bacillus subtilis (B29).

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Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia sebagai

memenuhi keperluan untuk ijazah Doktor Falsafah

PEMENCILAN SEBATIAN FITOKIMIA DARIPADA KAEMPFERIA

ANGUSTIFOLIA ROSC., ALPINIA CONCHIGERA GRIFF. DAN CURCUMA

MANGGA VAL. DAN VAN ZIJP. SERTA KEAKTIFAN BIOLOGINYA

Oleh

TANG SOOK WAH

April 2011

Pengerusi: Profesor Mohd. Aspollah Hj. Sukari, PhD

Fakulti: Sains

Zingiberaceae merupakan salah satu daripada famili herba yang paling penting dalam

hutan tropika. Dalam kajian ini, Kaempferia angustifolia Rosc. (Kunci pepet),

Alpinia conchigera Griff. (lengkuas ranting) dan Curcuma mangga Val. & Van Zijp.

(kunyit mangga) telah dipilih untuk kajian fitokimia dan bioaktiviti. Kandungan

kimia daripada beberapa kelas sebatian telah dipencilkan dan dikenalpasti dengan

teknik spektroskopi. Ini termasuklah sikloheksana epoksida, kalkon, terpena,

fenilpropanoid dan diarilheptanoid.

Kaempferia angustifolia telah menghasilkan krotepoksida (29), boesenboksida (30),

2'-hidroksi-4,4',6'-trimetoksikalkon (31), zeylenol (28), 6-metilzeylenol (32), abiet-

8(14)-enapenta-6,7,9,11,13-ol (kaempfolienol) (84), (24S)-24-metil-lanosta-9(11),25-

dien-3β-ol (80), pipoksida (25), β-sitosterol (82) dan β-sitosterol-3-O-β-D-

glukopiranosida (83). β-Sitosterol-3-O-β-D-glukopiranosida (83) juga diperoleh

daripada rizom Alpinia conchigera selain p-hidroksisinamaldehid (55), 1’-

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asetoksikavikol asetat (33) and trans-p-koumaril diasetat (53). Di samping itu, β-

sitosterol (82), kurkumin (59), demetoksikurkumin (60) dan 12,17-epoksi-3β,17-

dihidroksilabda-13-en-16,15-olida (curcumangganol) (94) juga didapati daripada

ekstrak Curcuma mangga. Struktur sebatian zeylenol (28) telah diubahsuai dengan

menggunakan tindak balas kimia untuk menghasilkan zeylenol diasetat (89),

zeylenol triasetat (90) and zeylenol epoksida (91). p-Hidroksisinamaldehid (55) juga

digunakan untuk mengsintesiskan p-metoksisinamaldehid (92) and p-

asetoksisinamaldehid (93).

5S,6S,7S,9S,10S,11R,13S-Abiet-8-enapenta-6,7,9,11,13-ol (Kaempfolienol) (84) dan

epimer 12,17-epoksi-3β,17-dihidroksilabda-13-en-16,15-olida (kurkumangganol I

dan II) (94) telah dipencilkan sebagai sebatian baru. Stereokimia bagi sebatian 84

telah ditentukan berdasarkan analisis kristallografi kristal tunggal sinar-X. Sebatian

80, 83 dan (+)-25 diasingkan daripada spesies berkenaan untuk kali pertama. Semua

sebatian fitokimia dan sebatian yang diperoleh daripada sintesis organik telah

dikenalpasti dengan pelbagai teknik spektroskopi seperti inframerah, spektrometri

jisim, resonans magnet nukleus serta berdasarkan perbandingan dengan literatur

dahulu.

Ekstrak mentah dan sebatian hasil telah dijalankan ujian sitotoksik ke atas empat sel

kanser (HL-60, HT-29, MCF-7 dan HeLa) dan ujian antimikrob. Ekstrak Kaempferia

angustifolia tidak aktif dalam ujian sitotoksik, dengan nilai IC50 melebihi 30 µg/mL.

Sebatian fitokimia daripada spesies ini menunjukkan rangsangan yang berbeza

terhadap sel kanser yang dikaji, dengan 2’-hidroksi-4,4’,6’-trimetoksikalkon (31) dan

(24S)-24-metil-lanosta-9(11),25-dien-3β-ol (80) menunjukkan aktiviti sitotoksik

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yang lebih tinggi (IC50 < 8 µg/mL) terhadap sel kanser tertentu. Bagi Alpinia

conchigera pula, ekstrak yang tidak polar dan sederhana polar menunjukkan

rangsangan sitotoksik yang sangat kuat ke atas sel leukemia manusia (HL-60), sel

kanser payudara manusia (MCF-7) serta sel servik manusia (HeLa). Di antaranya,

sebatian 1’-asetoksikavikol asetat (33) dan p-hidroksisinamaldehid (55)

menunjukkan kesan sitotoksik ke atas semua sel kanser yang dikaji. Selain itu,

aktiviti sitotoksik ekstrak kloroform Curcuma mangga dan juga sebatian yang

dipencilkan daripadanya iaitu 59, 60 dan 94 mempamerkan aktiviti sitotoksik yang

berpotensi dalam kajian ini.

Semua ekstrak dan sebatian daripada Kaempferia angustifolia tidak aktif dalam ujian

antimikrob. Sebaliknya, ekstrak heksana, kloroform dan etil asetat bagi Alpinia

conchigera sangat aktif terhadap Salmonella choleraesuis. Dalam ujian penyaringan

antifungal, ekstrak heksana dan etil asetat bagi Alpinia conchigera menunjukkan

aktiviti yang rendah ke atas Saccharomyces cerevisiae. p- Hidroksisinamaldehid (55)

menunjukkan aktiviti yang menonjol dalam ujian antimikrob dengan diameter zon

rencatan lebih daripada 20 mm. Ekstrak kloroform Curcuma mangga dan

demetoksikurkumin (60) menunjukkan keputusan negatif dalam ujian penyaringan

antimikrob dan antifungus tetapi sebatian kurkumangganol (94) mempamerkan

aktiviti yang sederhana terhadap Bacillus subtilis (B29).

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ACKNOWLEDGEMENTS

Without the help and support from so many people, my research and study would not

have been completed and come to the compilation of work in this thesis.

I would like to express my deepest gratitude to my supervisor, Prof. Dr. Mohd.

Aspollah Hj. Sukari for his guidance, encouragement, sharing of knowledge,

financial assistance from the post of research assistant and providing opportunities to

attend seminars and conferences throughout this research. My sincere thanks are also

extended to my co-supervisors, Prof. Mawardi Rahmani, Prof. Nordin Hj. Lajis and

Prof. Abdul Manaf Ali for their intellectual advices and ideas which help in problem

solving during research.

Besides, assistance from Prof. Dr. Sugeng Riyanto, Dr. Faridah Abas, Assoc. Prof.

Dr. Sidik Silong, Dr. Mohamed Ibrahim Mohamed Taher, Dr. Mohd Azlan Nafiah

(UPSI), Prof Dr. Mohd Ambar Yarmo (UKM), Dr. Neoh Bee Keat, Dr. Nor Azah

Mohamad Ali (FRIM) and Siti Hajar Kasim in obtaining experimental data and/or

materials for this study is highly appreciated.

My thanks are also conveying to Science Officers and laboratory assistants Mr.

Johadi Iskandar, Madam Rusnani Amirudin, Mr Zainal Kassim, Mr Abas Abd.

Rahman, Mr Ismail Yasin, Madam Rakina bt Manaf as well as Madam Norhaszalina

Md. Isa and Madam Norhafiza Azwa Ghozali (Institute of Bioscience) for their help

and co-operation.

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Thanks to Universiti Putra Malaysia for awarding the Graduate Research Fellowship

for three semesters during my study.

Not forgetting my labmates – Amy Yap Li Ching, Noor Haslizawati Abu Bakar,

Nurul Waznah Muhd Sharif, Rahayu Utami Umar, Mohd Zulkhairi Azid, Noorul

Adawiyah Mustahil, Halimatul Saadiah Mohd Noor and Sadikah Ahmad for their

suggestions and encouragement throughout this research. Special thanks to Kwong

Huey Chong for the help regarding single crystal X-ray crystallography and Dr. Tan

Pei Jean and Wen Yin Ping for helping in structural elucidation of compounds.

Last but not least, I appreciate my family members and friends for their unlimited

support in all kinds, thank you for giving me strength to accomplish my dream.

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I certify that a Thesis Examination Committee has met on 12 April 2011 to conduct the final examination of Tang Sook Wah on her thesis entitled "Isolation of Kaempferia angustifolia Rosc., Alpinia conchigera Griff. and Curcuma mangga Val. and van Zijp. Phytochemicals and their Bioactivities” 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 Doctor of Philosophy. Members of the Thesis Examination Committee were as follows: Faujan bin Hj Ahmad @ Hj Amat, PhD

Professor Faculty of Science Universiti Putra Malaysia (Chairman) Gwendoline Ee Cheng Lian, PhD Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Emilia Abd. Malek, PhD Senior Lecturer Faculty of Science Universiti Putra Malaysia (Internal Examiner) Hasnah Mohd Sirat, PhD Professor Department of Chemistry, Faculty of Science Universiti Teknologi Malaysia (External Examiner)

NORITAH OMAR, PhD Associate Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date: 26 July 2011

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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 Doctor of Philosophy.

The members of the Supervisory Committee were as follows:

Mohd. Aspollah Hj. Sukari, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Chairman)

Mawardi Rahmani, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Member)

Nordin Hj. Lajis, PhD

Professor

Faculty of Science

Universiti Putra Malaysia

(Member)

Abdul Manaf Ali, PhD

Professor

Deputy Vice Chancellor (Research & Innovation)

Universiti Sultan Zainal Abidin

(Member)

________________________________

HASANAH MOHD. GHAZALI, PhD

Professor and Dean

School of Graduate Studies

Universiti Putra Malaysia

Date:

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DECLARATION

I declare that the thesis is my original work except for quotations and citations which

have been duly acknowledged. I also declare that it has not been previously, and is

not concurrently, submitted for any other degree at Universiti Putra Malaysia or at

any other institution.

__________________

TANG SOOK WAH

Date: 12 April 2011

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TABLE OF CONTENTS

Page

ABSTRACT ii

ABSTRAK v

ACKNOWLEDGEMENTS viii

APPROVAL x

DECLARATION xii

LIST OF TABLES xvi

LIST OF FIGURES xviii

LIST OF ABBREVIATIONS xxvii

LIST OF APPENDICES xxx

CHAPTER

1 INTRODUCTION 1

1.1 General Introduction 1

1.2 Natural Products Research in Malaysia 3

1.3 Zingiberaceae 5

1.3.1 Uses and Commercial Importance 8

1.3.2 Kaempferia 10

1.3.3 Kaempferia angustifolia Rosc. 11

1.3.4 Alpinia 12

1.3.5 Alpinia conchigera Griff. 13

1.3.6 Curcuma 14

1.3.7 Curcuma mangga Valeton & van Zijp. 15

1.4 Bioassays 16

1.4.1 Cancer and Cytotoxic Drugs 17

1.4.2 Antimicrobial Agents 18

1.5 Problem statements 20

1.6 Objectives of the Study 20

2 LITERATURE REVIEW 21

2.1 Literature Review of Zingiberaceae Species 21

2.1.1 Phytochemical Studies of Zingiberaceae 21

2.1.2 Bioactivity Screenings of Zingiberaceae Species 24

2.1.3 Literature Review of Kaempferia Species 26

2.1.4 Literature Review of Kaempferia angustifolia 30

2.1.5 Literature Review of Alpinia species 33

2.1.6 Literature Review of Alpinia conchigera 35

2.1.7 Literature Review of Curcuma species 38

2.1.8 Literature Review of Curcuma mangga 40

2.2 Structural Modification of Natural Products 45

3 MATERIALS AND METHODS 48

3.1 Instruments 48

3.2 Chromatographic Methods 50

3.2.1 Column Chromatography and Thin-Layer

Chromatography

50

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3.2.2 Centrifugal Thin-Layer Chromatography

(Chromatotron)

51

3.3 Experimental Methods 52

3.3.1 Plant Materials 52

3.3.2 Extraction and Isolation Work on Kaempferia

angustifolia Rosc.

52

3.3.3 Structural Modification on Zeylenol (28) 69

3.3.4 Extraction and Isolation Work on Alpinia

conchigera Griff.

73

3.3.5 Structural Modification on p-Hydroxy-

cinnamaldehyde (55)

78

3.3.6 Extraction and Isolation Work on Curcuma mangga

Val. & van Zijp.

80

3.4 Bioassays 85

3.4.1 Cytotoxic Assay 86

3.4.2 Antimicrobial Assay 87

4 RESULTS AND DISCUSSION 89

4.1 Extraction and Isolation of Phytochemicals from

Kaempferia angustifolia Rosc.

89

4.1.1 Characterization of (24S)-24-Methyl-lanosta-9(11),

25-dien-3β-ol (80)

90

4.1.2 Characterization of β-Sitosterol (82) 108

4.1.3 Characterization of 2’-Hydroxy-4,4’,6’-trimethoxy-

chalcone (31)

114

4.1.4 Characterization of Crotepoxide (29) 125

4.1.5 Characterization of β-Sitosterol-3-O-β-D-

glucopyranoside (83)

137

4.1.6 Characterization of Boesenboxide (30) 148

4.1.7 Characterization of Abiet-8(14)-enepenta-

6,7,9,11,13-ol (Kaempfolienol) (84)

156

4.1.8 Characterization of Pipoxide (25) 188

4.1.9 Characterization of Zeylenol (28) 194

4.1.10 Characterization of 6-Methylzeylenol (32) 208

4.2 Structural Modification of Zeylenol (28) 215

4.2.1 Characterization of Zeylenol diacetate (89) 215

4.2.2 Characterization of Zeylenol triacetate (90) 228

4.2.3 Characterization of Zeylenol epoxide (91) 236

4.3 Extraction and Isolation of Phytochemicals from Alpinia

conchigera Griff.

247

4.3.1 Characterization of 1’-Acetoxychavicol acetate (33) 250

4.3.2 Characterization of trans-p-Coumaryl diacetate (53) 259

4.3.3 Characterization of p-Hydroxycinnamaldehyde (55) 265

4.3.4 Characterization of β-Sitosterol-3-O-β-D-

glucopyranoside (83)

271

4.4 Structural Modification of p-Hydroxycinnamaldehyde (55) 272

4.4.1 Characterization of p-Methoxycinnamaldehyde (92) 272

4.4.2 Characterization of p-Acetoxycinnamaldehyde (93) 277

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4.5 Extraction and Isolation of Phytochemicals from Curcuma

mangga Valeton & van Zijp.

282

4.5.1 Characterization of β-sitosterol (82) 282

4.5.2 Characterization of Curcumin (59) 283

4.5.3 Characterization of Demethoxycurcumin (60) 289

4.5.4 Characterization of 12,17-Epoxy-3β,17-dihydroxy-

labda-13-en-16,15-olide (Curcumangganol) (94)

302

4.6 Cytotoxic Screening 336

4.6.1 Cytotoxic Screening of Kaempferia angustifolia 336

4.6.2 Cytotoxic Screening of Alpinia conchigera 339

4.6.3 Cytotoxic Screening of Curcuma mangga 342

4.7 Antimicrobial Screening 343

4.7.1 Antimicrobial Screening of Kaempferia angustifolia 344

4.7.2 Antimicrobial Screening of Alpinia conchigera 345

4.7.3 Antimicrobial Screening of Curcuma mangga 348

5 CONCLUSIONS 350

REFERENCES 353

APPENDICES 367

BIODATA OF STUDENT 403

LIST OF PUBLICATIONS 404