UNIVERSITI PUTRA MALAYSIA

CHEMICAL CONSTITUENTS OF MICROMELUM MINUTUM AND ACRONYCHIA LAURIFOLIA (RUTACEAE)

YAP YUN HIN @ MD. TAUFIQ

FSAS 1994 3

CHEMICAL CONSTITUENTS OF MICROMELUM MINUTUM A ND A CRONYCHIA LAURIFOLIA (RUTACEAE)

By

YAP YUN HTN@MD. TAURQ

Thesis Presented in Fulfilment of the Requirements for the Degree of Master of Science

in the Faculty of Science and Enviromental Studies, Universiti Pertanian Malaysia

1 994

'To my bdovea father ana mother,

THE LATE YAPYOON SANG ARWAH ABDULLAH LIM HENG FUI SITI AISHAH CHIN SU KIA W

'To my brothers ana sister,

LIM YIT HIANG NORAINI LIM FUNG KUN ABDUL HALIM LIM YIT VUI

'To my tfearest Jrientfs SAIDAH BTE MAMAT NORIZAN BIN REM BUN

ana the memfjers of9{afiwan 9{ur WITHOUT WHOSE LOVE AND CONTINUED SUPPORT THIS THESIS WOULD NOT HAVE BEEN POSSIBLE.

A CKNOWLEDGEMENTS

I would l ike to express my sincere and deepest appreciation to Assoc.

Prof. Dr. Mawardi Rahmani for the i n va luable advi ses, gui dances,

encouragement, patient and support throughout the course of the work , Dr.

Hazar B .M. Ismai l , Dr. Mohd Aspollah Hj . Sukari , Assoc. Prof. Dr. Jambari

Hj . Al i , Assoc. Prof. Dr. Karen Badri and Dr. Mohd. Niyadz Khan for their

constant support.

I am also very grateful to my collegues, friends and staff of the

Chemistry Department of UPM, Dr. Faujan Hj . Ahmad, Dr. Mahiran Basri ,

En. Zainal Abidin Kasim , En. Zainal Zahari Zakaria, En. Nordin Ismail and En .

Zainuddin Samadi , whose help, advice and encouragement were i nval uable

during this research project.

My special thanks also goes to: Prof. P.G.Waterman of University of

Strachlyde for obtaining the IH- and 1 3C-NMR spectra of microminutinin, 6-

methoxymicrominutinin and MM5; Prof. T.W. Sam of USM and Dr. Hiroshi

Sugiyama of Kyoto University, for obtaining of IH- and 1 3C-NMR spectra of

microminutin and 6-methoxymicrominutinin; Prof. Latif Muhammad and En.

A . Zainuddin Ibrahi m of UKM , for identifying and col lecti ng the plant

materials; Assoc. Prof. Dr. S .C. Quah of UPM, for kindly supplying the

Dmwphila melanoga<;ter culture; Puan Noraini Abu Bakar, for typing and

ii

doing some editing work on the thesis; and Universiti Pertanian Malaysia for

providing facil ities and space for completion of the project.

Fi nancial support from Malaysian Goverment under the IRPA

programme i s gratefully acknowledged

iii

T ABLE OF CONTENTS

Page

ACKNOWLEDGEMENTS

LIST OF TABLES

LIST OF RGURES

LIST OF ABBREVIATIONS

ABSTRACT

11

vi i

IX

Xl

Xll

ABSTRAK XIV

PART I EXTRACTION AND ISOLATION OF CHEMICAL COMPONENTS FROM MICROMELUM MINUTUM AND A CRONYCHIA LA URIFOLIA (RUTACEA E)

INTRODUCTION AND LITERATURE REVIEW OF MICROMELUM MINUTUM (FROST. f.) SEEM AND ACRONYCHIA LA URIFOLIA B LUME. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2

OBJECTIVES OF STUDy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3

RESULTS A ND DISCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 4

Micromelum minutum (Frost. f.) Seem . . . . . . . . . . . . . . . . . . . 1 4

A cronychia laurif()lia B l ume . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 70

CONCLU SI O N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

iv

EXPERI MENTAL. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 79

EXTRACTION OF MICROMELUM MINUTUM ( K ELANTAN) . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . 8 1

I solation of Microminutinin (3 2) . . . . . . . . .. . . . . . . . . . . . . . . . . . . 82

I solation of6-Methoxymicrominutinin ( 3 5) . . . . . . . . . . . . . . . 83

Isolation of Scopoletin (13 ). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 84

Isolation of CHC13 extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 86

EXTRACTION OF MICROMELUM MINUTUM (PAHANG) . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . 86

Isolation of Squalene ( 3 6) . . . . . . . . . . . .................. ...... 86

Isolation of Micromelin(I) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ,. fn

Isolation of Scopoletin (13 ). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 88

Isolation of MM5 (37 ). . . . . . . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . 89

EXTRA CTION OF ACRONYCHIA LA URIFOLIA . . . . . . . . . 90

PART II

Isolation of Sitosterol (3 8) . . . . . . ..... .... ... .... .... ........ 91

Isolation of Sesel in (3 9). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92

I sol ati on of A P6. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93

PRELIMINARY BIOASSA YS AGAINST BRINE SHRIMP (Artemia salina) A ND VINEGAR FLY (Drosophila melanogaster)

INTRODUCTION .. . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 95

v

OBJECTIVES OF STUDy . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 00

RESULTS AND DI SCUSSION. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 00

CONCLlJ S I O N . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1

E XPER I M ENTA L. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1

Hatchin g of Brine Shrimp Cysts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1

Preparation of Assay Soluti on . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2

Bi oassay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 2

Analys is of Resul ts . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 3

Preparation of Culture Medium for Drosophila. . . . . .. . . . . . . 1 1 3

Preparation of D. melanogaster Larvae . . . . . . . . . . . . . . . . . . . . . . 1 1 4

Preparation of Assay Media . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 4

B i oassay Procedure . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 5

B I B LI O GRA PHy. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 6

A PPEN D I X . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 22

V ITA . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 23

vi

LIST OF TABLES

TABLE Page

Compounds from Micromelum sp. . . . . . . . . . . . . . . . . . . . . . . . . . . 9

2 IH-NMR Chemical Shifts (0) and Coupling Patterns of

the Protons in Various IH-NMR Techniques of (32) . .. . . . 25

3 13C-NMR Chemical Shift Data(ppm) of(3 2 ), (33) and(3 4 ). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30

4 13C-NMR Chemical Shift Data (ppm) of (32) and (3 5) . . . . . . . ..... .............. ........ ........... .......... ...... 40

5 13C-NMR Chemical Shift Data (ppm) of Micromelin (1) 56

6 Coupling of Protons of MM5 (37). . . . . . . . . . . . . . . . . . . . . . . . . . 66

7 13C-NMR Chemical Shift Data (ppm) and 13C-1 H Coupl ing of MM5. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 69

8 IH-NMR Spectral Data (ppm) of Seselin and Norbraylin with the Observed Data. . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . . . 76

9 Plant Extract Concentrations used in Brine Shrimp Bi oassay . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 0 1

1 0 Brine Shrimp Bioassays Results of Extracts (Pahang) and Compounds of M. minutum . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 02

11 Brine Shrimp Bioassays Results of CHC13 Extracts of A . laurifol ia. . . .. . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . 1 03

1 2 Diet Fonnulas for D. melanogaster. . . . . . .. . .. . . . . . . . . . . . . . . . . 1 05

1 3 Effect of M. minutum (Kelantan) Extract on D. melanogaster Larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 06

1 4 Effect of M. minutum (Kel an tan ) MeOH Extract on D. melanoga\· ter Larvae . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 07

vii

1 5 Effect of CHC13 Extract (leaves) of M. minutum (Pahang) Extract on D. melanoga'lter Larvae . . . . . . . . . . . . . . . . 1 08

1 6 Effect of Pure Compounds on D. melanoga'lter Larvae. . . . 1 09

viii

LIST OF FIGURES

FIGURE Page

Mass Spectrum of (32 ). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6

2 lR Spectrum of (32). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . 1 7

3 IH-NMR Spectrum of (32). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 9

4 COSY -45 Spectrum of (3 2 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 20

5 COSY -LR Spectrum of (3 2) . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . 2 1

6 Long-range Coupling of Various Protons Observed from COSY -LR Spectrum . . . . . . . . . . .. . . . . . . . . ... . . . . . . . . . . . . . . 22

7 NOESY Spectrum of (3 2 ) . . . . . . . . . . . . . . . . . . '" . . . . . . . . . . . . . . . 23

8 DEPT 13C-NMR Spectrum of (32 ) . . . . . . . . . . . . . . . . . . . . . . . . . 24

9 HC-COBI Spectrum of (32) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 26

1 0 HMBC Spectrum of (3 2) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27

1 1 HMBC Spectrum of (32) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 28

12 13C- IH Coupling Pattern Observed in HMBC Spectra of (32) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 29

1 3 Mass Spectrum of (35) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32

1 4 I R Spectrum of (35). . . . . . . . . . . . . . ... . . . . . . . . . . . . . . . . . . . . . . . . . 33

1 5 1H-NMR Spectrum of (3 5) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 35

1 6 COSY -45 Spectrum of (35). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 36

17 13C-NMR Spectrum of (35). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 37

18 DEPT 13C-NMR Spectrum of (3 5) . . . . . . . . . . . . . . . . . . . . . . . . . 39

1 9 Mass Spectrum of ( 13 ) . . . . . . . . . . .. . . . . . . . . . . . . . . . . .. . . .. . . . . . 42

ix

20 IR Spectrum of (13 ) . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . 43

2 1 IH-NMR Spectrum of (13)..... .. . . . . .. . . . . . . . . .. .. . . . .. . . .. 45

22 13C-NMR Spectrum of (13)................ . . . . . . . ... . . .. . . . 46

23 Mass Spectrum of (36). . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 47

24 IR Spectrum of ( 3 6) . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . 48

25 1 H-NMR Spectrum of (36).. . . . . . . . . . . . . . .. . . . . . . . . . . .. . . . . . 50

26 Mass Spectrum of ( 1) . .. . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . 52

27 TR Spectrum of (1). . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . 53

28 IH-NMR Spectrum of (1).................................... 54 29 MS Spectrum of (37). . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . 58

30 TR Spectrum of ( 37 ) . . . . . . . ... . .. . . . . . . .. . .. . . . . .. . . . . . . .. . . . .. 59

3 1 1 H-NMR Spectrum of (37) . .. . . .. . . . . . . . . . . . . . . .. . . . . . . .. . . . 6 1

32 COSY-45 Spectrum of ( 3 7 ) . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . 62

33 NOESY Spectrum of ( 3 7 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 63

34 DEPT 13C-NMR Spectrum of (37).. . . . . . . .. ...... ....... . 64

35 HC-COBT Spectrum of ( 3 7 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 65

36 13C-IH Coupling Pattern of MM5 ( 3 7 ). . . . . . . . . . . . . . . . . . . . 67

3 7 HMBC Spectrum of (37). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 68

38 IR Spectrum of (38) . . . . . . . . . . . . . . . . '" . . . . . . . . . . . . . . . . . . . .. . . 7 1

39 Mass Spectrum of ( 3 8) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 73

40 Mass Spectrum of ( 3 9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 74

41 IH-NMR Spectrum of ( 3 9) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 77

42 DEPT 13C-NMR Spectrum of ( 3 9).. . .. . . . . . . . . . . . . . . . . . . . . 78

x

br. CHCI3 CDC13 d dt dq 0-t:> IR I LC m m1 mg Me MeOH m .p . MS NMR PE PLC ppm q s TLC IN w

LIST OF ABBREVIATIONS

broad chlorofonn de ute rated chlorofonn doublet doublet of triplet double of quartet gram Infra Red litre Lethal Concentration medium mili litre mili gram methyl methanol melting point Mass Spectrum Nucleur Magnetic Resonance petroleum ether Preparative Thin Layer Chromatography part per million quartet strong Thin Layer Chromatography Ultra Violet weak

xi

Abstract of the thesis presented to the Senate of Universiti Pertanian Malaysia in fulfilment of requirements for the degree of Master of Science.

CHEMICAL CONSTITUENTS OF MICROMELUM MINUTUM A ND A CRONYCHIA LA URIFOLIA (RUTACEA E)

Chairman Faculty

By

YAP YUN HIN @ MD. TAUFIQ

April 1994

Associate Professor Dr. Mawardi Rahmani Science and Enviromental Studies

A chemotaxonomic i nvesti gation on the l eaves and stems of

Micromelum minutum (Rutaceae), has resulted in the i solation of si x

components. The structure of these compounds was elucidated by means of

spectroscopic methods i ncluding by the extensive used of vari ous NMR

techniques and al so compari son with previous studies. The use of High Field

NMR is essential in structural determination of these complex molecules. With

the aids of various NMR experimental techniques and other spectroscopic

methods such as MS, UV and JR, the correct structures of the pure i solated

compounds were establ ished.

M. minutum coll ected from Kelantan yielded two novel coumarins,

microminutinin and 6-methoxymi crominutin in , an d a known coum ann ,

xii

scopoletin . From another separate collection of the plant from Pahang, a novel

compound, MM5, squalene, and two known coumarin , m icromel in and

scopoletin have al so been i solated. A similar study on the l eaves and stems of

Acronychia laurifolia col l ected from Pulau S inga Besar, Langkawi Island

afforded three compounds, sitosterol , seselin and an unidentified compound.

The presence of bioactive compounds in these plants was detected by

the use of brine shrimp (Artemia salina ). With the aid of thi s bioassay system

the activity of either the crude plant extracts or pure i solated compounds could

be determined. The LC50 value of CHCl3 and MeOH extracts of M. minutum

(Pahang) gave no significant activity while CHCl3 extracts of leaves and stems

of A. laurifolia had LC50 of 2.4 and 22.6 ppm respectively. Microminutinin

gave strong test result with LC50 of 34.7 ppm and MM5 with LC50 of 1 65.5

ppm.

A more specific bioassay was also carried out on the crude and pure

compounds for development i nhibition activity by using fruit fly, Drosophila

melanogaster larvae. The MeOH extract of M. minutum (Kel antan) , at

concentration of 200 mg/2 g diet, completely retarded the l arval growth . For

the pure compounds, microminutinin has been shown to have a strong

development inhibition acti vity. At onl y 4 m gl2 g diet, i t caused 1 00%

mortal i ty against first/second instar l arvae of the insect, whereas no activities

was observed from MM5.

xiii

Abstrak tesi s yang dikemukakan kepada Senat Universi ti Pertanian Malaysia bagi memenuhi keperluan ljazah Master Sains.

KANDUNGAN KIMIA D ARI MICROMELUM MINUTUM D A N ACRONYCHIA LAURIFOLIA (RUTACEAE)

Pengerusi Fakulti

Oleh

YAP YUN fiN @ MD. TAUFIQ

April 1994

Professor Madya Dr. Mawardi Rahmani Sains dan Pengajian Alam Sekitar

Kajian k imotaksonomi ke atas ekstrak m entah daun dan ranti n g

Micromelum minutum (Rutaceae) telah memberikan enam komponen . Struktur

sebatian-sebatian ini telah dapat dikenal pastikan dengan menggunakan kaedah

spektroskopi tennasuk penggunaan yang meluas pelbagai teknik NMR dan juga

perbandingannya dengan kajian-kajian yang lepas. Penggunaan NMR berkuasa

tinggi adalah amat diperlukan untuk menentukan struktur yang rumit. Dengan

bantuan berbagai teknik in i dan juga kaedah spektroskopi yang lain seperti MS,

UV dan IR, struktur yang betul bagi sebatian tulen yang dipencilkan dapat

ditentukan .

M. minutum yang dikumpulkan dari Kelantan telah menghasi lkan dua

koumari n baru, mi krominuti n in dan 6-metoksimikrominutinin dan satu

xiv

koumarin yang telah dikenal i , skopoletin . Dari pengumpulan berasingan

(Pahang) pokok yang sarna telah memberikan satu sebatian baru, MM5,

skualena dan dua koumarin yang telah dikenal i , mikromelin dan skopoletin .

Kajian yan g serupa k e at as daun dan ranting Acronychia laurifolia yan g

dikurnpulkan dari Pulau Singa Besar, Pulau Langkawi telah rnenghasilkan tiga

sebatian , sitosterol , sesel in dan satu sebatian yang belum dikenalpasti .

Kehadiran sebatian bioaktif dalam tumbuhan-tumbuhan in i dapat

dikesan dengan menggunakan udang pepai (Artemia salina). Dengan bantuan

sistem biocerakinan in i , aktiviti sarna ada dan ekstrak rnentah turnbuhan atau

sebatian tulen dapat ditentukan . Nilai LC50 dari ekstrak CHC13 dan MeOH dan

M. minutum (Pahang) tidak membenkan keputusan yang bermakna sementara

ekstrak CHC13 bagi daun dan ranting A. laurifolia masing-masing memberikan

LCSO 2.4 dan 22.6 ppm. Mikrominutin in memberikan keputusan yan g kuat

dengan LC50 34.7 ppm dan MM5 dengan LC50 1 65.5 ppm.

Kaedah biocerakinan yang lebih spesifik juga telah dijalankan ke atas

ekstrak mentah dan sebatian tulen bagi akti viti penghalang perkembangan

dengan menggunakan larva Dmsophilamelannga. .. ter. Ekstrak MeOH bagi M.

minutum (Kelantan) pada kepekatan 200 m g/2 g diet telah sepenuhnya

menghalang perkembangan larva ini . Bagi sebatian tulen pula, mikrominutin in

telah menunjukkan aktiviti penghalang perkembangan. Pada kepekatan hanya 4

mg/2 g diet, mikrominutinin telah menyebabkan 1 00% kematian terhadap larva

xv

peringkat pertama dan kedua bagi serangga in i , sementara i tu tiada aktiviti yang

dikesan bagi MM5.

xvi

PART 1

EXTRACTION AND ISOLATION OF CHEMICAL COMPONENTS FROM

MICROMELUM MINUTUM AND

ACRONYCHIA LAURIFOLIA (RUTACEAE)

1

INTRODUCTION AND LITERATURE REVIEW OF

MICROMELUM MINUTUM (FROST. f.) SEEM A ND

ACRONYCHIA LAURIFOLIA BLUME

2

Ten species of Micromelum have been found throughout South-Eastern

Asia and the Pacific of which only two species occur in Peninsular Malaysia,

M. minutum and M. hirsutum. M. minutum (synonymous with M. puhescens

Blume) is a shrub or small tree of the family Rutaceae, which can grow up to 25

feet high . In Peninsul ar Malaysia, M. minutum which is local ly known as

"Chemama", "Cherek-cherek" or "Secherek" normally occurs in the hil ly parts

of the northern half and in the north-west area.

The plants are used medicinal ly. The leaves are normally pounded with

tamari nds and salt and appl i ed to the ski n to rel i eve pain . I n Pen insular

Malaysia, i t is tradi tional ly used in the treatment of fever and giddiness and a

poultice of the boiled roots is applied to treat ague (Burkill , 1 966) . The plant i s

also found widely throughout the Fij i Islands, where i t i s termed "Qiqila" and is

also used by the Fijians in traditional folk medicine (Craft and Toia, 1 989).

Previous phytochemical studies of Micromelum species reveal ed a

n umber of prenyl ated coum arins , a pyranoquino l i ne al kal o i d and

polyoxygenated flavanoids. In 1 966, Lamberton et al. i solated a neutral

consti tuent, micromel in ( 1) , C 15HL�06, m .p. 2 1 8-21 9.SoC from the MeOH

extract of M. minutum. Another coumarin, osthol (2) , m .p. 82-83 .5°C was

3

also found in the methylene chloride extract. Later Chatteljee et al. ( 1 967)

reported the isolation of coumarins, micropubescin , C1SH 1 404 (3) , m.p. 1 30-

1 3 1 °C and (1) from the stem bark of M. puhescens. Tn 1 974, Joshi et al.

i s o l a t e d a n o v e l c o um aTln, 6-(2,3-dihydroxy-3-methylbutyl )-7-

methoxycoumarin or ulopterol (4), ClsHlSOS, m .p . 1 39- 1 400C from M.

puhescens Blume.

MeO o

R

(1) R= (4) R = -CH2CH( O H)C( O H)(CH3h

(2) R = -CH2CH=C(CH3)2 (3) R = -�-CH2C(CH3)=CH2

o (5) R� ): J o 0

An investigation of M. minutum by Tantivatana et al. ( 1 982) yielded a

cytotoxic compound microminutin ( 5) , and a pyranoquinoline alkaloid,

flindersine (6). Microminutin , m .p. 1 54- 1 55°C was found to be inactive i n

KB cytotoxicity test but was reported to show weak activity (EDSO 3 .7 !lg/mL)

against the P-388 lymphocytic leukemia test system in vitro.

(6)

Further study on M. minutum collected in Diphu area of the Karbi

Anglong District, Assam, India by Das et al. (1984) afforded four known

coumarins, (1), ( 2) , murralongin ( 7 ) and murrangatin ( 8); and four new

coumarins, dihydromicromelin A and B , (9a a n d 9b), acetyl

dihydromicromelinA (9c), minumicrolinlmupanidin (10) and a new flavone,

7,12-ether of 5,7-dihydroxy-3,6,8,4'-tetramethoxyflavone (11).

( 7 )

( 8)

(10)

R

R = -C(CHO)=C(CH3h

H H I I

R = -C - C - C(CH3)=CH2 I I GH OH

H OH I I R = -y - y - C(CH3)=CH2

OHH

4

MeO

(9a) R = a-O H

(9b) R = �-OH

(9c) R = a-OAc

OH 0

(11 )

5

OMe

Tanti shaiyakul etal. (1986) undertook the exam ination of the stern bark

extract of M. minutum from which they reported the i solation of phebalosin

( 12), m .p. 120.5-121 .5°C. Phebalosin ( 14) was reported to be significantly

toxic to brine shrimp (LCso 47 ppm, ) and to signifi cantly i nhib i t the

development of crown gall tumors on potato disc but had insignificant cytotoxic

activity.

From the stem-leaf of M. integerrimum, Cassady et al. (1979)

discovered the occurrence of two known coumarins, (1) and scopoletin (13),

and both were demonstrated to have antitumor activities. 7- [4'-(41-methyl-5"-

6

oxo-2" ,5"-dihydro-2"-furyl)-3'-methyl-2'-butenyloxy )coumarin (14) , has

been i solated by De Silva et al. (1980) from the PE extract of the leaves of M.

zeylanicum Swingl e.

MeO

(12) (13)

D o 0 CH2- C(CH3)=CH-CH2 0

(14)

Tn another i nvestigation by Bowen and Perera (1981), the leaves and

stem s of M. zeylanicum have yie lded a n ew oxazol e al kal oi d , 0-

methylh al fordi nol (15 ) , c o u m ari n (1) and 5-hydroxy-3 ,3 ' ,4' ,7 ,8-

pentamethoxyflavone (16) . Tn addition , the stems al so contained 6-formyl-7-

methoxycoumarin , or crenulatin (17 ) and the leaves contained the carbazole

alkaloid koenigine (18) and �-sitostero1 .

OMe

(15 )

MeO

OH 0

(16)

OHC

(17)

M eO

HO

(18)

o

OMe

OMe

A nother investigation by Kong et at. (1988) on the roots of M.

jalcatum has revealed the occurrence of the indole alkaloid, yuehchukene (19),

the novel carbazole alkaloid, 5,6-pyranoglycozoline (20), as well as (1) and

(10). The various chemical components present in Micrmnelum sp. are l isted in

Table 1.

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