Ahmad, Kartini. Chemical constituents of Murraya Koenigii...
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UNIVERSITI PUTRA MALAYSIA CHEMICAL CONSTITUENTS OF MURRAYA KOENIGII (RUTACEAE) AND THEIR BIOLOGICAL ACTIVITIES KARTINI BINTI AHMAD FSAS 1999 25
Transcript of Ahmad, Kartini. Chemical constituents of Murraya Koenigii...
UNIVERSITI PUTRA MALAYSIA
CHEMICAL CONSTITUENTS OF MURRAYA KOENIGII (RUTACEAE) AND THEIR BIOLOGICAL ACTIVITIES
KARTINI BINTI AHMAD
FSAS 1999 25
CHEMICAL CONSTITUENTS OF MURRAYA KOENIGII (RUTACEAE) AND THEIR BIOLOGICAL ACTIVITIES
KARTINI BINTI AHMAD
MASTER OF SCIENCE
UNIVERSITI PUTRA MALAYSIA
1999
CHEMICAL CONSTITUENTS OF MURRA YA KOENIGII (RUTACEAE) AND THEIR BIOLOGICAL ACTIVITIES
By
KARTINI BINTI AHMAD
Thesis Submitted in Fulfilment of the Requirement for the Degree of Master of Science
in the Faculty of Science and Environmental Studies, Universiti Putra Malaysia
May 1 999
ACKNOWLEDGEMENTS
In the name of Allah, most Gracious, most Merciful. I would like to
convey my gratitude to my supervisors, Associate Professor Dr Mohd Aspollah
Hj. Sukari, Associate Professor Dr Mawardi Rahmani and Associate Professor
Dr Abdul Manaf Ali, for their guidance, concern, understanding and support
throughout the development of this project.
I would like to thank all the staff of Chemistry Department for bearing
with my demands and readily assisting me in moments of need.
My heartfelt thanks to all my friends, Kak Pah, Kak Ratna, Pak Sugeng,
Gaber and officemates for the precious help, encouragement and confidence
they gave me during the years of my studies.
Grateful appreaciation is also extended to my beloved husband (Mohd
Azlan Nafiah), my parents, brothers and sisters who were responsible in
completing my study in Universiti Putra Malaysia.
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TABLE OF CONTENTS
Page
ACKNO'VLEDGEMENTS................................................... II LIST OF TABLES. .. . . . . . . . . . . . . . . . . . . . .. ... ... . .. . . . . . . . . . . . . . . . . .. . . . . . . . . . .. v LIST OF FIGURES... ... ..... ...................... ............ ............... Vll LIST OF ABBREVIATIONS................................................ Xl ABSTRACT...................................................................... Xll ABSTRAK... ... . ..... ...... . ........ ... ......... ....................... ....... ... XIV
CHAPTER
I
II
III
IV
INTRODUCTION ........................................... . 1 Murraya koenigii and the Uses. . . .. . . . . . . . . . ......... . . . ..... 1 Screening of Bioactive Compounds... . . .... . . . . . . . . . . . . . . . . 3 Objectives... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . 5
LITERATURE REVIE'V .................................. . Previous Works on Murraya Species . . . . . . . . . . . . . . . . . . . . . . . . Biological Activity Assay ..... . . . . . . . . . . . . . . . . . . . . . . . . .. . . .. . .
EXPERIMENTAL ........................................... .
6 6
16
22 Extraction and Isolation Processes of Murraya koenigii.. 22
Materials and Methods....... . . . . .. . . ... . . . . . . . . . . . . .. 22 Extraction of Murraya koenigii (Stem Bark)...... 23 Extraction of Murraya koenigii (Leaves).. . . . . . . . 29 Extraction of Murraya koenigii (Roots) . . . . . .. . . .. 32
Biological Activity . . . . ... . . . . . . ..... . . . . ..... . . .... . . . . . . . . . . .. 35 Antimicrobial Activity.. . . . .. .. ... .... . ... . . . . . . . . .... 35 Cytotoxicity Assay. .. .. .. . . . . . . . . . .. .. . . . . . . . ... . .. . . . 36 Antitumor Promoting Activity Assay (Detection of the Inhibition of PMA Induced EBV -EA).. . . . . 37
RESULT AND DISCUSSION ............................ .. 41 Extraction and Isolation of Chemical Constituents from Murraya koenigii . . . ..... . . . . . .. . . . . .. . . . . . . . . . . . . . . . . . . .. .. . . .. 41 Isolation of Chemical Constituents from Murraya koenigii (Stem Bark)........................................... 43
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Mahanimbine (31). . . . ... . . . . . . .. . . . . .. . . . . . . . . . . .. . . .. 43 Girinimbine (32). . . . . . . . .. . . . . . . . .. . . ... . . ... . . . . .. . ... 54 Murrayanine (47)... . . . . . . . . . . . . . ... . . . . .. . . . . . . . . . . . . . 64 Murrayazoline (48). . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . ... 74 Murrayacine (49). . . . . . . . . . . . .. . . . . . . . . . . . .. . .. . . . . . . .. 86 Sucrose (50)... . . . . ... . . . . ... . . . . . . . . . .. . . . ... . . . . . . . . . . 96
Isolation of Chemical Constituents from Murraya koenigii (Leaves)..... . . . .... . . . . . . . . . . . . . . . .. . . . . .. . . . . . ... . . ... 102
Long Chain Mixture (51). . . ... . ..... . . . .. . . . . . . . .. . . . 102 Ethyl Octadecanoate (52). . . ... . . . . .... . . . . . . ... . . . . . . 108 Mahanine (53) . . . . . . . . . . . . . . . . . ... . . . .. . . . . . . . .. . . . . . . .. 111
Isolation of Chemical Constituents from Murraya koenigii (Roots). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 118
3-Methy1carbazole (54). . . . ... . . . . . . . . . .. . . . . . . . . .. . . . 118 Murrayafoline A (55). . .. . . . ... . .. . .. . . . . . .. . . . . . . . . .. 127
Biological Activities of Crude Extracts and Isolated Compounds of Murraya koenigii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 134
CONCLUSION ............................................... . 138
BIBLIOGRAPHY 140
VITA 145
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Table
1
2
3
4
5
6
7
8
9
LIST OF TABLES
JH, I3C NMR Chemical Shifts (8) and Coupling Patterns of the Protons in HMQC and HMBC Techniques of Mahanimbine (31) ......... . . . ... . ... . ...................... ....... . . . .
JH, I3C NMR Chemical Shifts (8) and Coupling Patterns of the Protons in HMQC and HMBC Techniques
f G· . . b' (i?) o IflnIm Ine __ . . . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
JH, I3C NMR Chemical Shifts (8) and Coupling Patterns of the Protons in HMQC and HMBC Techniques of Murrayanine (47) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
JH, I3C NMR Chemical Shifts (8) and Coupling Patterns of the Protons in HMQC and HMBC Techniques of Murrayazoline (48) . ............ . .. . .... . .... . . . . . . . ... . . ........ .... . .
lH, l3C NMR Chemical Shifts (8) and Coupling Patterns of the Protons in HMQC and HMBC Techniques of Murrayacine (49) ..... . . .... . ... . .... . ....... . . . ....... . .. . ........ . ... .
Chemical Shifts (8) of IH and 13C NMR of Mahanimbine (53) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Chemical Shifts (5) of IH and I3C NMR Of 3-Methylcarbazole (54) . . . .. . . . . . . .. . ........ . . . .. . . .. . ..... ... . .... . .
Chemical Shifts (8) of lH and Be NMR Of Murrayafoline A (55) . . .. . ... . .. . ... . .. . . . . . . ... . ... . .. . ....... . .... ..
Antimicrobial activity test results of crude extracts and the isolated pure compounds (diameter in mm) ............... .
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133
135
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11
Cytotoxicity activity of crude extracts and isolated pure compounds against CEM-SS cell line (T-lymphoblastic leukemia) . . . . . . . .. . . . . . . . .. . . . . . .. . . . . . . . . ..... . . . . . . . . . . . . . . . . .. . . . . . . . . . . .
In vitro antitumor promoting activity and Raji cell viability . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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LIST OF FIGURES
Figure
1 Schematic Diagram of the Isolated Compounds From Stern
Page
Bark of M. koenigii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . , . . , . . . . . . . . . . . . . . . . . . . . . 41
2 Schematic Diagram of the Isolated Compounds From Leaves of M. koenigii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 42
3 Schematic Diagram of the Isolated Compounds From Roots of M. koenigii . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43
4 IR Spectrum of Mahanimbine (31) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " '" 46
5 Mass Spectrum of Mahanimbine (31). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 47
6 lH-NMR Spectrum of Mahanimbine (32) in CDCl3 . . . . . . . . . . . . . . . . 48
7 13C-NMR Spectrum of Mahanimbine (32) in CDCh. . . . . . . . . . . . . . . 49
8 HMQC Spectrum of Mahanimbine (32) . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . 50
9 HMBC Spectrum of Mahanimbine (32). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51
1 0 13C_1 H Coupling Pattern Observed in HMBC Spectra of Mahanimbine (32). .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 52
11 Mass Fragmentations of Mahanimbine (32). . . . . . . . . . . . . . . . . . . . . . . .. 52
12 IR Spectrum of Girinimbine (31) . . . . . . . . . .. ... . ... . . . . . . . . . .. . . . . . . . . , 56
13 Mass Spectrum of Girinimbine (31). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 57
1 4 I H -NMR Spectrum o f Girinimbine (31) in CDCh. . . . . . . . . . . . . . . . . . 58
15 J3C-NMR Spectrum of Girinimbine (31) in CDCh . . . . . . . . . . . . . . . . . 59
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1 9
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3 1
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35
36
HMQC Spectrum of Girinimbine (31) . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . .
HMBC Spectrum of Girinimbine (31) . . . . . . . . . . . . . . . . . . . " . . , . . . . . . . .
13C_1H Coupling Pattern Observed in HMBC Spectra of Girinimbine (3 1 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mass Fragmentations of Girinimbine (31) . . . , . . . . , . . . . . . . . . . . . . . . . . .
IR Spectrum of Murrayanine (47) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . .
Mass Spectrum of Murrayanine (47) . . . . , . ' " . . . . . . . ' " . . . . . . . . . . . . . . .
lH-NMR Spectrum of Murrayanine (47) in CDCh .. . ... . . . . .. . ... .
13C-NMR Spectrum of Murrayanine (47) in CDCh . . ... . .... . ... . .
HMQC Spectrum of Murrayanine (47) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
HMBC Spectrum of Murrayanine (47) .............................. .
13C_1H Coupling Pattern Observed in HMBC Spectra of Murrayanine (47) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mass Fragmentations of Murrayanine (47) . . . . . . . . . . . . . . . . . . . . . . . . . . .
IR Spectrum of Murrayazoline (48) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Mass Spectrum of Murrayazoline (48) . . . . . . . . . . . . . . . . . . , . . . . . . . . . . . .
Mass Fragmentations of Murrayazoline (48) . . . . . . . . . . . . . . . .. . . . . . . .
IH-NMR Spectrum of Murrayazoline (48) in CDCh . . .. . . ..... ... .
13C-NMR Spectrum of Murrayazoline (48) in CDCh ... . ... . . . ... .
HMQC Spectrum of Murrayazoline (48) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
HMBC Spectrum of Murrayazoline (48) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
13C_1 H Coupling Pattern Observed in HMBC Spectra of Murrayazoline (48) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
IR Spectrum of Murrayacine (49) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
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60
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8 1
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37 Mass Spectrum of Murrayacine (49) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 89
38 IH-NMR Spectrum of Murrayacine (49) in CDCh. . . . . . . . . . . . . . . . . 90
39 13C-NMR Spectrum of Murrayacine (49) in CDCh. . . . . . . . . . . . . . . . 91
40 HMQC Spectrum of Murrayacine (49) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 92
4 1 HMBC Spectrum o f Murrayacine (49) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 93
42 13C_1H Coupling Pattern Observed in HMBC Spectra of Murrayacine (49) . . . . . . . . . . . . . , . . . . . . . . . . . , . . , . . . . . " . . . . . . . . . . . . . . . . . 94
43 IR Spectrum of Sucrose (50) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 98
44 Mass Spectrum of Sucrose (50) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . 99
45 IH-NMR Spectrum of Sucrose (50) in DMSO. . . . . . . . . . . . . . . . . . . . . . 100
46 13C-NMR Spectrum of Sucrose (50) in DMSO . . . . . . . . . . . . . . . . . . . ... 101
47 IR Spectrum of Long Chain Mixture (5 1 ) . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 04
48 IH NMR Spectrum of Long Chain Mixture (51) in CDCh. . . . . . . . 105
49 Mass Spectrum of Long Chain Mixture (51a) . . . . . . . . . . . . . . . . . . . . . .. 106
50 Mass Spectrum of Long Chain Mixture (SIb) . . . . . . . . . . . . . . . . . . . . . . . 107
51 IR Spectrum of Ethy l Octadecanoate (52) . . . . . . . . . . . . . . . . . . . . . . . . . . . 109
52 Mass Spectrum of Ethyl Octadecanoate (52). . . . . . . . . . . . . . . . . . . . . . . . 110
53 Mass Spectrum of Mahanine (53) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 113 54 IR Spectrum of Mah anine (53) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 14
55 IH-NMR Spectrum of Mahanine (53) in CDCh . . . . . . . . . . . .. . . . . . . . . 1 1 5
56 13C-NMR Spectrum of Mahanine (53) in CDC}3. . . . . . . . . . . ... . . . . . . 1 16
57 Mass Spectrum of 3-Methy1carbazole (54) . . . . . . . . . . . . . . . . . .. . . . . . . . . 120
58 IR Spectrum of 3-Methylcarbazole (54) . . . . . . . . . . . . . . . . . . . . . . . . . . ... . 121
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59 lH-NMR Spectrum of 3-Methy1carbazole (54) in CDCh . . . . . . . . . 1 22
60 COSY Spectrum of 3-Methy1carbazole (54) . . .. . . . . . . . . . . . . . . . . . . . . 123
6 1 13C NMR Spectrum of 3-Methy1carbazole (54) in CDCb . . . . . . . . . 1 24
62 DEPT 13C NMR Spectrum of 3-Methylcarbazole (54) . . . . . . . . . . . . . 125
63 IR Spectrum of Murrayafoline A (55) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 29
64 Mass Spectrum of Murrayafoline A (55) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 130
65 IH NMR Spectrum of Murrayafoline A (55) in CDCI3 . . . . . . . . . . . . 13 1
66 DC NMR Spectrum of Murrayafoline A (55) in CDC13 . . . . . . . . . . 132
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LIST OF ABBREVIATIONS
br broad
CC column chromatography
CDC13 deuterated chloroform
CHCh chloroform
d doublet
dd double doublet
ddd doublet of doublet of doublet
triplet
s s inglet
m multiplet
DMSO dimethyl sulphoxide
Pet. ether petroleum ether
MeOH methanol
m.p melting point
MS Mass Spectrum
NMR Nuclear Magnetic Resonance
TLC Thin Layer Chromatography
IR Infrared
UV Ultraviolet
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Abstract of the thesis presented to the Senate of Universiti Putra Malaysia in fulfilment of requirements for the degree of Master of Science
CHEMICAL CONSTITUENTS OF MURRAYA KOENIGII (RUTACEAE) AND THEIR BIOLOGICAL ACTIVITY
By
KARTINI BINTI AHMAD
May 1999
Chairman: Associate Professor Mohd. Aspollah Hj. Sukari, Ph.D.
Faculty : Science and Environmental Studies
The study on Murraya koenigii (curry leaf) involves extraction,
separations by using various chromatographic methods and structural
determination by spectroscopic techniques such as IR, NMR including 2D-NMR
and MS. The structures of the compounds were also elucidated by comparison
with the previous work. I solation works on stem bark of the plant yielded
mahanimbine (31) , girinimbine (32) , murrayanine (47), murrayazoline (48) ,
murrayacine (49) and sucrose (50). From the leaves, five compounds were
isolated and elucidated. Two of them were long chain compounds, Il-decyl-
henecosane (5 1 a) and methoxydotriacontane (51 b) , isolated as a mixture and the
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other compounds were mahanimbine (31) , ethyl octadecanoate (52) and
mahanine (53) .
Further study on the roots of the same plant afforded four compounds
including girinimbine (32), murrayanine (47) , together with 3-methylcarbazole
(54) and murrayafoline A (55).
Crude extract and i solated compounds from various part of this plant
were screened for antimicrobial activity using disc diffus ion method, cytotoxic
activity using rnicrotitration method and antitumor promoting activity using
Epstein B arr virus activation assay. The crude extracts of the roots and pure
isolated compound including mahanimbine (31 ) , girinimbine (32), mahanine
(53) and murrayafol ine A (55) exhibited significant cytotoxicity activity against
CEM-SS cell line with ICso 3 Ilg/ml . Girinimbine (32), although did not show
any cytotoxic activity against CEM-SS cell line, the compound however
inhibited EBV-activation (100%) in the antitumor promoting assay . The
pet. ether , CHCh and MeOH crude extracts of leaves , the methanol extract of
stem bark, mahanimbine (31 ) and g irinimbine (32) failed to show significant
antimicrobial activity, while the pet.ether and the chloroform crude extracts of
the stem bark exhibited weak antibacterial activity against Bacillus cereus.
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Abstrak tesis ysng dikemukakan kepada Senat Universiti Putra Malaysia sebagai memenuhi keperluan untuk ijazah Master Sains
KANDUNGAN KIMIA DAR! MURRAYA KOENIGII (RUTACEAE) DAN ACTIVITI BIOLOGINYA
Oleh
KARTINI BINTI AHMAD
Mei 1999
Pengerusi : Profesor Madya Mohd Aspollah Hj. Sukari, Ph.D.
Fakulti: Sains dan Pengajian Alam Sekitar
Kajian terhadap pokok Murraya koenigii (daun kari) melibatkan
pengekstrakan dan pengasingan menggunakan pelbagai kaedah kromatografi
dan pengenalpastian struktur dengan teknik-teknik spektroskopi seperti
inframerah, resonans magnet nukleus termasuk resonans magnet nukleus dua
dimensi dan spektroskopi jisim. Struktur sebatian yang dipenci1kan juga telah
dibuat perbandingan dengan kajian lepas. Pengasingan keatas kulit batang pokok
ini telah menghasilkan mahanimbin (31), girinimbin (32), murayanin (47),
murayazolin (48), murayasin (49) dan sukros (50). Lima sebatian telah
dipencilkan dan diasingkan daripada daun pokok yang sarna. Dua daripadanya
ialah sebatian rantai panJang, Il-dekil-henekosana (51a) dan
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metoksidotriakontana (51 b), diasingkan sebagai campuran dan sebatian lain
adalah mahanimbin (31). etil oktadekanoat (52) dan mahanin (53).
Kajian seterusnya dilakukan keatas akar pokok yang sama memberikan
empat komponen termasuk girinimbin (32), murayanin (47), bersama dengan 3-
metilkarbazol (54) dan murayafolin A (55).
Ekstrak mentah dan sebatian yang telah diasingkan dari pelbagai
bahagian pokok tersebut juga telah diuji untuk aktiviti-aktiviti antimikrob
menggunakan kaedah resapan cakera, sitotoksik dengan kaedah mikrotitratan
dan anti promosi barah menggunakan kaedah cerakinan pengaktifan virus
Epstein Barr. Ekstrak mentah dari akar dan sebatian yang telah dipencilkan
termasuk mahanimbin (31), girinimbin (32), mahanin (53) dan murayafolin A
(55) menunjukkan aktiviti- aktiviti sitotoksik yang kuat keatas sel CEM-SS
dengan IC50 3 ).lg/ml. Walaubagaimanapun, girinimbin (32) tidak menunjukkan
sebarang aktiviti sitotoksik keatas sei CEM-SS, sebatian ini telah menunjukkan
aktiviti anti promosi barah dengan 100% perencatan. Ekstrak mentah pet.eter,
kloroform dan metanol dari daun, ekstrak metanol dari kulit batang, mahanimbin
(31) dan girinimbin (32) gagal untuk menunjukkan sebarang aktiviti antimikrob,
manakala ekstrak mentah pet.eter dan kloroform dari kulit batang menunjukkan
aktiviti antimikrob yang lemah terhadap Bacillus cereus.
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CHAPTER I
INTRODUCTION
Murraya koenigii and the Uses
Murraya koenigii is a member of the large Rutaceae family and represented by
about 150 genera and 1600 species. However, in Malaysia, nearly 60 species of this
family can be found including two species of Murraya; namely Murraya koenigii and
lviurraya paniculata. Other Murraya species are M alata, M exotica, M kwangsiensi
and M microphylla, consisting mainly of trees or shrubs and a very small proportion of
herbs all of which flourish in the tropics and sUbtropics.
lvi koenigii (curry leaf) is one of the most widely used plants whose leaves are
added to curries to improves flavour. It is usually cultivated for its aromatic smell which
are used as natural flavourings in curries and souces as reported by Sastri in 1952 fu'1d
Brandis in 1971. The intensive pungent, aromatic leaves are best when fresh, but
adequately retain their potency for some time after picking.
2
This plant is originated from Tarai of Uttar Pradesh, India and distributed
throughout India and Andaman Islands (Sastri, 1 952). Curry leaf odoms every
houseyard of South India and is usually propagated by seeds and roots suckers.
However, attempts to propagate by air layering have not been successful (Phillip et al. 1 98 1 ).
This plant is an unarmed, semideciduous, aromatic, shrub or small tree of about
3-5 meter high with strong woody stem. The branches are covered with dark grey bark.
The woody stem has a closely crowded, shady crown. Leaves are 1 5-20 mm long, have
slightly pungent, bitter and feebly acidulous taste. The flowers are small, 1 cm long and
the small fruits occur in close clusters . It turned from green to red and ultimately black
on ripening (Joseph and Peter, 1 985) .
Various parts of thi s plant have many medicinal uses and values as raw material
for traditional medicine and popular in India (Kirthikar and Basu, 1935) . It have been
reported that the leaves and the roots can cure piles and heat removal of the body,
imflamation and itching, while the powdered leaf is used to aid healing of fresh cuts .
Drury (1978) reported that the green leaves could be eaten in raw for the
treatment of dysentery. As an external application, barks and roots are used to relieve
skin eruptions and the bites by poisonous animals (Dasturs, 1970).
3
Generally, this plant has also been used in the industry, where fresh leaves are
steam distilled under reduced pressure to yield volatile oils (curry leaf oil) which can be
used as a fixative for a heavy type of soap perfume (Joseph et al. 1 985).
Murraya paniculata or local ly known as "kemuning" is a shrub or small tree,
grow wild in many parts of Malays ia. found mainly in the drier parts of the North and
the East coast, or on limestone hills and also often planted in gardens. In appearance the
"kemuning" looks l ike a citrus on account of the dark green, upstanding leaflets . It is
evergreen and apparentl y night-flowering (Comer, 1 988) .
Screening of Bioactive Compounds
In Malaysia the plant remain a very important source of therapeutic traditional
medicine practices among the various ethnic groups. The tropical forest of Malaysia
comprises of more than 15,000 plants species many of which have been claimed to
possess medicinal properties (Ali e t al. 1 995).
Among the species commonly used in the preparation of traditional medicines
are those belonging to the families of Annonaceae, Apocynaceae, Araceae and
Rutaceae. Lately, a number of Rutaceous species have been reported to posses
pharmacological activities, which then lead to the isolation and characterization of their
active compounds (Ontengo et al. 1 993). The study of biologically active compound
from plants must be related to their medicinal values .
4
Biological activities of Malaysian plants were first reported by Nakashini et al.
m 1 965. More recently , a more systematic screening of medicinal plants for
antimicrobial activity against bacteria and fungi was reported by Ali et al. ( 1 995) .
Much effort in previous studies has been devoted in testing the bioactivities of
phytochemicals in the search of new and useful compounds.
The most important factor in the search of new bioactive substances is the
convenience and reliability of the bioassay system. Bioassay for screening purpose must
be inexpensive and rapid, have broad applications to numerous target organisms, be
reproducible and statistically valid . It also must require small amount of the test
substance .
In order to achieve meaningful and significant results, future works in natural
product chemistry must incorporate bioassays (Mc Laughlin, 1 990) . Rahmani et al.
have carried out a screening procedure of tropical plants for the presence of bioactive
compounds by using brine shrimp in 1 992.
Objectives
The objectives of this research were;
1 . To extract, i solate and purify chemical components from stem bark, leaves and roots
of Murraya koenigii.
2. To identify and elucidate the structure of the isolated compounds using modern
spectroscopic methods.
3 . To determine the antimicrobial , cytotoxicity and antitumor promoting activities
againts the extracts and the isolated compounds from Murraya koenigii.
4. To identify the compounds which contribute to the toxicity.
CHAPTER II
LITERA TURE REVIEW
Previous Works on Murraya Species
Many constituents have been reported from the extraction of various part of
1v1urraya species using different type of solvents such as petroleum ether, chloroform,
methanol and others. Several coumarins (Gray, 1978; Wu et al. 1980; and Wu, 1981),
carbazole alkaloids (Ganguly et al. 1978) and flavanoids (Dreyer, 1968) have been
isolated. Murraya koenigii has also been proven to be a rich source of carbazole
alkaloids.
Previous work by Furukawa et al. (1985) reported the isolation of several novel
alkaloids including simple carbazole alkaloids, dimeric carbazole alkaloids and
carbazole quinone from the roots and stem bark of Murraya euchrestifolia. Futher study
on the leaves constituents of the same plant by Wu et al. (1991) has resulted in the
isolation of two carbazole alkaloids, bis-7-hydroxygirinimbine-A (1) and -B (2). In the
same year Wu also isolated a few other carbazole alkaloids, including murrayamine-A
(3), -B (4), and -C (5). Murrayamine-A showed significant cytotoxicity in the KB tissue
culture assay at 3.0 ).lg mrl.
6
7
H
H
( 1 )
H
H
(2)
(3) : H (4) : Me (5) : OAc
8
In 1 984. Mahinda et al. reported the isolation and identification of coumarins,
murraglenin (6), mexoticine (7), sibrin (8) and pheabalosin (9) from the petrol extract of
Murraya glenei leaves, together with known compound such as meranzine hydrate (10) .
MeO �
O
CH2CHCHMe2 I I OHOH
Rl R' (6): OMe OMe (7): OMe H
( 1 0) : H H
