UNIVERSITI PUTRA MALAYSIA
PHYTOCHEMICAL STUDIES AND BIOACTIVITY TESTS OF MURRAYA PANICULATA JACK, AEGLE MARMELOS CORREA AND
ZINGIBER AMARICANS BLUME
SUGENG RIYANTO
FSAS 2003 3
PHYTOCHEMICAL STUDIES AND BIOACTIVITY TESTS OF MURRAYA PANICULATA JACK, AEGLE MARMELOS CORREA AND
ZINGIBER AMARICANS BLUME
By
SUGENG RIY ANTO
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for
the Degree of Doctor of Philosophy
January 2003
DEDICATION
%is tfiesis is tfecficatea to: my son, If(fisan)fcfi :Nugrofio ( I)f:N)
ana my aaugfiter, )fsefin :Nuru{ If(fitiarini (}f!NI)
ana my 6ewvea wife Nanief(Sufiaryani
11
Abstract of the thesis presented to the Senate ofUniversiti Putra Malaysia in fulfilment of the requirement for the degree of Doctor of Philosophy
PHYTOCHEMICAL STUDIES AND BIOACTIVITY TESTS OF MURRAYA PANICULATA JACK,AEGLE MARMELOS CORREA AND
ZINGIBER AMARICANS BLUME
By
SUGENG RIYANTO
January 2003
Chairman: Associate Professor Mohd. Aspollah Hj. Sukari, Ph.D.
Faculty: Science and Environmental Studies
South East Asian countries are very rich in biodiversity with thousands of
the plants species of which only a small proportion of the species have been
investigated in detail. This research was carried out to investigate the chemical
constituents and bioactivities of three well known medicinal plants Murraya
paniculata Jack, Aegle marmelos Correa (Rutaceae) and Zingiber amaricans
Blume (Zingiberaceae).
Leaves and stem bark of M paniculata; leaves, bark and root of A.
marmelos and rhizomes of Z. amaricans were separately extracted with various
solvents. The extracts and isolated pure compounds were examined for their
bioactivity against microbes and cancer cell line. Cytotoxic assay was perfonned
III
against CEM-SS (human T-Iymphoblastic leukaemia) cell line with MIT reagent,
while antimicrobial tests were conducted by diffusion method against a list of the
pathogenic bacteria and fungi. The structures of the isolated compounds were
elucidated by using spectroscopic methods.
Eight compounds were isolated from M. paniculata and identified as
auraptene, stigmasterol, �-sitosterol, sucrose and methoxylated flavones including
3,3' ,4' ,5,5 ' ,6,7 ,8-octamethoxyflavone, 3,3 ',4' ,5,5 ' ,6,7 -heptamethoxyflavone,
3,3 ' , 4',5,5 ',7 -hexamethoxyflavone and 3' ,4',5,5 ',7, -pentamethoxyflavone. The
study on A. marmelos afforded eleven compounds; �-sitosterol, stigmasterol and
hopane triterpenes including zeorin and dustanin; alkaloids aegeline and
skimmianin; epilupeol, lupenone and coumann derivates; auraptene,
epoxyauraptene and marmin. By employing the same procedures, the extracts of
Z. amaricans afforded zerumbone as the major constituent, together with
cholesterol, campesterol, stigmasterol and �-sitosterol.
The results of antimicrobial and cytotoxic tests revealed that some of the
extracts showed strong activities. Nevertheless, most of the isolated compounds
showed only weak activity. On the other hand, the cytotoxic test results on
dustanin and epilupeol showed moderate activity with ICso values of 1 . 19 10-2
Ilmol/ml and 1 .43 10-2 Ilmol/ml, respectively. The results obtained for other
compounds revealed weak cytotoxic activity as indicated by their ICso values;
zerumbone (6.88 10-2 J.1IDol/ml), zeorin (3.76 10-2 Ilmol/ml), lupenone (4.08 10-2 Il
mol/ml), marmin (6.66 10-2 Ilmol/ml) and aegeline (7.58 10-2 J.1IDollml).
IV
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia bagi memenuhi keperluan ijazah Doktor Falsafah
KAJIAN FITOKIMIA DAN UJIAN KEAKTIFAN BIOLOGI DARIPADA MURRAYA PANICULATA JACK,AEGLE MARMELOS CORREA AND
ZINGIBER AMARICANS BLUME
Oleh
SUGENGRIYANTO
Januari 2003
Pengerusi: Profesor Madya Mohd. Aspollah Hj. Sukari, Ph.D.
Fakulti: Sains dan Pengajian Alam Sekitar
Negara-negara di Asia tenggara mempunyai kepelbagaian biologi yang
kaya, ribuan spesies pokok tumbuh-tumbuhan wujud di daerah ini, tetapi hanya
sebahagian kecil spesies dikaji secara mendalam. Kajian ini telah dijalankan untuk
menyelidiki kandungan kimia dan keaktifan biologi daripada tiga tanaman ubatan
yang terkenal iaitu : Murraya paniculata Jack, Aegle marmelos Correa dan
Zingiber amaricans Blume.
Daun dan kulit batang daripada M paniculata; daun, kulit batang dan akar
daripada A. marmelos dan rizom daripada Z. amaricans diekstrak secara
berasingan dengan berbagai pelarut. Ekstrak dan sebatian yang berjaya
dipencilkan diperiksa keaktifan biologinya terhadap sel kanser dan mikrob. Ujian
sitotoksik dilakukan ke atas sel leukaemia T -lymfoblast manusia (CEM-SS)
v
dengan menggunakan reagen MTT, sedangkan untuk mengesan keaktifan
antimikrob dilakukan dengan kaedah peresapan cakera ke atas senarai bakteria
patogen dan fungi. Struktur molekul daripada sebatian yang telah dipencilkan
dikenal pasti dengan kaedah spektroskopi.
Lapan sebatian yang telah berjaya dipencilkan dari M paniculata dikenal
pasti sebagai: auraptene, stigmasterol, �-sitosterol, sukros and £lavon tennetoksi
iaitu 3,3 ' ,4' ,5,5 ' ,6,7 ,8-oktarnetoksi£lavon, 3,3' ,4' ,5,5 ' ,6,7 -hepta-metoksi£lavon,
3,3' ,4' ,5,-5 ' ,7-heksarnetoksi£lavon and 3 ' ,4' ,5,5 ' ,7 -pentarnetoksi-£lavon.
Penyelidikan ke atas A. marmelos memperolehi sebelas sebatian, yakni: �
sitosterol, stigmasterol dan triterpena hop ana iaitu zeorin dan dustanin; alkaloid,
aegeline and skimmianin; epilupeol, lupenon dan terbitan koumarin; auraptene,
epoxyauraptene and marmin. Z. amaricans diselidiki menggunakan kaedah yang
sarna menghasilkan zerumbone sebagai kandungan utarna, bersarna dengan
cholesterol, carnpesterol, stigmasterol and �-sitosterol.
Keputusan ujian antimikrob dan sitotoksik menunjukkan bahawa beberapa
ekstrak sangat berkesan. Namun begitu, kebanyakan sebatian yang telah
dipencilkan didapati mempunyai keaktifan lemah. Manakala keputusan ujian
sitotoksik mendapati bahawa dustanin dan epilupeol menunjukkan kesan
sederhana dengan nilai ICso masing-masing 1 . 19 10-2 JlmoVml dan 1 .43 1 0-2 Jl
moVml,. Keputusan yang diperolehi daripada sebatian lain menunjukkan kesan
sitotoksik lemah dengan nilai ICsQ, yaitu: zerumbon (6.88 10-2 JlmoVml), zeorin
VI
(3.76 10-2 /lmol/ml), lupenon (4.08 lO-2 /lmol/ml), marmin (6.66 110-2 /lmol/ml)
dan aegelin (7.58 lO-2/lmol/ml).
Vll
ACKNOWLEDGEMENTS
This work was carried out with a hope to contribute towards the expansion
of our currently limited knowledge on compounds isolated from medicinal plants
and its bioactivities. The completion of this thesis would have been impossible
without the assistance and direct involvement of so many kindhearted individual.
Thus, I am very much indebted to my previous mentors and I have no way of
repaying such a debt except to express my sincerest gratitude.
First and foremost, I am very grateful to my adviser Associate Professor
Mohd. Aspollah HJ.Sukari for his strong support, guidance, patience and for the
very enriching and thought provoking discussions and lectures which helped to
shape the thesis. He was always there to provide everything I needed in the
laboratory. I would also like to thank him for providing financial support during
the period of study through the IRP A research fund.
I am also grateful to Professor Mawardi Rahmani and Professor
Abdul Manaf Ali in their capacities as members of the Supervisory Committee.
Thank you for the comments and suggestion which contributed a lot towards the
improvement of the final manuscript. I am also indebted to the staff of the
Department of Chemistry Universiti Putra Malaysia for their help and
cooperation. My thanks are extended to Professor Norio Aimi and Associate
Professor Moriko Kitajima of Chiba University Japan and Professor Waterman
from Australia for their collaboration in spectroscopic analysis.
Vlll
Acknowledgment is also made for the thesis grant given by the SEAMEO
SEARCA of Los Banos, Philippines which has helped in certain ways to
accomplish my thesis.
In addition, I would like to express my appreciation to the Dean of Faculty
of Pharmacy and Rector of Gadjah Mada University for study leave which enable
me to pursue Ph.D. degree and gave me aid when I faced financial problem in
Malaysia. My thanks also go to my colleagues in the Department of
Pharmaceutical Chemistry for taking over my job since I started this study. I wish
to express thank to Prof. Dr. Mulyadi, Director of " Pasca Sarjana" UGM and Drs.
Edy Pramono, President Director ofPT Indofarma for the financial support.
IX
I certifY that an Exmnination Committee met on 2nd January 2003 to conduct the final examination of Sugeng Riyanto on his Doctor of Philosophy thesis entitled "Phytochemical Studies and Bioactivity Tests of Murraya paniculata Jack, Aegle marmelos Correa and Zing/her amarlcans Blume" in accordance with Universiti Pertanian Malaysia (Higher Degree) Act 1980 and Universiti Pertanian Malaysia Higher Degree) Regulations 1981. The oommittee recommends that the candidate be awarded the relevant degree. Members of the Examination Committee are as follows:
FAUJAN W. AHMAD, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Chairman)
MOIID. ASPOLLAH W. SUKARI, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia (Member)
MAWARDI RAHMANI, Ph.D. 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)
IKRAM M. SAID, Ph.D. Professor Faculty of Science and Tecbnology Universiti Kebangsaan Malaysia (Independent Examiner)
....oIlA.ll'Aa3.1lUllR MOBAMAD RAMADILI, Ph.D. Professor I Deputy Dean School of Graduate Studies Universiti Putra Malaysia
Date: a 7 JAN 2003
x
This thesis submitted to the Senate ofUniversiti Putra Malaysia has been accepted as fulfilment of the requirements to the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follows:
MORD. ASPOLLAH RJ. SUKARI, Ph.D. Associate Professor Faculty of Science and Environmental Studies Universiti Putra Malaysia. (Chairman)
MAW ARDI RAHMANI, Ph.D. 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)
AINI IDERIS, Ph.D. Professor / Dean School of Graduate Studies Universiti Putra Malaysia
Date : 1 3 MAR 2003
Xl
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 other degree at UPM or other institutions.
SUGENG RlYANTO Date: £.� - \ - 2Do�
xii
TABLE OF CONTENTS
page DEDICATION.... . . . . . . . . . . . . . ...... ........ ............. ........... ............ ..... U ABSTRACT ............. ........ .. .. ........ . . ........... . ... . . ....... .. . ..... . .. . '" iii ABSTRAK. . . . . . . .. ................. . ................. . . . .. . ........ . . . ........... ... v ACKNOWLEDGEMENTS. . . . . . . . . . ... .... ... . . . . . . . . ...... . . . . . . . .. .. . . . . . . . . . .. VUI APPROVAL SHEETS . . .. .. . .......... . . . . . ... .. .... . ... .. . . . . . . . . . . . . . . . . . .. . . . . . x DECLARATION FORM. . . . . . . . . . . . . . . .. . . . ...... . . . . . . . . . .... ... . . . . . . . . .. . ... . . . xu TABLE OF CONTENTS....... . . . . . . . ........ . . . . . . . . . ...... . . . . . . . . . . .. . .. . . . . ... XUI LIST OF TABLES. . . . . . . ... ......... . . . . . . .... . . ... .. .. .. . . .. . . . . . . . . . ...... . . . . . . . . XVI LIST OF FIGURES . . . . . . . . .. . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . .. . . . . ... . xvu LIST OF ABBREVIATIONS.... .... . . . . . . . . . .. . . . . .. . . . . . . . . . . . .... . . . . . . . . . . . . . . XXI
CHAPTER
I INTRODUCTION.. ..... . ........ . . . . . . . .. ...... . . . . . . . . . . . . . . . . . . . . .... ... . . . 1 History. . . .. ....................... ........ .......... . . .............. ... . ..... .... 1 Traditional Medicinal Plants.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2 The Modem Medicine Developed from Traditional Medicinal Plants. 3 Natural Products Research in Malaysia and Indonesia . . . . . . . . . . . . . . . . . . . 5 The Selected Medicinal Plants. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 7 The Objectives of the Research. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1
II LITERATURE REVIEW . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . '" 12 Murraya paniculata Jack........ . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . .. . . .. 12
Plants Description and Traditional Use.. . . . . . . . .. .. . .. .. . .. . . ... .... 12 Previous Work on M paniculata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3
Aegle marmelos Correa.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 30 Plants Discription and Traditional Use......... . . ...... . . . . . . . .. .... . 30 Previous Works on A. marmelos. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 32
Zingiber amaricans Bl. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 39 Plant Description and Traditional Uses. . . .. ...... . . . . . . . . . . . . . . . . . . . 39 Previous Works on Zingiber zerumbet. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 40
Biological Activity Assay. . . . . . . . . . . .. . ... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Antimicrobial Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 43 Cytotoxic Activity Assay.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 44
III EXPERIMENTAL. . . ........ . . . . . ..... . . . . ...... . . . . . . . .. . . . . . . . .. . ..... . . .... 46 Extraction, Isolation and Spectrocopic Analysis.. .. . . . . .... . . . . .... . . . . . . . 46
General Instrumentation.. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 46 Chromatographic Methods. . . . . . . . . . . . . . . . . .. . ... . ... . . . ... . . . . . . ... . . 47 Plant Material. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 48 Extraction and Fractionation of Extracts of Stem Bark of M paniculata from Ipoh, Malaysia. . . . . ... .. . . . . . . . . . . . . . . . . . . . . . . . . . 49 Isolation and Spectroscopic Analysis of Compounds from Petroleum Ether Extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 49
Xlll
Isolation and Spectroscopic Analysis of Compounds from Chloroform Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Isolation and Spectroscopic Analysis of Compounds from Methanol Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extraction and Fractionation of Extracts of Leaves of M. paniculata from Y ogyakarta, Indonesia . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Petroleum Ether Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " . .... .
Isolation and Spectroscopic Analysis of Compounds from Chloroform Extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Methanol Extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . " . .. . ... " . ............ .
Extraction and Fractionation of Extracts of Leaves of A. marmelos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Petroleum Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Chloroform Extract . . " ............................. . ... . ........... ... .
Isolation and Spectroscopic Analysis of Compounds from Methanol Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extraction and Fractionation of Extracts of Bark of A. marmelos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Petroleum Extract . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Chloroform Extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Methanol Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extraction and fractionation of extracts of roots of A. marmelos ......... . ...................................................... .
Isolation and Spectroscopic Analysis of Compounds from Petroleum Ether Extract. . . , . " ....... " ............................... .
Isolation and spectroscopic analysis of compounds from chloroform Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Methanol Extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Extraction and Fractionation of Extracts of Rhizomes of Z. amaricans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Hexane Extract. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Dichloromethane Extract . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Isolation and Spectroscopic Analysis of Compounds from Methanol and Acetone Extract. . . . . . . . . . . . . . . . . . . . . . . " ............. .
Biological Activity Tests . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Antimicrobial Activity Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Microorganism . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Preparation of Nutrient and Potato Dextrose Broth Culture . . . . . .
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62
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72 73 73 74 74
XIV
Disc Diffusion Method. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Cytotoxic Activity Test. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 75 Culture of Cells. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76 Cytotoxic Assay. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 76
N RESULTS AND DISCUSSION . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 78 Structure elucidation of isolated compounds from M paniculata.. . ... 78
3, 3' , 4',5 ' ,5,6,7,8-0ctamethoxyflavone (114)..................... 79 3, 3' ,4', 5 ' , 5, 6,7-Heptamethoxyflavone (3)........................ 85 Stigmasterol (115) and �-Sitosterol (116)........................... 95 3,3 ',4',5,5 ',7-Hexamethoxyflavone (45) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 101 Sucrose (117) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1 Auraptene (7-Geranyloxycoumarin)(42). . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 16 3 ',4',5,5',7-Pentamethoxyflavone (19).............................. 123 Comparison of Chemical Constituents of M. paniculata from Various Places . . . . . . . . . . . . . . . . . . . . '" . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 128
Structure elucidation of isolated compounds from A. mannelos. . . . .... 130 6a,22-Dihydroxyhopane (lIS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 3 1
1 5a,22-Dihydroxyhopane (119)......... . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 143 The Isolation ofHopane Triterpenes fromA. mannelos. . ... . . . . . 1 55 Aegeline (SS). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 157 20(29)-Lupene-3a-ol (S4). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 167 20(29)-Lupene-3-on (120)............................................. 1 74 Martnin (79)............................... .... . .... ....... . . ....... . . . . .. 1 79 6', T-Epoxyauraptene (121)............................................ 1 89 Skimmianine (SO). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 Comparison of Chemical Constituents of A. mannelos from Various Places . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 205
Structure Elucidation ofIsolated Compounds from Rhizomes of Z. amaricans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 207
2,6,9-Humulantrien-8-one (105). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 208 Phytosterol Mixtures. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 216
Biological Activities of Extracts and Isolated compounds. . . . . . . . . . . . . . . 220
V CONCLUSION. . . .. . ..... .. . . . . . . . . . . . .. . . . . . . . . . . .......... ......... ......... 227
BffiLIOGRAPHY. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 230 APPENDIX. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 240 BIODATA. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 242
xv
LIST OF TABLES
Table
1 . Concentration ofYuehchukene in Murraya species . . . . . . . . . . . . . . . . . .
2. Chemical Content of A. marmelos Fruits . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
3 . Sample Concentration in Well Plate o f Cytotoxic Test . . . . . . . . . . . . . . .
4. The Extracts of Stem Bark and Leaves of M. paniculata . . . . . . . . . . . . .
5 . The Assignment of lH and 13C-NMR Spectrum of 3 ,3 ' ,4' ,5 '5 ,6,7,8 Octamethoxyflavone (1 14) in CDCh . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. The Assignment of IH and 13C-NMR Chemical Shifts in CDCh of 3 , 3 ' ,4' , 5 ' , 5,6,7-Heptamethoxyflavone (3) ........................... .
7 . The Assignment of IH and 13C Chemical Shift in CDCh of 3,3' ,4' ,5,5' ,7-Hexamethoxyflavone (45). . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . .
8 . The Assignment of I H and 13C_NMR Spectrum in CDC13 and COSY Correlation of Auraftene (42) .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
9. Chemical Shifts (ppm) of l C-NMR of 3 ' ,4',5,5 ',7-Pentamethoxy-flavone (19) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 0. Chemical Constituents of M paniculata Collected from Various Places . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 1 . The Extracts of Leaves, Bark and Root of A. marmelos . . . . . . . . . . . . . .
12 . The Assignment ofNMR of 6a,22-Dihydroxy hopane (1 19) . . . . . . . .
1 3 . The Assignment ofNMR of 1 5a,22-Dihydroxyhopane (120) . . . . . . .
1 4. NMR Spectral Data of Aegeline (S5). . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 5 . The Assignment of IH and 13C-NMR of20(29)-lupene-3a-ol
(S4)and 20(29)-lupene-3-on (121) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 6. NMR Spectral Data of Marmin (79) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 7 . NMR Spectral Data of 6 ' ,7' -Epoxyauraptene (122) . . . . . . . . . . . . . . . . .
1 8 . Chemical Shift of IH and 13C_NMR of Skimianine (SO) . . . . . . . . . . . . . .
1 9. Chemical Constituents of A. marmelos Collected from Various Places . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20. The Extracts of Rhizome of Z. amaricans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
2 1 . Compounds Isolated from Z. amaricans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
22. NMR Spectroscopic Data of 2,6,9-Humulatrien-8-one (105) . . . . . . . . 23. Chemical Constituents of Phytosterol Mixtures . . . . . . . . . . . . . . . . . . . . . .
24. Antimicrobial and Cytotoxic Activities of Extracts from Stem Bark of M. paniculata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
25. Antimicrobial and Cytotoxic Activities of Extracts from Leaves of M. paniculata . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
26. Cytotoxic Test Results of Isolated Compounds from A. marmelos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
Page
1 7 36 77 78
82
94
1 1 0
1 22
1 27
129 1 30 142 1 54 1 66
1 73 1 88 1 99 204
206 207 207 2 1 5 2 1 6
220
22 1
224
XVI
LIST OF FIGURES
Figure
1 . Murraya paniculata tree . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . ... . . . . . . . . .
2. The leaves of Muraya paniculata . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
3 . Pro1ipid . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . .
4. Aegle marmelos tree (wild) . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . .
5 . Leaves o f Aegle marmelos . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
6. Zingiber amaricans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ..
7. Rhizome of Zingiber amaricans . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
8. Mass Spectrum of 3,3 ' ,4' ,5 ' ,5 ,6,7,8-0ctamethoxyflavone (1 14) . . .
9. IR Spectrum of3, 3 ' , 4' ,5 ' ,5 ,6,7,8-0ctamethoxyflavone (114) . . . . 1 0. IH-NMR Spectrum of3,3 ' ,4' ,5 ' ,5 ,6,7,8-0ctamethoxyflavone (114) ... . .
1 1 . 13C_NMR Spectrum of 3, 3 ' , 4' ,5 ' ,5 ,6,7,8-0ctamethoxyflavone (1 14) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
12 . Mass Fragmentation of 3, 3 ' , 4' ,5 ' ,5 ,6,7,8-0ctamethoxyflavone (1 14) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
1 3 . Mass Fragmentation Pattern of Flavone . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
14. IR spectrum 3, 3 ' ,4' , 5 ' , 5 , 6,7-Heptamethoxyflavone (3) . . . . . . . . . . 1 5 . Mass Spectrum of 3 , 3 ' ,4' , 5 ' , 5 , 6,7-Heptamethoxyflavone (3) . . . . 1 6. IH-NMR Spectrum of3, 3 ' ,4' , 5 ' , 5 , 6,7-Heptamethoxyflavone
(3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
17 . I 3C-NMR Spectrum of3 , 3 ',4', 5 ' , 5 , 6,7-Heptamethoxyflavone (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . .
1 8 . HMQC Spectrum of3 , 3 ' ,4' , 5 ' , 5 , 6,7-Heptamethoxyflavone (3). 1 9. Expanded HMQC Spectrum of3, 3 ' ,4' , 5 ' , 5 , 6,7-Heptamethoxy-
flavone(3) . . . . . . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
20. HMBC Spectrum of 3, 3 ' ,4', 5 ' , 5, 6,7-Heptamethoxy-flavone (3) 2 1 . Expanded HMBC Spectrum of 3 , 3 ' ,4' , 5 ' , 5 , 6,7-Heptamethoxy-
flavone (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . . . . . . . . . . . . . . . . . ... .
22. Fragmentation Pattern of 3 , 3 ' ,4' , 5 ' , 5, 6,7-Heptamethoxy-flavone (3) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... .
23 . IR Spectrum of Mixture of Stigmasterol (115) and �-Sitostero1 (116) ....................................................................... .
24. Gas-Chromatogram of the Mixture of Substances and Mass Spectrum of Component 1 (Positive Chemical Ionisation) . . . . . . .. .
25. Mass Spectrum of Component 2 (Positive Chemical Ionisation) . . . 26. IH-NMR Spectrum ofthe Mixture of Stigmasterol (1 15) and
�-Sitostero1 (116) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 27. 3C-NMR Spectrum of Mixture of Stigmasterol (1 15) and
�-Sitosterol (116) . . . . . .. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
28. Mass Fragmentation of Stigmasterol (1 15) . . . . . . . . . . . . . . . . . . . . . . . . . . . .
29. Mass Fragmentation of Sitosterol (116) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .
30. IR Spectrum of 3,3 ' ,4' ,5,5 ' ,7-Hexamethoxyflavone (45) . . . . . . . . . ..
3 1 . Mass Spectrum of 3,3 ' ,4',5,5' ,7-Hexamethoxyflavone (45) . . . . . . ..
32. I H-NMR Spectrum of 3,3' ,4' ,5,5' ,7-Hexamethoxyflavone (45) . . .
33. 1 3C-NMR Spectrum of 3,3' ,4' ,5,5 ' ,7-Hexamethoxyflavone (45) . .
Page
8 8 8 9 9
1 0 1 0 79 80 80
8 1
83 84 85 86
87
87 88
89 90
9 1
93
95
96 96
97
98 99
1 00 101 1 02 1 03 1 04
XVll
34. HMQC Spectrum of 3,3 ' ,4' ,S,S' ,7-Hexamethoxyflavone (45) . . . . . l OS 35 . HMBC Spectrum of 3,3 ' ,4' ,S,S' ,7-Hexamethoxyflavone (45) . . . . . 1 06 36. Expanded HMBC Spectrum of 3 ,3 ' ,4' ,5,5 ',7-Hexamethoxy-
flavone (45) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 07 37 . Observed HMBC Correlation Between Protons and Carbons in
3 , 3 ' , 4',S, S' ,7-Hexamethoxyflavone (45) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 08 38 . Mass fragmentation of 3,3 ' ,4',S,S',7-Hexamethoxyflavone (45) .. . 1 09 39. IR Spectrum of Sucrose (1 17) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 1 40. Mass Spectrum of Sucrose (117) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 12 4 1 . IH-NMR Spectrum of Sucrose (1 17) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 13 42. 1 3C_ NMR (DEPT) Spectrum of Sucrose (1 1 7) . . . . . . . . . . . . . . . . . . . . . . . . 1 14 43. Mass Fragmentation Pattern of Sucrose (1 1 7) . . . . . . . . . . . . . . . . . . . . . . . . lIS 44. IR Spectrum of Auraptene (42) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 16 4S. Mass Spectrum of Auraptene (42) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 1 7 46. IH-NMR Spectrum of Auraptene (42) . . . . . . . . . . . . . . . . . . . . . . . . . . . . .. . .. 1 1 8 47. Expanded IH-NMR Spectrum of Auraptene (42) . . . . . . . . . . . . . . . . . . . . . 1 19 48. I H_I H COSY Spectrum of Auraptene (42) . . . . . . . . . . . . . . . . . . . . . . . . . . . . 120 49. 1 3C-NMR Spectrum of Auraptene (42) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 12 1 50 . IR Spectrum of 3 ' ,4',S,S' ,7-Pentamethoxyflavone (19) . . . . . . . . . . . . . 1 23 5 1 . Mass Spectrum of 3 ' ,4' ,5 ,5 ' ,7-Pentamethoxyflavone (19) . . . . . . . . . 1 24 52. IH-NMR Spectrum of 3 ',4',5,5 ',7-Pentamethoxyflavone (19) . . . . . 125 53 . 1 3C-NMR Spectrum of 3 ' ,4',5,5 ' ,7-Pentamethoxyflavone (19) . . . . 1 26 54. Mass Fragmentation of 3 ' ,4',S,5 ' ,7-Pentamethoxyflavone (19) . . . 1 27 5S. IR Spectrum of 6a,22-Dihydroxyhopane (1 18) . . . . . . . . . . . . . . . . . . . . . 1 3 1 56. Mass Spectrum of 6a,22-Dihydroxyhopane (1 18) . . . . . . . . . . . . . . . . . . . 1 32 57 . IH-NMR Spectrum of 6a,22-Dihydroxyhopane (1 18) . . . . . . . . . . . . . . . 1 33 58 . IH_IH COSY Spectrum of 6a,22-Dihydroxyhopane (118) . . . . . . . . . 1 34 59. IH_IH NOESY Spectrum of 6a,22-Dihydroxyhopane (1 18) . . . . . . . 1 35 60. 13C-NMR Spectrum (DEPT) of 6a,22-Dihydroxyhopane (1 18) . . . 1 36 6 1 . HMQC Spectrum of 6a,22-Dihydroxyhopane (1 18) . . . . . . . . . . . . . . . . 1 38 62. HMBC Spectrum of 6a,22-Dihydroxyhopane (1 18) . . . . . . . . . . . . . . . . 1 39 63 . Biosynthesis ofLupane and Hopane Derivatives . . . . . . . . . . . . . . . . . . . . . 141 64. Mass Fragmentation of 6a, 22-Dihydroxyhopne (1 18) . . . . . . . . . . . . 143 65. IR Spectrum of 1 5a,22-Dihydroxyhopane (1 19) . . . . . . . . . . . . . . . . . . . . . 144 66. Mass Spectrum of 1 5a,22-Dihydroxyhopane (1 19) . . . . . . . . . . . . . . . . . . 145 67. I H-NMR Spectrum of ISa,22-Dihydroxyhopane (119) . . . . . . . . . . . . . 146 68. IH_IH COSY Spectrum of 1 5a,22-Dihydroxyhopane (1 19) . . . . . . . . 147 69. NOESY Spectrum of 1 5a,22-Dihydroxyhopane (119) . . . . . . . . . . . . . . 149 70. 1 3C-NMR Spectrum (DEPT) of 1 5a,22-Dihydroxyhopane (119) . . 1 50 7 1 . HMQC Spectrum of 1 5a,22-Dihydroxyhopane (1 19) . . . . . . . . . . . . . . . lSI 72. HMBC Spectrum of 1 5a,22-Dihydroxyhopane (1 19) . . . . . . . . . . . . . . . 1 53 73 . Mass Fragmentation of ISa, 22-Dihydroxyhopane ( 119) . . . . . . . . . . 1 56 74. IR Spectrum of Aegeline (85) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 57 75 . Mass Spectrum of Aegeline (85) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 58 76. IH-NMR Spectrum of Aegeline (85)(Expanded) . . . . . . . . . . . . . . . . . . . . 1 59 77. I H_I H COSY Spectrum of Aegeline (85) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 60 78. 13C-NMR Spectrum of Aegeline (85) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 6 1
XV111
79. HMQC Spectrum of Aegeline (85) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 63 80. HMBC Spectrum of Aegeline (85) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 64 8 1 . Observed HMBC Correlation of Aegeline (85) . . . . . . . . . . . . . . . . . . . . . 1 64 82. Mass Fragmentation of Aegeline (85) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .... 1 65 83 . IR Spectrum of 20(29)-Lupene-3a-ol (84) . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 67 84. Mass Spectrum of 20(29)-Lupene-3a-ol (84) . . . . . . . . . . . . . . . . . . . . . . . 1 68 85. IH-NMR Spectrum of 20(29)-Lupene-3a-ol (84) . . . . . . . . . . . . . . . . . . . 1 69 86. The Expanded I H-NMR Spectrum of 20(29)-Lupene-3a-ol (84) . 1 70 87. I 3C-NMR Spectrum of20(29)-Lupene-3a-ol (84) . . . . . . . . . . . . . . . . . . 1 7 1 8 8 . Mass Fragmentation of 20(29)-Lupene-3a-ol (84) . . . . . . . . . . . . . . . . . . 1 72 89. IR Spectrum of20(29)-Lupene-3-on (120) .. . . . . . . . . . . . . . . . . . . . . .. . . 1 74 90. Mass Spectrum of 20(29)-Lupene-3-on (120) ... . . . . . . . . . . . . . . . . . . . . . 1 75 9 1 . IH-NMR Spectrum of20(29)-Lupene-3-on (120) .... . . . . . . . . . . . . . . .. 1 75 92. I 3C NMR and DEPT Spectrum of 20(29)-Lupene-3-on (120) .. . . . 1 77 93 . Mass Fragmentation of 20(29)-Lupene-3-on (120) . . . . . . . . . . . . . . . .. 1 78 94. IR Spectrum of Marmin (79) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . ... 1 79 95. Mass Spectrum ofMarmin (79) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 80 96. Expanded I H-NMR Spectrum ofMarmin (79) . . . . . . . . . . . . . . . . . . . . . . 1 8 1 97. I H_IH COSY Spectrum ofMarmin (79) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 82 98. 1 3C_NMR Spectrum ofMarmin (79) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 83 99. HMQC Spectrum of Marmin (79) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1 84
1 00. HMBC Spectrum of Marmin (79) . . . . . . . . . . . .. . . . . . . .. . . . . . . . . . . . . . . . . 1 85 1 0 1 . Mass Fragmentation Pattern of Marmin (79) . . . . . . . . . . . . . . . . . . . . . . . . 1 87 1 02. IR Spectrum of 6',T-Epoxyauraptene (121) . . . . . . . . . . . . . . . . . . . . . . . .. 1 89 1 03 . Mass Spectrum of 6' ,T-Epoxyauraptene (121) . . . . . . . . . . . . . . . . . . . . . . 1 90 1 04. I H-NMR Spectrum of 6' ,7'-Epoxyauraptene (121) . . . . . . . . . . . . . . . . . . 1 9 1 1 05 . IH_IH COSY Spectrum of 6',T-Epoxyauraptene (121) . . . . . . . . . . . . 1 92 1 06. I H_IH NOESY Spectrum of 6' ,T-Epoxyauraptene (121) . . . . . . . . . . . 1 93 1 07. I 3C-NMR Spectrum of 6' ,T-Epoxyauraptene (121) . . . . . . . . . . . . . . . .. 1 94 1 08. DEPT Spectrum of 6' ,T-Epoxyauraptene (121) . . . . . . . . . . . . . . . . . . . . . 1 94 1 09. HMQC Spectrum of 6',T-Epoxyauraptene (121) . . . . . . . . . . . . . . . . . . . . 1 96 1 1 0. HMBC Spectrum of 6',T-Epoxyauraptene (121) . . .. . . . . . . . . . . . . . . . . . 1 97 1 1 1 . IR Spectrum of Skimmianine (80) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 200 1 1 2. Mass Spectrum of Skimmianine (80) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 201 1 1 3 . IH-NMR Spectrum of Skimmianine (80) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 202 1 14. I 3C-NMR Spectrum of skimmianine (80) . . . . . . . . . . . . . . . . . . . . . . . . . . . 203 1 1 5 . Mass Fragmentation of Skimmianine (80) . . . . . . . . . . . . . . . . . . . . . . . . . . . 204 1 1 6. IR Spectrum of2,6,9-Humulatrien-9-one (l05) . . . . . . . . . . . . . . . . . . . . . 208 1 1 7. Mass Spectrum of 2,6,9-Humulatrien-9-one (105) . . . . . . . . . . . . . . . 209 1 1 8 . IH-NMR Spectrum of2,6,9-Humulatrien-9-one (105) . . . . . . . . . . . . . 2 1 0 1 19. I 3C-NMR (DEPT) Spectrum of 2,6,9-Humulatrien-9-one (105) 2 1 1 1 20. HMQC Spectrum of2,6,9-Humulatrien-9-one (105) . . . . . . . . . . . . . . . 2 1 3 1 2 1 . HMBC Spectrum of 2,6,9-Humulatrien-9-one (105) . . . . . . . . . . . . . . . 2 14 1 22. Mass Fragmentation of 2,6,9-Humulatrien-9-one (105) . . . . . . . . . . . . 2 1 5 1 23 . Gas-Chromatogram of Phytosterol Mixtures . . . . . . . . . . . . . . . . . . . . . . . . 2 16 1 24. Mass Spectrum of Peak 1 (Cholesterol) (122) . . . . . . . . . . . . . . . . . . . . . . . . 2 1 7 1 25 . Mass Spectrum of Peak 2 (Campesterol) (123) . . . . . . . . . . . . . . . . . . . . . . . 2 1 8 1 26. Mass Spectrum (EI) of Peak 3 (Stigmasterol) (1 15) . . . . . . . . . . . . . . . .. 2 1 8
XIX
1 27. Mass Spectrum (EI) of Peak 4 (P-Sitosterol) (1 16) . . . . . . . . . . . . . . . . . . . 2 19
xx
LIST OF ABBREVIATIONS
8 chemical shift E molar absorption '" wavelength
"'max maximum wavelength
/-l mIcro }.lmol micro molar AcONa sodium acetate AICl) aluminum chloride °c degree in Celcius CDCl) deuterated chloroform CHCh chloroform cm-I per centi meter COSY Correlated Spectroscopy d doublet dd doublet of doublet ddd doublet of doublet of doublet DMSO dimethyl sulphoxide EDso Effective Dose (50%mortality) EtOAc ethyl acetate FAB Fast Atom Bombardment g gram IH proton HMBC Heteronuc1ear Multiple Bond Connectivity HMQC Heteronuc1ear Multiple Quantum Coherent HPLC High Performance Liquid Chromatography Hz hertz ICso Inhibition Concentration (50%mortality) IR Infrared lU international unit J coupling constant kg kilogram Lit. literature m multiplet M+ molecular ion MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide m.p. melting point MeOH methanol MeO methoxy(l) MeONa sodium methoxide ml mililitre MS (Electron Impact) Mass Spectroscopy rnJz mass per zarah NCI Negative Chemical Ionisation
XXI
n.hexane nm NMR NOESY ppm s t TLC TMS UV
nonnal hexane nanometer Nuclear Magnetic Resonance Nuclear Overhauser and Exchange Spectroscopy parts per million singlet triplet Thin Layer Chromatography Tetramethylsilane Ultraviolet
XXll
CHAPTER I
INTRODUCTION
History
The use of plants as medicines started since the early time. Literature
evidence of man utilizing plants for the treatment of various diseases have been
documented by the great civilization of the ancient China, India and Egypt. The
prominent scholar in ancient Greece, named Theophratus (372-278 BC) has been
described by some as the father of botany who studied over five hundred plants
from the Mediterranean valley and also introduced the virtues of medicinal plants.
At that time the scholars classified and gave the description of the plants, which
aided the identification process. In the 1 9th century man started to isolate the
active substances from medicinal plants and it was marked by the discovery of an
alkaloid, quinine from Cinchona bark. Such discovery led scientists to search and
collect the plants from jungles for sources of medicines from the natural
resources. Some of natural products isolated from plants are still in use to day,
such as quinine from Cinchona bark, morphine and codeine from the Papaver
somniverum, digoxin from Digitalis purpurea leaves, atropine and hyoscine from
Atropa belladonna.
Traditional Medicinal Plants
Tropical rainforest of southeast Asia, particularly in Malaysia stores large
collection of plant species which provide an important source of traditional
medicine. There are over 20,000 species of tropical plants in the country and
about 500 genera containing 1 5,000 species in Peninsula Malaysia, of which 1 6%
are claimed to have medicinal properties (Zakaria et al. , 1 994). The plants have
been used for generations in various traditional medicine practices in Malaysia,
Indonesia and its surrounding areas. Traditional medicine is well known for its
high nutritional value, as well as its ability to cure various ailments. There are vast
similarities between local Malay traditional medicine practices compared to the
Indonesian traditional medicines. Probably a more scientific and modem approach
may earn traditional medicine a more respectable place in the community.
Traditional medicine is part of our national heritage and must be accorded its
proper place. As it has been realized in some countries which introduced and
promoted their traditional medicinal plant such as in Korea with Panax ginseng,
India with Rauwolfia serpentina (Apocynaceae) and China with Rheum palmatum
(Rhamnaceae). Nowadays, generally public has accepted these plants as one of the
sources of modem medicines. Malaysia for example has the resource and capacity
to investigate and promote certain well-known medicinal plants such as Eurycoma
longifolia (Tongkat Ali) (Simarubaceae) and the other promising herbs for
commercial exploitation. In Indonesia, traditional medicine has become a
lucrative commercial industry bringing in large profits to entrepreneurs and
farmers. Some medicine manufacturers treat the traditional medicinal plants as
2
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