UNIVERSITI PUTRA MALAYSIA
CHEMICAL CONSTITUENTS OF ANAXAGOREA JAVANICA AND THEIR ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES
KHAIRANA BINTI HUSAIN
IB 2008 11
CHEMICAL CONSTITUENTS OF ANAXAGOREA JAVANICA AND THEIR ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES
By
KHAIRANA BINTI HUSAIN
Thesis Submitted to the School of Graduate Studies, Universiti Putra Malaysia, in Fulfilment of the Requirements for
the Degree of Doctor of Philosophy
December 2008
Abstract of thesis presented to the Senate of Universiti Putra Malaysia in
fulfillment of the requirements for the degree of Doctor of Philosophy
CHEMICAL CONSTITUENTS OF ANAXAGOREA JAVANICA AND THEIR
ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES
By
KHAIRANA BINTI HUSAIN
December 2008
Chairman : Professor Nordin H. Lajis, PhD Institute : Bioscience Anaxagorea javanica Blume (synonym A. scortechinii King) or locally known as
‘bunga pompun’, ‘kekapur’, ‘larak lecek’, ‘akar angin’, ‘kinchong’ or ‘atis’ is an
endermic plant in Malaysia. The plant belongs to Annonaceae family and is
traditionally used for the treatment after childbirth.
Phytochemical investigation on the bark and root of this plant yielded seventeen
pure compounds, comprising steroids, fatty acids, simple phenolics, alkaloids,
flavonoids and sesquiterpenes. In total, seven alkaloids were isolated, out of
which two new derivatives of copyrine alkaloid were 4,11-
dimethoxyeupolauridine (87) and 2-methoxy-3-hydroxyeupolauridine (88) in
addition to four known compounds, eupolauridine (22), 11-
i
methoxyeupolauridine (86), sampangine (95) and 3-methoxysampangine (96).
Another known alkaloid came from oxoaporphine was lysicamine (91). The
isolation of copyrine and oxoaporphine alkaloids was a first time from the
genus. Other known compounds isolated, included a mixture of β-sitosterol (49)
and stigmasterol (94), quercetin (46), 7α-hydroxystigmasterol (92), 7-
oxostigmasterol (93), 4-hydroxybutanamide (95), tetradecanoic acid (96),
syringic aldehyde (97) and 2,3-dihydroxy-1-(4’-hydroxy-3’,5’-
dimethoxyphenyl)-1-propanone (98). A novel sesquiterpene identified as 2,2,9-
trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (85) (nordine) was
also isolated. All structures were elucidated by spectroscopic techniques and by
comparison with data available from literature.
Studies on the biological activities related to inflammation were conducted
using nitric oxide (NO) and 5-lipoxygenase (5-LOX) inhibitory assay as a
priminary screening as well as antioxidant assay. The results showed that both
hexane and dichloromethane extracts from the barks possessed weak inhibition
on NO production at the highest concentration of 200 µg/ml without cytotoxic
effect. Meanwhile, inhibition of lipoxygenase activity was shown only by
methanolic and acetone extracts from roots at 100 µg/ml with an IC50 of 48.21
and 63.87 µg/ml, respectively. However, all the crude extracts from barks and
roots are poor radical scavenger inhibitors. Further in vitro investigation on the
selected pure compounds showed that only sampangine (89) and 3-
ii
methoxysampangine (90) significantly inhibited NO production with IC50 values
of 18.17 and 32.25 μg/ml, respectively, at the highest concentration of 25 µg/ml
tested.
iii
Abstrak tesis yang dikemukakan kepada Senat Universiti Putra Malaysia
sebagai memenuhi keperluan untuk ijazah Doktor Falsafah
SEBATIAN KIMIA KE ATAS ANAXAGOREA JAVANICA SERTA
AKTIVITI ANTIOKSIDA DAN ANTI-INFLAMASI
Oleh
KHAIRANA BINTI HUSAIN
Disember 2008 Pengerusi : Professor Nordin H. Lajis, PhD Institut : Biosains Anaxagorea javanica Blume (sinonim A. scortechinii King) atau dikenali sebagai
‘bunga pompun’, ‘kekapur’, ‘larak lecek’, ‘akar angin’, ‘kinchong’ atau ‘atis’
merupakan tumbuhan endemik di Malaysia. Tumbuhan ini tergolong di dalam
famili Annonaceae dan kegunaan tradisionalnya untuk merawat perempuan
selepas bersalin.
Penyelidikan fitokimia ke atas bahagian kulit batang dan akar tumbuhan ini
menghasilkan tujuhbelas sebatian tulen, yang mengandungi steroid, asid lemak,
fenolik ringkas, alkaloid, flavonoid dan seskuiterpena. Sejumlah tujuh alkaloid
yang telah dipencilkan, dua daripadanya merupakan terbitan baru daripada
alkaloid kopirina yang dinamakan sebagai 4,11-dimetoksieupolauridina (87)
iv
dan 2-metoksi-3-hidroksieupolauridina (88) bersama-sama dengan tiga sebatian
yang diketahui, eupolauridina (22), 11-metoksieupolauridina (86), sampangina
(95) dan 3-metoksisampangina (96). Sebatian alkaloid lain yang diketahui
daripada alkaloid oxoaporfina dinamakan sebagai lisikamina (91). Sebatian lain
yang diketahui yang telah ditulenkan, termasuklah campuran daripada β-
sitosterol (49) and stigmasterol (94), kuersetin (46), 7α-hidroksistigmasterol (92),
7-oxostigmasterol (93), 4-hidroksibutanamida (95), asid tetradekanoik (96),
aldehid siringik (97) dan 2,3-dihidroksi-1-(4’-hidroksi-3’,5’-dimetoksifenil)-1-
propanon (98). Seskuiterpena novel dikenalpasti sebagai 2,2,9-trimetil-5-
metilena-12-oxa-bisiklo[6.3.1]dodekana-4-9-diol atau dinamakan sebagai
nordina (85) turut juga dipencilkan. Struktur kesemua sebatian dielusidasi
berdasarkan kaedah spektroskopi dan perbandingan dengan data spectrum
daripada literatur.
Kajian penyaringan awal ke atas aktiviti biologi yang berkaitan inflamasi
dilakukan dengan menggunakan asai perencatan nitrik oksida (NO), 5-
lipoksigenase (5-LOX) dan antioksida. Keputusan ujian menunjukkan kedua-
dua ekstrak heksana dan diklorometana daripada kulit batang menunjukkan
aktiviti yang lemah ke atas pengeluaran nitrik oksida pada kepekatan tertinggi
200 μg/ml tanpa kesan ketoksikan. Manakala, aktiviti perencatan lipoksigenase
pada 100 μg/ml menunjukkan hanya ekstrak metanol dan aseton daripada akar
dengan nilai IC50 48.21 dan 63.87 μg/ml, masing-masing. Walau bagaimanapun,
v
semua ekstrak mentah daripada bahagian kulit batang dan akar adalah lemah
terhadap perencatan skavenger radikal. Kajian selanjutnya ke atas anti-inflamasi
ke atas sebatian-sebatian tulen terpilih menunjukkan hanya sampangina (89)
and 3-metoksisampangina (90) secara signifikan merencat pengeluaran NO
pada kepekatan tertinggi 25 μg/ml dengan nilai IC50 18.17 dan 32.25 μg/ml,
masing-masing.
vi
ACKNOWLEDGEMENTS First and foremost, I would like to thank ALLAH (SWT) for allowing me to
successfully finish this task and giving me the opportunity to make the thesis a
reality.
I also wish to express my sincere thanks and deep appreciation to my
supervisor, Prof Dr Nordin Lajis, for his scientific guidance, encouragement,
constructive comments and advice throught out these years.
I am also very grateful to members of my supervisory committee, Assoc. Prof.
Dr Khozirah Shaari, who has always been helpful on NMR problem; Prof. Dr
Daud Ahmad Israf Ali for his guidance in the performance of in vitro nitric
oxide assay and Dr Intan Safinar Ismail for her discussion and professional
assistance in my research.
I would like to extend my heartfelt gratitude to Mr Samsul Khamis (for plant
collection and identification), Mr Salahudin and Mrs Mazina of 500 MHz NMR
and Mass Spectroscopy, respectively; thank you so much for their most helpful
services. To all my labmates in Laboratory of Natural Products, Instiute
Biosciences, UPM, especially Uwik, Pei Jin, Faridah, Reena, Ayu and Kak
vii
Salmah, thanks for our friendship and helpful. Special thanks to Ayie, Ka Heng
and also Uwik who helped with some bioassays, thank you so much.
I wish to thank Assoc. Prof. Dr Jalifah Latip (Faculty of Sciences and
Technology, UKM, Bangi) for running 400 MHz-NMR sample in UKM, as well
as Dr Chistian Paetz (University of Kagawa, Japan) for their helpful services of
700 and 600 MHz-NMR, and High Resolution Mass Spectroscopy.
I am indebted to Skim Latihan Akademik Bumiputra (SLAB) and Universiti
Kebangsaan Malaysia for the Ph.D sponsorship; Prof. Dr Ibrahim Jantan (Dean
of Faculty of Pharmacy), and to all my officemates in Faculty of Pharmacy,
UKM, especially to Assoc. Prof. Dr Jamia Azdina Jamal and Dr Juriyati Jalil for
their scientific guidance, support, friendship and for assistance during my study
leave.
Finally, but never the least, I wish to express my sincere thanks and gratitude to
my husband, Khairunnizam Sulaiman; my son Alif Ikhwan and my two
daughters (Amirah and Athirah) for their love, care, encouragement, sacrifice
and their support. To all my sisters, brother and younger brother, thank you for
your moral support. And lastly to my mother, Hajjah Aaduyah Mat Yatim, and
also to my late father, Hj Husain Salleh who have always prayed for my success.
Thank you and I loved you all so much.
viii
I certify that a Thesis Examination Committee has met on 24th December 2008 to conduct the final examination of Khairana binti Husain on her thesis entitled “Chemical Constituents of Anaxagorea javanica and Their Antioxidant and Anti-inflammatory Activities” in accordance with the Universities and University Colleges Act 1971 and the Constitution of the Universiti Putra Malaysia [P.U.(A) 106] 15 March 1998. The Committee recommends that the student be awarded the Doctor of Philosophy. Members of the Examination Committee are as follows: Mohd Aspollah Sukari , PhD Professor Faculty of Science Universiti Putra Malaysia (Chairman) Mawardi Rahmani, PhD Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Faujan Ahmad, PhD Associate Professor Faculty of Science Universiti Putra Malaysia (Internal Examiner) Chan Kit Lam, PhD Professor Faculty of Pharmacy Universiti Sains Malaysia (External Examiner) __________________________ BUJANG KIM HUAT, PhD Professor and Deputy Dean School of Graduate Studies Universiti Putra Malaysia Date:
ix
This thesis submitted to the Senate of Universiti Putra Malaysia and has been accepted as fulfillment of the requirement for the degree of Doctor of Philosophy. The members of the Supervisory Committee are as follow: Md. Nordin Hj. Lajis, PhD Professor Institute of Bioscience Universiti Putra Malaysia (Chairman) Khozirah Shaari, PhD Associate Professor Institute of Bioscience Universiti Putra Malaysia (Member) Intan Safinar Ismail, PhD Lecturer Institute of Bioscience Universiti Putra Malaysia (Member) Daud Ahmad Israf Ali, PhD Professor Institute of Bioscience Universiti Putra Malaysia (Member)
_________________________________ HASANAH MOHD. GHAZALI, PhD Professor and Dean
School of Graduate Studies Universiti Putra Malaysia
Date:
x
xi
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. ___________________________________ KHAIRANA BINTI HUSAIN Date:
TABLES OF CONTENTS Page
ABSTRACT i ABSTRAK iv ACKNOWLEDGEMENTS vii APPROVAL ix DECLARATION xi TABLE OF CONTENTS xii LIST OF TABLES xvi LIST OF FIGURES xviii LIST OF SCHEMES xxvi LIST OF ABBREVIATIONS xxviii CHAPTER
I INTRODUCTION
1
1.1 General Introduction 1 1.2 Objectives of Research 4
II LITERATURE REVIEW
6
2.1 Annonaceae Family 6 2.2 Medicinal Properties and Economic Value of the
Annonaceae Species 9
2.3 The Annonaceae of Malaysia 11 2.4 Chemotaxonomic and Biological Studies of the
Annonaceae 14
2.5 Genus Anaxagorea 21 2.6 Anaxagorea javanica Blume 24 2.7 Phytochemical Review of Anaxagorea Genus 27 2.8 Biological Activities Related to Inflammation 33 2.8.1 Inflammation 33 2.8.2 Nitric Oxide (NO) Inhibition 33 2.8.3 5-Lipoxygenase (5-LOX) Inhibitory
Activity 35
2.8.4 Antioxidant 36 2.8.5 Radical-Scavenging of Stable DPPH
Radical
37
xii
2.8.6 A Review of Previous Anti-Inflammatory and Antioxidant Works on Anaxagorea
37
III RESEARCH EXPERIMENTAL
39
(A) PHYTOCHEMICAL ANALYSIS
39
3.1 General Instrumentation 39 3.2 Chromatographic Methods 40 3.3 Use of Solvents 43 3.4 Plants Materials 43 3.5 Extraction and Fractionation of Crude Extracts of
Anaxagorea javanica 43
3.6 Isolation of Compounds from Barks of Anaxagorea javanica
45
3.6.1 Pure Compounds from the Dichloromethane Fraction (Barks)
47
3.6.2 Pure Compounds from the Ethyl Acetate Fraction (Barks)
52
3.6.3 Pure Compounds from the Acetone Fraction (Barks)
55
3.7 Isolation of Compounds from Roots 57 3.7.1 Pure Compounds from the
Dichloromethane Fraction (Roots) 57
3.7.2 Pure Compounds from the Ethyl Acetate Fraction (Roots)
59
3.7.3 Pure Compounds from the Acetone Fraction (Barks)
59
3.8 Chemical Experimental Results 62 3.8.1 2,2,9-Trimethyl-5-methylene-12-oxa-
bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
62
3.8.2 Eupolauridine (AJB 4, 22) 63 3.8.3 11-Methoxyeupolauridine (AJB 6x, 86) 63 3.8.4 4,11-Dimethoxyeupolauridine
(AJB 6y, 87) 64
3.8.5 2-Methoxy-3-hydroxyeupolauridine (AJB 24, 88)
65
3.8.6 Sampangine (AJB 2, 89) 66 3.8.7 3-Methoxysampangine (AJB 3, 90) 67 3.8.8 Lysicamine (AJB 14, 91) 68 3.8.9 Quercetin (AJB 22b, 46) 69 3.8.10 7α-Hydroxystigmasterol (AJB 23, 92) 70
xiii
3.8.11 7-Oxostigmasterol (AJB 27, 93) 71 3.8.12 Mixture of sterols [AJB 1, β-Sitosterol (49)
and stigmasterol (94)] 72
3.8.13 4-Hydroxylbutanamide (AJB 26, 95) 73 3.8.14 Tetradecanoic acid (AJB 5, 96) 74 3.8.15 Syringic aldehyde (AJB 7, 97) 74 3.8.16 2,3-dihydroxy-1-(4’-hydroxy-3’,5’-
dimethoxyphenyl)-1-propanone (AJB 17, 98)
75
(B) BIOASSAY PROCEDURES OF THE PLANTS
AND PURE COMPOUNDS
76
3.9 Anti-inflammatory Activities 76 3.9.1 Nitric Oxide Inhibition Activity (Griess
Assay) 76
Cell Culture 76 Measurement of Nitrite 77 Cell Viabillity (MTT Assay) 77 3.9.2 5-Lipoxygenase (5-LOX) Inhibitory
Activity 78
3.10 Antioxidant Activity 79 1,1-Diphenyl-2-picrylhydrazyl (DPPH) Free Radical
Scavenging Activity 79
3.11 Plant Extract and Pure Compounds 79 3.12 Statistical Analysis 79
IV DISCUSSION ON THE CHEMICAL ASPECT 80 4.1 General Overview of the Types of Compounds
Isolated 80
4.1.1 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
80
4.1.2 Eupolauridine (AJB 4, 22) 94 4.1.3 11-Methoxyeupolauridine (AJB 6x, 86) 106 4.1.4 4,11-Dimethoxyeupolauridine
(AJB 6y, 87) 117
4.1.5 2-Methoxy-3-hydroxyeupolauridine (AJB 24, 88)
128
4.1.6 Sampangine (AJB 2, 89) 139 4.1.7 3-Methoxysampangine (AJB 3, 90) 150 4.1.8 Lysicamine (AJB 14, 91) 160 4.1.9 Quercetin (AJB 22b, 46) 170
xiv
xv
4.1.10 7α-Hydroxystigmasterol (AJB 23, 92) 180 4.1.11 7-Oxostigmasterol (AJB 27, 93) 191 4.1.12 Mixture of sterols [β-Sitosterol (49) and
stigmasterol (94), AJB 1] 202
4.1.13 4-Hydroxybutanamide (AJB 26, 95) 207 4.1.14 Tetradecanoic acid (AJB 5, 96) 215 4.1.15 Syringic aldehyde (AJB 7, 97) 221 4.1.16 2,3-dihydroxy-1-(4-hydroxy-3,5-
dimethoxyphenyl)-1-propanone (AJB 17, 98)
229
V RESULTS AND DISCUSSION ON THE BIOLOGICAL
ASPECT
237
5.1 Anti-inflammatory Activity 237 5.1.1 Nitric Oxide Inhibition Activity on Crude
Extracts 237
5.1.2 Nitric Oxide Inhibition Activity on Selected of Pure Compounds
239
5.1.3 5-LOX Inhibition Activity on Crude Extracts
242
5.2 Antioxidant Activity 243 5.2.1 DPPH Free Radical-scavenging Activity
on Crude Extracts 243
VI CONCLUSION
245
BIBLIOGRAPHY
248
BIODATA OF THE STUDENT 254
LIST OF TABLES Table Page 1.1 Some Malaysian plants yielding clinically useful modern
drugs
3
2.1 The list of genera and approximate number of species (indicated in bracket) in the Annonaceae
7
2.2 List of genera and species in the family Annonaceae in Malaysia
12
2.3 List of selected alkaloidal compounds from Annonaceae species
14
2.4 List of selected non-alkaloidal compounds from Annonaceae species
18
2.5 List of Annonaceae species worldwide
23
4.1 13C, 1H, HSQC and HMBC NMR data for AJB 13 (CDCl3)
86
4.2 13C, 1H, HSQC and HMBC NMR data for AJB 4 (CDCl3)
99
4.3 13C, 1H, HMQC and HMBC NMR data for AJB 6x (CDCl3)
109
4.4 13C, 1H, HMQC and HMBC NMR data for AJB 6y (CDCl3)
121
4.5 13C, 1H, HMQC and HMBC NMR data for AJB 24 (CDCl3 + CD3OD)
132
4.6 13C, 1H, HMQC and HMBC NMR data for AJB 2 (CD3OD)
143
4.7 13C, 1H, HMQC and HMBC NMR data for AJB 3 (CDCl3)
154
4.8 13C, 1H, HSQC and HMBC NMR data for AJB 14 (CDCl3)
164
4.9 The assignment of protons and carbons of AJB 22b (CD3OD) and comparison with literature data
174
4.10 Assignment of 7α-hydroxystigmasterol (AJB 23) in CDCl3 and comparison with literature data of stigmasterol
185
xvi
xvii
4.11 Assignment of 7-oxostigmasterol (AJB 27) in CDCl3
195
4.12 1H-NMR data for a mixture of β-sitosterol and stigmasterol (AJB 1) in CDCl3 and comparison with literature data
204
4.13 13C, 1H, HSQC and HMBC NMR data for AJB 26 (CDCl3)
209
4.14 1H and 13C-NMR data of tetradecanoid acid (AJB 5) in CDCl3
217
4.15 13C, 1H, HSQC and HMBC NMR data for AJB 7 (CDCl3)
224
4.16 13C, 1H, HSQC and HMBC NMR data for AJB 17 (CD3OD)
232
5.1 Percent inhibition of NO-released from RAW 264.7 murine macrophage stimulated with E. coli LPS (10 µg/ml) and IFN-γ (200 U/ml) by crude extracts of A. javanica
238
5.2 Percent inhibition of NO-released from RAW 264.7 murine macrophage stimulated with E. coli LPS (10 µg/ml) and IFN-γ (200 U/ml) by selected of pure compounds of A. javanica
240
5.3 Percent inhibition of lipoxygenase by crude of A. javanica at concentration 100 µg/ml
243
5.4 DPPH free radical scavenging activity of crude extract and standard (Quercetin)
244
LIST OF FIGURES Figure Page 2.1 Anaxagorea javanica Blume
25
2.2 Anaxagorea javanica Blume: Leaves, flowers and leaves
25
2.3 Morphology of Anaxagorea javanica Blume
26
2.4 Biosynthesis of nitric oxide
34
2.5 Conversion of radical DPPH to DPPH molecule from antioxidant
37
3.1 HPLC chromatogram of AJB 6x (86) and AJB 6y (87) with UV detector at 254 nm
53
3.2 HPLC chromatogram of AJB 6x (86) and AJB 6y (87) with UV detector at 365 nm
53
4.1 Chemical structure and numbering systems of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (85)
80
4.2 Selected HMBC, TOCSY and COSY correlations for 2,2,9-trimethyl-5- methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
84
4.3 EIMS spectrum of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
87
4.4 Infra red spectrum of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
87
4.5 1H-NMR spectrum of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
88
4.6 13C-NMR spectrum of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
89
4.7 HSQC spectrum of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
90
xviii
4.8 HMBC spectrum of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
91
4.9 COSY spectrum of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
92
4.10 TOCSY spectrum of 2,2,9-trimethyl-5-methylene-12-oxa-bicyclo[6.3.1]dodecane-4,9-diol (AJB 13, 85)
93
4.11 Chemical structure and numbering systems of eupolauridine (22)
94
4.12 Selected HMBCand COSY correlation for eupolauridine (AJB 4, 22)
98
4.13 EIMS spectrum of eupolauridine (AJB 4, 22)
100
4.14 Infra red spectrum of eupolauridine (AJB 4, 22)
100
4.15 1H-NMR spectrum of eupolauridine (AJB 4, 22)
101
4.16 13C-NMR spectrum of eupolauridine (AJB 4, 22)
102
4.17 HMBC spectrum of eupolauridine (AJB 4, 22)
103
4.18 COSY spectrum of eupolauridine (AJB 4, 22)
104
4.19 HSQC spectrum of eupolauridine (AJB 4, 22)
105
4.20 Chemical structure and numbering systems of 11-methoxyeupolauridine (86)
106
4.21 Selected HMBC and NOESY correlation for 11-methoxyeupolauridine (AJB 6x, 86)
109
4.22 EIMS spectrum of 11-methoxyeupolauridine (AJB 6x, 86)
110
4.23 Infra red spectrum of 11-methoxyeupolauridine (AJB 6x, 86)
110
4.24 1H-NMR spectrum of 11-methoxyeupolauridine (AJB 6x, 86)
111
xix
4.25 13C-NMR spectrum of 11-methoxyeupolauridine (AJB 6x, 86)
112
4.26 HMQC spectrum of 11-methoxyeupolauridine (AJB 6x, 86)
113
4.27 HMBC spectrum of 11-methoxyeupolauridine (AJB 6x, 86)
114
4.28 COSY spectrum of 11-methoxyeupolauridine (AJB 6x, 86)
115
4.29 NOESY spectrum of 11-methoxyeupolauridine (AJB 6x, 86)
116
4.30 Chemical structure and numbering systems of 4,11-dimethoxyeupolauridine (87)
117
4.31 Selected HMBC and NOESY correlation for 4,11-dimethoxyeupolauridine (AJB 6y, 87)
120
4.32 EIMS spectrum of 4,11-dimethoxyeupolauridine (AJB 6y, 87)
122
4.33 Infra red spectrum of 4,11-dimethoxyeupolauridine (AJB 6y, 87)
122
4.34 1H-NMR spectrum of 4,11-dimethoxyeupolauridine (AJB 6y, 87)
123
4.35 13C-NMR spectrum of 4, 11-dimethoxyeupolauridine (AJB 6y, 87)
124
4.36 HMQC spectrum of 4,11-dimethoxyeupolauridine (AJB 6y, 87)
125
4.37 HMBC spectrum of 4,11-dimethoxyeupolauridine (AJB 6y, 87)
126
4.38 NOESY spectrum of 4,11-dimethoxyeupolauridine (AJB 6y, 87)
127
xx
4.39 Chemical structure and numbering systems of 2-methoxy-3-hydroxyeupolauridine (88)
128
4.40 Selected HMBC and COSY correlation for 2-methoxy-3-hydroxyeupolauridine (AJB 24, 88)
131
4.41 EIMS spectrum of 2-methoxy-3- hydroxyeupolauridine (AJB 24, 88)
133
4.42 Infra red spectrum of 2-methoxy-3-hydroxyeupolauridine (AJB 24, 88)
133
4.43 1H-NMR spectrum of 2-methoxy-3-hydroxyeupolauridine (AJB 24, 88)
134
4.44 13C-NMR spectrum of 2-methoxy-3-hydroxyeupolauridine (AJB 24, 88)
135
4.45 HMQC spectrum of 2-methoxy-3-hydroxyeupolauridine (AJB 24, 88)
136
4.46 HMBC spectrum of 2-methoxy-3-hydroxyeupolauridine (AJB 24, 88)
137
4.47 COSY spectrum of 2-methoxy-3-hydroxyeupolauridine (AJB 24, 88)
138
4.48 Chemical structure and numbering systems of sampangine (89)
139
4.49 Selected HMBC and NOESY correlation for sampangine (AJB 2, 89)
142
4.50 EIMS spectrum of sampangine (AJB 2, 89)
144
4.51 Infra red spectrum of sampangine (AJB 2, 89)
144
4.52 1H-NMR spectrum of sampangine (AJB 2, 89)
145
4.53 APT-13C-NMR spectrum of sampangine (AJB 2, 89)
146
4.54 NOESY-NMR spectrum of sampangine (AJB 2, 89)
147
xxi
4.55 HMBC spectrum of sampangine (AJB 2, 89) (expanded I)
148
4.56 HMBC spectrum of sampangine (AJB 2, 89) (expanded II)
149
4.57 Chemical structure and numbering systems of 3-methoxysampangine (90)
150
4.58 Selected HMBC and NOESY correlations for 3-methoxysampangine (AJB 3, 90)
153
4.59 EIMS spectrum of 3-methoxysampangine (AJB 3, 90)
155
4.60 Infra red spectrum of 3-methoxysampangine (AJB 3, 90)
155
4.61 1H-NMR spectrum of 3-methoxysampangine (AJB 3, 90)
156
4.62 APT-13C-NMR spectrum of 3-methoxysampangine (AJB 3, 90)
157
4.63 HMBC spectrum of 3-methoxysampangine (AJB 3, 90)
158
4.64 NOESY-NMR spectrum of 3-methoxysampangine (AJB 3, 90)
159
4.65 Chemical structure and numbering systems of lysicamine (91)
160
4.66 Selected HMBC and COSY correlation for lysicamine (AJB 14, 91)
163
4.67 EIMS spectrum of lysicamine (AJB 14, 91)
165
4.68 Infra red spectrum of lysicamine (AJB 14, 91)
165
4.69 1H-NMR spectrum of lysicamine (AJB 14, 91)
166
4.70 HSQC spectrum of lysicamine (AJB 14, 91)
167
4.71 HMBC spectrum of lysicamine (AJB 14, 91)
168
4.72 COSY spectrum of lysicamine (AJB 14, 91)
169
xxii
4.73 Chemical structure and numbering systems of quercetin (46)
170
4.74 Meta and ortho coupling aromatic protons of quercetin (AJB 22b, 46)
172
4.75 Selected HMBC correlations for quercetin (AJB 22b, 46)
173
4.76 EIMS spectrum of quercetin (AJB 22b, 46)
175
4.77 Infra red spectrum of quercetin (AJB 22b, 46)
175
4.78 1H-NMR spectrum of quercetin (AJB 22b, 46)
176
4.79 13C-NMR spectrum of quercetin (AJB 22b, 46)
177
4.80 HSQC spectrum of quercetin (AJB 22b, 46)
178
4.81 HMBC spectrum of quercetin in CD3OD (AJB 22b, 46)
179
4.82 Chemical structure and numbering systems of 7α-hydroxystigmasterol (92)
180
4.83 Selected HMBC and COSY correlation for of 7α-hydroxystigmasterol (AJB 23, 92)
184
4.84 EIMS spectrum of 7α-hydroxystigmasterol (AJB 23, 92)
186
4.85 Infra red spectrum of 7α-hydroxystigmasterol (AJB 23, 92)
186
4.86 1H-NMR spectrum of 7α-hydroxystigmasterol (AJB 23, 92)
187
4.87 13C-NMR spectrum of 7α-hydroxystigmasterol (AJB 23, 92)
188
4.88 HMBC spectrum of 7α-hydroxystigmasterol (AJB 23, 92)
189
4.89 COSY spectrum of 7α-hydroxystigmasterol (AJB 23, 92)
190
4.90 Chemical structure and numbering systems of 7-oxostigmasterol (93)
191
xxiii