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UNIVERSITI PUTRA MALAYSIA CHEMICAL CONSTITUENTS OF ANAXAGOREA JAVANICA AND THEIR ANTIOXIDANT AND ANTI-INFLAMMATORY ACTIVITIES KHAIRANA BINTI HUSAIN IB 2008 11

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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

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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