Seriphidium Quettense, Vincetoxicum Stocksii and an...
Transcript of Seriphidium Quettense, Vincetoxicum Stocksii and an...
Discovery of Novel Bioactive Secondary Metabolites from
Seriphidium Quettense, Vincetoxicum Stocksii and an
Endophytic Fungus Cryptosporiopsis Sp.
A Dissertation Submitted to
Fulfill the Requirement for the Award of Degree of Doctor of Philosophy in Chemistry
By
Muhammad Imran Tousif (M.Sc., M.Phil.)
Department of Chemistry
The Islamia University of Bahawalpur 63100-Bahawalpur, Pakistan
January 2015
DECLARATION
I Muhammad Imran Tousif, hereby declare that the research work,
entitled, “Discovery of Novel Bioactive Secondary Metabolites from
Seriphidium quettense, Vincetoxicum stocksii and an Endophytic Fungus
Cryptosporiopsis Sp.” was carried out in the Chemistry Department of
The Islamia University of Bahawalpur, Pakistan. Most of the work has
been published by us in peer reviewed journals. I undertake that I have
not submitted the same work to any other institution or university for
the degree of Doctor of Philosophy (Ph.D.) and will not, in future,
submit for obtaining the similar degree to any other university.
Muhammad Imran Tousif January 2015
CERTIFICATE
It is certified that Muhammad Imrn Tousif S/O Muhammad Tousif
has carried out the research work, embodied in this thesis, under my
supervision in the Department of Chemistry, the Islamia University of
Bahawalpur. He has submitted a thesis entitled, “Discovery of Novel
Bioactive Secondary Metabolites from Seriphidium quettense,
Vincetoxicum stocksii and an Endophytic Fungus Cryptosporiopsis Sp.”
to earn Ph.D. degree. I have evaluated the thesis and found appropriate
in its present form. This is also verified that no part of the thesis is
plagiarized (report attached). It is, therefore, forwarded for further
processes please.
Dr. Muhammad Saleem Chairman (Supervisor) Department of Chemistry Associate Professor The Islamia University of Bahawalpur Department of Chemistry Pakistan The Islamia University of Bahawalpur Pakistan
ACKNOWLEDGEMENTS
All praises for Almighty ALLAH, the most merciful and the most
beneficent who showered upon me His blessings throughout my efforts for the
completion of this research work. All respects and praises for the Holy Prophet
MUHAMMAD (PBUH) who enabled us to recognize our creator, showed us the
right path and gave a complete code of life.
It is with a profound sense of gratitude, I would like to express my
sincere thanks to my supervisor and esteemed guide Dr. Muhammad Saleem,
The Islamia University of Bahawalpur, for his personal care, understanding,
and stimulating discussion with humble and inspiring guidance throughout
the entire studies.
I would like to express my sincere thanks to my teachers, Dr. Abdul
Jabbar and Dr. Naheed Riaz for their suggestions and valuable assistance
throughout my research work.
I must pay thanks to Prof. Dr. Faiz-ul-Hassan Nasim, Chairman
Chemistry Department and Prof. Dr. Muhammad Asghar Hashmi, Dean Faculty
of Sciences for their kind support in documentation processes. I also
acknowledge the Director ICCBS, University of Karachi for providing
spectroscopic analysis facilities.
I am very thankful to Prof. Dr. Muhammad Ashraf, Department of
Biochemistry and Biotechnology for performing various bioassays of my pure
compounds.
I also acknowledge the HEC for financial support both in terms of
scholarship and lab support. I also wish to thank the technical staff, non-
teaching and clerical staff of the deparment, for their constant support and
timely help.
How I can say thanks to my hard-working parents who provided
unconditional love and care. I love them so much, and I would not have made
it this far without them. Therefore, I offer my sincere thanks to my Parents and
parents-in-law. Besdies, my siblings also have been source of my inspiration
due to their love and moral support. I would like to thank all my friends and
every single soul that has made a contribution to my life.
The best outcome from previous years is my soul-mate, my wife
Shagufta Parveen. These past several years have not been an easy ride, both
academically and personally. Her constant support made it possible; therefore, I
truly thank Shagufta for sticking by my side, even when I was depressed. I feel
that what we both learned a lot about life and strengthened our commitment
and determination to each other and to live life to the fullest.
I take this opportunity, to express my heartfelt thanks to Jallat, Akram,
Basharat sb, Abdul Ghaffar, Iftikhar, Shahnawaz, Dr. Nusrat, Dr. Bushra, Dr.
Shehla, Dr. Naseem, Rizwana, Dr. Sara, and Dr. Shabir sb for their kind
suggestions and generous help rendered throughout my Ph. D. studies.
I will forever be thankful to my friends Dr. Muhammad Arshad, Khalid
Nazir, Hafiz Asadullah and Hafiz Gulzar who really contributed a lot in my life,
my real friends. I would like also say thanks to Sir Wheed Anwer and all my
school staff for being so kind to me during this period.
Muhammad Imran Tousif
Dedicated to
My Loving parents
and
The Victims of Sad Incident of APS Peshawar
CONTENTS
Titles Page No.
Summary I
CHAPTER 1
INTRODUCTION OF NATURAL PRODUCTS AND THEIR ROLE IN
DRUG DISCOVERY
01
1.1. NATURAL PRODUCTS AND EVOLUTION OF MODERN MEDICINE
SYSTEM
02
1.2. NATURAL PRODUCT RESEARCH ON MEDICINAL PLANTS; PAST,
PRESENT AND FUTURE
05
1.3. ADVANTAGES OF NATURAL PRODUCT RESEARCH 10
1.4. BIOLOGICALLY ACTIVE NATURAL PRODUCTS 11
1.5. SOURCES OF NATURAL PRODUCTS 12
1.5.1. Natural Products from Plant Sources 12
1.5.2. Natural Products Isolated from Microbial Sources 15
1.5.3. Natural Products Isolated from Animal Sources 18
1.5.4. Natural Products Isolated from Marine Sources 19
RESEARCH QUESTION AND AIM OF THE PRESENT STUDY 23
HYPOTHESIS OF THE RESEARCH 24
CHAPTER 2
RESULTS AND DISCUSSION: PREVIOUS RESEARCH ON THE
VINCETOXICUM GENUS AND ISOLATION OF SECONDARY
METABOLITES FROM VINCETOXICUM STOCKSII
25
2.1. THE GENUS VINCETOXICUM 26
2.2. PREVIOUS PHYTOCHEMICAL INVESTIGATION OF THE GENUS VINCETOXICUM
26
2.3. VINCETOXICUM STOCKSII 28
2.3.1. Classification of the Vincetoxicum Stocksii 28
2.4. RESULTS AND DISCUSSION: CHARACTERIZATION OF SECONDARY METABOLITES ISOLATED FROM V. STOCKSII
29
2.4.1. Characterization of Vincetolate (69) 30
2.4.2. Characterization of Vincetoside (70) 32
2.4.3. Characterization of Vincetetrol (71) 35
2.4.4. Characterization of Vincetomine (72) 37
2.4.5. Characterization of Apocynin (73) 39
2.4.6. Characterization of 4-Hydroxy-3-methoxyphenyl-1-(9-
hydroxy-propan-7-one) (74)
40
2.4.7. Characterization of 4-Hydroxy-3-methoxyphenyl-7,8,9,-
propanetriol (75)
42
2.4.8. Characterization of Feruloyl-6'-O--D-glucopyranoside (76) 43
2.4.9. Characterization of 7-Megastigmene-2,3,5,9-tetrol (77) 45
2.4.10. Characterization of 4-Hydroxyphenylacetic Acid (78) 47
2.4.11. Characterization of 4-Hydroxy-3,5-dimethoxybenzoic Acid
(79)
48
2.4.12. Characterization of 4'-Methoxy Kaempferol-3-O--D- glucopyranoside (80)
50
2.4.13. Characterization of 4'-Methoxy Kaempferol-3-O--L-rhamnopyranoside (81)
51
2.4.14. Characterization of Quercetin-3-O--L-rahmnopyranoside (82)
53
2.4.15. Characterization of 4'-Methoxy-quercetin-3-O--L- rehmnopyranoside (83)
54
2.4.16. Characterization of Quercetin-3-O--D-glucopyranoside (84)
and Quercetin-3-O--D-galactopyranoside (85)
55
2.4.17. Characterization of 7-Hydroxy-3',4',5-trimethoxyflavone (86) 56
2.4.18. Characterization of β-sitosterol (87) 58
2.4.19. Characterization of ß-sitosterol-3-O-D-glucopyranoside (88) 59
2.5. BIOLOGICAL STUDIES OF THE EXTRACT OF VINCETOXICUM STOCKSII AND THE COMPOUNDS PURIFIED FROM THE SAME
EXTRACT
61
2.8. EXPERIMENTAL 64
2.8.1. General Experimental Procedures 64
2.8.2. Plant Material 65
2.8.3. Extraction, Isolation and Identification 65
2.8.4. Spectroscopic Data of Isolated Compounds 68
2.8.4.1. Spectroscopic data of vincetolate (69) 68
2.8.4.2. Spectroscopic data of vincetoside (70) 68
2.8.4.3. Spectroscopic data of vincetetrol (71) 69
2.8.4.4. Spectroscopic data of vincetomine (72) 69
2.8.4.5. Spectroscopic data of apocynin (73) 70
2.8.4.6. Spectroscopic data of 4-Hydroxy-3-methoxyphenyl-1-
(9-hydroxy-propan-7-one) (74) 70
2.8.4.7. Spectroscopic data of 4-Hydroxy-3-methoxyphenyl-
7,8,9-propanetriol (75)
71
2.8.4.8. Spectroscopic data of Feruloyl-6'-O--D-glucopyranoside (76)
71
2.8.4.9. Spectroscopic data of 7-Megastigmene-2,3,5,9-tetrol
(77)
72
2.8.4.10. Spectroscopic data of 4-Hydroxyphenylacetic acid
(78)
72
2.8.4.11. Spectroscopic data of 4-hydroxy-3,5-
dimethoxybenzoic acid (79)
73
2.8.4.12. Spectroscopic data of 4'-Methoxy kaempferol-3-O--D-glucopyranoside (80)
73
2.8.4.13. Spectroscopic data of 4'-Methoxy kaempferol-3-O--L-rhamnopyranoside (81)
74
2.8.4.14. Spectroscopic data of quercetin-3-O--L-rahmnopyranoside (82)
74
2.8.4.15. Spectroscopic data of 4'-Methoxy qurcetin-3-O--L-rehmnopyranoside (83)
75
2.8.4.16. Spectroscopic data of quercetin-3-O--D- glucopyranoside (84)
76
2.8.4.17. Spectroscopic data of quercetin-3-O--D-galactopyranoside (85)
76
2.8.4.18. Spectroscopic data of 7-Hydroxy-3',4',5-
trimethoxyflavone (86)
77
2.8.4.19. Spectroscopic data of β-sitosterol (87) 77
2.8.4.20. Spectroscopic data of ß-sitosterol-3-O-D-
glucopyranoside (88) 78
2.8.5. Acid Hydrolysis of Compounds 79
2.8.6. Bioassays 80
2.8.6.1. Determination of antioxidant activity (NO scavenging
method) 80
2.8.6.2. Determination of antiurease (Berthelot method) 80
2.8.6.3. Antibacterial activity 81
CHAPTER 3
THE GENUS SERIPHIDIUM AND LITERATURE REVIEW 82
3.1. THE GENUS SERIPHIDIUM 83
3.2. PHARMACEUTICAL IMPORTANCE OF THE SERIPHIDIUM SP. 83
3.3. PREVIOUS PHYTOCHEMICAL STUDIES OF THE GENUS SERIPHIDIUM
84
CHAPTER 4
STRUCTURE ELUCIDATION OF SECONDARY METABOLITES
ISOLATED FROM SERIPHIDIUM QUETTENSE
90
4.1. SERIPHIDIUM QUETTENSE 91
4.1.1. CLASSIFICATION OF THE SERIPHIDIUM QUETTENSE 91
4.2. RESULTS AND DISCUSSION 91
4.2.1. Characterization of Seriphilidine (136) 92
4.2.2. Characterization of 6-hydroxy-8(10)-oplopen-14-one (137) 95
4.2.3. Characterization of salvigenin (138) 97
4.2.4. Characterization of cirsumaritin (139) 99
4.2.5. Characterization of p-Coumaric Acid (140) 100
4.2.6. Characterization of β-sitosterol (87) 101
4.2.7. Characterization of β-sitosterol-3-O-β-D-glucopyranoside (88) 102
4.2.8. Characterization of oleanolic acid (141) 102
4.3. EXPERIMENTAL 104
4.3.1. General Experimental Procedures 104
4.3.2. Collection and Identification of Plant Material 104
4.3.3. Extraction 105
4.3.4. Isolation and Identification 105
4.3.5. Spectroscopic Data of Purified Compounds 108
4.3.5.1. Spectroscopic data of seriphilidine (136) 108
4.3.5.2. Spectroscopic data of -hydroxy-8(10)-oplopen-14-
one (137)
108
4.3.5.3. Spectroscopic data of salvigenin (138) 109
4.3.5.4. Spectroscopic data of cirsumaritin (139) 110
4.3.5.5. Spectroscopic data of p-Coumaric acid (140) 110
4.3.5.6. Spectroscopic data of oleanolic acid (141) 111
CHAPTER 5
PURIFICATION AND CHARACTERIZATION OF SECONDARY
METABOLITES FROM ENDOPHYTIC FUNGUS CRYPTOSPORIOPSIS SP.
112
5.1. ENDOPHYTES AND ENDOPHYTIC FUNGI 113
5.2. BIODIVERSITY OF FUNGAL ENDOPHYTES 114
5.3. ADVANTAGE OF MICROBIAL STUDIES IN DRUG DISCOVERY 114
5.4. BIOACTIVE COMPOUNDS FROM ENDOPHYTIC FUNGI 115
5.4.1. Antibacterial Agents from Endophytic Fungi 115
5.4.2. Anti-Cancer Agents from Endophytic Fungi 118
5.4.3. Anti-Fungal Agents from Endophytic Fungi 120
5.4.4. Anti-Viral Agents from Endophytic Fungi 122
5.4.5. Immunosuppressant Agents from Endophytic Fungi 123
5.5. TAXONOMY OF THE ENDOPHYTIC FUNGUS CRYPTOSPORIOPSIS 124
5.6. LITERATURE SURVEY ON THE GENUS CRYPTOSPORIOPSIS 125
5.6.1. Previous Investigation of Cryptosporiopsis Sp. 125
5.7. RESULTS AND DISCUSSION 126
5.7.1. Structure Elucidation of the Isolated Compounds from Endophytic Fungus Cryptosporiopsis Sp.
126
5.7.1.1. Characterization of cryptosporioptide (192) 127
5.7.1.2. Characterization of cryptosporioptide A (193) 132
5.7.1.3. Characterization of cryptosporioptide B (194) 134
5.7.2. Computational Section for Compound 192 136
5.7. 4. Biological Activities of Compounds 192-194 136
5.8. EXPERIMENTAL 138
5.8.1. General Experimental Procedures 138
5.8.2 Culture, Extraction and Isolation 139
5.8.3. Spectroscopic Data of Isolated Compounds 141
5.8.3.1. Spectroscopic data of cryptosporioptide (192) 141
5.8.3.2. Spectroscopic data of cryptosporioptide A (193) 142
5.8.3.3. Spectroscopic data of cryptosporioptide B (194) 142
5.8.4. Enzyme Inhibitory Assays 143
5.8.4.1.-Glucosidase inhibition assay 143
5.8.4.2. Acetylcholinesterase assay 144
5.8.4.3. Butyrylcholinesterase assay 145
5.8.4.4. Lipoxygenase assay 145
5.8.5. Agar Diffusion Test for Antimicrobial Activity 146
Refernce 147
List of Tables
Titles Page No.
Table.1.1: All anti-infective drugs from 1981 to 2012 from source 09
Table.1.2: Bioactive compounds isolated from different source 11
Table. 2.1: 1H and 13C-NMR data of 69 (CD3OD; 500 and 125 MHz) 32
Table. 2.2: 1H and 13C-NMR data of 70 (CD3OD; 500 and 125 MHz) 34
Table. 2.3: 1H and 13C-NMR data of 71 (CD3OD; 500 and 125 MHz) and
72 (CD3OD; 600 and 150 MHz) 39
Table. 2.4: 1H and 13C-NMR data of 73 and 74 (CD3OD; 500 and 125
MHz) 41
Table. 2.5: 1H and 13C-NMR data of 75 (CD3OD; 600 and 150 MHz) and
76 (CD3OD; 400 and 100 MHz) 45
Table. 2.6: 1H and 13C-NMR data of 77 (CD3OD; 600 and 150 MHz), 78
(CD3OD; 400 and 100 MHz) and 79 (CD3OD; 500 and125 MHz)
49
Table. 2.7: 1H and 13C-NMR data of 80 (CD3OD; 500 and 125 MHz), 81
(CD3OD; 600 and 150 MHz)
52
Table. 2.8: 1H and 13C-NMR data of 82 and 83 (CD3OD; 600 and 150
MHz) 55
Table. 2.9: 1H and 13C-NMR data of 84 and 85 (CD3OD; 400 and 100
MHz) 56
Table. 2.10: 1H and 13C-NMR data of 86 (CDCl3 500 and 125 MHz) 58
Table. 2.11: 1H and 13C-NMR data of 87 (CD3OD; 500 and 125 MHz) 59
Table. 2.12: 1H and 13C-NMR data of 88 (CD3OD; 500 and 125 MHz) 60
Table. 2.13: Antioxidant and antiurease activity of isolated compounds 63
Table. 2.14: Antibacterial activity of isolated compounds 63
Table. 4.1: 1H and 13C-NMR data of 136 (CD3OD; 500 and 125 MHz) 95
Table. 4.2: 1H and 13C-NMR data of 137 (CD3OD; 500 and 125 MHz) 97
Table. 4.3: 1H and 13C-NMR data of 138 and 139 (CD3OD; 500 and 125
MHz)
100
Table. 4.4: 1H and 13C-NMR data of 140 (CDCl3, 400 and 100 MHz) 101
Table. 4.5: 1H and 13C-NMR data of 141 (CD3OD; 500 and 125 MHz) 103
Table. 5.1: 1H and 13C-NMR data of 192 (CDCl3; 500 and 125 MHz) 193
and 194 (CD3OD; 500 and 125 MHz) 134
Table. 5.2: Enzyme inhibitory activity of compounds 192-194 137
List of Figure
Titles Page No.
Figure. 1.1: Graphical representation of chemical scaffolds obtained from different source.
07
Figure. 1.2: Percentage of natural product and natural product derivatives by
year wise contribution in drug discovery from 1981-2010.
08
Figure. 1.3: All new approved drugs by source from 1981 to 2010. 08
Figure. 1.4: All anticancer drugs 1940s-2010 by source. 09
Figure. 2.1: 1H-1H COSY ( ) and 1H-13C HMBC ( ) interactions observed
in the spectra of 69.
31
Figure. 2.2: 1H-1H COSY ( ) and 1H-13C HMBC ( ) interactions
observed in the spectra of 70.
34
Figure. 2.3: 1H-13C HMBC correlations observed in compound 71. 36
Figure. 2.4: Proposal of structure a and b for compound 71, based on NMR
data.
36
Figure. 2.5: 1H-1H COSY ( ) and 1H-13C HMBC ( ) interactions observed
in the spectra of 72.
38
Figure. 2.6: 1H-1H COSY ( ) and 1H-13C HMBC ( ) interactions observed
in the spectra of 74.
42
Figure. 2.7: 1H-1H COSY ( ) and 1H-13C HMBC ( ) interactions observed
in the spectra of 77.
47
Figure. 2.8: Important 1H-13C HMBC ( ) interactions observed in the
spectra of 78.
48
Figure. 4.1: COSY (─) and important HMBC ( ) correlations observed in compound 136.
94
Figure. 4.2: NOESY correlation observed in compound 136. 94
Figure. 5.1: Joining of various fragments of cryptosporioptide identified
through HMBC correlation.
129
Figure. 5.2: Solid lines: experimental UV-vis (top), CD spectra (bottom) of (+)-cryptosporioptide (CH3CN, 6.28 mM, 0.01 cm cell). Dotted line:
calculated CD spectrum of (2R,3S,12R,13R,14R)-
cryptosporioptide with B3LYP/TZVP//B3LYP-6-311G+(d,p)
method, as Boltzmann average of 9 low-energy structures at
300K. The calculated spectrum was obtained as sum of Gaussian
bands with 0.38 eV exponential half-width, and red-shifted by 50 nm.
131
Figure. 5.3: Lowest-energy structure calculated for (2R,3S,12R,13R,14R)-
Cryptosporioptide with B3LYP-6-311G+(d,p) method.
132
Figure. 5.4: NOESY correlation observed in compound 193. 134
List of Schemes
Titles Page No.
Scheme. 2.1: Extraction and isolation of secondary metabolites from Vincetoxicum Stocksii.
67
Scheme. 4.1: Purification protocol of secondary metabolites from Seriphidium quettense.
107
Scheme. 5.1: Extraction and isolation of secondary metabolites from Cryptosporiopsis (internal strain no. 8999) endophytic
fungal sp
140
[Pick the date] Summary
I
SUMMARY
Natural product research gained importance as it discovered
chemicals of diverse structural classes with potential pharmacological
actions. Among various natural sources, plants and their guest
endophytic fungi are two potential pools for the discovery of new leads.
In the present work, I investigated two medicinal plants: Seriphidium
quettense, Vincetoxicum stocksii and an endophytic fungus
Cryptosporiopsis sp. to explore for their bioactive secondary
metabolites. The whole dissertation has been divided into five chapters.
Chapter 1 offers comprehensive overview of role of natural products in
human health and also provides a mini review of important bioactive
secondary metabolites and drugs derived from different natural
sources. In Chapter 2 a brief phytochemical survey of various species
of the genus Vincetoxicum has been incorporated, besides, isolation and
characterization the secondary metabolites of Vincetoxicum stocksii
have also been discussed in this chapter. Chromatographic purification
of ethyl acetate soluble frcation of methanolic extract of this plant
yielded four new; vincetolate (69), vincetoside (70), vincetetrol (71) and
vincetomine (72) along with sixteen known compounds; apocynin (73),
4-hydroxy-3-methoxyphenyl-1-(9-hydroxy-propan-7-none) (74), 1-(4-
hydroxy-3-methoxyphenyl)-1,2,3,-propanetriol (75), feruloyl-6-O--D-
glucopyranoside (76), 7-megastigmene-3,5,6,9-tetrol (77), 4-
hydroxyphenylacetic acid (78), 4-hydroxy-3,5-dimethoxybenzoic acid
(79), 4'-methoxy-kaempferol-3-O--D-glucopyranoside (80), 4'-methoxy-
[Pick the date] Summary
II
kaempferol-3-O--L-rhamnopyranoside (81), qurcetain-3-O--L-
rahmnopyranoside (82), 4'-methoxy-qurcetin-3-O--L-
rehmnopyranoside (83), qurcetin-3-O--D-glucopyranoside (84),
qurcetin-3-O--D- galactopyranoside (85), 7-hydroxy-3',4',5-
trimethoxyflavone (86), β-sitosterol (87), and ß-sitosterol-3-O-D-
glucopyranoside (88), which have been identified due to a combination
of 1D, 2D NMR and HREIMS and HRFABMS techniques and in
comparison with the literature. Among these isolates, compounds 73,
75, 76, 79 and 84 were screened for NO inhibition scavenging
potential. These compounds exhibited significant inhibition with IC50
values of 80.17±0.33, 150±0.5, 64.1±0.09, 56.42±0.04, 89.33±0.1
µg/ml respectively and compounds 69, 70, 75, 77, 79, 80 and 84 were
also evaluated for antiurease activity, which showed moderate to
significant inhibition with IC50 values of 85.8±0.01, 105.47±0.01,
22.1±0.43, 74.8±0.04, 93.4±0.01, 23.9±0.51 and 88.6±0.01 µg/ml
respectively.
O
O
OH
OH
69
1
2
3
45
6
7 8
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13'
14'
1
2
3
1'
2'3'
4'
5'6'
O
HO
OH
OH
OHO
2526
27
28
21
70
CH3
31
H3CO
O
O
OH
O CH3
71
1
2 4
5
OH3C
72
H3CO
NH2
1
3
5
7
4'
5'
6'
1'
2'
3'
14
2
6
4
[Pick the date] Summary
III
Chapter 3 deals with the botanical description and literature survey of
the genus Seriphidium, which revealed that Seriphidium sp. are mostly
producing flavonoids. In the present work, Seriphidium quettense was
investigated for its bioactive phytochemicals and as a result The ethyl
acetate fraction of methonalic extract yielded seriphilidine (136) as a
new compound along with seven known compounds; 6-hydroxy-8(10)-
oplopen-14-one (137), salvigenin (138), cirsumaritin (139), p-coumaric
acid (140), β-sitosterol (87), β-sitosterol-3-O-D-glucopyranoside (88)
and oleanolic acid (141). The discussion on their structure elucidation
has been provided in Chapter 4, which covres the detail of their
purification process and spectroscopic techniques.
OH3CO
H3C
CH3H3C
CH3
OH
1
2
34
56
7
89
1011
12
1314
1516
136
Other part of my research work deals with the purification and
characterization of secondary metabolites from endophytic fungus
cryptosporiopsis Sp., which has been described in chapter 5. However,
the same chapter also describes the biodiversity of fungal endophytes,
its advantages in drug discovery and some details of bioactive
compounds from endophytic fungi. The characterization of novel
bioactive polyketide 192 was accompalished due to 1D, 2D NMR
spectroscopy and high resolution mass spectrometry. The absolute
[Pick the date] Summary
IV
stereochemistry of 192 was fully established through electronic circular
dichroism (CD). This compound was published in a peer-reviewed
journal [1], whereas, re-culturing and investigation of the fungal extract
resulted in the isolation of two derivatives; 193 and 194 of polyketide
192. Compounds 192-194 were tested against four enzymes
acetylcholinesterase, butyrylcholinesterase, -Glucosidase and
lipoxygenase and compound 192 was found significantly active against
the enzyme lipoxygenase with an IC50 value of 49.15±0.17 M and
enzyme -glucosidase with an IC50 value of 50.59±0.28 M, whereas it
was inactive against other two enzymes and it also showed anti-Bacillus
megaterium activity. Compounds 193 and 194 showed moderate
activity against -glucosidase (IC50 = 44.93±0.26 and 41.42±0.14
respectively) and lipoxygenase (IC50 = 134.38±1.65 and 149.62±1.53
respectively), whereas, remained inactive against other two enzymes.
The latter derivatives 193 and 194 have been published in another
peer-reviewed journal [2].
O
OOHO
H3CO
HO Me
OH
OHOH
Me
194
2
3 14
16
15
1312
H
1
4
56
7
89
1011
O
OOHO
H3CO
HN Me
OH
OOH
Me
O
O
2
3
1918
14
16
17
15
1312
H
1
4
56
7
89
1011
192
O
OOHO
HO
HN Me
OH
OOH
Me
O
O
2
3
1918
14
16
17
15
1312
H
1
4
56
7
89
1011
193
1. Muhammad Saleem, Muhammad Imran Tousif, Naheed Riaz, Ishtiaq Ahmed, Barbara Schulz, Muhammad Ashraf, Rumana Nasar, Gennaro Pescitelli, Hidayat Hussain, Abdul Jabbar, Nusrat Shafiq and Karsten Krohn, Cryptosporioptide: A novel bioactive polyketide produced by an endophytic fungus Cryptosporiopsis sp. Phytochemistry, 93, 199-202 (2013).
[Pick the date] Summary
V
2. Muhammad Imran Tousif, Natasha Shazmeen, Naheed Riaz, Nusrat Shafiq, Tasneem Khatoon, Barbara Schulz, Muhammad Ashraf, Ayesha Shaukat, Hidayat Hussain,
Abdul Jabbar & Muhammad Saleem, -Glucosidase and lipoxygenase inhibitory
derivatives of cryptosporioptide from the endophytic fungus Cryptosporiopsis sp. Journal of Asian Natural Products Research, 16, 1068–1073 (2014).
The references have been provided in the end of the dissertation,
whereas, the re-prints of the two publications (1 and 2) have been
incorporated as annexure-A and B.
Chapter 1 Introduction of Natural Product
1
CHAPTER 1
INTRODUCTION OF NATURAL PRODUCTS AND THEIR
ROLE IN DRUG DISCOVERY
Chapter 1 Introduction of Natural Product
2
1.1. NATURAL PRODUCTS AND EVOLUTION OF MODERN MEDICINE SYSTEM
Natural products are bio-chemical substances obtained from
natural sources like plants, animals, microorganisms and usually have
a pharmacological or biological activity. In natural product chemistry,
we study the structure, uses and purposes of these bio-chemical
substances for the benefit of human. Natural products are secondary
metabolites, including terpenoids, flavonoids, alkaloids, steroids,
polyketides and other compounds of diverse structure. These chemicals
are produced due to the environmental stress and other challenges
faced by organisms for survival. Keeping in view this idea scientists
study plants and other organisms for their important metabolites that
may also help human to survive against environmental and other
challenges (Townsend, C. A. and Ebizuka, Y. 1999).
Health is the prime priority of man, who spends most of his
sources to save it from environmental dangers. His search for new and
novel source of medicine is ever growing. In this way, he explored
natural sources for their bioactive chemicals. His search of medicine is
a continuous process and nature is one who acts as guide in this
process (Bungihan et al., 2011).
In the past, man depended upon the local flora for his existence.
There are countless documentary proofs which confirmed that different
countries, culture and civilization from all over the world were familiar
with natural sources, for their uses as medicine. Archeological evidence
showed that Shanidar (in Iraq) were using plants (holly- hock, yarrow,
Chapter 1 Introduction of Natural Product
3
grape hyacinth) for healing purposes. The first documentary proof with
respect to the history of medicine was found in Egyptian dynasties was,
“Papyri” a book of medicine (De Pasquale 1984). The “Papyri” first time
appeared in 1873 offered by G. Ebers, but in fact it was sold by Arab to
G. Ebers and was discovered in 1862 in Thebes. It was titled by
following word "Here begins the book on preparation of medicines for all
parts of human body”. Approximately 160 vegetable drugs were listed
in this book, which were used for the treatment of digestive disorder
and ulcer. In 1493 B.C Queen Hatchepsut of 18th dynasties, during her
period a plant Ammi mujus, its roots were eaten by Caravaneers to
protect themselves from sun burning. It was discovered later that it
contains 8-methoxypsoralen which stimulates pigment formation and
produced an artificial Suntan (De Pasquale 1984). Trade was carried
out between Egypt and India in 3rd millennium B.C either by the
mountain of Elbuz in North of Iran or from the South of Baluchistan,
Pakistan. In this way, the exchange of knowledge and drugs was taken
place between various regions (Thorwald 1962). In Europe, the Greek,
Roman and Hippocrates were most representative figure in field of
medicinal plants. The “Pendanius Dicscorides” a great Greek physician
and pharmacologist described about 500 drugs of vegetable origin and
106 drugs of animal origin. In Asia, “Serapione the young and Ibn
Baithar” in 13 century described about 1400 drugs of vegetable origin.
At that time Arab schools were the active center of learning for
scientific knowledge. In China (1122-221 B.C), the Chou period was
known for medical therapy. They used “Ephedria Sinica” for asthma
Chapter 1 Introduction of Natural Product
4
and pulmonary diseases and “Dichroa Febrifuga, against malaria. The
use of the phytotherapy was more prominent in India and China and
both were the leading countries for phytotherapy in Asia because this
region was blessed with many important medicinal plants. Some rural
regions still have no access to modern facilities and dependent on the
Ayurvedic medicines. Therefore, Indian Ayurvedic medicines are still
famous all over the world (Anderson 1977; Farnsworth 1979; Stone
1962; De Pasquale 1984).
A Swiss physician, Paracelsus at the start of 17th century,
introduced the process of extraction of drugs from plants for the first
time. Meanwhile, the invention of printing press also favored the
spreading of knowledge all over the world. The new exotic drugs made
their appearance in Europe and starting point of investigation was
plant extracts. The precious work of using plant parts as medicine was
started but no defined chemical was isolated from plant till the end of
18th century and knowledge of active principle of bio-chemicals was not
studied properly. The scientists were not familiar with extraction of
chemical substance until Carl Wilhelm Scheele separated the organic
acids (Shellard 1980-1981; De Pasquale 1984). At the beginning of the
19th century William Withering published his work on Digitalis
purpurea plant and extracted the cardiotonic foxglove, use for the
treatment of heart patients (Aronson 1985). Further, work on this plant
led to the discovery of digoxin, a drug marketed as Lanoxin
(GlaxoSmithKline, Research Triangle Park, North Carolina) for heart
Chapter 1 Introduction of Natural Product
5
patients (Smith 1930). After that Freidrich Serturne isolated morphine
in 1806 and Charles Derosne isolated narcotine in 1803 (Schmitz
1985). Pelletier and Caventou isolated strychnine in 1818 from the
seeds of the Strychnos vomica a woody tree found in South America
(Woodward et al., 1954). Quinine from cinchona was isolated in 1820.
Pierre-Jean Robiquet (1780-1840), a French pharmacist, made an
important contribution and isolated the cantharidin from cantharides,
caffeine from coffee and amygdalin from bitter almonds. Nativelle
(1868), crystallized digitalin from foxglove (Warolin 1999). The isolation
of salicylic acid from willow bark and synthesis of aspirin from salicylic
acid was another breakthrough (Vane 1992) in natural drug discovery
field.
The above overview revealed that progress in natural product
chemistry played major role as a contributor in the development of
biological lead and their derivative into drugs. Knowledge of active
principle led to the discovery of life saving drugs from medicinal plants.
In this way natural product research began its contribution in human
welfare.
1.2. NATURAL PRODUCT RESEARCH ON MEDICINAL PLANTS; PAST, PRESENT AND FUTURE
The discovery of active compounds from the crude extract or
crude drugs of plant origin are major focus in this field. Scientists are
interested in study of active principles of these sources and their use as
medicine. Research on medicinal plants began all over the world to
discover the active leads of biological interest, with their all-possible
Chapter 1 Introduction of Natural Product
6
derivatives and try to find out their structure-activity relationship, to
increase their activity, lower the side effect and toxicity (Robbers and
Tyler 1999).
These facts demand the enhancement of natural product
research on promising medicinal plants towards the development of
new drugs. Therefore, presently many research groups from all over the
world are working on the isolation of bioactive compounds from
different natural sources. They study antimicrobial, antioxidant, anti-
inflammatory and other activities of plants extracts, isolation of their
active components and their use in drug discovery. These research
groups have different criteria for their studies like; some groups study
selected plants of medicinal importance as per reported from different
region of the world, some groups select plants according to their
geographical occurrence and climate habitat, whereas, some groups
use to select the plants on the basis of information from folk and
traditional medicine system (Keen et al., 2005).
With respect to future research on medicinal plant activities,
whether new results or known, the most important thing is the
reproducibility of results to evaluate the quality of products and their
use in human health care. Obviously, this type of study is of immense
interest for the future use of plants and crude extracts in medicine
(Cordell et al., 1993). New improved drugs are needed because
appearance of life threatening diseases and emergence of bacteria that
have developed resistance to antimicrobial drugs, for example, US
Chapter 1 Introduction of Natural Product
7
Center for Disease Control and Prevention has reported a strain of
Mycobacterium tuberculosis that was resistant to all approved drugs to
date (Morbidity and Mortality, Weekly Report 2006). Therefore
tremendous increase in bacterial, fungal and viral infections all over
the world has been observed.
On the other hand, nature is beautiful source to answer his own
problems. The hub of the diseases that endanger the human health,
their best solution is also given by nature. These entire human health
problems are needed to be addressed but how? Therefore, a question
arises; where do drug come from? The answer came from the fact that
“natural products were the origin of all the medicine with 80% of
medicinal compounds came from natural material” (Sneader 1996).
Nearly 83% of the chemical scaffolds used for drug development come
from natural sources while 17% come from synthesis (figure 1.1).
Figure. 1.1: Graphical representation of chemical scaffolds obtained from different source
animals
microbes
synthesis
mineral
plants
Chapter 1 Introduction of Natural Product
8
The natural product contribution in drug development over
period of 1981-2010 (figure 1.2) was mile stone. (Newman and Cragg
2012).
Figure. 1.2: Percentage of natural product and natural product derivatives by year wise contribution in drug discovery from 1981-2010.
According to the data above, 1355 drugs were introduced in the
market during a period 1981-2010, in which 41% were of natural origin
while 20% were synthetic derivatives of natural product. The figure 1.3
shows the percentage of all approved drugs during 1981-2010.
Figure. 1.3: All new approved drugs by source from 1981 to 2010.
25.6
38.5
23.6
34.9
46.7
32.7
46.4
36.731.6
28.220
41.74039.5
28.921.1
12.2
27.3
38.935.1
45.5
35.333.338.939.4
23.8
42.337.5
20.8
50
19
81
19
82
19
83
19
84
19
85
19
86
19
87
19
88
19
89
19
90
19
91
19
92
19
93
19
94
19
95
19
96
19
97
19
98
19
99
20
00
20
01
20
02
20
03
20
04
20
05
20
06
20
07
20
08
20
09
20
10
percent N/ND by year from 1981 to 2010
percent N/ND by year from 1981 to 2010
64
299268
442
80
202
4 22 20 33 6 15
NP ND S/NM S V B
All new approved drugs from 1981 to 2010 by source
No. of all new approved drug %age of all new approved drugs
Chapter 1 Introduction of Natural Product
9
During the period of 1940-2012, natural products contribution in
the development of cancer therapeutic drugs was of great significance
(figure 1.4). Out of total 206 anticancer drugs marketed during this
period, 85 came from natural product and 121 drugs were contributed
from all other sources (Newman and Cragg 2012).
Figure. 1.4: All anticancer drugs 1940s-2010 by source.
Anti-infective drugs like antibacterial, antifungal, antiparasitic
and antiviral are mostly small molecules of natural origin and their
derivatives. The total number of anti-infective drugs approved till 2012
are given in the table 1.1.
Table. 1.1: All anti-infective drugs from 1981 to 2012 from source.
Indication Total N ND S S/NM V
Antibacterial 118 10 67 26 1 14
Antifungal 29 1 3 22 3 - Antiparasitic 14 2 5 6 - 1
Antiviral 109 14 4 32 12 47
Total 270 27 79 86 16 62
Percentage 100 4.4 29.3 31.9 5.8 23
N= Natural product ND= Natural product derivate S= synthetic S/NM= Synthetic of natural product minic
V= Vaccine
NP ND S S/ND V B
28
57
44 46
5
26
13
2821 23
2
13
All anticancer drugs 1940s to 2010 by source
No. of anti cancer drug by source % of anti cancer drug by source
Chapter 1 Introduction of Natural Product
10
The above data revealed that the role of natural product in drug
discovery is a continuous process and natural products are the only
source, which provide a bulk of leads to scientific community
continuously that will be used in drug designing process.
1.3. ADVANTAGES OF NATURAL PRODUCT RESEARCH
The advantage of natural product over the synthetic chemistry is
its ability to produce the novel structure of diverse classes. The
published data revealed that 40% of the chemical skeleton came from
natural product research were not present in the synthetic library.
Thus the natural product isolation is successful process in the
screening of new compounds of novel skeleton (Henkel et al., 1999).
The main advantages are discussed briefly.
Natural products produce chemicals of diverse structural classes
and are excellent source of biologically active metabolites
(Verdine 1996).
Natural products are produced due to adverse effect of nature so
they have specific biological interactions between organism and
environment, that’s why they have drug like properties (Nisbet
and Moore 1997).
Natural product resources are largely unexplored and are the
main source of pharmacophores (Bram et al., 1993).
Natural product leads are complementary to synthetic and
combinatorial libraries (Henkel et al., 1999).
Chapter 1 Introduction of Natural Product
11
Natural product led to discovery of active principle and novel
mechanisms of action (Urizar et al., 2002).
Natural products act as pathfinder for molecular biology (Hung et
al., 1996).
Synthesis of compounds were designed with help of natural
product guide lines (Breinbauer et al., 2002).
1.4. BIOLOGICALLY ACTIVE NATURAL PRODUCTS
According to a review published in 2012, approximately 500,000
compounds have been isolated from natural sources with about 70,000
metabolites from microbial sources. With respect to biological
properties, 16500 bioactive compounds were reported as antimicrobial
agents isolated from all natural sources. Table 1.2 describes the total
number of important antimicrobial natural products along with other
chemotherapeutic agents (Berdy 2005; Berdy 2012).
Table. 1.2: Bioactive compounds isolated from different source.
Antibacterial compounds Antifungal Compounds G. positive 11000-12000 Yeasts 3000-3500
G. negative 5000-5500 phytopathogenic 1600-1800
Mycobacteria 800-1000 Other fungi 3800-4000
Antitumor 5000-5500 Antiviral 1500-1600
Enzyme inhibitor 3000-3200 Immunological 800≈ Biochemical 1000≈ Anti-inflammatory 2000-2500
Chapter 1 Introduction of Natural Product
12
1.5. SOURCES OF NATURAL PRODUCTS
There are mainly four types of natural product sources:
1. Plant source
2. Animal source
3. Microbial source
4. Marine source
1.5.1. Natural Products from Plant Sources
Plants are known as a primary source of medicines. They provide
active ingredients of medicines. There is no exact figure about the total
number of medicinal plants present on earth and their percentage as
their number vary from region to region depending upon the climate
condition (Schippmann 2001). It was estimated that 35000-40000
medicinal plants species are present worldwide (Schippmann et al.,
2002; Shiva 1996). According to scholarly medical systems relatively
few medicinal plants (500–600) are being used in Traditional Chinese
Medicine, with a total estimated number worldwide as 6000.
Active ingredients from medicinal plants that are used in Western
medicine are even fewer. An article published in 1991 shows that 121
plant-based drugs were used in America (Farnsworth and Soejarto
1991). However, in current scenario, medicinal herbs are grown on
large scale in U.S.A, U.K. Canada, France and Turkey and are being
marketed to earn foreign exchange. It was estimated that 200 tons per
annum of herbs were used only in Indian region. About 188 tons are
Chapter 1 Introduction of Natural Product
13
used for culinary purposes and nearly 12 tons are consumed for
medicinal and cosmetic preparations. The largest global markets for
medicinal plants are China, France, Germany, Italy, Japan, Spain, UK
and US. Japan has the highest per capital consumption of botanical
medicines in the world. The plant base drugs of early time are aspirin
(1), digitoxin (2), morphine (3), quinine (4) and pilocarpine (5).
OH
OAc
OO
H
OH
H
H
HO1
2
OH
OH
OH
OH
O
HO
OH
OH
N
HOH N
H
H3CO
O
HO
H
NCH3N
N
OO
H3CCH3
3 4
5
An antimalarial drug approved as arteether (6) was derivative of
natural lead artemisinin (7) and was marketed by Artecef BV (Central
Drug Institute) (Graul 2001). Alzheimer’s disease drug galantamine (8),
manufacture by Johnson & Johnson was isolated from plant Galanthus
spp. (Heinrich and Teoh 2004). Miglustat (9) drug for type 1 Gaucher
disease and lead compound 1-deoxynojirimycin (10) was isolated from
Streptomyces trehalosaticus.
Chapter 1 Introduction of Natural Product
14
CH3O
O CH3
H
HCH3
O
OH3C
CH3O
H
HCH3
O
OH3C
O
6 7
O
H3CO
OH
N
CH3
8
N
CH3
OH
OHHO
HO
9
HN
OH
OHHO
HO
10
M6G (11, morphine-6-glucuronide) is a pain killer and was
introduced in market by CeNeS. Exatecan (12), an anticancer synthetic
drug was derived from camptothecin (13), which was isolated from the
plant Camptotheca acuminate. Sativex a mixture of dronabinol (14) and
cannabidol (15) was introduced by G. W. Pharmaceuticals derived from
the Cannabis plant for neuropathic pain relief in multiple sclerosis
(Wade et al., 2003; Nurmikko et al., 2007). Rubitecan (16) an
anticancer isolated from the plant Camptotheca acuminate introduced
by SuperGe.
O
CH3
OH
H3CCH3
12 13 14
N
N
O
O
O
CH3
H3C
N
N
O
O
O
CH3
H3C
H3C
F
NH2
CH3O
HO
H
NCH3O
HOHO
OH
HO2C
11
O
16
H3CO2C
NH
N CH3
FFH
H
NH
OH
N
CH3
OAc
CO2CH3CH3
H3CO
17
N
N
O
O
O
CH3
H3C
NO2
OH
CH3
O
HO
15
CH3
CH3
Chapter 1 Introduction of Natural Product
15
Vinflunine (17) and vinorelbine (19) is also an anticancer semi
synthetic drug derived from vinblastine (18), which was originally
isolated from the plant Catharanthus roseus (Butler 2004).
H3CO2C
NH
N CH3
OH
H
NH
OH
N
CH3
OAc
CO2CH3CH3
H3CO
18
H3CO2C
NH
N CH3
H
NH
OH
N
CH3
OAc
CO2CH3CH3
H3CO
19
1.5.2. Natural Products Isolated from Microbial Sources
Over 50,000 secondary metabolites have been reported from
microbial sources, with 22000 to 23000 are bioactive compounds.
Approximately 17,000 are antibiotic; unfortunately, very small
percentage from them has been further processed as natural product
drug (Berdy 2005; Knight et al., 2003).
The discovery of penicillin by Alexander Fleming in 1928 and its
development by Chain and Florey in the 1940s was a gateway to the
isolation of several bioactive compounds from microbes. Streptomycin
(20), chloramphenicol (21), chlortetracycline (22), cephalosporin C (23),
Pestacin (24) and vancomycin (25) are other common examples
(Newman et al., 2000).
Chapter 1 Introduction of Natural Product
16
O
OHHO
HO
HN
CH3O
O
CHOHO
O
20
O2N
OH
HN CHCl2
O
OH
21
Cl
OH O
HOH
OH O
CONH2
OH
NCH3
H3C
H
22
N
S
CO2H
OCOCH3
O
HN
O
H
23
24
OHNH
HN
HO OHNH2
HN
NH2
NH
OH
HO
O
HO
HO
OH
CO2HH2N
Another milestone in the history of medicine was development of
the immunosuppressant drugs cyclosporine A (26) and rapamycin (27)
from microbial metabolites (Demain 1998).
O O
Cl
O
Cl
HO
ONH
O
O
O
NH
H3C
CH3
CH3
HN
O
NH
O
O
O
OH
HO
HO O
OH
NH2
CH3
OH
OH
O
CH3
O
HN
OOH
25
OHHO
Chapter 1 Introduction of Natural Product
17
N
CH3
O
N
O
N
CH3 O
N
CH3
O
N
O
N CH3
O
HN
O
N
O
HN
O
NH
CH3
CH3
H3C CH3
CH3
CH3H3CCH3H3C
O
H3C
H3C
N
O
O
H3C
O
OH
CH3
OH
H3COH
HOO
H3C
CH3
OH
CH3
26 27
CH3
H3C CH3
OO
O
H3C CH3
CH3 CH3 CH3
Anti-hyperlipidemics lovastatin (28) and guggulsterone (29) are
other good examples of microbial natural products (Urizar et al., 2002).
Fumagillin (30) lunched by Sanofi-Aventis as an antimicrobial lead is
capable of inhibiting the proliferation of endothelial cells. It was
isolated from Aspergillus fumigates (Mccowen et al., 1951).
Pleuromutilin (31) is an antibacterial agent that exhibits antibacterial
activity by inhibiting protein synthesis in bacteria (Daum et al., 2007).
Retapamulin (32) developed by GlaxoSmithKline, use in impetigo
caused by Gram-positive Staphylococcus aureus or Streptococcus
pyogenes. Lisdexamfetamine (33) was designed by New River
Pharmaceuticals to help attention deficit hyperactivity disorder (ADHD).
It consists of dextroamphetamine coupled with the essential amino acid
L-lysine (Popovic et al., 2009).
Chapter 1 Introduction of Natural Product
18
O
H3C
O
H3C
CH3CH3
O
OHO
O
CH3CH3
HH3C
O
(E)-form
28 29
CH3
CH3
CH3
O
O H
O
O
HN
OCH3
NH2
30
33
CH2 CH3 OH
CH3
O
H
H3C
H3C
O
O
HO
31
CH2 CH3 OH
CH3
O
H
H3C
H3C
O
O
SH3CN
32
COOH
H2N
1.5.3. Natural Products Isolated from Animal Sources
The natural products of animal origin are of great importance.
The antihypertensive agents; captopril (34), ramipril (35) and quinapril
(36) are derived from the snake venom peptide teprotide. Ilepatril (37) is
also under clinical evaluation (Davidson et al., 2007).
N
OHS
CH3
NH
HO
HO
O
O
O
CH3
N
O
OH
O
HN
O
O
CH3
CH3 N
ONH
O
SAc
H3C
H3C
CO2H
H
34
37
35 36
CH3
O
OH
An important metabolite trodusquemine (38) is a sulfated
aminosterol isolated from the dogfish shark (Rao et al., 2000).
Chapter 1 Introduction of Natural Product
19
Epibatidine (39), obtained from the skin of an Ecuadorian poisonous
frog, is ten time more potent than morphine (Spande et al., 1992).
Ziconotide (40) is an N-type voltage sensitive calciumchannel blocker
which was isolated from the toxin of Conus magus, is a synthetic form
of the peptide -conotoxin (Mcgivern 2007). Exenatide (41) was isolated
from the oral secretions of the poisonous lizard Heloderma suspectum.
It was marketed by Amylin Pharmaceuticals and can mimic the
antidiabetic or glucose-lowering properties of incretins (Bunck et al.,
2009).
NClHN
39
38
NH
NH
HN
NH
H2N
H3CCH3
OSO3H
HGEGTFTSDLSKQMEEEAVRLFIEWLKNGGPSSGAPPPS
41
H2N-CKGKGAKCSRLMYDCCTGSCRSGKC-CONH2
40
1.5.4. Natural Products Isolated from Marine Sources
Natural products from marine derived fungi show promising
biological and pharmaceutical properties. Saleem et al, in a review
article, described 103 marine secondary metabolites of biological
importance during the period of 2000-2006, whereas, only 15
secondary metabolites isolated from marine sources are in clinical trials
Chapter 1 Introduction of Natural Product
20
(Saleem et al., 2007). In another review published in November 2010 by
Mostafa E. Rateb describes 690 secondary metabolites of biological
importance covering period of 2006 to mid-2010 which shows the
marine source contribution in drug discovery or its role in finding the
new lead structure for drug development (Ratebab and Ebel 2011).
The important secondary metabolites isolated from marine
sources are, the epicoccone (42) was isolated from marine brown alga
Fucus Vesiculosus and was found potentially active, showing radical
scavenging effect at 25 g/ml (Abdel-lateff etal., 2003). Evariquinone
(43) exhibits antiproliferative activity and was found active against KB
and NCI-460 cells with concentration 3.16 µg/ml (Bringmann et al.,
2003). Varitriol (44) and varioxirane (45) show the potency against
selected renal, breast cancer and CNS cell lines, whereas, virixanthone
(46) shows activity against Gram-positive and Gram-negative bacteria
(Malmstrom et al., 2002). Trichodermamides A (47) and B (48) having
cyclic O-alkyl-oxime functionality show significant in vitro cytotoxicity
against HCT-116 human colon with an IC50 value of 0.32 µg/ml (Garo
et al., 2003).
Chapter 1 Introduction of Natural Product
21
O
O
OH
CH3
CH3
H3C
O
H3C
OH
O
O
H3C
46
NO
OH
OH
OH
NH
O
O
O
OCH3
OCH3
NO
OHCl
OH
NH
O
O
O
OCH3
OCH3
47 48
CH3
HO
HO
OH
O
O
CH3
OCH3O
O
OH
HO
HO
42 43
OCH3
OH
O
CH3
OH
HO
OCH3
OH
CH3
OH
CH3
HO
4445
In short natural products and natural product chemistry have
revolutionized the world of medicine with antibiotics like penicillin (49),
tetracycline (50), erythromycin (51), antimalarials e.g. quinine (4),
artemisinin (52), antiparasitics drugs like avermectin (53), lipid
controlling agents like lovastatin (28) and its analogs,
immunosuppressants for organ transplants, e.g. cyclosporine A (26),
rapamycins (27) and anticancer drug like doxorubicin (54) etc. (Harvey
2008).
Chapter 1 Introduction of Natural Product
22
O
CH3
CH3
COOH
HHNR
O
OOHOH
OH O O
NH2
OH
NCH3
CH3HO
5051
49
CH3
O
O
O
OH
H3C
HO
CH3
H3C
H3C
O O CH3
CH3
H3C OCH3
O
OOH
OH
N
CH3
CH3
H3C
52
O
CH3
O
CH3
O
H3COO
O
O
CH3
OCH3
OH
O O
OO
CH3
H3CO
CH3OCH3
OH
HO
H
53
O
OH
OH
OH
O
OOCH3 O
OH
54
O
NH2
CH3
OCH3
CH3H3C
The success of natural product drug development was
commercially excellent. Out of 20 best-selling non-protein drugs in
2000, nine were natural products or either derived from them. Their
combined annual sales were over US$16 billion. Many new
developments from natural products are in pipeline (Landry and Gies
2008; Harvey 2001).
Research Problem and hypothesis
23
RESEARCH QUESTION AND AIM OF THE PRESENT STUDY
About 500 medicinal plant species out of a total 5700 are
estimated to exist in Pakistan, whereas, it is estimated that 80% of the
rural population of Pakistan depends on traditional medicines for their
primary healthcare needs. Despite of the facts that Pakistan is blessed
with large number of medicinal plants, about 90% of the country’s
medicinal herbs are imported (Saqib and Sultan 2005). Therefore, there
is a crucial need to investigate local flora for their medicinal potential
and to provide a systematic data base to the community to use these
plants and their extracts as medicine. These facts motivated us to
investigate Pakistani indigenous medicinal plants to discover novel
compounds of pharmaceutical importance and to establish a scientific
data base of the local treasure. Therefore, for the present study, I
selected two unexplored plants: Seriphidium quettense and
Vincetoxicum stocksii to investigate them for their bioactive secondary
metabolites.
On the other hand, the discovery of new drug candidates from
endophytic fungi is relatively a new and attractive area of drug
development. Unfortunately, this modern and important area of drug
discovery has been totally ignored in Pakistan. Therefore, as an
initiative, and in cooperation with German scientists, I included
investigation on an endophytic fungus Cryptosporiosis sp. in my plan of
Ph.D. research.
Research Problem and hypothesis
24
HYPOTHESIS OF THE RESEARCH
Due to their use in folk medicine system and previous study on
the related plants, it was hypothesized that Seriphidium quettense and
Vincetoxicum stocksii must be synthesizing bioactive secondary
metabolites, which can be lead for drug development, while various
species of the endophytic fungi of the genus Cryptosporiopsis have been
reported to produce potent antibacterial, antifungal and insecticides;
therefore, the species isolated from Vibrum tanus must also be
producing novel bioactive compounds.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
25
CHAPTER 2
RESULTS AND DISCUSSION: PREVIOUS RESEARCH ON THE VINCETOXICUM GENUS AND ISOLATION OF
SECONDARY METABOLITES FROM VINCETOXICUM STOCKSII
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
26
2.1. THE GENUS VINCETOXICUM
The genus Vincetoxicum is among various genera of plant family
Asclepiadaceae. It is a small genus of 10-20 species, mainly distributed
in Europe and Asia. In Pakistan, it is represented by only six species
including V. arnottianum, V. canescens, V. cardiostephanum, V.
hirundinaria, V. sakesarense and V. stocksii (Ali et al., 1982).
2.2. PREVIOUS PHYTOCHEMICAL INVESTIGATION OF THE GENUS VINCETOXICUM
Literature search revealed that a very little phytochemical work
has been done on the species of this genus. Fewer reports were
published in literature. Lavault et. al., isolated 10-(−)-antofine N-oxide
(55), 10-(−)-antofine N-oxide (56), (−)-antofine (57), (−)-6-O-
demethylantofine (58) and (−)-14-hydroxyantofine (59) from V.
hirundinaria (Lavault et al., 1994).
N
OCH3
H3CO
H3CO
N
OCH3
H3CO
H3CO
O
H
N
OCH3
H3CO
H3CO
H
O
H
N
OCH3
H3CO
HO
H
55 56 57 58
The pregnane glycosides, cynatratosides E (60) and C (61),
hirundigoside B-D (62-64) were isolated from the roots of V.
hirundinaria (Lavault et al., 1999).
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
27
N
OCH3
H3CO
H3CO
OH
59
O
OO
H
CH3
OH
R1O
R2H O
OO
H
CH3
O
R1O
R2H
60 R1 = b-D-glu (1-4) a-D-ole (1-4) b-D-digit (1-4) b-D-ole; R2 = H
61 R1= a-D-ole (1-4) b-D-digit (1-4) b-D-ole; R2 = H
62 R1= b-D-glu (1-4) a-D-ole (1-4) b-D-digit (1-4) b-D-ole; R2 = OH
63 R1= a-D-ole (1-4) b-D-digit (1-4) b-D-ole; R2 = OH
64 R1= a-D-ole (1-4) b-D-digit (1-4)
The hancokinol (65) was isolated from V. officinale (Nowaka and
Kisielb 2000), while compounds 66-68 were obtained from V. pumilum
(Staerk et al., 2005).
68
N
OCH3
MeO
H3CO
H
66
N
OCH3
H3CO
67
O
H
N
OCH3
H3CO
O
OH
HO
65
H3C
CH3
Above are the fewer reports published on phytochemicals of the
relatives of this genus. Among these plants, V. stocksii has never been
explored for its secondary metabolites. Therefore, my research work, for
the first time emphasizes on the isolation of bioactive metabolites from
this plant.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
28
2.3. VINCETOXICUM STOCKSII
Vincetoxicum stcoksii is an important member of the genus
Vincetoxicum. It is a perennial climbing leafy vine growing in
Baluchistan, Pakistan. Vincetoxicum stocksii is known for its poisonous
as well as its medicinal properties. It is used to treat the external
cancers, wounds and injuries in man and animals (Simandi et al.,
1996). In local medicine system, this plant has been reported to
possess antibacterial and antifungal activities. In addition, the crude
extract of the V. stcoksii shows antidiarrheal and antispasmodic
potential (Zaidi and Crow 2005).
2.3.1. Classification of the Vincetoxicum Stocksii
Family Asclepiadaceae
Subfamily Periplocoideae
Class Asclepiadeae
Genus Vincetoxicum
Specie Vincetoxicum stocksii
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
29
2.4. RESULTS AND DISCUSSION: CHARACTERIZATION OF
SECONDARY METABOLITES ISOLATED FROM V. STOCKSII
The methanolic extract of Vincetoxicum stocksii was divided into
n-hexane, ethyl acetate and water fractions. The ethyl acetate fraction
exhibited several biological activities (Table 2.13 and 2.14), thus it was
proceeded for the isolation of secondary metabolites. As a result of
chromatographic purification, four new; vincetolate (69), vincetoside
(70), vincetetrol (71), vincetomine (72) and sixteen know compounds;
apocynin (73), 4-hydroxy-3-methoxyphenyl-1-(9-hydroxy-propan-7-one)
(74), 4-hydroxy-3-methoxyphenyl-7,8,9-propanetriol (75), feruloyl-6'-O-
-D-glucopyranoside (76), 7-megastigmene-2,3,5,9-tetrol (77), 4-
hydroxyphenylacetic acid (78), 4-hydroxy-3,5-dimethoxybenzoic acid
(79), 4'-methoxy-kaempferol-3-O--D-glucopyranoside (80), 4'-methoxy-
kaempferol-3-O--L-rhamnopyranoside (81), quercetin-3-O--L-
rhamnopyranoside (82), 4'-methoxy-quercetin-3-O--L-
rhamnopyranoside (83), quercetin-3-O--D-glucopyranoside (84),
quercetin-3-O--D- galactopyranoside (85), 7-hydroxy-3',4',5-
trimethoxyflavone (86), β-sitosterol (87) and ß-sitosterol-3-O-D-
glucopyranoside (88) were obtained.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
30
2.4.1. Characterization of Vincetolate (69)
Compound 69 was isolated as white amorphous powder. The
EIMS of 69 showed the
molecular ion peak at
m/z 364, while the
molecular formula
C22H36O4 with five
double bond equivalence (DBE) was calculated through HR-EIMS which
exhibited the molecular ion peak at m/z 364.2416. The IR spectrum
showed diagnostic absorption bands at 3505, 1730, 1610 and 1580, for
hydroxyl function, ester moiety and aromatic system, respectively.
The 1H-NMR spectrum of 69 (Table 2.1) showed two ortho-
coupled doublets at δ 7.02 (J = 8.5 Hz) and 6.73 (J = 8.5 Hz) in the
aromatic region. The splitting pattern (A2B2) of these two doublets
indicated the presence of a p-substituted benzene ring in 69. The
spectrum further displayed signals for three methylenes at δ 3.49 (s),
4.07 (t, J = 6.5 Hz) and 3.63 (t, J = 6.5 Hz). The first methylene signal
at δ 3.49 was correlated in HSQC spectrum with the carbon at δ 41.8,
which indicated that this methylene must be present between two sp2
hybridized centers. The other two methylene protons at δ 4.07 and 3.63
showed COSY correlation with a signal of four hydrogen (two
methylene) at δ 1.58 (m) and 1.50 (m), which in turn was correlated
with a broad singlet of several hydrogen at δ 1.40-1.22. This data
indicated that compound 69 must have a p-hydroxy phenylethanoate
O
O
OH
OH
69
1
2
3
45
6
7 8
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13'
14'
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
31
system coupled with a hydrocarbon moiety ending with a primary
alcoholic function.
The 13C-NMR (Table 2.1) substantiated the above deduction as it
displayed signals for carbonyl at δ 174.2, aromatic moiety at δ 158.4,
131.7, 130.8 and 116.3, oxymethylenes at δ 65.8 and 64.6 and
remaining methylenes at δ 41.8, 33.9, 26.0 with an intense signal at δ
30.0 for several aliphatic methylenes.
The above discussed data led to the structure of 69 as 14-
hydroxytetradecyl-2-(4-hydroxyphenyl) acetate, which was further
confirmed through HMBC spectral analysis (Figure 2.1), in which the
aromatic proton at δ 7.02 was found to correlate with the carbons at δ
158.4 (C-4), 131.7 (C-6) and 41.8 (C-7). In turn the proton resonating
at δ 3.49 showed HMBC correlation with the carbons at δ 131.7 (C-1),
130.8 (C-2, 6) and 174.2 (C-8), whereas, oxymethylene at δ 4.07
showed HMBC correlation with the carbonyl carbon at δ 174.2 (C-8). All
other important HMBC and COSY correlation are shown in figure 2.1.
O
O
OH
OH
69
Figure. 2.1: 1H-1H COSY ( ) and 1H-13C HMBC ( ) interactions
observed in the spectra of 69.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
32
On the basis of these observations compound 69 was finally
identified as 14-hydroxytetradecyl-2-(4-hydroxyphenyl) acetate which is
a new addition to the natural product list and is named as vincetolate.
Table. 2.1: 1H and 13C-NMR data of 69 (CD3OD; 500 and 125 MHz).
Position δ H (J in Hz) δ c
1 - 130.8
2 7.02 (1H, d, 8.5) 131.7
3 6.73 (1H, d, 8.5) 116.3
4 - 158.4
5 7.02 (1H, d, 8.5) 131.7
6 6.73 (1H, d, 8.5) 116.3
7 3.49 (2H, s) 41.8
8 - 174.2
1' 4.07 (2H, t, 6.5) 65.8
2' 1.58 (2H, m) 26.0
3'-12' 1.40-1.22 (20H, br., s) 30.0
13' 1.50 (2H, m) 33.9
14' 3.63 (1H, t, 6.5) 64.6
14'-OH - -
2.4.2. Characterization of Vincetoside (70)
Compound 70 was obtained as white amorphous powder, whose
FD-MS displayed the
molecular ion at m/z 612.
The HR-FAB-MS in
negative mode exhibited a pseudo-molecular ion peak at m/z 611.5263
corresponding to the molecular formula as C37H72O6 with two DBE. The
IR spectrum displayed absorption bands at 3385 and 1655 cm-1 for
hydroxyl and olefinic functions, respectively. An intense absorption
band at 1300 cm-1 was attributed to a hydrocarbon chain.
The 1H-NMR spectrum (Table 2.2) of 70 showed two signals at δ
5.31 (1H, br., m, W1/2 = 20.8 Hz) and 5.27 (1H, br., m, W1/2 = 20.8 Hz)
1
2
3
1'
2'3'
4'
5'6'
O
HO
OH
OH
OHO
2526
27
28
21
70
CH3
31
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
33
due to a trans double bond. The above two protons showed COSY
correlation with two aliphatic methylene at δ 1.92 (2H, m), 1.90 (2H,
m), respectively. One of the methylene at δ 1.90 was further correlated
with another methylene at δ 1.30, which in turn was correlated in the
same spectrum with a triplet methyl at δ 0.80 (J = 6.6 Hz). These
observations helped to fix the position of double bond. An oxymethylene
signal resonated at δ 3.99 (t, J = 6.9 Hz), which showed COSY
correlation with another methylene at δ 1.70 (m), which in turn was
correlated in COSY spectrum with a broad singlet of several aliphatic
protons δ 1.30-1.16. This data indicated a hydrocarbon chain in the
molecule. A doublet methine proton resonating at δ 4.20 (J = 7.0 Hz)
could be attributed to anomeric center of a sugar moiety, whereas,
other sugar protons resonated at δ 3.66 (1H, t, J = 9.0 Hz), 3.16 (1H,
m), 3.95 (1H, dt, J = 9.0, 6.0 Hz), 3.46 (1H, m), 3.78 (1H, dd, J = 12.0,
2.6 Hz) and 3.76 (1H, dd, J = 12.0, 6.6 Hz).
The 13C-NMR spectrum (Table 2.2) of 70 was in complete
agreement with the proton and mass data as it displayed the signals for
hydrocarbon part at δ 130.6, 129.7, 68.6, 35.2, 34.3, 31.8, 29.6-25.8,
22.6, 14.0 and sugar moiety at δ 102.9, 76.2, 74.3, 73.3, 72.2, 62.2.
The anomeric proton (δ 4.20) exhibited HMBC correlation with
oxymethylene carbon at δ 68.6 and vice versa. This observation
established the attachment of aliphatic chain with sugar moiety. Other
important COSY and HMBC correlations found in spectrum are shown
in figure 2.2.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
34
O
HO
OH
OH
OHO
21
70
CH3
Figure. 2.2: 1H-1H COSY ( ) and 1H-13C HMBC ( ) interactions observed in the spectra of 70.
The above discussed data led to the structure of 70 as (E)-2-
(hentriacont-27-enyloxy)-6-(hydroxymethyl)-tetrahydro-2H-pyran-
3',4',5'-triol.
Table. 2.2: 1H and 13C-NMR data of 70 (CD3OD; 500 and 125 MHz).
Position δH (J in Hz) δ c
1 3.99 (2H, t, 6.9) 68.6
2 1.70 (2H, m) 34.3
3-25 1.16-1.30 (46H, br., s) 29.6-25.8
26 1.90 (2H, m) 35.2
27 5.31 (1H, br., m, W1/2 = 20.8) 130.6 28 5.27 (1H, br., m, W1/2 = 20.8) 129.7
29 1.92 (2H, m) 31.8
30 1.30 (2H, m) 22.6
31 0.80 (3H, t, 6.6) 14.0
1' 4.20 (1H, d, 7.0) 102.9 2' 3.66 (1H, t, 9.0) 74.3
3' 3.16 (1H, m) 73.3
4' 3.95 (1H, dt, 9.0, 6.0) 72.2
5' 3.46 (1H, m) 76.2
6' 3.78 (1H, dd, 12, 2.6),
3,76 (1H, dd, 12, 6.6)
62.2
The sugar could be identified as -glucose due to acid hydrolysis
of 70 that provided two products, which were separated by solvent
extraction. The ethyl acetate layer contains hydrocarbon portion,
whereas, the glycone part was separated from the aqueous layer and
purified using preparative thin-layer chromatography (TLC) using a
solvent system of EtOAc:MeOH:H2O:AcOH (4:2:2:2) and the Rf value
was compared with the authentic sample. It was further confirmed due
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
35
to the value of optical rotation []D26 +50.2 (Mukhtar et al., 2004). The
combination of above spectroscopic data finally led to the structure of
70 as (E)-hentriacont-4-ene-1-O-β-D-glucoside, which we reported as a
new secondary metabolite and is named as vincetoside.
2.4.3. Characterization of Vincetetrol (71)
Compound 71 was also purified as white amorphous powder,
whose EIMS spectrum displayed molecular ion
peak at m/z 240, whereas, the molecular
formula C11H12O6 with six DBE was calculated
through HR-EIMS. The IR spectrum displayed
absorption bands at 3510 (O-H), 1760 (C=O)
1605 and 1570 (aromatic system) cm-1. Aromatic region of the 1H-NMR
spectrum (Table 2.3) showed signals for two singlet protons at δ 7.25
and 6.66 due to a 1,2,4,5-tetrasubstitited benzene ring. The spectrum
also displayed resonance due to a methoxyl proton (δ 3.84) and a
singlet signal of six hydrogen at δ 1.90, which was attributed to two
equivalent methyls of acetyl moieties.
The 13C-NMR (Table 2.3) displayed resonances of two methine
carbons at δ 105.2 and 104.5, one methoxy at δ 56.8, two methyl at δ
23.7 and five quaternary carbon atoms at δ 171.4, 149.7, 149.4, 138.2,
138.2. Based on the above data and HMBC correlations (Figure 2.3),
two structures a and b (Figure 2.3) were proposed for compound 71.
H3CO
O
O
OH
O CH3
71
1
2 4
5
OH3C
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
36
H3CO
O
O
OH
O CH3
a
H3CO
HO
O
O
O CH3
b
CH3
O
OH3C
Figure. 2.3: 1H-13C HMBC correlations observed in compound 71.
However, structure “a” most fitted on the data as the 13C-NMR
experimental shifts were close to the theoretical shifts with the
positions of various substituents as in structure “a” (Figure 2.4).
H3CO
O
O
OH
O CH3
a
1
2 4
5
H3CO
HO
O
O
O CH3
b
1
2 4
5
CH3
O
OH3C
Figure. 2.4: Proposed structures a and b for compound 71, based on NMR data.
Combination of the experimental and theoretical observations,
finally led to the structure of compound as 71a, which is a new
metabolite and is named as vincetetrol.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
37
72
H3CO
NH2
1
3
5
7
4'
5'
6'
1'
2'
3'
14
2
6
4
2.4.4. Characterization of Vincetomine (72)
Compound 72 was isolated as white amorphous powder, which
exhibited absorption bands in IR spectrum for
primary amine at 3260, 1601 cm-1 and at
1605 and 1570 for aromatic system. The
EIMS spectrum showed molecular ion peak at
m/z 213, whereas, the molecular formula
C14H15NO with eight DBE, was established
through HR-EIMS (m/z 213.1254).
The 1H-NMR spectrum (Table 2.3) of compound 72 displayed the
most downfield signal at 8.49 (2H, s), which was attested for an
amino group. In the aromatic region other resonances at δ 7.39 (1H, J =
8.6 Hz), 7.32 (1H, d, J = 8.5), 7.10 (1H, d, J = 8.6 Hz) and 7.08 (1H, d, J
= 8.5 Hz) were observed for aromatic systems. The A2B2 splitting
pattern of these signals was attributed to two p-substituted benzene
rings in 72. In addition, the 1H-NMR spectrum displayed a signal due
to methylene proton at δ 3.78 (s) and methoxyl proton at δ 3.62.
The 13C-NMR spectrum (Table 2.3) of 72 showed nine carbon
signals, which were identified as three methine (δ 128.8, 118.2 and
116.4), one methylene (δ 39.9), one methyl (δ 51.4) and four quaternary
carbon atoms (δ 153.9, 137.6, 135.5, 134.8). The NMR shifts (δH 3.78
and δC 39.9) of methylene group indicated its presence between two
aromatic systems, which was substantiated through HMBC experiment
(Figure 2.5), in which methylene proton exhibited HMBC correlations
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
38
with the aromatic carbons at 135.5 (C-1), 134.8 (C-1'), 128.8 (C-2, 6,
3', 5'). The position of methoxyl group was fixed due to its HMBC
correlation with the aromatic at 153.9, whereas, amino proton
displayed its HMBC correlation with the carbon signals resonated at
137.6 and thus was placed at C-4'.
H3CO
NH2
72
Figure. 2.5: 1H-1H COSY ( ) and 1H-13H HMBC ( ) interactions
observed in the spectra of 72.
Combination of the above spectroscopic data helped to establish
structure of 72 as 4-(4-methoxybenzyl) benzenamine, which is named
as vincetomine.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
39
Table. 2.3: 1H and 13C-NMR data of 71 (CD3OD; 500 and 125 MHz) and 72
(CD3OD; 600 and 150 MHz)
71 72
Position δH (J in Hz) δC δH (J in Hz) δC
1 - 138.2 - 135.5
2 - 149.7 7.39 (1H, d, 8.6) 128.8
3 7.25 (1H, s) 104.5 7.10 (1H, d, 8.6) 116.4
4 - 138.2 - 153.9
5 - 149.4 7.10 (1H, d, 8.6) 116.4
6 6.66 (1H, s) 105.2 7.39 (1H, d, 8.6) 128.8 7 - 171.4 3.78 (2H, s) 39.9
8 - 171.4 - -
1 - - - 134.8
2 - - 7.08 (1H, d, 8.5) 118.2
3 - - 7.32 (1H, d, 8.5) 128.8
4 - - - 137.6
5 - - 7.32 (1H, d, 8.5) 128.8
6 - - 7.08 (1H, d, 8.5) 118.2
14-OMe - - 3.62 (3H, s, OMe) 51.4
1-NH2 - - 8.49 (1H, s) -
3-OMe 3.84 (3H, s, OMe) 56.8 - -
7, 8-Me 1.90 (3H, s, CH3) 23.7 - -
2.4.5. Characterization of Apocynin (73)
Compound 73 was obtained as white amorphous powder, which
showed the molecular ion peak in EIMS spectrum
at m/z 166, whereas, the molecular formula
C9H10O3 with five DBE was calculated through
HR-EIMS (m/z 166.1052 [M]+). The IR spectrum
of 73 displayed prominent absorption bands at 1705, 1590 and 1565
cm-1 for carbonyl function and aromatic moiety respectively.
In the aromatic region, the 1H-NMR spectrum of 73 (Table 2.4)
showed three resonances at δ 7.14 (1H, dd, J = 8.4, 2.0 Hz), 7.24 (1H,
d, J = 2.0 Hz) and 6.50 (1H, d, J = 8.4 Hz). This ABX splitting pattern
revealed a tri-substituted benzene ring in 73. In addition, signals for
two singlet methyls appeared at δ 3.93 and 2.54. The chemical shifts of
HO
OCH3
CH3
O
73
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
40
HO
OCH3
O
74
OH
these signals depicted the acetyl and methoxyl nature of the two
methyls respectively.
The 13C-NMR spectrum (Table 2.4) displayed 9 carbon signals,
which were identified as two methyl (δ 26.2, 56.0), three methine, (δ
124.0, 113.7, 109.6) and four quaternary carbons (δ 195.0, 149.0,
145.0, 129.0) due to DEPT experiment. This data led to the idea for the
structure of compound 73 as derivative of acetophenone, which was
finally confirmed through HMBC analysis of 73, in which the aromatic
proton at δ 7.24 and 7.14 showed HMBC interaction with carbonyl
carbon at δ 195.0) and acetyl methyl displayed HMBC correlation with
aromatic carbon at 129.0. This information helped to fix the position
of acetyl moiety. The methoxyl proton at δ 3.93 shows HMBC with the
aromatic carbon at δ 145.0 and hence was placed at C-3. Combination
of the whole spectroscopic data and comparison with the reported data
compound 73 was identified 1-(4-hydroxy-3-methoxyphenyl) ethanone,
which is known as apocynin, a previously reported phytochemical (Kim
et al., 2011).
2.4.6. Characterization of 4-Hydroxy-3-methoxyphenyl-1-(9-
hydroxy-propan-7-one) (74)
Compound 74 was isolated as white amorphous powder, whose
EIMS spectrum showed the molecular ion
peak at m/z 196, whereas, the HR-EIMS
analysis (m/z 196.2061 [M]+) depicted the
molecular formula C10H12O4 with five DBE.
The IR spectrum showed absorption bands at 1700 cm-1 due to a
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
41
conjugated ketonic function and bands at 3460, 1610 and 1560 cm-1
were attested for hydroxyl group and aromatic ring respectively.
Three proton signals in the aromatic region of the 1H-NMR
spectrum (Table 2.4) of 74 appeared as ABX system at δ 7.58 (1H, dd, J
= 8.0, 2.0 Hz), 7.54 (1H, d, J = 2.0 Hz) and δ 6.86 (1H, d, J = 8.0 Hz)
and were attributed to a tri-substituted benzene ring. Further, the
resonances due to two methylene were observed at δ 3.94 (t, J = 6.0 Hz)
and 3.16 (t, J = 6.0 Hz) which were found to correlate with each other
in COSY spectrum. The same spectrum also afforded signals for a
methoxyl proton at 3.93.
The 13C-NMR data (Table 2.4) showed total 10 signals, three
methine at δ 124.7, 115.7 and 111.8, two methylene at δ 58.8 and
41.6, four quaternary carbons at δ 199.6, 153.3, 149.0 and 130.6 and
a methoxyl carbon at 56.6.
Table. 2.4: 1H and 13C-NMR data of 73 and 74 (CD3OD; 500 and 125 MHz).
73 74
Position δH (J in Hz) δc δH (J in Hz) δc
1 - 129.0 - 130.6
2 7.24 (1H, d, 2.0) 109.6 7.54 (1H, d, 2.0) 111.8 3 - 145.0 - 149.0
4 - 149.0 - 153.3
5 6.93 (1H, d, 8.4) 113.7 6.86 (1H, d, 8.0) 115.7
6 7.14 (1H, dd, 8.4, 2.0) 124.0 7.58 (1H, dd, 8.0, 2.0) 124.7
7 - 195.0 - 199.6 8 - - 3.16 (2H, t, 6.0) 41.6
9 - - 3.94 (2H, t, 6.0) 58.8
3-OMe 3.93 (3H, s, OMe) 56.0 3.89 (3H, s, OMe) 56.6
7-Me 2.54 (3H, s, CH3) 26.2 - -
Various connectivities were established through HSQC, COSY
and HMBC (Figure 2.6) experiments and finally compound 74 was
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
42
identified as 4-hydroxy-3-methoxyphenyl-1-(9-hydroxy-propan-7-one)
which is also a known metabolite (Kim et al., 2011) but has been
isolated for the first time from our investigated source.
HO
OCH3
O
74
OH
Figure. 2.6: 1H-1H COSY ( ) and 1H-13H HMBC ( ) interactions observed in the spectra of 74.
2.4.7. Characterization of 4-Hydroxy-3-methoxyphenyl-7,8,9- Propanetriol (75)
Compound 75 was also obtained as white amorphous powder
and was found to be the derivative of 74. The EIMS spectrum of 75
exhibited the molecular ion peak at m/z 214,
the molecular formula C10H14O5 with four DBE
could be determined through HR-EIMS. The IR
spectrum missed the absorption band for a
ketonic function as was observed in the spectrum of 74, however, the
absorption bands for hydroxyl function and aromatic moiety were seen
nearly at the same positions. The 1H-NMR spectrum (Table 2.5) showed
the similar signals in aromatic region as were observed for compound
74, with the difference that the spectrum showed two oxymethine
signals at δ 4.51 (1H, d, J = 6.0 Hz) and δ 3.72 (1H, q, J = 5.8). Both
the methines showed mutual correlation in the COSY spectrum,
whereas, the oxymethine at δ 3.72 was further correlated with an
oxymethylene at δ 3.58 (1H, dd, J = 11.4, 7.2) and 3.47 (1H, dd, J =
OH
OH
OH
HO
OCH3
75
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
43
11.4, 4.2). This data indicated that ketonic group in 74 has been
reduced to an alocoholic function in 75. This deduction was
substantiated through the 13C-NMR spectrum (Table 2.5), which
showed five signals for methine carbons at δ 121.0, 115.8, 111.8, 77.6
and 75.0, one signal for oxymethylene carbon at δ 64.5, three
quaternary carbon at δ 148.8, 147.1 and 134.8 and one signal for
methoxyl carbon at 56.6. This data confirmed that ketonic function in
74 has been reduced to secondary alcohol in 75, whereas, one
methylene in 74 has been oxidized to another hydroxyl function. Based
on these observations, compound 75 was finally characterized as 4-
hydroxy-3-methoxyphenyl-7,8,9-propanetriol which is also a known
phytochemical (Dellagreca et al., 1998; Warashina et al., 2005) but has
been purified for the first time from Vincetoxicum stocksii.
2.4.8. Characterization of Feruloyl-6'-O--D-glucopyranoside (76)
Compound 76 showed characteristic IR absorption bands at
3442, 1710, 1655, 1608 and 1540
for hydroxyl function, carbonyl
group, olefinic system and aromatic
moiety respectively. The EIMS
spectrum of 76 showed the molecular ion peak at m/z 356, while the
HR-EIMS analysis of the same ion peak (m/z 356.1646) depicted the
molecular formula C16H20O9 with seven DBE.
The 1H-NMR (Table 2.5) showed few similar signals splitted at the
same ABX pattern in aromatic region as was observed for 74, in
O
OCH3
HOO
HOHO
OHOH
O
76
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
44
addition two doublets resonated at δ 7.63 (1H, d, J = 16.0 Hz) and δ
6.41 (1H, d, J = 16.0 Hz) were attested for a trans double bond. This
observation indicated the caffeoyl-derived molecular nature of 76. The
spectrum also displayed a doublet due to an anomeric proton at δ 5.10
(1H, J = 7.0 Hz), which was attributed to a β-hexose moiety. The other
signals of sugar moiety displayed their positions at δ 3.95 (1H, d, J =
6.5), 3.74 (1H, dd, J = 13.0, 4.9 Hz), 3.66 (1H, dd, J = 13.0, 2.6 Hz),
3.56 (1H, t, J = 7.0 Hz), 3.46 (1H, m), 3.16 (1H, m), whereas, a
resonance due to methoxyl proton was seen at 3.89.
The 13C-NMR spectrum (Table 2.5) showed total 16 signals which
were distinguished as ten methine (δ 146.9, 124.5, 116.9, 115.6, 111.7,
98.2, 78.3, 77.6, 75.5, 72.0), one methylene (δ 64.6) and four
quaternary carbon (δ 169.1, 150.6, 147.3 and 127.7) based on the
DEPT experiment. Various positions of functional groups were carefully
identified and connected through HMBC and COSY correlations. The
methylene 3.74 and 3.66 of sugar moiety showed the HMBC
correlation with the carbonyl carbon at δ 169.1 (C-9), which confirmed
the aglycon and glycon linkage at C-6 of sugar moiety instead of at C-1.
This observation resolved the issue of most of the double signals in
NMR spectra that anomerization in solution form was responsible for
the resonances of all double signals. However, the sugar moiety was
finally identified as glucose due to acid hydrolysis of 76 and its
comparison with the standard sample of sugar as is described in
compound 70. On the base of above discussion and comparison with
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
45
77
H3CCH3
CH3
OHOH
CH3
OH
HO
the reported data the compound 76 was identified as feruloyl-6-O--D-
glucopyranoside, which is a known phytochemical (Dallacqua and
Innocenti 2004), however, has been isolated first time from our
investigated source.
Table. 2.5: 1H and 13C-NMR data of 75 (CD3OD; 600 and 150 MHz) and 76
(CD3OD; 400 and 100 MHz). 75 76
Position δH (J in Hz) δc δH(J in Hz) δc
1 - 134.8 - 127.7
2 6.99 (1H, d, 2.0) 111.8 7.17 (1H, d, 2.0) 111.7
3 - 148.8 - 147.3
4 - 147.1 - 150.6
5 6.74 (1H, d, 8.0) 115.8 6.85 (1H, d, 8.0) 116.9
6 6.81 (1H, dd, 8.0, 2.0) 121.0 7.06 (1H, d, 8.0, 2.0) 124.5
7 4.51 (1H, d, 6.0) 75.0 7.63 (1H, d, 16.0) 146.9
8 3.72 (1H, q, 5.8) 77.6 6.41 (1H, d, 16.0) 115.6
9 3.58 (1H, dd, 11.4, 7.2),
3.47 (1H, dd, 11.4, 4.2)
64.5 - 169.1
3-OMe 3.89 (3H, s, OMe) 56.6 3.92 (3H, s, OMe) 56.8
1' 5.10 (1H, d, 7.0) 98.2
2' 3.56 (1H, t, 7.0) 75.5
3' 3.16 (1H, m) 72.0
4' 3.95 (1H, d, 6.5) 77.6
5' 3.46 (1H, m) 78.3
6' 3.74 (1H, dd, 13, 4.9),
3.66 (1H, dd, 13, 2.6)
64.6
2.4.9. Characterization of 7-Megastigmene-2,3,5,9-tetrol (77)
Compound 77 was obtained as white
amorphous solid, which exhibited diagnostic
IR bands at 3350 and 1655 cm-1 due to
hydroxyl function and double bond
respectively. The EIMS spectrum showed the
molecular ion peak at m/z 244, whereas, the molecular formula
C13H24O4 with two DBE was calculated through HR-EIMS (m/z
244.1742 [M]+).
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
46
The 1H-NMR spectrum (Table 2.6) showed the most down field
signals at δ 6.07 (1H, d, J = 15.6 Hz) and 5.80 (1H, dd, J = 15.6, 6.4
Hz) attested for a trans-olefin. The spectrum also showed signals for
two oxymethine protons at δ 4.34 (1H, m), 4.07 (1H, m). The first
oxymethine signal was correlated in COSY spectrum with two
methylene δ 1.75 (2H, br., d) and 1.63 (1H, dd, J = 14.4, 6.0 Hz) and
1.44 (1H, dd, J = 14.4, 3.6 Hz), whereas, other showed correlation with
a secondary methyl at δ 1.25 (d, J = 6.6 Hz). In addition, signals for
three tertiary methyls were see at δ 1.12, 1.21 and 0.83.
The 13C-NMR (Table 2.6) showed 13 signals, which were identified
as four methine (δ 136.1, 131.2, 69.5 and 65.2), two methylene (δ 46.4
and 45.7), four methyl (δ 27.5, 27.0, 26.2 and 24.1) and three
quaternary carbons (δ 79.4, 78.0 and 40.0). This data led to the
tentative structure of 77 as an ionol derivative (Murai et al., 1992;
Otsukaa et al., 2003). However, the structure was finally confirmed
through HMBC experiment (Figure 2.7), in which two tertiary methyls (
1.21 and 0.83) were correlated with each other and with other two
common carbons ( 78.0, 65.2 and 40.0) indicating their geminal
nature and presence at C-1. The secondary methyl was correlated in
HMBC spectrum with the oxymethine carbon at 69.5 (C-9) and
olefinic methine carbon at 136.1 (C-8). This observation helped to
identify the position of double bond. The tertiary methyl ( 1.12)
exhibited HMBC correlations with two oxygenated quaternary carbons
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
47
OH
O
OH
78
at 79.4 and a methylene at 45.7. This analysis fixed the position of
two hydroxyl groups at C-2 and C-3.
H3CCH3
CH3
OHOH
CH3
OH
HO
77
Figure. 2.7: 1H-1H COSY ( ) and 1H-13H HMBC ( ) interactions observed in the spectra of 77.
Combination of above spectroscopic data led to structure of 77 as
7-megastigmene-2,3,5,9-tetrol, which is a known metabolite and has
been isolated for the first time from this plant (Murai et al., 1992;
Otsukaa et al., 2003).
2.4.10. Characterization of 4-Hydroxyphenylacetic Acid (78)
Compound 78 was obtained as white crystalline
solid, which exhibited a broad absorption band at
2420-3410 cm-1 due to chelated hydroxyl of a
carboxylic acid function, with carbonyl carbon
absorbed at 1720 cm-1. The HR-EIMS spectrum showed
the molecular ion peak at m/z 152.4216 corresponding to the
molecular formula C8H8O3 with five DBE.
Aromatic region of the 1H-NMR (Table 2.6) showed two signals at
δ 7.08 (2H, d, J = 8.4 Hz) and 6.08 (2H, d, J = 8.4 Hz), which were
attributed to a p-substituted benzene ring. In addition, the spectrum
displayed resonance of a singlet methylene at δ 3.47.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
48
The 13C-NMR (Table 2.6) fully supported the mass and 1H-NMR
data as it showed total 8 signals at δ 176.2, 157.4, 131.3, 126.8, 116.2
and 41.1. The HMBC correlations (Figure 2.8) of methylene at δ 3.47
with the carbons at δ 176.2, 131.3 and 126.8 confirmed its presence
between aromatic system and carbonyl carbon. Also the HMBC
interaction of an aromatic proton at δ 7.08 with the methylene carbon
at δ 41.1 substantiated the above deduction.
OH
O
OH
78
Figure. 2.8: Important 1H-13H HMBC ( ) interactions observed in the spectra of 78.
Based on the above data, the compound 78 was identified as 4-
hydroxyphenylacetic acid, which is a known metabolite (Hareland et al.,
1975) but has been isolated for the first time from this plant.
2.4.11. Characterization of 4-Hydroxy-3,5-dimethoxybenzoic Acid
(79)
The IR spectrum of 79 was also an indicative of its carboxylic
acid nature as it showed absorption bands at
2410-3400 and 1705 cm-1 due to carboxylic acid
function. Other characteristic absorptions were
observed at 1605 and 1570 cm-1 due to an
aromatic system. The EIMS of 79 showed molecular ion peak at m/z
OH
O
HO
H3CO
OCH3
79
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
49
198, whereas, the HR-EIMS (m/z 198.2134) depicted the molecular
formula as C9H10O5 with five DBE. The 1H-NMR spectrum of 79 (Table
2.6) displayed only two singlets at δ 7.32 and 3.88 corresponding to an
aromatic system and methoxyl units. The 13C-NMR (Table 2.6) showed
total seven signals appeared at δ 170.3, 148.8, 142.2, 122.4, 108.4 and
56.8. This data indicated that compound 79 was derivative of gallic
acid, therefore the whole data was compared with the reported values
(Tung et al., 2007) and the compound was finally identified as 4-
hydroxy-3,5-dimethoxybenzoic acid, which has been reported for the
first time from our investigated source.
Table. 2.6: 1H and 13C-NMR data of 77 (CD3OD; 600 and 150 MHz), 78 (CD3OD;
400 and 100 MHz) and 79 (CD3OD; 500 and125 MHz).
77 78 79
Position δH (J in Hz) δc δH(J in Hz) δc δH(J in Hz) δc
1 - 40.0 - 126.8 - 122.4
2 - 78.0 7.08 (1H, d, 8.4) 131.3 7.32 (1H, s) 108.4
3 - 79.4 6.08 (1H, d, 8.4) 116.2 - 148.8
4 1.75 (2H, br., d) 45.7 - 157.4 - 142.2 5 4.07 (1H, m) 65.2 6.08 (1H, d, 8.4) 116.2 - 148.8
6 1.63 (1H, dd, 14.4, 6.0),
1.44 (1H, dd, 14.4, 3.6)
46.4 7.08 (1H, d, 8.4) 131.3 7.32 (1H, s) 108.4
7 6.07 (1H, d, 15.6) 131.2 3.47 (2H, s) 41.1 - 170.3
8 5.80 (1H, dd, 15.6, 6.4) 136.1 - 176.2 9 4.34 (1H, m) 69.5
10-Me 1.25 (3H, d, 6.6, CH3) 24.1
11-Me 1.12 (3H, s, CH3) 27.0
12-Me 1.21 (3H, s, CH3) 26.2
13-Me 0.83 (3H, s, CH3) 27.5
3,5-OMe
3.88 (6H, s, 2OMe)
56.8
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
50
2.4.12. Characterization of 4'-Methoxy Kaempferol-3-O--D-
glucopyranoside (80)
Compound 80 was obtained as yellowish amorphous powder,
which exhibited absorption maxima
at 236, 257, 245, 314 and 357 nm
as a characteristic pattern of
kaempferol or quercetin glycoside
(Mabry et al., 1970; Saleh et al.,
1990). The IR spectral analysis revealed hydroxyl function and aromatic
system, while the molecular formula C22H23O11 was calculated through
HR-FAB-MS (Positive mode) due to a pseudo-molecular ion peak at m/z
463.4123.
The 1H-NMR (Table 2.7) showed four signals in the aromatic
region in which two pairs of ortho coupled protons resonated at δ 8.05
(2H, d, J = 8.5 Hz) and 6.89 (2H, d, J = 8.5 Hz) and two pairs of meta
coupled protons appeared at δ 6.23 (1H, d, J = 2.0 Hz) and 6.20 (1H, d,
J = 2.0 Hz). The same spectrum also displayed resonance due to an
anomeric proton at δ 5.25 (1H, d, J = 8.4 Hz) with other sugar protons
displayed their positions at δ 3.60 (1H, m), 3.53 (2H, dd, J = 10.5, 4.5
Hz), 3.41 (1H, d, J = 6.6 Hz), 3.40 (1H, m), 3.35 (1H, dd, J = 10.5, 2.9
Hz) and 3.20 (1H, m). A signal of three hydrogen at δ 3.62 (s) was
attributed to a methoxyl group.
The 13C-NMR (Table 2.7) of compound 80 showed 20 carbon
signals, which were attested for 22 carbons. Aromatic methine carbon
O
OH
HO
O
OCH3
OOH
OHHO
O
OH
80
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
51
signals were observed at δ 132.2, 116.0, 99.9 and 94.7, while the sugar
carbons displayed their positions at δ 104.1, 78.4, 78.0, 75.7, 71.4,
63.0. The methoxyl carbons resonated at δ 51.4. The remaining nine
quaternary carbons displayed their positions at δ 179.1, 165.0, 162.0,
159.8, 159.0, 158.0, 134.0, 124.0 and 105.0. This data was
superimposable to the data reported for 4'-methoxy-kaempferol-3-O--
D-glucopyranoside, thus compound 80 was found to be the same
(Calderón-Montañ et al., 2011).
2.4.13. Characterization of 4'-Methoxy Kaempferol-3-O--L-rhamnopyranoside (81)
The IR and UV data of compound 81 was identical to that of
compound 80 as an indication of
flavonoid glycoside nature of this
compound. The molecular formula
C23H25O10 was calculated through
positive HR-FAB-MS, which exhibited
pseudo-molecular ion peak at m/z 447.4308.
The 1H-NMR data (Table 2.7) of 81 was also identical to that of
80 with the only difference that in compound 80, the sugar was found
to be glucose, whereas, in 81, rhamnose was observed due to the
resonance of a doublet methyl at δ 1.23 (J = 7.2 Hz). The anomeric
proton appeared as a broad singlet to support -L-rhamnoside.
The acid hydrolysis of 81 provided two products, which were
separated by solvent extraction. The EtOAc layer contains aglycone part
O
OH
HO
O
OCH3
81
OOH
OH
O
CH3
HO
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
52
and water soluble glycone part was separated and purified by using
preparative thin layer chromatography developing at solvent system
EtOAc:MeOH:H2O:AcOH (4:2:2:2). It was identified as -rhamnose due
to comparison of the retention times of their trimethylsilyl (TMS) with
that of the standards in gas chromatography (GC) and their optical
rotation values [α]D23 +7.7˚ (c, 0.2 in H2O) (Liocharova et al., 1989).
Finally the data was compared with the literature values and 81 was
identified as 4'-methoxy-kaempferol-3-O--L-rhamnopyranoside
(Calderon-Montan et al., 2011).
Table. 2.7: 1H and 13C-NMR data of 80 (CD3OD; 500 and 125 MHz), 81
(CD3OD; 600 and 150 MHz). 80 81
Position δH(J in Hz) δc δH(J in Hz) δc
1 - - - -
2 - 158.0 - 159.1 3 - 134.0 - 135.3
4 - 179.1 - 179.1
4a - 105.0 - 105.1
5 159.8 163.2
6 6.20 (1H, d, 2.0) 99.9 6.18 (1H, d, 2.0) 100.1 7 - 165.0 - 165.8
8 6.23 (1H, d, 2.0) 94.7 6.35 (1H, d, 2.0) 94.9
8a - 159.0 - 158.2
1' - 124.0 - 122.1
2' 8.05 (1H, d, 8.5) 132.2 7.76 (1H, d, 8.4) 131.8
3' 6.89 (1H, d, 8.5) 116.0 6.93 (1H, d, 8.4) 116.5 4' - 162.0 - 162.9
5' 6.89 (1H, d, 8.5) 116.0 6.93 (1H, d, 8.4) 116.5
6' 8.05 (1H, d, 8.5) 132.2 7.76 (1H, d, 8.4) 131.8
1'' 5.25 (1H, d, 8.4) 104.1 5.34 (1H, br., s) 103.5
2'' 3.20 (1H, m) 78.4 3.31 (1H, m) 71.9 3'' 3.40 (1H, m) 75.7 3.71 (1H, m) 72.1
4'' 3.41 (1H, d, 6.6) 71.4 3.33(1H, m) 73.2
5'' 3.60 (1H, m) 78.0 3.38 (1H, m) 72.0
6'' 3.53 (1H, dd, 10.5, 4.5),
3.35 (1H, dd, 10.5, 2.9)
63.0 1.23 (1H, d, 7.2) 17.6
4'-OMe 3.62 (3H, s, OMe) 51.4 3.93 (3H, s, OMe) 56.4
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
53
O
OH
HO
O
OH
82
OOH
OH
O
CH3
HO
OH
2.4.14. Characterization of Quercetin-3-O--L-rhamnopyranoside
(82)
Compound 82 was also obtained as yellowish amorphous
powder, whose molecular formula
C22H25O11 was calculated through
positive HR-FAB-MS (m/z 449.3816
[M+H]+). The IR and UV data was
identical to that of compounds 80 and
81 telling the same nature of 82.
The aromatic region of the 1H-NMR spectrum (Table 2.8) of 82
showed five signals at 7.32 (1H, d, J = 2.4 Hz), 7.30 (1H, dd, J = 8.5,
2.4 Hz), 6.90 (1H, d, J = 8.5 Hz), 6.35 (1H, d, J = 1.8 Hz) and 6.18 (1H,
d, J = 1.8 Hz). First three signals splitted at ABX pattern were
attributed to ring B of flavonoid skeleton, while others were attested for
ring A. This information revealed that compound 82 must have a
quercetin moiety as aglycon part, whereas, the sugar was characterized
as rhamnose due to the resonance of anomeric proton at δ 5.34 (1H, s)
and methyl doublet at δ 0.94 (d, J = 6.8 Hz). The spectrum showed four
signals of oxymethine were observed at δ 4.21(1H, br, s), δ 3.75 (1H,
dd, J = 9.0, 4.5 Hz), δ 3.34 (1H, m) and a doublet was present at δ 3.41
(1H, d, J = 6.0 Hz). The 13C-NMR spectrum (Table 2.8) supported the
above information as it showed 23 carbon signals at δ 179.6, 165.8,
163.2, 159.3, 158.9, 149.7, 146.4, 136.2, 123.0, 122.9, 117.0, 116.4,
105.9, 103.5, 99.8, 94.7, 73.3, 72.1, 72.0, 71.9 and 18.2. The whole
data was identical to that of reported for qurcetain-3-O--L-
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
54
rhamnopyranoside (Poumale et al., 2008), therefore, compound 82 was
found to be the same.
2.4.15. Characterization of 4'-Methoxy Quercetin-3-O--L-
rhamnopyranoside (83)
Compound 83 was found to be 4'-methoxy-derivative of 82 as it
displayed similar signals in NMR spectra
(Table 2.8) with the additional signal of
methoxyl group (δH 3.92 and δC 56.3),
which was further confirmed due to the
determination of molecular formula as
C22H23O11 based on the positive HR-FAB-MS, (m/z 463.4261 [M+H]+).
It was also a known phytochemical (Bolzani et al., 1996) but has
been isolated for the first time from our investigated source.
O
OH
HO
O
OCH3
83
OOH
OH
O
CH3
HO
OH
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
55
Table. 2.8: 1H and 13C-NMR data of 82 and 83 (CD3OD; 600 and 150 MHz).
82 83
Position δH (J in Hz) δc δH (J in Hz) δc
1 - - - -
2 - 159.3 - 159.3
3 - 136.2 - 136.2
4 - 179.6 - 179.6 4a - 105.9 - 105.9
5 163.2 - 163.2
6 6.18 (1H, d, 1.8) 99.8 6.19 (1H, d, 1.8) 99.8
7 - 165.8 - 165.9
8 6.35 (1H, d, 1.8) 94.7 6.36 (1H, d, 1.8) 94.7
8a - 158.9 - 158.5 1' - 123.0 - 122.9
2' 7.32 (1H, d, 2.4) 117.0 7.33 (1H, d, 2.0) 116.9
3' - 146.4 - 147.6
4' - 149.7 - 151.6
5' 6.90 (1H, d, 8.5) 116.4 6.90 (1H, d, 8.4) 116.3 6' 7.30 (1H, dd, 8.5, 2.4) 122.9 7.30 (1H, dd, 8.4, 2.0) 122.8
1'' 5.34 (1H, s) 103.5 5.34 (1H, s) 103.5
2'' 4.21 (1H, br., s) 71.9 4.11 (1H, br., s) 71.9
3'' 3.75 (1H, dd, 9.0, 4.5) 72.1 3.73 (1H, dd, 9.3, 4.2) 72.1
4'' 3.34 (1H, m) 73.3 3.30 (1H, m) 73.2
5'' 3.41 (1H, d, 6.0) 72.0 3.40 (1H, d, 5.8) 72.0 6'' 0.94 (1H, d, 6.8, CH3) 18.2 0.93 (1H, d, 6.4) 17.6
4'-OMe - - 3.92 (3H, s, OMe) 56.3
2.4.16. Characterization of Quercetin-3-O--D- glucopyranoside (84)
and Quercetin-3-O--D- galactopyranoside (85)
Compounds 84 and 85 were also found to be the glycosides of
quercetin as their NMR spectra (Table 2.9) displayed identical signals to
that of 82 and 83. The difference was that compound 84 was found to
be quercetin-3-O-glucoside, whereas, compound 85 was characterized
as quercetin-3-O-galactoside due to their hydrolysis and comparison of
the physic-chemical data of sugar moieties. Both the metabolites have
already been isolated from various natural sources (Islam et al., 2012;
Arora et al., 2005).
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
56
O
OH
HO
O
OH
85
OH
O
OH
OHHO
O
HO
O
OH
HO
O
OH
OOH
OHHO
O
OH84
OH
Table. 2.9: 1H and 13C-NMR data of 84 and 85 (CD3OD; 400 and 100 MHz).
84 85
Position δH(J in Hz) δc δH(J in Hz) δc
1 - - - -
2 - 159.0 - 159.0 3 - 135.6 - 135.6
4 - 179.6 - 179.5
4a - 105.7 - 105.4
5 162.9 163.0
6 6.18 (1H, d, 2.0) 99.9 6.39 (1H, d, 2.0) 99.9
7 - 165.9 - 166.1 8 6.37 (1H, d, 2.0) 94.7 6.20 (1H, d, 2.0) 94.7
8a - 158.8 - 158.5
1' - 123.2 - 122.9
2' 7.70 (1H, d, 2.4) 117.6 7.70 (1H, d, 2.4) 117.5
3' - 145.8 - 145.9 4' - 149.8 - 149.8
5' 6.86 (1H, d, 8.5) 116.0 6.87 (1H, d, 8.8) 116.0
6' 7.57 (1H, dd, 8.5, 2.4) 122.8 7.59 (1H, dd, 8.8, 2.4) 123.1
1'' 5.23 (1H, d, 7.6) 104.3 5.14 (1H, d, 7.6) 104.3
2'' 3.47 (1H, m) 75.1 3.47 (1H, m) 75.7
3'' 3.34 (1H, m 71.2 3.34 (1H, m) 71.2
4'' 3.40 (1H, d, 6.0) 78.1 3.41 (1H, d, 2.9) 78.1 5'' 3.33 (1H, m) 78.3 3.33 (1H, m) 78.4
6'' 3.64 (1H, dd, 12.0, 4.8)
3.36 (1H, dd, 12.0, 2.9)
62.5 3.64 (1H, dd, 12.0, 4.8)
3.36 (1H, dd, 12.0, 2.9)
62.5
2.4.17. Characterization of 7-Hydroxy-3',4',5-trimethoxyflavone (86)
Compound 86 was also found to
be a flavonoid as it was obtained as
yellowish amorphous powder, which
exhibited molecular ion peak in the
EIMS spectrum at m/z 328. The
O
OMe
OOMe
HOOMe
86
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
57
molecular formula C18H16O6 was calculated through HR-EIMS (m/z
328.3257 [M]+).
The 1H-NMR spectrum of compound 86 (Table 2.10) showed
similar signals in aromatic region as were observed for quercetin
derivatives, however, an additional singlet was observed at δ 7.25
attested for H-3 of flavones nucleus. However, sugar signals were
missing, instead three methoxyl groups appeared at δ 4.06, 3.97 and
3.94. The 13C-NMR spectrum (Table 2.10) substantiated the above
information as it displayed signals for a flavone nucleus and three
methoxyl groups. The positions of the three methoxyl groups were fixed
due to their HMBC correlations with the quaternary carbons at δ 149.5
(C-4'), 149.0 (C-3') of ring B and 164.0 (C-5) of ring A. Further, this
data was closely similar to the reported data for 7-hydroxy-3',4',5-
trimethoxyflavone, which is a known phytochemical (Lu et al., 2012),
therefore, compound 86 was found to be the same, which has been
reported for the first time from Vincetoxicum stocksii.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
58
Table. 2.10: 1H and 13C-NMR data of 86 (CDCl3 500 and 125 MHz).
Position δH (J in Hz) δc
1 - -
2 - 160.0
3 7.25 (1H, s) 105.0
4 - 179.8 4a - 104.3
5 - 164.0
6 6.48 (1H, d, 2.0) 100.0
7 - 166.0
8 6.21 (1H, d, 2.0) 95.0
8a - 159.0 1' - 123.0
2' 7.51 (1H, d, 2.0) 120.2
3' - 149.0
4' - 149.5
5' 6.94 (1H, d, 8.5) 117.0 6' 7.01 (1H, dd, 8.5, 2.0) 113.0
5-OMe 4.06 (3H, s, OMe) 56.0
4'-OMe 3.94 (3H, s, OMe) 57.1
5'-OMe 3.97 (3H, s, OMe) 57.4
2.4.18. Characterization of β-sitosterol (87)
Compound 87 was purified as shining needles, which exhibited
absorption bands in IR spectrum at
3350 and 1655 cm-1 due to hydroxyl
function and olefinic system. The
molecular ion peak at m/z 414 was
observed in EIMS spectrum with other
characteristic fragment ions at m/z 399, 396, 381,329, 275, 273 and
255 for a ∆5-sitosterol nucleus (Kamboj and Saluja 2011).
The 1H-NMR spectrum (Table 2.11) of 87 showed three secondary
methyl signals at δ 0.95 (J = 6.1 Hz, 26-Me), 0.86 (J = 6.6 Hz, 27-Me)
and 0.91 (J = 6.0 Hz, 21-Me), two tertiary methyl singlets at δ 0.77 (18-
Me) and 0.94 (19-Me) and a primary methyl triplet at δ 0.86 (J = 7.3 Hz,
29-Me). The spectrum also displayed an olefinic proton resonance at δ
HO
87
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
59
5.35 (1H, d, J = 5.0 Hz) and an oxymethine at δ 4.56 (m) as
characteristic information for β-sitosterol.
The 13C-NMR spectrum (Table 2.11) of 87 was in complete
agreement with mass and 1H-NMR data. Along with this information
and its compariative TLC with standard sample of β-sitosterol,
compound 87 was identified as β-sitosterol (Kamboj and Saluja 2011).
Table. 2.11: 1H and 13C-NMR data of 87 (CD3OD; 500 and 125 MHz).
87
Position δ H (J in Hz) δ c Position δ H (J in Hz) δ c
1 1.26 (2H, m) 38.3 16 1.58 (2H, m) 28.4
2 1.77 (2H, m) 32.8 17 1.51 (1H, m) 55.2
3 4.45 (1H, m) 78.4 18 0.77 (3H, s, Me) 12.9
4 2.40 (2H, m) 44.4 19 0.94 (3H, s, Me) 19.8 5 - 142.2 20 0.11 (1H, m) 36.7
6 5.35 (1H, d, 5.0) 122.1 21 0.91 (3H, d, 6.0, Me) 19.4
7 1.91 (4H, m) 33.1 22 1.76 (2H, m) 34.5
8 - 32.6 23 1.29 (2H, m) 29.6
9 1.42 (1H, m) 50.4 24 1.36 (1H, m) 50.8 10 - 37.6 25 1.66 (1H, m) 27.2
11 1.97 (4H, m) 20.1 26 0.95 (3H, d, 6.1, Me) 18.4
12 1.13 (2H, m) 41.3 27 0.86 (3H, d, 6.6, Me) 19.6
13 - 43.6 28 1.17 (2H, m) 24.1
14 1.16 (1H, m) 57.8 29 0.86 (3H, t, 7.3, Me) 12.9
15 1.10 (2H, m) 25.3
2.4.19. Characterization of ß-sitosterol-3-O-D-glucopyranoside (88)
Compound 88 was isolated as a white amorphous powder, whose
EIMS showed the
molecular ion peak [M]+ at
m/z 576. The IR spectrum
exhibited absorption
bands at 3455 cm-1 (OH)
and 1615 cm-1 (C=C).
OHO
HOOH
O
OH
88
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
60
The 1H-NMR spectrum (Table 2.12) of 88 showed similar signals
as were observed in the spectrum of 87. However, addition signals were
seen due to a sugar moiety at δ 4.51 (1H, d, 7.8), 3.65 (1H, m, H-2'),
3.21 (1H, m, H-3'), 3.31 (1H, m, H-4'), 3.10 (1H, d, H-5') and methylene
at δ 3.12 (1H, m, H-6'), 3.52 (1H, m, H-6'). The 13C-NMR spectrum of 88
showed total 35 signals (Table 2.12), which were similar to the
spectrum observed for β-sitosterol (87). These observations and
comparision with the published values, compound 88 was found to be
β-sitosterol-3-O-β-D-glucopyranoside, which is a known phytochemical
(Akhtar et al., 2010).
Table. 2.12: 1H and 13C-NMR data of 88 (CD3OD; 500 and 125 MHz).
88
Position δ H (J in Hz) δ c Position δ H (J in Hz) δ c
1 1.24 (2H, m) 37.3 19 0.95 (3H, s, Me) 19.9 2 1.72 (2H, m) 31.8 20 0.10 (1H, m) 36.8
3 4.56 (1H, m) 79.4 21 0.99 (3H, d, 6.0, Me) 19.6
4 2.39 (2H, m) 44.4 22 1.73 (2H, m) 34.7
5 - 142.4 23 1.25 (2H, m) 29.8
6 5.35 (1H, d, 5.0) 121.6 24 1.33 (1H, m) 50.7
7 1.89 (4H, m) 34.1 25 1.69 (1H, m) 26.9 8 - 32.6 26 0.94 (3H, d, 6.1, Me) 18.5
9 1.39 (1H, m) 50.6 27 0.84 (3H, d, 6.6, Me) 19.7
10 - 37.1 28 1.16 (2H, m) 24.6
11 1.92 (4H, m) 20.5 29 0.87 (3H, t, 7.3, Me) 13.0
12 1.09 (2H, m) 41.6 1' 4.51 (1H, d, 7.8) 102.4 13 - 44.6 2' 3.65 (1H, m) 76.4
14 1.14 (1H, m) 58.8 3' 3.21 (1H, m) 73.4
15 1.08 (2H, m) 24.9 4' 3.31 (1H, m) 71.7 16 1.56 (2H, m) 28.6 5' 3.10 (1H, m) 76.0
17 1.49 (1H, m) 55.4 6' 3.12 (H, m), 3.52
(1H, m)
62.4
18 0.74 (3H, s, Me) 13.0
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
61
2.5. BIOLOGICAL STUDIES OF THE EXTRACT OF VINCETOXICUM STOCKSII AND THE COMPOUNDS PURIFIED FROM THE SAME
EXTRACT
The hexane and ethyl acetate soluble fractions of methanolic
extract were evaluated for antiurease, antioxidant and antimicrobial
activity. The ethyl acetate fraction was found significantly active (Table
2.13 and 2.14). Therefore, pure compounds isolated from ethyl acetate
fraction were also evaluated for their antiurease, antioxidant and
antimicrobial activities.
In an in vitro NO inhibition scavenging bioassay, the ethyl acetate
soluble fraction showed antioxidant and antiurease activities with IC50
values of 41±0.04 and 29±0.03 µg/ml (Table 2.13) respectively.
Therefore, the purified compounds which were obtained in sufficient
amounts 73, 75, 76, 79 and 84 were screened for NO inhibition
scavenging potential. These compounds exhibited significant inhibition
with IC50 values of 80.17±0.33, 150±0.5, 64.1±0.09, 56.42±0.04,
89.33±0.1 µg/ml respectively (Table 2.13). It was therefore, concluded
that the antioxidant activity of ethyl acetate fraction could be due to the
presence of these metabolites, especially apocynin (73), feruloyl-6-O--
D-glucopyranoside (76) and derivate of gallic acid (79), which have
already been reported as antioxidant in literature (Dallacqua and
Innocenti 2004; Tung et al., 2007).
Further, compounds 69, 70, 75, 77, 79, 80 and 84 were also
evaluated for antiurease activity, which showed moderate to significant
inhibition with IC50 values of 85.8±0.01, 105.47±0.01, 22.1±0.43,
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
62
74.8±0.04, 93.4±0.01, 23.9±0.51 and 88.6±0.01 µg/ml respectively
(Table 2.13). Remaining compounds were not evaluated for their
activity due to the low amount.
As part of Structure-Activity Relationship (SAR), it is observed
that a catechol or resorcinol-derived nucleus is required for antioxidant
activity. However, substitution of hydroxyl group with methoxyl moiety
causes a decrease in the activity. On the other hand, the metabolites
like derivatives of ferulic acid, a conjugated carboxylate function is also
important for the activity, which otherwise may decrease, as in case of
compounds 75 and 76.
For aniturease activity, an uncertain trend was observed. Among
69, 73, 75, 76 and 79, compound 75 exhibited significant activity
when compared to other related phenolics. This observation revealed
that a carboxylate or keto group is not necessary for the activity. In
case of flavonoids (80 and 84), catechol moiety seems insignificant and
however, a p-substituted aromatic ring B may play important role in
inhibiting the enzyme activity.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
63
Table. 2.13: Antioxidant and antiurease activity of the isolated compounds.
Sample code Antioxidant Antiurease
IC50 (ug/ml) IC50 (ug/ml)
Ethyl acetate fraction 41±0.04 29±0.03
69 Nil 85.8±0.01
70 Nil 105.47±0.01
73 80.17±0.33 Nil 75 150±0.5 22.1±0.43
76 64.1±0.09 Nil
77 Nil 74.8±0.04
79 56.42±0.04 93.4±0.01
80 Nil 23.9±0.51 84 89.33±0.1 88.6±0.01
Vit.C* 75.01±0.05 -
Thioure* - 23.3±0.01
*standard drug
All the measurements were done in triplicate and statistical analysis was performed by Microsoft Excel 2003.
Results are presented as mean sem.
Six of the isolated compounds; 69, 70, 73, 79, 84 and 85 were
further screened for their antibacterial activity. These compounds
showed weak activity (Table 2.14). When compared to the activity of the
crude ethyl acetate extract. The other compounds were not evaluated
for their activity because of low amount.
Table. 2.14: Antibacterial activity of isolated compounds.
Sample Code
%age activity at 100 ug/ml
Bacillus Staphlococ
cus E.coli Psedumonas shigella Salmonella
Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD Mean±SD
EtOAc-fraction
31 31 36 215 118 128
69 -2.73+0.03 7.94+0.002 15.44+ 0.03 5.42+0.002 9.51+0.0
05 15.02+0.000
7
70 1.34+0.01 2.95+0.007 8.23+0.01 7.83+0.007 25.16+0.
01 21.76+0.03
73 1.89+0.01 13.6+0.001 21.30+0.001 14.60+0.04 32.19+0 21.82+0.007
79
-5.00+0.032 10.0+0.002 6.42+ 0.003 6.75+0.03
10.0+70.001 12.77+0.004
84
-1.88+0.033 5.65+0.007 22.33+ 0.02 7.24+0.02 -3.76+0 9.59+0.002
85 -7.0+0.002 7.11+0.000
7 29.09+ 0.002 3.46+0.01 9.96+0.0
007 12.84+0.005
Streptomycin* 65+0.01 74+0.005 71+0.005 65+0.06 80+0.006 66+0.04
* standard drug
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
64
2.6. EXPERIMENTAL
2.6.1. General Experimental Procedures
Column chromatography (CC) was performed with silica gel (230-
400 mesh, E-Merck). Various fractions obtained from CC were monitored
through TLC on aluminum sheets precoated with silica gel 60 F254 (20×20
cm, 0.2 mm thickness; E-Merck). Further, the fractions and pure
compounds were also monitored under UV lamp (254 and 366 nm) and by
spraying with ceric sulfate followed by heating.
The optical rotation values were measured on JASCO DIP-360
polarimeter. The UV spectra were recorded in ethanol on JASCO V-650
spectrophotometer (max in nm). IR spectra were measured on Shimadzu
460 spectrometer (Duisburg, Germany). The 1H and 2D NMR (HMQC,
HMBC and COSY) spectra were measured on Bruker spectrometer of
AMX-400, 500 and 600 instrument using TMS as an internal reference.
13C-NMR spectra were also recorded on the same machines operating at
100, 125 and 200 MHz frequency. The EIMS, HR-EIMS and HR-FAB-MS
were recorded on Finnigan (Varian MAT, Waldbronn, Germany) JMS H X
110 with a data system and JMSA 500 mass spectrometers. The GC was
performed on a Shimadzu gas chromatograph (GC-9A; Noisiel, France).
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
65
2.6.2. Plant Material
The aerial parts (leaves and branches) of Vincetoxicum stocksii
were collected from Ziarat, Baluchistan, in September 2011 and were
identified by Prof. Dr. Rasool Bakhsh Tareen, Department of Botany,
University of Baluchistan, Quetta, Pakistan. Where a voucher specimen
(RBT-VS-11) has been deposited in the herbarium.
2.6.3. Extraction, Isolation and Identification
The plant was dried under shade for two weeks and was ground to
semi-powder (8 kg). It was then extracted by dipping (thrice 12.0 L) in
methanol at room temperature for 7 days. The extract was concentrated
under educed pressure to get dark brown gummy material (200 g). The
crude extract was evaluated for antiurease, antioxidant and antimicrobial
activity. It was then suspended in distilled water (2.0 L) and was extracted
with hexane and ethyl acetate. The n-hexane (40 gm) and ethyl acetate
fraction (60 gm) was evaluated for antioxidant and antiurease activity and
ethyl acetate showed significant activity that's why it was subjected to
column chromatography over silica gel eluting with n-hexane, n-hexane–
ethyl acetate, ethyl acetate, ethyl acetate–methanol in increasing order of
polarity to get twelve (V1-V12) fractions based on TLC profile.
The first two fractions V1 and V2 mainly contained lipid material and
were not processed further, however, fraction V3 obtained at n-
hexane:ethyl acetate (7:3) on further chromatography under same
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
66
conditions gave compound 73 (26 mg). Other main fraction V4 obtained at
n-hexane:ethyl acetate (6:4) was also chromatographed on silica gel
column eluting in increasing order of polarity to get compound 74 (15 mg)
and 87 (25 mg). Main fraction V5 and V6 obtained at n-hexane:ethyl
acetate (5:5) and (4:6) when further chromatographed on silica gel eluting
at n-hexane:ethyl acetate (4:6) fraction V5 gave 75 (25 mg) and fraction V6
give compounds 69 (25 mg) and 71 (10 mg) as yellowish spot on T.L.C
plate after using locating reagent. Fraction V7 obtained at n-hexane:ethyl
acetate (3:7) was further chromatographed on silica gel eluting with n-
hexane:ethyl acetate to get five fraction subfractions VR1-VR5. The
subfraction VR4 which was obtained at n-hexane:ethyl acetate (4:6) was
chromatographed on silica gel eluting with n-hexane:ethyl acetaye in
increasing order of polarity to give compounds 70 (30 mg) and 72 (18 mg)
and VR5 gave compounds 84 (25 mg) and 85 (32 mg).
Fraction V8 which was obtained at n-hexane:ethyl acetate (2:8) was
chromatographed on silica gel in increasing order of polarity to get
compounds 76 (34 mg), 88 (34 mg) and 80 (27 mg). The fraction V9
obtained at n-hexane:ethyl acetate (1:9) was further chromatographed on
silica gel in increasing order of polarity to get subfrctions VS1-VS3. The
subfractions VS2 and VS3 which were obtained at n-hexane:ethyl acetate
(3.5:6.5) and (3:7) on eluting n-hexane:ethyl acetate in increasing order of
polarity gave subfraction VS2 gave compounds 81 (17 mg), 82 (16 mg) and
subfraction VS3 gave compound 86 (14 mg).
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
67
Main Fraction V10 eluting with ethyl acetate:methonal (9.5: 0.5) give
two fractions VX1 and VX2 which was chromatographed on silica gel
eluting in increasing order of polarity to get compounds 77 (25 mg), 78
(34 mg) and V11 give 83 (20 mg) and 79 (34 mg).
Methanolic extract of vincetoxicum stocksii
Suspended in water
Hexane partWater part
Hexane
EtOAc
EtOAc partWater part
V1-Fr. V2 -Fr.
V3 -Fr. V4-Fr.V5 -Fr. V6-Fr.
V7 -Fr.
V8 -Fr. V9 -Fr.
V10-Fr.
V11-Fr.
V12-Fr.
73 74
75 76
69 71
70 72
VS2 VS384 85
76 80
VR4 VR5
82 86
VX1 VX2
77 78
83
81
87
88
79
Scheme. 2.1: Extraction and isolation of secondary metabolites from
Vincetoxicum Stocksii: V1. Fraction obtained in n-hexane:ethyl acetate(9:1), V2. Fraction obtained in n-hexane:ethyl acetate(8:2), V3. Fraction obtained in n-hexane:ethyl acetate(3:7), V4. Fraction obtained in n-hexane:ethyl acetate(6:4), V5. Fraction obtained in n-hexane:ethyl acetate(5:5), V6. Fraction obtained in n-hexane:ethyl acetate(4:6), V7. Fraction obtained in n-hexane:ethyl acetate (3:7), V8. Fraction obtained in n-hexane:ethyl acetate (2:8), V9. Fraction obtained in n-hexane:ethyl acetate (1:9), V10. Fraction obtained in ethyl acetate(100%), V11. Fraction obtained in ethyl acetate: MeOH (9:1), V12. Fraction obtained in ethyl acetate: MeOH (8:2).
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
68
O
O
OH
OH
69
1
2
3
45
6
7 8
1'
2'
3'
4'
5'
6'
7'
8'
9'
10'
11'
12'
13'
14'
1
2
3
1'
2'3'
4'
5'6'
O
HO
OH
OH
OHO
2526
27
28
21
70
CH3
31
2.6.4. Spectroscopic Data of Isolated Compounds
2.6.4.1. Spectroscopic data of vincetolate (69): white amorphous
powder (25 mg); IR max (KBr) cm-1: 3505, 1730, 1610-1580; 1H-NMR
(CD3OD; 500 MHz): δ 7.02
(1H, d, J = 8.5 H-2, 6),
6.73 (1H, d, J = 8.5, H-3,
5), 4.07 (2H, t, J = 6.5 Hz,
H-1'), 3.63 (2H, t, J = 6.5 Hz, H-14'), 3.49 (2H, s, H-7), 1.58 (2H, m, H-2'),
1.50 (2H, m, H-13'), 1.40-1.22 (20H, br., s); 13C-NMR (CD3OD;125 MHz): δ
174.2 (C-8), 158.4 (C-4), 131.7 (C-2, 6), 130.8 (C-1), 116.3 (C-3, 5), 65.8
(C-1'), 64.6 (C-14'), 41.8 (C-7), 33.9 (C-13'), 30.0 (C-3',12'), 26.0 (C-2');
EIMS: m/z 364 [M]+; HR-EIMS: m/z 364.2416 [M]+ (364.2611 calcd. for
C22H36O4).
2.6.4.2. Spectroscopic data of vincetoside (70): white amorphous
powder (30 mg); IR max (KBr) cm-1: 3385, 1655, 1300; 1H-NMR (CD3OD;
500 MHz): δ 5.31 (1H, br., m,
W1/2 = 20.8 Hz, H-27), 5.27
(1H, br., m, W1/2 = 20.8 Hz, H-
28), 4.20 (1H, d, J = 7.0 Hz, H-1'), 3.99 (2H, t, J = 6.9 Hz, H-1), 3.95 (1H,
dt, J = 6.0, 9.0 Hz, H-4'), 3.78 (1H, dd, J = 12.0, 2.6 Hz, H-6'), 3.76 (1H,
dd, J = 12.0, 6.6 Hz, H-6'), 3.66 (1H, t, J = 9.0 Hz, H-2'), 3.46 (2H, m, H-
5'), 3.16 (1H, m, H-3'), 1.92 (2H, m, H-29), 1.90 (2H, m, H-26), 1.70 (2H,
m, H-2), 1.30 (2H, m, H-30), 1.16-1.30 (br., s, H-3-25), 0.80 (3H, t, J = 6.6
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
69
H3CO
O
O
OH
O CH3
71
1
2 4
5
OH3C
Hz, Me); 13C-NMR (CD3OD; 125 MHz): δ 130.6 (C-27), 129.7 (C-28), 102.9
(C-1'), 76.2 (C-5'), 74.3 (C-2'), 73.3 (C-3'), 72.2 (C-4'), 68.6 (C-1), 62.2 (C-
6'), 34.3 (C-2), 31.8 (C-29), 29.6-25.8 (C-3 to 26), 22.6 (C-30), 14.0 (C-31);
FD-MS: m/z 612 [M]+; HR-FAB-MS: m/z 611.5239 [M-H]+ (611.4287
calcd. for C37H72O6).
2.6.4.3. Spectroscopic data of vincetetrol (71): white amorphous
powder (10 mg); IR max (KBr) cm-1: 3510, 1760, 1605-1570; 1H-NMR
(CD3OD; 500 MHz): δ 7.25 (1H, s, H-3), 6.66 (1H, s,
H-6), 3.84 (OMe), 1.90 (Me); 13C-NMR (CD3OD; 125
MHz): δ 171.4 (C-7, 8), 149.7 (C-2), 149.4 (C-5),
138.2 (C-1, 4), 105.2 (C-6), 104.5 (C-3), 56.8 (OMe),
23.7 (CH3); EIMS: m/z 240 [M]+; HR-EIMS: m/z 240. 2134 [M]+ (240.2646
calcd. for C11H12O6).
2.6.4.4. Spectroscopic data of vincetomine (72): white crystalline (18
mg); IR max (KBr) cm-1: 3260, 1605-1570, 1601;
1H-NMR (CD3OD; 600 MHz): δ 7.39 (2H, d, J =
8.6 Hz, H-2, 6), 7.32 (1H, d, J = 8.5 Hz, H-2', 6'),
7.10 (2H, d, J = 8.6 Hz, H-3, 5), 7.08 (1H, d, J =
8.5 Hz, H-3', 5') 3.78 (2H, s, H-7), 3.62 (OMe), 8.49 (NH, s); 13C-NMR
(CD3OD; 150 MHz): δ 153.9 (C-4), 137.6 (C-4'), 135.5 (C-1), 134.8 (C-1'),
128.8 (C-2, 6, 2', 6'), 118.2 (C-3, 5), 116.4 (C-3', 5'), 39.9 (C-7), 51.4
72
OCH3
H2N
1
3
5
7
4'
5' 6'
1'
2'3'
14
2
6
4
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
70
1
2
3
4
5
6
7
HO
OCH3
CH3
O
73
1
2
3
4
5
6
78
HO
OCH3
O
74
OH
9
(OMe); EIMS: m/z 213 [M]+; HR-EIMS: m/z 213. 1254 [M]+ (213.1346
calcd. for C14H15NO).
2.6.4.5. Spectroscopic data of apocynin (73): white amorphorus powder
(26 mg); IR max (KBr) cm-1: 1715, 1590-1565 cm-1;
1H-NMR (CD3OD; 500 MHz): δ 7.24 (1H, d, J = 2.0
Hz, H-2), δ 7.14 (1H, dd, J = 8.4, 2.0 Hz, H-6), 6.50
(1H, d, J = 8.4 Hz, H-5), 3.93 (OMe), 2.54 (Me); 13C-
NMR (CD3OD;125 MHz): δ 195.0 (C-7), 149.0 (C-4), 145.0 (C-3), 129.0 (C-
1), 124.0 (C-6), 113.7 (C-5), 109.6 (C-2), 56.0 (OMe), δ 26.2 (Me); EIMS:
m/z 166 [M]+; HR-EIMS: m/z 166.1052 [M]+ (m/z 166.1456 calcd. for
C9H10O3).
2.6.4.6. Spectroscopic data of 4-Hydroxy-3-methoxyphenyl-1-(9-
hydroxy-propan-7-one) (74): white amorphous powder (15 mg); IR max
(KBr) cm-1: 3750, 1700, 1610-1560; 1H-NMR
(CD3OD; 500 MHz): δ 7.58 (1H, dd, J = 8.0, 2.0
Hz, H-6), 7.54 (1H, d, J = 2.0 Hz, H-2), 6.86 (1H,
d, J = 8.0 H-5), 3.94 (2H, t, J = 6.0 Hz, H-8), 3.89
(OMe), 3.16 (2H, t, J = 6.0 Hz, H-9); 13C-NMR (CD3OD;125 MHz): δ 199.6
(C-7), 153.3 (C-4), 149.0 (C-3), 130.6 (C-1), 124.7 (C-6), 115.7 (C-5), 111.8
(C-2), 58.8 (C-9), 41.6 (C-8), 56.6 (OMe); EIMS: m/z 196 [M]+; HR-EIMS:
m/z 196.2061 [M]+ (calcd. for C10H12O4, 196.2042).
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
71
2.6.4.7. Spectroscopic data of 4-Hydroxy-3-methoxyphenyl-7,8,9-
propanetriol (75): white amorphous powder (25 mg); 1H-NMR (CD3OD;
600 MHz): δ 6.99 (1H, d, J = 2.0 Hz, H-2), 6.81
(1H, d, J = 8.0, 2.0 Hz, H-6), 6.74 (1H, d, J = 8.0
Hz, H-5), 4.51 (1H, d, J = 6.0 Hz, H-7), 3.72 (1H,
q, J = 5.8, H-8), 3.58 (1H, dd, J = 11.4, 7.2), 3.47
(2H, dd, J = 11.4, 4.2 Hz, H-9), δ 3.89 (OMe, s); 13C-NMR (CD3OD; 150
MHz): δ 148.8 (C-3), 147.1 (C-4), 134.8 (C-1), 121.0 (C-6), 115.8 (C-5),
111.8 (C-2), 77.6 (C-8), 75.0 (C-7), 64.5 (C-9), 56.6 (OMe); EIMS: m/z 214
[M]+; HR-EIMS: m/z 214. 2246 [M]+ (214.2745 calcd. for C10H14O5).
2.6.4.8. Spectroscopic data of Feruloyl-6'-O--D-glucopyranoside (76):
white amorphous powder (34 mg); IR
max (KBr) cm-1: 3542, 1710, 1655,
1608-1540; 1H-NMR (CD3OD; 400
MHz): δ 7.63 (1H, d, J = 16.0 Hz, H-7),
7.17 (1H, d, J = 2.0 Hz, H-2), 7.06 (1H, d, J = 8.0, 2.0 Hz, H-6), 6.85 (1H,
d, J = 8.0 Hz, H-5), 6.41 (1H, d, J = 16.0 Hz, H-8), 5.10 (1H, d, J = 7.0 Hz.
H-1'), 3.95 (1H, d, J = 6.5), 3.74 (1H, dd, J = 13, 4.9), 3.66 (1H, dd, J = 13,
2.6), 3.56 (1H, t, J = 7.0), 3.46 (1H, m), 3.16 (1H, m); 13C-NMR (CD3OD;
100 MHz): δ 169.1 (C-9), 150.6 (C-4), 147.3 (C-3), 146.9 (C-7), 127.7 (C-1),
124.5 (C-6), 116.9 (C-5), 115.6 (C-8), 111.7 (C-2), 98.2 (C-1'), 78.3 (C-5'),
77.6 (C-4'), 75.5 (C-2'), 72.0 (C-3'), 64.6 (C-6'). 56.8 (OMe); EIMS: m/z 356
[M]+; HR-EIMS: m/z 356.1646 [M]+ (356.1486 calcd. for C16H20O9).
12
3
4
5
6
78
9
OH
OH
OH
HO
OCH3
75
1
2
3
4
5
6 7
89
O
OHO
HOOH
OH
O
76
1'2'
3'
4'5'
6'
OCH3
HO
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
72
1
2
3
4
5
6
7 8 OH
O
OH
78
2.6.4.9. Spectroscopic data of 7-Megastigmene-2,3,5,9-tetrol (77):
white amorphous powder (25 mg); IR max (KBr)
cm-1: 3350, 1655; 1H-NMR (CD3OD; 600 MHz): δ
6.07 (1H, d, J =15.6 Hz, H-7), 5.80 (1H, dd, J
=15.6, 6.4 Hz, H-8), 4.34 (1H, m, H-9), 4.07 (1H,
m, H-5), 1.75 (2H, br., d, H-4), 1.63 (1H, dd, J = 14.4, 6.0 Hz), 1.44 (1H,
dd, 14.4, 3.6 Hz), 1.25 (3H, d, J = 6.6 Hz, CH3), 1.21(3H, s, CH3),1.12 (3H,
s, CH3), 0.83(3H, s, CH3); 13C-NMR (CD3OD; 150 MHz): δ 136.1 (C-8),
131.2 (C-7), 79.4 (C-3), 78.0 (C-2), 69.5 (C-9), 65.2 (C-5), 46.4 (C-6), 45.7
(C-4), 40.0 (C-1), 27.5 (C-13 Me), 27.0 (C-11 Me), 26.2 (C-12 Me), 24.1 (C-
10 Me); EIMS: m/z 244 [M]+; HR-EIMS: m/z 244.1742 [M]+ (244.1437
calcd. for C13H24O4).
2.6.4.10. Spectroscopic data of 4-Hydroxyphenylacetic acid (78):
white amorphous powder (34 mg); IR max (KBr) cm-1:
2420-3410, 1720; 1H-NMR (CD3OD; 400 MHz): δ 7.08 (1H,
d, J = 8.4 Hz, H-2, 6), 6.08 (1H, d, J = 8.4 Hz, H-3, 5), 3.47
(2H, s); 13C-NMR (CD3OD;100 MHz): δ 176.2 (C-8), 157.4
(C-4), 131.3 (C-2, 6), 126.8 (C-1), 116.2 (C-3, 5), 41.1 (C-
7); EIMS: m/z 152.4365 [M]+.
H3CCH3
CH3
OHOH
CH3
OH
HO
77
12
3
45
6
7
89
10
11
12
13
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
73
12
3
4
5
6
7 OH
O
HO
H3CO
OCH3
79
1
2
34
5
6
78 1'
2'
3'
4'
5'
6'
O
OH
HO
O
OCH3
OOH
OHHO
O
OH
80
4a
8a
1''2''
3''
4''5''
6''
2.6.4.11. Spectroscopic data of 4-Hydroxy-3,5-dimethoxybenzoic acid
(79): white amorphous powder (34 mg); IR max (KBr)
cm-1: 2410-3400, 1705, 1605 and 1570; 1H-NMR
(CD3OD; 500 MHz): δ 7.32 (2H, s), 3.88 (6H, s); 13C-
NMR (CD3OD; 125 MHz): δ 170.3 (C-7), 148.8 (C-3,
5), 122.4 (C-1), 142.2 (C-4), 108.4 (C-2, 6), 56.8 (OMe); EIMS: m/z 198
[M]+; HR-EIMS: m/z 198.2134 [M]+ (198.4261 calcd. for C9H10O5).
2.6.4.12. Spectroscopic data of 4'-Methoxy kaempferol-3-O--D-
glucopyranoside (80): yellowish
amorphous powder (27 mg); UV max
(max, 6.28 mM in CH3CN) nm (104M–1cm–
1): 236, 257, 245, 314 and 357 nm; IR
max (KBr) cm-1: 3480, 1615–1520; 1H-
NMR (CD3OD; 500 MHz): δ 8.05 (2H, d, J = 8.5 Hz, H-2',6'), 6.89 (2H, d, J
= 8.5 Hz, H-3', 5'), 6.23 (1H, d, J = 2.0 Hz, H-8), 6.20 (1H, d, J = 2.0 Hz, H-
8), 5.25 (1H, s), 3.88 (3H, s), 3.60 (1H, m), 3.53 (2H, dd, J = 10.5, 4.5),
3.41(1H, d, J = 6.6), 3.40 (1H, m), 3.35 (1H, dd, J = 10.5, 2.9) 3.20 (1H,
m); 13C-NMR (CD3OD;125 MHz): δ 179.1 (C-4), 165.0 (C-7), 162.0 (C-4'),
159.8 (C-5), 159.0 (C-8a), 158.0 (C-2), 134.0 (C-3), 124.0 (C-1'), 132.2 (C-
2', 6'), 116.0 (C-3'. 5'), 105.0 (C-4a), 104.1 (C-1''), 99.9 (C-6), 94.7 (C-8),
78.4 (C-2''), 78.0 (C-5''), 75.7 (C-3''), 71.4 (C-4''), 63.0 (C-6''), 51.4 (OMe);
HR-FAB-MS: m/z 463.4123 [M+H]+ (463.4986 calcd. for C22H23O11).
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
74
1
2
34
5
6
78 1'
2'3'
4'
5'
6'
4a
8a
1''2''
3''
4''5''
6''
O
OH
HO
O
OH
82
OOH
OH
O
CH3
HO
OH
2.6.4.13. Spectroscopic data of 4'-Methoxy kaempferol-3-O--L-
rhamnopyranoside (81): yellowish
amorphous powder (17 mg); IR max
(KBr) cm-1: 3480, 1615–1520; 1H-NMR
(CD3OD; 600 MHz): δ 7.76 (2H, d, J = 8.4
Hz, H-2',6'), 6.93 (2H, d, J = 8.4 Hz, H-3',
5'), 6.35 (1H, d, J = 2.0 Hz, H-8), 6.18 (1H, d, J = 2.0 Hz, H-8), 5.34 (1H,
br., s), 3.93 (OMe, s), 3.31 (1H, m), 3.71(1H, m), 3.38(1H, m), 3.33 (1H,
m), 0.91 (3H, d, J = 7.2 Hz); 13C-NMR (CD3OD; 150 MHz): δ 179.1 (C-4),
165.8 (C-7), 163.2 (C-5), 162.9 (C-4'), 159.1 (C-2), 158.2 (C-8a), 135.3 (C-
3), 131.8 (C-2', 6'), 122.1 (C-1'), 116.5 (C-3'. 5'), 105.1 (C-4a), 103.5 (C-1''),
100.1 (C-6), 94.9 (C-8), 73.2 (C-4''), 72.1 (C-3''), 72.0 (C-5''), 71.9 (C-2''),
17.6 (C-6''), 56.4 (OMe); HR-FAB-MS: m/z 447.4308 [M+H]+ (447.4248
calcd. for C23H25O10).
2.6.4.14. Spectroscopic data of qurcetain-3-O--L-rhamnopyranoside
(82): yellowish amorphous powder (16 mg); 1H-NMR (CD3OD; 600 MHz): δ
7.32 (1H, d, J = 2.4 Hz, H-2'), 7.30 (1H,
dd, J = 8.5, 2.4 Hz H-6'), 6.90 (1H, d, J =
8.5 Hz, H-5'), 6.35 (1H, d, J = 1.8 Hz, H-
8), 6.18 (1H, d, J = 1.8 Hz H-6), 5.34 (1H,
br., s), 4.21 (1H, br., s), 3.75 (1H, dd, J =
9.0, 4.5 Hz), 3.34 (1H, m), 3.41 (1H, d, J = 6.0), 0.94 (1H, d, J = 6.8); 13C-
1
2
34
5
6
78 1'
2'
3'
4'
5'
6'
4a
8a
1''2''
3''
4''5''
6''
O
OH
HO
O
OCH3
81
OOH
OH
O
CH3
HO
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
75
NMR (CD3OD; 150 MHz): δ 179.6 (C-4), 165.8 (C-7), 163.2 (C-5), 159.3 (C-
2), 158.9 (C-8a), 149.7 (C-4'), 146.4 (C-3'), 136.2 (C-3), 123.0 (C-1'), 122.9
(C-6'), 117.0 (C-2'), 116.4 (C-5'), 105.9 (C-4a), 103.5 (C-1''), 99.8 (C-6),
94.7 (C-8), 73.3 (C-4''), 72.1 (C-3''), 72.0 (C-5''), 71.9 (C-2''), 18.2 (C-6'');
HR-FAB-MS: m/z 449.3816 [M+H]+ (449.3765 calcd. for C22H25O11).
2.6.4.15. Spectroscopic data of 4'-Methoxy quercetin-3-O--L-
rhamnopyranoside (83): yellowish amorphous powder (20 mg); 1H-NMR
(CD3OD; 600 MHz): δ 7.33 (1H, d, J =
2.0 Hz, H-2'), 7.30 (1H, dd, J = 8.4, 2.0
Hz, H-6'), 6.90 (1H, d, J = 8.4 Hz, H-5'),
6.36 (1H, d, J = 1.8 ), 6.19 (1H, d, J =
1.8 Hz, H-8), 5.34 (1H, s), 3.92 (3H, s),
4.11 (1H, br., s), 3.73 (1H, dd, 9.3, 4.2), 3.40 (1H, d, 5.8), 3.30 (1H, m),
0.93 (3H, d, J = 6.4); 13C-NMR (CD3OD; 150 MHz): δ 179.6 (C-4), 165.9 (C-
7), 163.2 (C-5), 159.3 (C-2), 158.5 (C-8a), 151.6 (C-4'), 147.6 (C-3'), 136.2
(C-3), 122.9 (C-1'), 122.8 (C-6'), 116.9 (C-2'), 116.3 (C-5'), 105.9 (C-4a),
103.5 (C-1''), 99.8 (C-6), 94.7 (C-8), 73.2 (C-4''), 72.1 (C-3''), 72.0 (C-5''),
71.9 (C-2''), 17.6 (C-6''), 56.3 (OMe); HR-FAB-MS: m/z 463.4261 [M+H]+
(463.2265 calcd. for C22H23O11).
1
2
34
5
6
78 1'
2'3'
4'
5'
6'
4a
8a
1''2''
3''
4''5''
6''
O
OH
HO
O
OCH3
83
OOH
OH
O
CH3
HO
OH
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
76
1
2
34
5
6
78 1'
2'3'
4'
5'
6'
4a
8a
1''2''
3''
4''5''6''
O
OH
HO
O
OH
85
OH
O
OH
OHHO
O
HO
2.6.4.16. Spectroscopic data of quercetin-3-O--D- glucopyranoside
(84): yellowish amorphous powder (25
mg); 1H-NMR (CD3OD; 400 MHz): δ 7.70
(1H, d, J = 2.0 Hz, H-2'), 7.57 (1H, dd, J
= 8.5, 2.4 Hz, H-6'), 6.86 (1H, d, J = 8.5
Hz, H-5'), 6.37 (1H, d, J = 2.0 Hz H-8),
6.18 (1H, d, J = 2.0 Hz, H-6), 5.23 (1H, d, J = 7.6 Hz), 3.64 (1H, dd, J =
12.0, 4.8), 3.47 (1H, m), 3.40 (1H, d, J = 6.0), 3.36 (1H, dd, J = 12.0, 2.9),
3.34(1H, m), 3.33 (1H, m); 13C-NMR (CD3OD; 100 MHz): δ 179.6 (C-4),
165.9 (C-7), 162.9 (C-5), 159.0 (C-2), 158.8 (C-8a), 149.8 (C-4'), 145.8 (C-
3'), 135.6 (C-3), 123.2 (C-1'), 122.8 (C-6'), 116.0 (C-5'), 117.6 (C-2'), 105.7
(C-4a), 104.3 (C-1''), 99.9 (C-6), 94.7 (C-8), 78.3 (C-5''), 78.1 (C-4''), 75.1
(C-2''), 71.2 (C-3''), 62.5 (C-6''); HR-FAB-MS: m/z 463.3561 [M+H]+
(463.4325 calcd. for C21H26O12).
2.6.4.17. Spectroscopic data of quercetin-3-O--D-galactopyranoside
(85): yellowish amorphous powder (32
mg); 1H-NMR (CD3OD; 400 MHz): δ
7.70 (1H, d, J = 2.4 Hz, H-2'), 7.59 (1H,
dd, J = 8.8, 2.4 Hz, H-6'), 6.87 (1H, d, J
= 8.8 Hz, H-5'), 6.39 (1H, d, J = 2.0 Hz
H-8), 6.20 (1H, d, J = 2.0 Hz, H-6), 5.14 (1H, d, J = 7.6 Hz), 3.64 (1H, dd, J
= 12, 4.8), 3.47 (1H, m), 3.41 (1H, d, J = 2.9), 3.36 (1H, dd, J = 12.0, 2.9),
3.34 (1H, m), 3.33 (1H, m); 13C-NMR (CD3OD; 100 MHz): δ 179.5 (C-4),
1
2
34
5
6
78 1'
2'3'
4'
5'
6'
4a
8a
1''2''
3''
4''5''
6''
O
OH
HO
O
OH
OOH
OHHO
O
OH
84
OH
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
77
12
34
5
6
7
89
10
11
12
13
14
15
16
17
HO
87
18
19 20
21 22
2324
2829
25
26
27
166.1 (C-7), 163.0 (C-5), 159.0 (C-2), 158.5 (C-8a), 149.8 (C-4'), 145.9 (C-
3'), 135.6 (C-3), 129.9 (C-1'), 123.1 (C-6'), 117.5 (C-2'), 116.0 (C-5'), 105.4
(C-4a), 104.3 (C-1''), 99.9 (C-6), 94.7 (C-8), 78.4 (C-5''), 78.1 (C-4''), 75.7
(C-2''), 71.2 (C-3''), 62.5 (C-6''); HR-FAB-MS: m/z 463.3561 [M+H]+
(463.4325 calcd. for C21H26O12).
2.6.4.18. Spectroscopic data of 7-Hydroxy-3',4',5-trimethoxyflavone
(86): yellowish amorphous powder (14 mg); 1H-NMR (CD3OD; 500 MHz): δ
7.51 (1H, d, J = 2.0 Hz, H-2'), 7.25 (1H, s),
7.01 (1H, dd, J = 8.5, 2.0 Hz, H-6'), 6.94 (1H,
d, J = 8.5 Hz, H-5'), 6.48 (1H, d, J = 2.0 Hz,
H-6), 6.21 (1H, d, J = 2.0 Hz, H-8), 4.06 (3H,
OMe), 3.97(3H, OMe), 3.94 (3H, OMe); 13C-
NMR (CD3OD; 125 MHz): δ 179.8 (C-4), 166.0 (C-7), 160.1 (C-2), 164.0 (C-
5), 159.0 (C-8a), 149.5 (C-4'), 149.0 (C-3'), 123.0 (C-1'), 120.2 (C-2'), 117.0
(C-5'), 113.0 (C-6'), 105.0 (C-3), 104.3 (C-4a), 100.0 (C-6), 95.0 (C-8), 56.0
(OMe), 57.1 (OMe), 57.4 (OMe); EIMS: m/z 328 [M]+; HR-EIMS: m/z
328.3257 [M]+ (328.4256 calcd. for C18H16O6).
2.6.4.19 Spectroscopic data of β-sitosterol (87): white amorphous
powder (25 mg); 1H-NMR (CD3OD; 500
MHz): δ 5.35 (1H, d, 5.0), 4.56 (1H, m),
2.40 (2H, m),1.97 (4H, m), 1.91 (4H, m),
1.77 (2H, m), 1.76 (2H, m), 1.66 (1H, m),
1
2
34
5
6
78 1'
2'3'
4'
5'
6'
4a
8a O
OCH3
OOCH3
HOOCH3
86
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
78
12
34
5
6
7
89
10
11
12
13
14
15
16
17
1'2'
3'
4'5'
6'
O
18
19 20
21 22
2324
2829
25
26
27
O
OH
OH
88
HOHO
1.58 (2H, m), 1.51 (1H, m), 1.42 (1H, m), 1.36 (1H, m), 1.29 (2H, m), 1.26
(2H, m), 1.17 (2H, m), 1.13 (2H, m), 1.16 (1H, m), 1.10 (2H, m), 0.95 (3H,
d, 6.1, Me), 0.94 (3H, s, Me), 0.91 (3H, d, 6.0, Me), 0.86 (3H, d, 6.6, Me),
0.86 (3H, t, 7.3, Me), 0.77 (3H, s, Me), 0.11 (1H, m); 13C-NMR(CD3OD;
125 MHz): δ142.2 (C-5), 122.2 (C-6), 78.4 (C-3), 57.8 (C-14), 55.2 (C-17),
50.4 (C-9), 50.8 (C-24), 43.6 (C-13), 44.4 (C-4), 41.3 (C-12), 38.3 (C-1),
37.6 (C-10), 36.7 (C-20), 34.5 (C-22), 33.1 (C-7), 32.8 (C-2), 32.6 (C-8),
29.6 (C-23), 28.4 (C-16), 27.2 (C-25), 25.3 (C-15), 24.1 (C-28), 20.1 (C-11),
19.8 (C-19),19.6 (C-27), 19.4 (C-21), 18.4 (C-26), 12.9 (C-29) and 12.9 (C-
18); EIMS: m/z 414, 399, 396, 381, 329, 275, 273, 255 [M]+.
2.6.4.20 Spectroscopic data of ß-sitosterol-3-O-D-glucopyranoside
(88): white amorphous powder
(34 mg); IR max (KBr) cm-1:
3455, 1615; 1H-NMR (CD3OD;
500 MHz): δ 5.35 (1H, d, 5.0),
4.56 (1H, m), 4.51 (1H, d, 7.8),
3.65 (1H, m), 3.52 (1H, m), 3.21 (1H, m), 3.31 (1H, m), 3.12 (H, m), 3.10
(1H, m), 2.39 (2H, m), 1.92 (4H, m), 1.89 (4H, m), 1.73 (2H, m), 1.72 (2H,
m), 1.69 (1H, m), 1.56 (2H, m), 1.49 (1H, m), 1.39 (1H, m), 1.33 (1H, m),
1.25 (2H, m), 1.24 (2H, m), 1.16 (2H, m), 1.14 (1H, m), 1.09 (2H, m), 1.08
(2H, m), 0.99 (3H, d, 6.0, Me), 0.95 (3H, s, Me), 0.94 (3H, d, 6.1, Me), 0.87
(3H, t, 7.3, Me), 0.84 (3H, d, 6.6, Me), 0.74 (3H, s, Me), 0.10 (1H, m); 13C-
NMR (CD3OD; 125 MHz): δ 142.4 (C-5), 121.6 (C-6), 79.4 (C-3), 58.8 (C-
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
79
14), 55.4 (C-17), 50.6 (C-9), 50.7 (C-24), 44.6 (C-13), 44.4 (C-4), 41.6 (C-
12), 37.3 (C-1), 37.1 (C-10), 36.8 (C-20), 34.7 (C-22), 34.1 (C-7), 32.6 (C-
8), 31.8 (C-2), 29.8 (C-23), 28.6 (C-16), 26.9 (C-25), 24.9 (C-15), 24.6 (C-
28), 20.5 (C-11), 19.7 (C-27), 19.9 (C-19), 19.6 (C-21), 18.5 (C-26), 13.0
(C-29) and 13.0 (C-18), 102.4 (C-1'), 76.4 (C-2'), 73.4 (C-3'), 71.7 (C-4'),
76.0 (C-5'), 62.4 (C-6'); EIMS: m/z 576.4 [M]+.
2.6.5. Acid Hydrolysis
A solution of the compounds under investigated (6 mg each) in
MeOH (5 ml) containing 1N HCl (5 ml) was prepared and it was refluxed
for 4 hour. After reflux it was concentrated under reduced pressure and
diluted with H2O (10 ml). Extraction of extract was done with EtOAc and
the residue recovered from the organic phase was found to be aglycone
part of compounds. The aqueous part was concentrated and purified
using preparative TLC using the solvent system (EtOAc:MeOH:H2O:AcOH,
4:2:2:2) and identified as D-glucose, D-galactose and L-rhamnose by the
sign of their optical rotations [α]D26 +50.2, [α]D23 +78 and [α]D23 +7.7˚ (c,
0.2 in H2O), respectively. These sugars were also confirmed by the
retention time of their TMS ether (D-glucose, -anomer, 5.0 min; D-
galactose, -anomer, 3.9 min; and L-rhamnose, 8.3 min) with the
standards.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
80
2.6.6. Bioassays
2.6.6.1. Antioxidant (NO scavenging) assay
The 1mg/ml sample solution was prepared. In wells of plate the 178
ml phosphate buffer solution along with 20 ml of sodium nitroprusside
were added. These walls of plates were incubated at 37 ºC for 2 hours. The
20 ml Griess reagent was added in each wells of plates for color
development and this mixture was kept at room temperature for 20
minutes. Absorbance was measured after color development on ELISA
reader by using Gen 5 software at 490 nm and percentage inhibition was
calculated by using the following formulae (Garret 1964).
Percentage Inhibition = O.D of control - O.D of sample
O.D of controlx100
2.6.6.2. Antiurease assay (Berthelot method)
The 6 ml of phosphate buffer was added in 20 ml of urease enzyme
and were dispended in wells of plates. It was incubated for 10 minutes at
25 ºC and 5 ml of the test compound was added in it. the mixture was
further incubated at room temperature and after that 15 ml of 20mM Urea
was added. It was again incubated for 10 minutes at 25 ºC and 100 ml
RGT 2 was added. After incubation at room temperature for 25 minutes,
absorbance was measured on ELISA reader by using Gen 5 software at
630 nm and percentage inhibition was calculated by using the following
formulae.
Chapter 2 Characterization of the Compounds Isolated from V. stocksii
81
Percentage Inhibition = O.D of control - O.D of sample
O.D of controlx100
*Control: No sample was added.
*Negative control: No urea was added
2.6.6.3. Antibacterial assay
The antibacterial activity was carried out in sterile 96-wells
microplates. The method is based on the principle that microbial cell
number increases as the microbial growth proceeds in the log phase of
growth which results in increased absorbance of broth medium (Kaspady
et al., 2009; Patel et al., 2009). Six bacterial strains were included in this
study, Bacillus, Staphlococcus, E.coli, Psedumonas, shigella and
Salmonella. The organisms were maintained on stock culture agar. The
test samples were dissolved in suitable solvent. The initial absorbance of
the culture was strictly maintained between 0.12-0.19 at 540 nm. The
total volume in each well was kept to 200 µl. The incubation was done at
37 oC for 16-24 hours with lid on the microplate. The absorbance was
measured at 540 nm using Synergy HT Bio-Tek, USA microplate reader,
before and after incubation and the difference was noted as an index of
bacterial growth. The percent inhibition was calculated using the formula:
Inhibition (%) = 100 (X–Y)/X
where “X” is absorbance in control with bacterial culture and Y is
absorbance in test sample. Streptomycin were taken as standard drug.
Chapter 3 Literature Review of Genus Seriphidium
82
CHAPTER 3
THE GENUS SERIPHIDIUM AND LITERATURE REVIEW
Chapter 3 Literature Review of Genus Seriphidium
83
3.1. THE GENUS SERIPHIDIUM
The genus Seriphidium belongs to the plant family Asteraceae. This
genus consists of 140 species occurring predominantly throughout
Europe, North Africa, Temperate Asia and Western North America.
Literature search revealed that the two genera Seriphidium and Artemisia
have identical genetic properties, but they are isolated and consider being
different genera of the family Asteraceae (Deng et al., 2004; Watson et al.,
2002).
3.2. PHARMACEUTICAL IMPORTANCE OF THE SERIPHIDIUM SP.
Many members of the genus Seripidium are important component of
folk medicine system. In traditional medical system they are used for
treatment of various diseases like antihelminthics, gastrointestinal,
disorders diabetes and high blood pressure (Hayat et al., 2009). S. annua
and S. indica are source of artemisinin (7), an anti-malarial drug, isolated
from the aerial parts of these plants. It was found active against all the
drug resistant strain of malarial parasites, plasmodium. The S. dubia has
been used as anti-ulcer and purgative agents. It is also used to cure
asthma and skin diseases like scabies. S. absinthium L. is source of
anthelmintic, antifungal and antimicrobial agents. S. brevifolia is widely
used as anthelmintic agent in ethno-veterinary medicinal system of
Pakistan (Mahmood et al., 2011). S. kurramense is commercially used for
extraction of santonin and transported to many countries for this purpose
(Gilani et al., 2010).
Chapter 3 Literature Review of Genus Seriphidium
84
3.3. PREVIOUS PHYTOCHEMICAL STUDIES OF THE GENUS SERIPHIDIUM
Literature search revealed several species of this genus have been
studied for their secondary metabolites. For example S. santolium have
been investigated for its secondary metabolites and was found to produce
variety of compounds like apigenin-7-O-β-D-glucopyranoside-(3'-O-7")-
quercetin-3"-methyl ether (89), apigenin (90), luteolin (91), chrysoeriol
(92), tricin (93), hispidulin (94), pectolinarigenin (95), eupatilin (96), 5,7-
dihydroxy-6,3',4',5'-tetramethoxyflavone (97), 5,7,4'trihydroxy-6,3',5'-
trimethoxyflavone (98), 3,6-O-dimethylquercetagetin-7O-β-D-glucoside
(99), 6-hydroxy-5,7-dimethoxy-coumarin (100), taraxerol (101), taraxeryl
acetate (102), β-sitosterol (103), stigmasterol (104), a series of the n-alkyl
trans-p-coumarates (105-107), series of the n-alkyl trans-ferulates (108-
110), 2-hydroxy-4, 6, di-methoxyacetophenone (111), 4-hydroxy-2,6-
dimethoxyphenol-1-O-β-Dglucopyranoside (112) and 2-hydroxycinnamoyl-
β-D-glucopyranoside (113) (Deng et al., 2004).
Chapter 3 Literature Review of Genus Seriphidium
85
O
OH
OH O
OH
OHO
HOOH
O
OH
99
H3CO
HO
HO
101
OO
CH3
102
HO
103 104
92
O
OH
HO
OH O
OCH3 O
OH
HO
OH O
OCH3
OCH3
93
O
OH
HO
OH O
O
OH
HO
OH O
OH
O
OH
OH O
O O
OH
OH O
OMe
OH
OHO
HOOH
O
OH
89 90
91
OHO
OH O
H3CO
94
OHO
OH O
H3CO
OH OCH3
OHO
OH O
H3CO
OCH3
9596
OCH3
OHO
OH O
H3CO
OCH3
OCH3
OCH3
97
OHO
OH O
H3CO
OH
OCH3
OCH3
98
O
HO
H3CO
100
O
OCH3
Chapter 3 Literature Review of Genus Seriphidium
86
HO
O
O(CH2)19 CH3
HO
105
CH3
HO
O
O(CH2)21 CH3
106
HO
O
O(CH2)23 CH3
107
H3CO
O
O(CH2)19 CH3
OHH3CO
O
O(CH2)21 CH3
108 109
H3CO
O
O(CH2)23 CH3
OHOH
OOH
OCH3
110
111
CH3
OOH
OCH3
OHO
HOOH
O
OH
112
O
113
OHO
OHOH
HOO
OH
Luteolin (91) has been reported as anti-microbial, anti-oxidant and
anti-inflammatory agent. In traditional medicinal system it is used against
lung cancer, gastric cancer and acute myocardial infarction (Lopez-Lazaro,
2008). Compound 93 acts as chemo-preventive agent (Deng et al., 2004;
Jiao, et al., 2007), while hispidulin (94) was reported as anti-inflammatory
and anti-oxidant agent (He et al., 2011). Pectolinarigenin (95) exhibit anti-
inflammatory and anti-allergic effect (Lim et al., 2008), whereas, eupatilin
(96) displayed potent cytotoxicity and antitumor activity (Shawi et al.,
2011). β-sitosterol (103) and stigmasterol (104) have been reported as
neutralizer for viper and cobra venom (Lim et al., 2008), while 4-
hydroxy,2,6-dimethoxyphenol-1-O-ß-D-glucopyranoside (112) has been
reported as anti-estrogenic agent (Luecha et al., 2009).
Chapter 3 Literature Review of Genus Seriphidium
87
Balchanin (114), hydroxy costunolide (115), costunolide (116),
balchanolide (117), balchanolide acetate (118) and hydroxy balchanolide
(119) were reported from S. balchanorum Krasch. This plant contains
another important metabolite vachanic acid (120), whereas, S. fragrans
and S. taurica produced tauremisin (121) (Geissman and Irwin).
O
CH2
O
CH3
OH
CH3 O
CH2
O
CH3
CH3 O
CH2
O
CH3
CH3
OH
OO
CH3
CH3
OH
CH3
OO
CH3
CH3
OH
OH
CH3
OO
CH3
CH3
O
CH3
O CH3
O
CH3OH
CH2
HOHO CH3 O
CH3
O
O
CH3HO
114 115 116117
118 119 120 121
Cumambrin A (122), B (123) and deoxycumambrin (124) were
isolated from S. tripartia and S. nova. Other Seriphidium species; S.
tridentata, S. cana pursh, S. juncea, S. lercheana and S. leucods are known
to produce matricarin (125), deacetylmatricarin (126) and
deacetyloxymatricarin (127) (Geissman and Irwin).
Chapter 3 Literature Review of Genus Seriphidium
88
H3C OH
CH3 O
O
OH
H3C OH
CH3 O
O
CH2
CH3 O
O
CH2
CH3O
O CH3
OCH3 O
O
CH3O
OH
CH3CH3 O
O
CH3O
O CH3
O
122 123 124
125 126 127
H3C
H3C OH
O
CH3
O
O
O
CH2 CH2
CH3
Gallic acid (128), gallic acid methyl ester (129), 1,2,4,6-O-tetra-
gallolyl-β-D-glucpyranoside (130), 1,2,3,4,6-O-penta-galloyl-β-D-
glucopyranoside (131), quecetin-3-O-β-D-(2ʺ -galloyl)-glucopyranoside
(132), quercetin-3-O-β-D-glucopyranoside (133), quercetin-3-O- -L-
arabinopyranoside (134) and kaempferol-3-O-β-D-glucopyranoside (135)
were isolated from S. oliverianum. Compounds 129 and 131 exhibited
strong DPPH radical scavenging activity with IC50 values of 3.0 and 2.8
µM, respectively (Ho et al., 2012).
Chapter 3 Literature Review of Genus Seriphidium
89
HO
OH
OH
O OH
HO
OH
OH
O OCH3
OO
HOO
O
O
OH
OHHO
OO
HO
OH
HO
HO
OH
OH
O
HO
OH
OH
OO
OO
O
O
O
OH
OHHO
OO
HO
OH
HO
HO
OH
OH
O
HO
OH
OH
OO
HO
OH
OH
128 129
130 131
O
OH
HO
O
OH
O
OH
O
O
HO
OH
O
HO
OH
OH
O
OH
HO
O
OH
OOH
OHHO
O
OH
OH
OH
O
OH
HO
O
OH
OOH
OHHO
O
OH
O
OH
HO
O
OH
OOH
OHHO
O
OH
132
133
134 135
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
90
CHAPTER 4
STRUCTURE ELUCIDATION OF SECONDARY METABOLITES ISOLATED FROM SERIPHIDIUM QUETTENSE
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
91
4.1. SERIPHIDIUM QUETTENSE
Seriphidium quettense is a woody shrub, growing up to 40-60 cm
high. It is found in Hazarganji, Balochistan, Pakistan (Ghafoor 2002).
4.1.1. Classification of the Seriphidium Quettense
Kingdom Plantae
Phylum Tracheophyta
Class Magnoliopsida
Order Asterales
Family Asteraceae
Genus Seriphidium
Specie Seriphidium quettense
4.2. RESULTS AND DISCUSSION
The ethyl acetate fraction of methonalic extract of Seriphidium
quettense was subjected to column chromatography which yielded
seriphilidine (136) as a new compound along with seven known
compounds; 6-hydroxy-8(10)-oplopen-14-one (137), salvigenin (138),
cirsumaritin (139), p-coumaric acid (140), β-sitosterol (87), β-sitosterol-3-
O-D-glucopyranoside (88) and oleanolic acid (141).
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
92
4.2.1. Characterization of Seriphilidine (136)
Compound 136 was isolated as white amorphous powder, whose
EIMS spectrum showed molecular ion peak at m/z 268 and molecular
formula C16H28O3 with three double bond equivalents (DBE) was
calculated through HR-EIMS (m/z 268.2435).
The IR spectrum showed characteristic
absorption bands at 3410 and 1645 cm-1 for
hydroxyl and olefinic functions respectively.
The 1H-NMR spectrum (Table 4.1) of 136 showed two signals at δ
5.53 (1H, ddd, J = 15.5, 9.5, 1.0 Hz) and 5.45 (1H, dd, J = 15.5, 7.0 Hz).
The larger J value of these two nuclei depicted a trans nature of the
olefinic system. Further, resonances due to four methine protons were
observed at δ 5.05 (t, J = 5.0 Hz), 3.41 (br., s), 2.30 (m) and 1.75 (d, J =
9.5). These protons were correlated in HSQC spectrum with the carbons at
δ 105.5, 83.5, 57.2 and 31.4 respectively. This data revealed that the first
methine could be an anomeric center, whereas, the second could be part
of furan ring system. In addition, the same spectrum displayed signals of
six protons at δ 1.89 (1H, d, J = 5.5 Hz), 1.85 (1H, d, J = 4.5 Hz), 1.78 (2H,
m), 1.51 (1H, d, J = 4.5 Hz) and 1.37 (1H, d, J = 5.5 Hz) attested for three
methylenes, whereas, resonances due to four methyl groups were
observed at δ 1.08 (s), 1.02 (s), 0.97 (d, J = 6.5 Hz) and 0.96 (d, J = 6.5
Hz). The doublet methyls showed COSY correlation with a methine
multiplet at δ 2.30 and were attested for an isopropyl group. Another
OH3CO
H3C
CH3H3C
CH3
OH
1
2
34
56
7
89
1011
12
1314
1516
136
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
93
signal of three hydrogen appeared at δ 3.93, which was attributed to a
methoxyl group.
The 13C-NMR spectrum (Table 4.1) of 136 was in full agreement
with the 1H-NMR and mass data as it showed signals for six methine δ
142.0, 122.5, 105.5, 83.5, 57.2 and 31.4, three methylene at δ 47.8, 39.5
and 21.6, one methoxy at δ 56.0, four methyl at δ 31.0, 22.7, 22.7 and
15.7 and two quaternary carbons at δ 72.3 and 46.0.
Most of the part of this molecule could be established due to several
COSY correlations (figure 4.1), whereas, the C-H connectivities were
accomplished due to HSQC experiment. The position of various functional
groups were fixed through HMBC analysis, in which two methyl groups (δ
0.97, Me-13 and 0.96, Me-14) showed the HMBC correlations with C-12 (δ
31.4), C-11 (δ 142.0) and also with each other. This information confirmed
the presence of an isoprene unit in 136. The HMBC correlation of
anomeric proton at δ 5.05 with the carbon at δ 83.5 (C-9) and 46.0 (C-4)
confirmed the furan moiety. The HMBC correlation of methyl proton at δ
1.02 (Me-16) with the carbon at δ 83.5 (C-9), 57.2 (C-5), 46.0 (C-4) and
47.8 (C-3) and HMBC interaction of H-5 with Me-16 (δ 15.8) and C-11 (δ
142.0) confirmed the potion of Me-16 and isopropyl moiety at C-5 (δ 57.2).
Other important HMBC correlations are shown in figure 4.1.
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
94
OH3CO
H3C
CH3H3C
CH3
OHH
136
Figure. 4.1: COSY (─) and important HMBC ( ) correlations observed in compound 136.
The relative stereochemistry at various chiral centers was
established by detail examination of NOESY spectrum and deriding model.
In the NOESY spectrum, (figure 4.2) H-2 (δ 5.05) showed the correlations
with H-9 (δ 3.41) indicated that these groups or atoms were oriented on
same side. Accordingly, Me-15 showed NOESY correlation with H-5 (δ
1.75), which in turn exhibited NOESY interaction with H-9 (δ 3.41) set the
orientation of these atoms or groups on the same side of molecule. The
combination of all spectroscopic data helped us to establish structure 136
and was identified as new natural product to which we propose the name
seriphilidine.
Figure. 4.2: NOESY correlation observed in compound 136.
OH3CO
H3C
CH3H3C
CH3
OH
H
H H
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
95
Table. 4.1: 1H and 13C-NMR data of 136 (CD3OD; 500 and 125 MHz).
Position 136
δH (J in Hz) δc
1 - -
2 5.05 (1H, t, 5.0) 105.5 3 1.89 (1H, d, 5.5), 1.37 (1H, d, 5.5) 47.8
4 - 46.0
5 1.75 (1H, d, 9.5) 57.2
6 - 72.3
7 1.85 (1H, d, 4.5), 1.51 (1H, d, 4.5) 39.5
8 1.78 (2H, m) 21.6 9 3.41 (1H, br., s) 83.5
10 5.53 (1H, ddd, 15.5, 9.5, 1.0) 122.5
11 5.45 (1H, dd, 15.5, 7.0) 142.0
12 2.30 (1H, m) 31.4
13-Me 0.97 (3H, d, 6.5) 22.7 14-Me 0.96 (3H, d, 6.5) 22.7
15-Me 1.08 (3H, s, CH3) 31.0
16-Me 1.02 (3H, s, CH3) 15.8
2-OMe 3.93 (3H, s, CH3) 56.0
4.2.2. Characterization of 6-Hydroxy-8(10)-oplopen-14-one (137)
Compound 137 was isolated as a white
crystalline solid, which exhibited diagnostic
absorption bands at 1715 and 3450 cm-1 for
ketonic and hydroxyl groups respectively (Marco et
al., 1993; Paolini et al., 2008). The EIMS spectrum of 137 showed
molecular ion at m/z 236, whereas, the molecular formula C15H24O2 with
four DBE was established through HR-EIMS (m/z 236.1780 [M]+).
The 1H-NMR spectrum (Table 4.2) of compound 137 showed two
most downfield resonances at δ 4.68 (1H, br s) and δ 4.59 (1H, br s),
which were correlated in HSQC spectrum with a methylene carbon at δ
105.2 and thus were attested for an exocyclic double bond. The spectrum
CH3
O OH
H
H
CH3H3C
137
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
96
also displayed five signals for methine at δ 3.53 (dt, J = 9.5, 4.5 Hz), 2.88
(dd, J = 12.5, 9.5 Hz), 2.02 (m), 1.70 (dd, J = 9.5, 5.0 Hz) and 1.36 (t, J =
10.0 Hz). Further, the same spectrum displayed a signal at δ 2.17 (3H, s)
for a methyl of acetyl group. The two doublet methyl, displayed their
positions at δ 1.09 (J = 7.5 Hz) and 0.82 (J = 7.5 Hz), were correlated in
COSY spectrum with a methine at δ 1.49 (m) indicating the presence of an
isopropyl moiety in 137. This data revealed a sesquiterpenoids skeleton of
137.
The 13C-NMR spectrum (BB and DEPT, Table 4.2) of 137 fully
supported the 1H-NMR data as it displayed fifteen carbon signals at δ
213.9 (C), 148.4 (C), 105.2 (CH2), 73.6 (C-H), 56.2 (C-H), 55.8 (C-H), 52.5
(C-H), 50.2 (C-H), 46.6 (CH2), 31.1 (C-H), 30.3 (CH2), 29.5 (CH3), 27.8
(CH2), 23.8 (CH3) and 16.8 (CH3). This data was quite similar to the data
reported for oplopanone type sesquiterpenoids (Marco et al., 1993).
Further confirmation of the structure of compound 137 was achieved by
comparison of 1H, 13C-NMR and mass spectral data with the published
data of oplopanone (Marco et al., 1993; Paolini et al., 2008).
This compound was already isolated in our group (Shafiq et al.,
(2014), where its absolute stereochemistry was determined by measuring
the electric circular dichroism spectrum of compound 137.
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
97
O
O
OCH3
OH
H3CO
H3CO
138
Table. 4.2: 1H and 13C-NMR data of 137 (CD3OD; 500 and 125 MHz).
Position 137
δH (J in Hz) δc
1 2.12 (1H, m), 1.57 (1H, m) 30.3
2 1.76 (1H, m), 1.54 (1H, m) 27.8
3 2.88 (1H, dd, 12.5, 9.5) 56.2
4 1.70 (1H, dd, 9.5, 5.0) 50.2
5 1.36 (1H, t, 10.0) 55.8
6 3.53 (1H, dt, 9.5, 4.5) 73.6 7 2.54 (1H, dd, 12.0, 4.5) 46.6
8 - 148.4
9 2.02 (1H, m) 52.5
10 4.68 (1H, br., s), 4.59 (1H, br., s) 105.2
11 1.49 (1H, m) 31.1
12-Me 1.09 (3H, d, 7.5) 23.8
13-Me 0.82 (3H, d, 7.5) 16.8 14 - 213.9
15-Me 2.17 (3H, s, CH3) 29.5
4.2.3. Characterization of Salvigenin (138)
Compound 138 was obtained as yellowish crystalline solid, which
exhibited UV absorption maxima at 235,
259, 246, 315 and 356 nm, the
characteristic pattern of kaempferol or
quercetin glycoside (Mabry et al., 1970;
Saleh et al., 1990). The EIMS spectrum displayed molecular ion peak at
m/z 328, whereas, high resolution of the same ion (m/z 328.2413)
depicted the molecular formula as C18H16O6.
The 1H-NMR spectrum (Table 4.3) of compound 138 showed two
doublets at δ 7.78 (2H, J = 9.0 Hz) and 6.69 (2H, J = 9.0 Hz) indicating the
presence of a p-substituted benzene ring system. The spectrum also
displayed two singlets in the aromatic region at δ 6.51 (1H) and 6.48 (1H)
which were found to be correlated with carbons at δ 104.0 and 90.5 in
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
98
HSQC spectrum respectively and thus were attested for H-3 of ring C and
H-8 of ring A respectively. Three methoxyl signals were found at δ 3.92,
3.88 and 3.85. The most down fielded signal at δ 12.73 (1H, s) was
attributed to a chelated hydroxyl function, which confirmed its position at
C-5. The 13C-NMR spectrum (Table 4.3) of 138 displayed total 16 signals
in which four signals at δ 127.9, 114.4, 104.0 and 95.0 were attested for
six methine carbons. The three methoxyl carbons resonated at δ 60.8,
56.2 and 55.5, while nine quaternary carbon nuclei displayed their
positions at δ 182.6, 163.9, 162.6, 158.7, 153.1, 153.0, 132.6, 123.4 and
106.1. This data indicated a substituted flavone nucleus. The positions of
the methoxyl groups were fixed due to HMBC correlations in which the
methoxyl proton resonating at δ 3.85, 3.92 and 3.88 showed the HMBC
correlation with C-4' (δ 162.6), C-5 (δ 132.6) and C-6 (δ 153.0)
respectively. The singlet proton at δ 6.51 showed HBMC correlation with
C-4a (δ 106.1), C-4 (δ 182.6) and C-1' (δ 123.4) and thus was assigned as
H-3. The combination of the whole spectroscopic data and comparison
with the reported values (Ragasa et al., 1999), compound 138 was found
to be salvigenin which is a known metabolite but has been isolated from
this plant for the first time.
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
99
O
O
OCH3
OH
H3CO
HO
139
4.2.4. Characterization of Cirsumaritin (139)
Compound 139 was also isolated as yellowish crystalline solid,
which exhibited similar UV data as that of
138, indicating its flavone nature. The
EIMS showed molecular ion peak at m/z
314, whereas, the molecular formula
C17H14O6 was calculated through the HR-EIMS that exhibited molecular
ion peak at m/z 314.2947.
Aromatic region of the 1H-NMR spectrum (Table 4.3) of compound
139 was nearly super imposable to that of 138 that depicted a p-
substituted ring B, H-3 of ring C and H-8 of ring A. The only difference
was the resonances of two methoxyl group at δ 4.02 and 3.89 instead of
three. The 13C-NMR spectrum (Table 4.3) of 139 displayed 15 signals,
which were differentiated as four methine (δ 128.3, 114.6, 103.8 and
95.9), two methyls (δ 56.7 and 55.5) and nine quaternary carbons (δ
180.5, 163.2, 162.4, 161.1, 157.9, 154.1, 130.6, 123.0 and 105.1), based
on DEPT experiment.
The methoxyl group present at δ 3.89 could be fixed at C-4' due to
its HMBC correlation with quaternary carbon at δ 163.2 and that of H-2'
with the same carbon. The other methoxyl group was placed at C-7 due to
its HMBC correlation with another quaternary carbon at δ 161.1.
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
100
All connectivities were confirmed through careful analysis of HSQC
and HMBC spectra and the whole data was comparable to the reported
data of cirsumaritin, which has already been reported from different
biological sources like Cirsium spp., Teucrium spp. and Digitalis ferruginea,
Drimys winteri (Imre et al., 1973; Cruz and Silva 1973).
Table. 4.3: 1H and 13C-NMR data of 138 and 139 (CD3OD; 500 and 125 MHz).
4.2.5. Characterization of p-Coumaric Acid (140)
The EIMS of 140 showed the molecular ion peak at m/z 164 and the
HR-EIMS analysis of the same ion depicted the
molecular formula as C9H8O4 with six DBE. The
1H-NMR spectrum of 140 (Table 4.4) displayed
only four signals in aromatic region at δ 7.64
(1H, d, J = 16.5 Hz), 7.40 (2H, d, J = 8.5 Hz), 6.64 (2H, d, J = 8.5 6.35 (1H,
Position 138 139
δH (J in Hz) δc δH (J in Hz) δc
1 - - - -
2 - 162.6 - 162.4
3 6.51 (1H, s) 104.0 6.61 (1H, s) 103.8
4 - 182.6 - 180.5
4a - 106.1 - 105.1
5 - 153.1 - 154.1 6 - 132.6 - 130.6
7 - 153.0 - 161.1
8 6.48 (1H, s) 95.0 6.45 (1H, s) 95.9
8a - 158.7 - 157.9
1' - 123.4 - 123.0 2' 7.78 (1H, d, 9.0) 127.9 7.94 (1H, d, 8.8) 128.3
3' 6.69 (1H, d, 9.0) 114.4 7.05 (1H, d, 8.8) 114.6
4' - 163.9 - 163.2
5' 6.69 (1H, d, 9.0) 114.4 7.05 (1H, d, 8.8) 114.6
6' 7.78 (1H, d, 9.0) 127.9 7.94 (1H, d, 8.8) 128.3
6-OMe 3.92 (3H, s, OMe) 56.2 4.02 (3H, s, OMe) 56.7 7-OMe 3.88 (3H, s, OMe) 60.8 - -
4'-OMe 3.85 (3H, s, OMe) 55.5 3.89 (3H, s, OMe) 55.5
OH
O
HO
140
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
101
d, J = 16.5 Hz). The signals splitted at higher J values (J = 16.5 Hz) were
attributed to a conjugated trans olefinic system, whereas, the other two
signals of four hydrogen were attested for a p-substituted benzene ring.
This data quite fitted with a p-coumaric acid structure, which was
substantiated due to the 13C-NMR spectrum (Table 4.4), in which seven
signals were seen as four methines (δ 142.3, 128.6, 116.4 and 114.2) and
three quaternary carbons (δ 169.4, 158.1 and 124.1). Combination of
above data and comparison with published literature (Ahmad et al., 2009),
compound 140 could be confirmed as p-coumaric acid, which is an
important known phytochemical but has been isolated for the first time
from Seriphidium quettense.
Table. 4.4: 1H and 13C-NMR data of 140 (CDCl3, 400 and 100 MHz).
Position δH (J in Hz) δc
1 - 124.1 2 7.40 (2H, d, 8.5) 128.6
3 6.64 (2H, d, 8.5) 116.4
4 - 158.1
5 6.64 (2H, d, 8.5) 116.4
6 7.40 (2H, d, 8.5) 128.6
7 7.64 (1H, d, 16.5) 142.3 8 6.35 (1H, d, 16.5) 114.2
9 - 169.4
4.2.6. Characterization of β-sitosterol (87)
The compound 87 was isolated as a white amorphous powder and
structure elucidation of this compound has already been discussed in
chapter 2 section 2.4.18. (Kamboj and Saluja 2011).
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
102
4.2.7. Characterization of β-sitosterol-3-O-β-D-glucopyranoside (88)
The compound 88 was isolated as a white amorphous powder and
structure elucidation of this compound 88 has already been discussed in
chapter 2 section 2.4.19. (Akhtar et al., 2010).
4.2.8. Characterization of Oleanolic Acid (141)
Compound 141 was isolated as white
amorphous powder, which exhibited
molecular ion peak in EIMS spectrum at m/z
456, while the HR-EIMS (m/z 456.3601)
depicted the molecular formula as C30H48O3.
The IR spectrum of compound 141 displayed characteristic absorption
bands at 2570-3500 and 1710 cm-1 for a carboxylic acid function.
The 1H-NMR spectrum (Table 4.5) displayed a triplet signal at 5.26
(1H, J = 3.6 Hz) due to a double bond and a doublet of doublet at 3.21
(1H, J = 11.0, 5.0 Hz) was attributed to an oxymethine. The spectrum also
displayed seven methyl singlets at 1.14, 0.98, 0.96, 0.94, 0.86, 0.75 and
0.74 characteristic of a pentacyclic triterpenoidal skeleton of oleanane
series. The 13C-NMR (BB and DEPT) revealed the presence of seven
methyls at 33.6, 29.6, 25.5, 24.1, 18.4, 16.3 and16.1, ten methylenes at
45.2, 37.9, 33.7, 33.4, 32.4, 27.6, 27.1, 23.7, 22.1 and 18.4, five
methines at 122.9, 78.5, 55.5, 49.6 and 40.2 and eight quaternary
carbons at 182.6, 142.5, 46.2, 41.3, 41.0, 38.7, 37.6 and 31.1. This data
HO
OH
O
141
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
103
was suggestive of oleanolic acid and its comparison with the reported data
(Ragasa and Lim 2005) confirmed the compound 141 to be oleanolic acid,
which is an important phytochemical and isolated first time from this
source.
Table. 4.5: 1H and 13C-NMR data of 141 (CD3OD; 500 and 125 MHz).
Position δH (J in Hz) δc Position δH (J in Hz) δc
1 1.71 (1H, m), 0.96 (1H, m) 37.9 16 1.98 (1H, m), 1.62 (1H, m) 22.1
2 1.61 (1H, m), 1.55 (1H, m) 27.1 17 - 46.2 3 3.21 1H, dd, 11.0, 5.0) 78.5 18 2.79 (1H, dd, 13.6, 3.6) 40.2 4 - 38.7 19 1.62 (1H, m), 1.14 (1H, m) 45.2 5 0.73 (1H, t, 6.8) 55.5 20 - 31.1 6 1.53 (1H, m), 1.36 (1H, m) 18.4 21 1.30 (1H, m), 1.20 (1H, m) 33.4 7 1.42 (1H, m), 1.27 (1H, m) 33.7 22 1.78 (1H, m), 1.57 (1H, m) 32.4
8 - 41.0 23 0.98 (3H, s, Me) 29.6 9 1.51 (1H, m) 49.6 24 0.86 (3H, s, Me) 16.3 10 - 37.6 25 0.74 (3H, s, Me) 16.1 11 1.87 (1H, m), 0.92 (1H, m) 23.7 26 0.75 (3H, s, Me) 18.4 12 5.26 (1H, t, 3.6) 122.9 27 1.14 (3H, s, Me) 25.5 13 - 142.5 28 - 182.6 14 - 41.3 29 0.94 (3H, s, Me) 33.6 15 1.70 (1H, m), 1.11 (1H, m) 27.6 30 0.96 (3H, s, Me) 24.1
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
104
4.3. EXPERIMENTAL
4.3.1. General Experimental Procedures
Column chromatography was performed with silica gel (230-400
mesh). The aluminum sheets precoated with silica gel 60 F254 (20×20 cm,
0.2 mm thick; E. Merck) for thin layer chromatography. The UV lamp (254
and 366 nm) was used to check unsaturation in compounds and ceric
sulfate was used as locating reagent for UV inactive metabolites. The UV
spectra were recorded in ethanol on JASCO V-650 spectrophotometer
(max in nm). IR spectra were measured on Shimadzu 460 spectrometer
(Duisburg, Germany). The 1D (1H NMR) and 2D NMR (HMQC, HMBC and
COSY) spectra were measured on Bruker spectrometer of AMX-400, 500
instrument, using TMS as an internal reference. The 13C-NMR spectra
were also recorded on the same machines operating at 100 and 125 MHz.
The EIMS and HR-EIMS were calculated on Finnigan (Varian MAT) JMS
H×110 with a data system and JMSA 500 mass spectrometers.
4.3.2. Collection and Identification of Plant Material
The whole plant material (stem, leaves and branches) of
Serphidium quettense was collected from Ziarat, Baluchistan, in
September 2011 and was identified by Prof. Dr. Rasool Bakhsh Tareen,
Department of Botany, University of Baluchistan, Quetta, Pakistan, where
a voucher specimen (RBT-SQ-11) has been deposited in the herbarium.
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
105
4.3.3. Extraction
The plant material was dried under shade for two weeks and was
ground to semi-powder (5.0 kg). It was then extracted with methanol (8.0
L) at room temperature for 6 days (twice). The combined concentrated
extract (150 gm) was suspended in water (1.0 L) and was extracted with n-
hexane (65 gm) and ethyl acetate (40 gm). The ethyl acetate fraction
showed significant antioxidant and antiurease activity and thus was
subjected to purification.
4.3.4. Isolation and Identification
Ethyl acetate fraction of methnolic extract was subjected to column
chromatography (Scheme 4.1) over silica gel eluting with n-hexane, n-
hexane–chloroform, chloroform, chloroform–methanol in increasing order
of polarity to obtain eight fractions (SQ1-SQ8) based on the TLC profiles.
The main fraction SQ1 and SQ2 mostly contain hydrocarbon part
and thus were not processed. The fraction SQ3 obtained at n-
hexane:chloroform (7:3) on silica gel chromatography give two
subfractions; S3-1 and S3-2, which on eluting with n-hexane:chlorofrom in
increasing order of their polarity give compound 137 (30 mg). Another
fraction SQ4 obtained at n-hexane:chlorofrom (6:4) was chromatographed
further to get compound 87 (25 mg). Main fraction SQ5 obtained at n-
hexane:chlorofrom (4:6) when further purified on silica gel eluting with n-
hexane:chlorofrom (4.5:5.5) to get five subfractions SQ5-1-SQ5-5. The
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
106
subfractions SQ5-4 and SQ5-5 were further subjected to purification on
silica gel column eluting at n-hexane:chlorofrom in increasing order of
polarity to get compounds 141 (25 mg) and 140 (32 mg). The fraction SQ6
obtained with n-hexane:chlorofrom (3:7) was re-chromatographed on silica
gel column using same eluting system to get compounds 136 (20 mg) and
88 (24 mg). Fraction SQ7 obtained at n-hexane:chlorofrom (1:9) when
further chromatographed with n-hexane:chlorofrom (3:7) yielded three
subfractions S7-1-S7-3. The subfractions S7-2 and S7-3 on eluting with n-
hexane:chlorofrom in increasing order of polarity through a silica gel
column to give compounds 138 (25 mg) and 139 (23 mg).
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
107
Methanolic extract of Seriphidium Quettense
Suspended in water
Hexane partWater part
Hexane
EtOAc
EtOAc partWater part
SQ2 SQ3 SQ4 SQ5SQ1 SQ6 SQ7SQ8
137 87
141 140
136 88
138 139
9:1 8:2 3:7 6:4 4:6 3:7 1:9 CHCl3
n-hexane: chloroform
Scheme. 4.1: Purification protocol of secondary metabolites from Seriphidium quettense.
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
108
4.3.5. Spectroscopic Data of Purified Compounds
4.3.5.1. Spectroscopic data of seriphilidine (136): white amorphous
powder (20 mg); IR max (KBr) cm-1: 3410,
1645; 1H-NMR (CD3OD; 500 MHz): 5.53 (1H,
ddd, J = 15.5, 9.5, 1.0 Hz, H-10), 5.45 (1H, dd,
J = 15.5, 7 Hz, H-11), 5.05 (1H, t, J = 5.0 Hz,
H-2), 3.93 (3H, s, 2-OMe), 3.41 (1H, br., d, H-
9), 2.30 (1H, m, H-12), 1.89 (1H, d, J = 5.5 Hz, H-3), 1.85 (1H, d, J = 4.5
Hz, H-7), 1.78 (2H, m, H-8), 1.75 (1H, d, J = 9.5 Hz, H-5), 1.51 (1H, d, J =
4.5 Hz, H-7), 1.37 (1H, d, J =5.5 Hz, H-3), 1.08 (3H, s, Me-14), 1.02 (3H, s,
Me-16), 0.97 (3H, d, J = 6.5 Hz, Me-13) and 0.96 (3H, d, J = 6.5 Hz, Me-
14); 13C-NMR (CD3OD; 125 MHz): δ 142.0 (C-11), 122.5 (C-10), 105.5 (C-
2), 83.5 (C-9), 72.3 (C-6), 57.2 (C-5), 47.8 (C-3), 46.0 (C-4), 39.5 (C-7),
31.4 (C-12), 31.0 (C-15), 22.7 (C-13, 14), 21.6 (C-8), 15.8 (C-16) and 56.0
(2-OMe); EIMS: m/z 268 [M]+; HR-EIMS: m/z 268.2435 [M]+ (calcd.
268.4375 for C16H28O3).
4.3.5.2. Spectroscopic data of -Hydroxy-8(10)-oplopen-14-one
(137): White crystalline solid (30 mg); IR max
(KBr) cm-1: 3450 and 1715; 1H-NMR (CD3OD;
500 MHz): δ 4.68 (1H, br., s, H-10), 4.59 (1H,
br., s, H-10), 3.53 (1H, dt, J = 9.5, 4.5 Hz, H-
6), 2.88 (1H, dd, J = 12.5, 9.5 Hz, H-3), 2.54 (1H, dd, 12.0, 4.5 Hz, H-7),
OH3CO
H3C
CH3H3C
CH3
OH
1
2
34
56
7
89
1011
12
1314
1516
136
1
2
3 45
6
789
10
11
1213
14
CH3
O OH
H
H
CH3H3C
137
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
109
1
2
34
5
6
78
O
O
OCH3
OH
H3CO
H3CO
138
4a
8a1'
2'
3'4'
5'
6'
2.17 (3H, s, Me-15), 2.12 (1H, m, H-1), 2.02 (1H, m, H-9), 1.76, 1.54 (1H,
m, H-2), 1.70 (1H, dd, J = 9.5, 5.0 Hz, H-4), 1.57 (1H, m, H-1), 1.49 (1H,
m, H-11), 1.36 (1H, t, J = 10.0 Hz, H-5), 1.09 (3H, d, J = 7.5 Hz, Me-12)
and 0.82 (3H, d, J = 7.5 Hz, Me-13); 13C-NMR (CD3OD; 125 MHz): δ 213.9
(C-14), 148.4 (C-8), 105.2 (C-10), 73.6 (C-6), 56.2 (C-3), 55.8 (C-5), 52.5
(C-9), 50.2 (C-4), 46.6 (C-7), 31.1 (C-11), 30.3 (C-1), 29.5 (C-15), 27.8 (C-
2), 23.8 (C-12) and 16.8 (C-13); EIMS: m/z 236 [M]+; HR-EIMS: m/z
236.1780 (calcd. 236.3548 for C16H28O3).
4.3.5.3. Spectroscopic data of salvigenin (138): Yellowish crystalline
solid (25 mg); UV max (max, 6.28 mM in CH3CN) nm (104M–1cm–1): 235,
259, 246, 315 and 356; 1H-NMR (CD3OD;
500 MHz): δ 7.78 (1H, d, J = 9.0 Hz, H-2',
6'), 6.69 (1H, d, J = 9.0 Hz, H-3', 5'), 6.51
(1H, s, H-3), 6.48 (1H, s, H-7), 12.73 (1H, s,
OH), 3.92 (3H, s, OCH3), 3.88 (3H, s, OCH3) and 3.85 (3H, s, OCH3); 13C-
NMR (CD3OD; 125 MHz): δ 182.6 (C-4), 163.9 (C-4'), 162.6 (C-2), 158.7
(C-8a), 153.1 (C-5), 153.0 (C-7), 132.6 (C-6), 106.1 (C-4a), 127.9 (C-2', 6'),
123.4 (C-1'), 114.4 (C-3', 5'), 104.0 (C-3), 95.0 (C-8), 60.8 (OMe), 56.2
(OMe) and 55.5 (OMe); EIMS: m/z 328 [M]+; HR-EIMS: m/z 328.2413 [M]+
(calcd. 328.2365 for C18H16O6).
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
110
1
2
34
5
6
78
O
O
OCH3
OH
H3CO
HO
139
4a
8a1'
2'
3'4'
5'
6'
1
2
3
4
5
67
89 OH
O
HO
140
4.3.5.4. Spectroscopic data of cirsumaritin (139): Yellowish crystalline
solid (23 mg); 1H-NMR (CD3OD; 500 MHz):
δ 7.94 (1H, d, J = 8.8 Hz, H-2', 6'), 7.05
(1H, d, J = 8.8 Hz, H-3', 5'), 6.61 (1H, s, H-
3), 6.45 (1H, s, H-7), 4.02 (3H, s, OCH3),
3.89 (3H, s, OCH3); 13C-NMR (CD3OD; 500 MHz): δ 180.5 (C-4), 163.2 (C-
4'), 162.4 (C-2), 161.1 (C-7), 157.9 (C-8a), 154.1 (C-5), 103.8 (C-3), 130.6
(C-6), 128.3 (C-2', 6'), 123.0 (C-1'), 114.6 (C-3', 5'), 105.1 (C-4a), 95.9 (C-
8), 56.7 (OMe) and 55.5 (OMe); EIMS: m/z 314 [M]+; HR-EIMS: m/z
314.2947 [M]+ (calcd. 314.2843 for C17H14O6).
4.3.5.5. Spectroscopic data of p-Coumaric acid (140): Yellow
amorphous powder (32 mg); 1H-NMR (CDCl3,
400 MHz): δ 7.64 (1H, d, J = 16.5 Hz, H-7), 7.40
(2H, d, J = 8.5 Hz, H-2, 6), 6.64 (2H, d, J = 8.5
Hz, H-3, 5) and 6.35 (1H, d, J = 16.5 Hz, H-8); 13C-NMR (CDCl3, 100 MHz):
δ 169.4 (C-9), 158.1 (C-4), 142.3 (C-7), 128.6 (C-2, 6), 124.1 (C-1), 116.4
(C-3, 5), 114.2 (C-8); EIMS: m/z 164 [M]+; HR-EIMS: m/z 164.0466 [M]+
(calcd. 164.0473 for C9H8O4).
Chapter 4 Structure Elucidation of Secondary Metabolites from S. Quettense
111
4.3.5.6. Spectroscopic data of oleanolic acid (141): White amorphous
powder (25 mg); IR max (KBr) cm-1: 1710,
2570-3500; 1H-NMR (CD3OD; 500 MHz): δ
5.26 (1H, t, 3.6, H-12), 3.21 (1H, dd, 11.0,
5.0, H-3), 2.79 (1H, dd, 13.6, 3.6, H-18),
1.98, 1.62 (1H, m, H-22), 1.87, 0.92 (1H, m,
H-11), 1.78, 1.57 (1H, m, H-22), 1.71, 0.96 (2H, m, H-1), 1.70, 1.11 (1H,
m, H-15), 1.62, 1.14 (1H, m, H-19), 1.61, 1.55 (2H, m, H-2), 1.53, 1.36
(2H, m, H-6), 1.51 (1H, m, H-9), 1.42, 1.27 (2H, m, H-7), 1.30, 1.20 (1H,
m, H-21), 1.14 (3H, s, Me-27), 0.98 (3H, s, Me-23), 0.96 (3H, s, Me-30),
0.94 (3H, s, Me-29), 0.86 (3H, s, Me-24), 0.75 (3H, s, Me-26), 0.74 (3H, s,
Me-25), 0.73 (1H, t, 6.8, H-5); 13C-NMR (CD3OD; 125 MHz): δ 182.6 (C-
28), 142.5 (C-13), 122.9 (C-12), 78.5 (C-3), 55.5 (C-5), 49.6 (C-9), 46.2 (C-
17), 45.2 (C-19), 41.3 (C-14), 41.0 (C-8), 40.2 (C-18), 38.7 (C-4), 37.9 (C-
1), 37.6 (C-10), 33.7 (C-7), 33.6 (C-29), 33.4 (C-21), 32.4 (C-22), 31.1 (C-
20), 29.6 (C-23), 27.6 (C-15), 27.1 (C-2), 25.5 (C-27), 24.1 (C-30), 23.7 (C-
11), 22.1 (C-16), 18.4 (C-6), 18.4 (C-26), 16.3 (C-24), 16.1 (C-25); EIMS:
m/z 456 [M]+; HR-EIMS: m/z 456.3601 [M]+ (calcd. 456.8473 for
C17H14O6).
1
2
34
5
6
7
89
10
11
1213
14
15
16
1718
19 20 21
22
23 24
28
29
25 26
27
HO
OH
O
143
30
Chapter 5 Endophytic Fungi
112
CHAPTER 5
PURIFICATION AND CHARACTERIZATION OF SECONDARY METABOLITES FROM ENDOPHYTIC FUNGUS
CRYPTOSPORIOPSIS SP.
Chapter 5 Endophytic Fungi
113
5.1. ENDOPHYTES AND ENDOPHYTIC FUNGI
The term ‘endophyte’ was firstly introduced by A. De Bary in 1866 to
describe all microorganisms including fungi, bacteria, cyanobacteria, and
actinomycetes that are found in plant tissues (De Bary 1866). Among
these organisms, endophytic fungi are a diverse group of microorganisms
and found more abundant in nature then bacterial endophytes.
Endophytic fungi have gain importance due to two reasons, firstly they are
found in all the plants, and secondly the secondary metabolites produced
by endophytic fungi help plants to improve their growth and resistance to
the environmental condition (including all the biotic and abiotic factors).
Secondary metabolites that provide resistance and survival to the plant, if
they are isolated and characterized, may found to be potential lead for
drug discovery. In drug discovery prospectus finding of new drugs for the
treatment of newly appearing diseases in human, animal and plants is
always a great aspect. In recent years, a massive investigation on
endophytes has been done with respect to biotechnological and ecological
aspects in recent years for the development of new source and products of
agriculture and pharmaceutical importance (Strobel et al., 2004; Chagas
et al., 2013).
Chapter 5 Endophytic Fungi
114
5.2. BIODIVERSITY OF FUNGAL ENDOPHYTES
Endophytes live in mutualistic association with plants and spend
their whole life living in plant tissues causing no damage to the host.
Fungal endophytes are the more diverse group of microorganisms that
they have been found everywhere, like in geothermal soils, deserts,
oceans, rainforests and coastal forests. They are important component of
microbial biodiversity. In green land plants, where they help the host to
develop offence against threats and other environmental circumstances,
which make their ability to produce fascinating and structurally complex
natural products (Debbab et al., 2010). Endophytic fungi are hosted by
algae, bryophytes, pteridophytes, gymnosperms and angiosperms. They
have been isolated from the shoot, root and leaves including all aerial
parts in every plant studied to date (Kharwar et al., 2011).
5.3. ADVANTAGE OF MICROBIAL STUDIES IN DRUG DISCOVERY
There are many advantages of microbial studies in drug discovery
process. Fungi and bacteria are existing on earth from million of year, as
they have adopted unique biosynthetic pathways and mechanism to
synthesis the secondary metabolites of diverse structural classes. These
secondary metabolites interact with different enzymes and help the
organism to survive in harsh weather conditions and other challenges.
Secondly the yield of different bioactive compounds can be increased in
certain organism, if culture conditions are optimized. The required
Chapter 5 Endophytic Fungi
115
microorganism can be stored for infinite time period, which ensure the
availability of the target product at any time.
There is a problem associated with plant derived drug that potent
metabolites are produced in very low amount, for example plant Taxus
contain only 0.01-0.03% of paclitaxel by dry phloem weight. In case of
microorganisms, this problem can be solved by culturing of the organisms
at any scale (Kharwar et al., 2011).
5.4. BIOACTIVE COMPOUNDS FROM ENDOPHYTIC FUNGI
Endophytes have represented a promising source of bioactive
compounds and their production can be increased at lower cost by large
scale fermentation and cultivation of the organisms. A bulk of leads has
been isolated and characterized from endophytes, which includes anti-
bacterial, anticancer, antifungals, antivirals and immunosuppressants.
5.4.1. Antibacterial Agents from Endophytic Fungi
About 25 years before, the antibiotic cephalosporin C (142) was
introduced in the pharmaceutical market as antibacterial drug (Newton
and Abraham 1955). Guanacastepenes A (143), obtained from
unidentified endophytic fungus was active against drug-resistant strains
of Enterococcus faecium and Staphylococcus aureus (Brady et al., 2001).
Chaetoglobosin A (144) was isolated from endophytic Chaetomium
globosum and rhizotonic acid (145) obtained from Rhizoctonia sp., both
Chapter 5 Endophytic Fungi
116
the compounds showed potent activity against Helicobacter pylori a
bacterium involved in the gastric ulcer (Tikoo et al., 2000; Ma et al., 2004).
Altersetin (146) isolated from Alternaria sp. was found active against
Gram-positive bacteria (Hellwig et al., 2002).
N
S
O
COOH
O CH3
O
HN
O
HOOC
NH2
HH
O
O
O
CH3
OH
CH3
H3C
O
CH3
O COOH
OHOO
H3C CH3
OH
H3C
NH
NH
O
CH3
H3C
OH
OH3C
O
ONH
HO
O
H3COH
CH3
H
HH3C
142
143
144 145 146
Marinopyrroles A (147) and B (148) were found active against
methicillin-resistant S. aureus (Hughes et al., 2008). Ascochytatin (149), a
marine-derived fungal metabolite isolated from Ascochyta sp. NGB4, has
been reported to kill Gram-positive bacteria and yeast C. albicans (Kanoh
et al., 2008). Lynamicins B (150) and C (151) are two halogenated bis-
indole derivatives, which have reported to be active against S. aureus
(MSSA, MRSA: methicillin resistant), Staphylococcus epidermidis and
Enterococcus faecalis (Mcarthur et al., 2008).
Chapter 5 Endophytic Fungi
117
OHO
NCl
Cl
HN
Cl
Cl
OOH
OHO
NCl
Cl Br
HN
Cl
Cl
OOH
151
147
O O
OH
O
OH
OH
OH
148
NH
HN
NH
O
OCH3
H3C
Cl
Cl
NH
HN
NH
Cl
Cl
Cl
Cl
149
150
Bionectins A-C (152-154), verticillin D (155) and G (156) exhibited
antibacterial activity against S. aureus, methicillin-resistant S. aureus
(MRSA) and quinolone-resistant S. aureus (QRSA). These compounds were
isolated from the fungus Bionectra byssicola F120 (Donnell and Gibbons
2007; Zheng et al., 2006; Zheng et al., 2007).
HN
NNH
NO
O
R
H
OH
S S
152 R =H
153 R = CH(OH)CH3
HN
NNH
NO
O
SCH3
H
OH
SCH3
154
NNH
NO
O
CH(OH)CH3
H
OH
S S
NHN
NO
O
CH(OH)CH3
HO
SS
155
NNH
NO
O
H
OH
S
NHN
NO
O
H
HO
SS
O
HS
156
Chapter 5 Endophytic Fungi
118
5.4.2. Anti-Cancer Agents from Endophytic Fungi
Compound 149 showed cytotoxicity as it inhibited the growth of
A549 (human lung carcinoma cell line) cell lines with EC values of 4.8 and
Jurkat cells (human leukaemia cell line) with EC values of 6.3 µM (Kanoh
et al., 2008). Camptothecin (157) and its analogues, 9-
methoxycamptothecin (158) and 10-hydroxycamptothecin (159), were
potentially active against human cancer cell lines A549 (lung cancer),
HEP-2 (liver cancer). These compounds were isolated from an endophytic
fungus Fusarium solani (Kusari et al., 2009).
NN O
O
O
HO
H3C
NN O
O
O
HO
H3C
OCH3
NN O
O
O
HO
H3C
OH
157 158 159
Cytoglobosins C (160) and D (161), two novel alkaloids, were
isolated from Chaetomium globosum which showed cytotoxicity with IC50
values of 2.26 and 2.55 µM against the tumor cell line (A549) (Cui et al.,
2010).
Chapter 5 Endophytic Fungi
119
NH
HN
H3C OH
CH3
OO
HO
OH
CH3
CH3
NH
HN
H3C
CH3
OO
HO
OH
CH3
CH3
160 161
Chaetominine (162) another alkaloid, obtained from endophytic
fungus Chaetomium sp. IFB-E015, was found active against the human
leukemia K562 and colon cancer cell lines SW1116 with IC50 values of
21.0 and 28.0 µM (Jiao et al., 2006). Brefeldin A (163) was isolated from
Acremonium, and has been reported potentially active against epidermoid
cancer of the mouth KB, small-cell lung cancer NCI-H187 and breast
cancer cell line BC-1 with IC50 values of 0.18, 0.11 and 0.04 µM,
respectively (Chinworrungsee et al., 2008). Radicicol (164) was purified
from Chaetomium chiversii, which inhibited MCF-7 cell lines (breast cancer
cell line) with an IC50 value of 0.03 µM (Turbyville et al., 2006).
Chaetoglobosin U (165) is known as the metabolite of the endophytic
fungus Chaetomium globosum IFB-E019. It showed potent cytotoxic
activity against the human nasopharyngeal epidermoid tumor KB cell line
with an IC50 value of 16.0 µM (Ding et al., 2006).
Chapter 5 Endophytic Fungi
120
N N
O
H
N N
O
O
OH
162
OO
HO
O
O
HH O
OH
HO
Cl
163 164 NH
NH
O
H
O
O
H
OOH
CH3
H
165
5.4.3. Anti-Fungal Agents from Endophytic Fungi
The first antifungal drug from filamentous fungus isolated was
griseofulvin (166) (Grove et al., 1952), afterwards, several new metabolites
were isolated from fungal sources. For example, anidulafungin marketd
with name of Eraxis, (167) and caspofungin lunched as Cancidas (168)
are two antifungal drugs obtained from Aspergillus rugulovalvus and
Glarlozoyensis (Butler 2004). Ambuic acid, (169), an antifungal
metabolite, was isolated from Pestalotiopsis microspora. It is a highly
functionalized cyclohexenone-derived natural product (Harper et al., 2001;
Harper et al., 2003; Harper et al., 2003). Cryptocin (170) is another
antifungal compound isolated from Cryptosporiopsis quercina. It has a
tetramic acid skeleton that possesses potent activity against Pyricularia
oryzae (Li et al., 2000). Oocydin A (171), a chlorinated macrocyclic lactone
was a potent antifungal compound, isolated from Serratia marcescens
(Strobel and Daisy 2003).
Chapter 5 Endophytic Fungi
121
O
H
H
COOH
CH3
CH3H H
O
H
OHH
OHH
H
170
H
H3C
CH3
CH3
NO
O
CH3
OH
CH3
H
H169
OH
OH
H CH3
COOH
O
HH
H
Cl
H3C
OAc O
171
CH3
HO
HO
OH
NH
O
HO
H3C
N
O
H3C
HONH
O
HO OH
NH
R
O
HN
O
N
CH3
OH
HN
O
OH
O
H3C
R =
167
R =
168
O
O
O
O
O CH3
H3COCl
H3CO
OCH3
165
H3C
(172) an antifungal lead isolated from fungus Aspergillus sydowii, is a
derivative of deoxymulundocandin (173) (Keating and Figgitt 2003;
Igarashi et al., 1956).
HO
OH
NH
O
HO
H3C
N
O
H3C
HONH
O
HO OH
NH
O
HN
O
N
CH3
OH
HN
O
OH
O
H3C
H3C
173HO
OH
NH
O
HO
H3C
N
O
H3C
HONH
O
HO NH
NH
O
HN
O
N
CH3
OH
HN
O
OH
O
O
H3C
NH2
172
Chapter 5 Endophytic Fungi
122
A fungal polyketide metabolite chaetocyclinones A (174) was
isolated from cultures of Chaetomium sp. This compound was found active
against Phytophthora Infestans (Losgen et al., 2007). Antifungal partricin A
(175) is a 38-membered heptaene polyene and its derivative (176) is also a
new antifungal agent. PLD-118 (BAY-10-8888) (177) is a cyclic -amino
acid and other compound cispentacin (178); both the compounds are in
clinical trial for the development of antifungal agent (Butler 2005).
HN
O
H3C
OH
H3C
H3C
OH
OOHOHOHOHO
HO
O
O
O
OH
R1
O
OHO OH
NHR2
HN
NCH3
CH3
R1
R2
O
N
CH3
CH3
176
COOHH2NCOOHH2N
178177O
O
O
H3CO
OH
OCH3
CH3
H3CO O
174
CH3
R1 OH
R2 H
175
5.4.4. Anti-Viral Agents from Endophytic Fungi
Cytonic acids A (179) and B (180) were isolated from the endophytic
fungus Cytonaema sp. and were reported as human cytomegalovirus
Chapter 5 Endophytic Fungi
123
protease inhibitors (Guo et al., 2000). Xanthoviridicatins E (181) and F
(182) obtained from endophytic Penicillium chrysogenum based on novel
quinone-related metabolites, blocked the cleavage reaction of HIV-1
integrase (Singh et al., 2003).
O
OO
OO
COOH
OHOH
OHHO
CH3
CH3
CH3
O
OO
OO
COOH
OHOH
OHHO
CH3
CH3
CH3
O
O
OCH3H3C
OH
O
O
CH3
CH3CH3
H3CO
O
O
OCH3H3C
OH
O
O
CH3
CH3CH3
H3CO
181 182
179 180
5.4.5. Immunosuppressant Agents from Endophytic Fungi
The first immunosuppressive drug cyclosporine A (26) was isolated
from Tolypocladium inflatum and was used as immunosuppressant during
organ transplantations (Borel and Kis 1991). Mycophenolic acid (183) is
known as the metabolite of the fungi Penicillium, Aspergillus,
Byssochlamys and Septoria species (Larsen et al., 2005). It is a potential
immunosuppressive drug that is used during the organ transplantations.
Chapter 5 Endophytic Fungi
124
Subglutinol A (184) another immunosuppressant, was isolated from an
endophytic Fusarium subglutinans (Lee et al., 1995a).
CH3
CH3
H3CO
CH3
HOOCO
O
O
CH3
CH3
OHO
H
O
O
CH3
H
H3C
H
H
H
CH3
CH3
184183
5.5. TAXONOMY OF THE ENDOPHYTIC FUNGUS CRYPTOSPORIOPSIS
Kingdom: Fungi
Phylum: Ascomycota
Class: Leotiomycetes
Subclass: Leotiomycetidae
Order: Helotiales
Family: Dermateaceae
Genus: Cryptosporiopsis
Chapter 5 Endophytic Fungi
125
5.6. LITERATURE SURVEY ON THE GENUS CRYPTOSPORIOPSIS
5.6.1. Previous Investigation of Cryptosporiopsis Sp.
Cryptosporiopsis is known to produce a unique lipopeptide cryptocandin
(185), which is a potent antimycotic agent. This compound was first time
isolated and reported from Cryptosporiopsis cf. quercina. The most
impressive antifungal activity of 185 was against Candida albicans with
MIC value 0.03 g/ml, Candida parapsilosis with MIC value of 2.5 g/ml
and Histoplasma capsulatum with MIC value of 0.01 g/ml (Strobel et al.,
1999). Another antimycotic agent cryptocin (170) isolated from the same
endophytic fungus bears a unique tetramic acid structure was found
active against many fungal strains, Pythium ultimum, Phytophthora
cinnamoni, Phytophthora citrophthora, Sclerotinia sclerotiorum, Pyricularia
oryzae, Rhizoctonia solani, Geotrichum candidum and Fusarium oxysporum
with MIC (g/ml) value 0.78, 0.78, 1.56, 0.78, 0.39, 6.25, 1.56 and 1.56
respectively (Li et al., 2000). In recent study two important polyketides,
cryptosporiopsin A (186) and hydroxypropan-2',3'-diol orsellinate (187), a
cyclic pentapeptide (188) and several other compounds (189-191) have
been isolated from Cryptosporiopsis sp., associated with the leaves and
branches of Zanthoxylum leprieurii (Rutaceae). These metabolites were
found active against zoospores of pathogen Plasmopara viticola with MIC
value 10-25 g/ml. They displayed potent inhibitory activity against
Pythium ultimum, Aphanomyces cochlioides and a fungus Rhizoctonia
solani (Talontsi et al., 2012).
Chapter 5 Endophytic Fungi
126
OCH3
HO
Cl
O
O
O
O
OH
HO
Cl
O
O
O
O
HO
OH
O
O
CH3
OH
OH
NH
NH
NHHN
HN
O
O
O
Leu2
Leu1
lleLeu3
O
O
O
188 R = CH(CH3)2
189 R = Phe, Leu instead of Ile
N
HOHO
O
H2N
O
O
HO
OH
OOH
N
OH
O CH3
OHO
NH
OH
OH
HN
O
O14185
ROH
186
187
190
191
5.7. RESULTS AND DISCUSSION
5.7.1. Structure Elucidation of the Isolated Compounds from Endophytic Fungus Cryptosporiopsis Sp.
The culture media of the fungus Cryptosporiopsis sp was extracted
with ethyl acetate, which on chromatographic purification yielded three
novel bioactive polyketide, cryptosporioptide (192), cryptosporioptide A
(193) and cryptosporioptide B (194), which were characterized due to
several spectroscopic analyses analysis including 1D (1H, 13C), 2D NMR
(HSQC, HMBC, COSY) techniques and mass spectrometry (FABMS,
HRFABMS).
Chapter 5 Endophytic Fungi
127
5.7.1.1. Characterization of cryptosporioptide (192)
Cryptosporioptide (192) was isolated as yellow amorphous powder,
whose molecular formula (C19H19NO10) with eleven double bond
equivalents (DBE) was established due
to (+ve) HR-FABMS (m/z 444.0781,
[M+Na]+). The IR spectrum displayed
absorption bands at 3465 for hydroxyl
group, 3105, 2945 (C-H), 1732, 1695-
1680 (broad for carbonyl group) and 1590-1565 (for double bond) cm-1.
The 1H NMR spectrum of 192 (Table 5.1) showed two ortho-coupled
doublets in the aromatic region at 7.40 (1H, J = 8.5 Hz) and 6.43 (1H, J
= 8.5 Hz), which were attributed to a tetra-substituted benzene ring. It
also displayed two methines at 5.62 (s) and 3.42 (s), two doublets for a
methylene at 3.44 (1H, J = 15.5 Hz) and 3.39 (1H, d, J = 15.5 Hz) and
methoxyl proton resonance at 3.66. The upfield region of the same
spectrum displayed displayed two singlet methyls at 1.70 and 1.60. The
most downfield signals at 14.12 (1H, s) and 11.78 (1H, s) were attributed
to amide hydrogen and chelated hydroxyl functions, respectively. The
relatively downfield shift of amide hydrogen could be due to an anisotropic
effect of carbonylic oxygen of the chromone moiety.
The 13C NMR spectrum of 192 (Table 5.1) displayed altogether 19
carbon signals which were identified as three methyls ( 52.5, 28.4, 18.0),
O
OOHO
H3CO
HN Me
OH
OOH
Me
O
O
2
3
1918
14
16
17
15
1312
H
1
4
56
7
89
1011
192
Chapter 5 Endophytic Fungi
128
one methylene ( 41.1), four methines ( 140.4, 108.4, 73.1, 55.8) and
eleven quaternary carbons ( 187.7, 169.8, 166.5, 165.3, 158.9, 157.1,
117.1, 105.9, 104.2, 78.8, 58.6). The positions of various functionalities
were fixed based on the HSQC, COSY and HMBC (Figure 5.1) information.
The methyl carboxylate function was placed at C-6 due to HMBC
correlation of H-7 ( 7.40) with the carbons at 117.1 (C-6) and 166.5
(C-11). The same hydrogen was further correlated with C-5 ( 158.9) and
C-9 ( 157.1). The chelated hydroxyl proton ( 11.78) showed long-range
interaction with the carbons C-5 ( 158.9), C-6 ( 117.1) and C-10 (
105.9), the proton H-8 ( 6.43) was found to interact with the carbons C-6
( 117.1), C-9 ( 157.1), C-10 ( 105.9) and have a weak correlation with
C-4 ( 187.7). The proton resonating at 3.42 (H-3) exhibited HMBC
coupling with C-2 ( 104.2), C-10 ( 105.9), C-12 ( 78.8), C-13 ( 73.1), C-
14 ( 58.6) and C-15 ( 18.0), whereas, the acylated methine H-13 ( 5.62)
showed HMBC correlation with C-17 ( 165.3), C-2 ( 104.2), C-3 ( 55.8),
C-12 ( 78.8), C-14 ( 58.6), C-16 ( 28.4) and C-15 ( 18.0). The above
data indicated the presence of a chromone nucleus merged with a 5-
membered ring in cryptosporioptide (192) which covers eight DBE,
whereas, the remaining three DBE could be attributed to a malonate
moiety 165.3 (C-17), 41.1 (C-18) and 169.8 (C-19), which was acylated at
C-2 and 13 to form a bridged 8-membered ring. The amide hydrogen (
14.12) that exhibited HMBC correlation with carbon at 169.8 (C-19),
Chapter 5 Endophytic Fungi
129
104.2 (C-2), and 55.8 (C-3) also confirmed this 8-memberd bridged ring
formation. The important HMBC correlations were shown in figure 5.1 to
join various fragments of cryptosporioptide.
78
O
Me
OH
OOH
Me
13 O
OOHO
OCH3
HN Me
OH
OOH
Me
O
O
H
HN Me
OH
OOH
Me
O
O
H
2
3
19
18
14
1617
15
13
12
56
9
10
10143
2 3
2
17
Figure. 5.1: Joining of various fragments of cryptosporioptide identified through HMBC correlation.
The relative stereochemistry at the chiral centers was confirmed by
NOESY correlations and molecular model. In NOESY spectrum, the
correlations between Me-15 ( 1.60) and Me-16 ( 1.70) with H-3 ( 3.42)
and H-13 ( 5.62) indicated that these groups or atoms were on the same
side of the 5-membered ring, and that the malonate moiety is attached at
C-2 and C-13 with cis- junctions. The absence of NOESY interaction of
amide proton and on C-18 methylene protons with any other hydrogen
also supported the assigned structure. The above discussed data led to
structure 192 as a novel polyketide and is named cryptosporioptide
(Saleem et al., 2013).
The unique structure of cryptosporioptide precludes the use of
reference compound to assign its absolute configuration. Therefore, the
Chapter 5 Endophytic Fungi
130
electronic circular dichroism (CD) spectrum of cryptosporioptide (192) was
recorded in solution (Figure 5.2, solid lines) and compared with that
calculated using time-dependent density functional theory (TDDFT) after a
computational procedure (described in the Computational Section 5.7.2)
was employed to generate input structures (having initially arbitrary
(2R,3S,12R,13R,14R) configuration) (Pescitelli et al., 2011; Autschbach et
al., 2012). Nine low-energy conformers were found with relative internal
energies within 2 kcal/mol computed at B3LYP/6-311G+(d,p) level. The
lowest-energy structure is shown in Figure 5.3 and, together with a very
similar one differing only of the rotation of 14-OH group, accounted for
52% Boltzmann population at 300K. This structure is in keeping with
NOESY results discussed above. All low-energy conformers were subdued
to CD calculations with B3LYP/TZVP (CAM-B3LYP functional gave a
consistent result), and the resultant calculated CD spectra were
Boltzmann-averaged at 300K. The calculated average CD spectrum is
shown in Figure 5.2 (dotted line). The good agreement with the
experimental ones, apart from a systematic wavelength shift, allowed us to
assign the absolute configuration of cryptosporioptide as (+)-
(2R,3S,12R,13R,14R).
Chapter 5 Endophytic Fungi
131
Figure. 5.2: Solid lines: experimental UV-vis (top), CD spectra (bottom) of (+)-
cryptosporioptide (CH3CN, 6.28 mM, 0.01 cm cell). Dotted line:
calculated CD spectrum of (2R,3S,12R,13R,14R)-cryptosporioptide
with B3LYP/TZVP//B3LYP-6-311G+(d,p) method, as Boltzmann
average of 9 low-energy structures at 300K. The calculated spectrum
was obtained as sum of Gaussian bands with 0.38 eV exponential
half-width, and red-shifted by 50 nm.
Chapter 5 Endophytic Fungi
132
Figure. 5.3: Lowest-energy structure calculated for (2R,3S,12R,13R,14R)-
Cryptosporioptide with B3LYP-6-311G+(d,p) method.
5.7.1.2. Characterization of cryptosporioptide A (193)
Cryptosporioptide A (193) was also obtained as yellow amorphous
powder, which exhibited a pseudo-
molecular ion at m/z 430.0721 [M+Na]+
in (+ve) HR-FABMS, corresponding to
the molecular formula C18H17NO10 with
eleven DBE. The IR spectrum was
indicative of the presence of carboxylic acid function as it exhibited a
broad absorption band at 2420-3410 cm-1. The spectrum also displayed a
shoulder band at 3405 cm-1 due to secondary amide with other prominent
absorption bands at 1715, 1685, 1590 and 1565 cm-1 for carbonyl
function and aromatic moiety.
The 1H NMR spectral (Table 5.1) of 193 was nearly identical to that
of 192 as it showed two doublets at δ 7.39 (1H, J = 8.3 Hz) and 6.50 (1H,
J = 8.3 Hz), two singlet methine at δ 3.57 and δ 5.67, methylene protons
at δ 3.43 (1H, J = 15.6 Hz) and δ 3.40 (1H, J = 15.6 Hz) and two singlet
O
OOHO
HO
HN Me
OH
OOH
Me
O
O
2
3
1918
14
16
17
15
1312
H
1
4
56
7
89
1011
193
Chapter 5 Endophytic Fungi
133
methyl at δ 1.62 and δ 1.53. The main difference was that the spectrum of
193 was missing the signal for methoxyl group, and thus afforded a
carboxylic acid function instead of methyl carboxylate as was observed in
the data of compound 192.
The 13C-NMR spectrum (Table 5.1) substantiated the above
deduction as it displayed 18 carbon signals, which were identified as two
methyl (δ 28.4, 18.7), one methylene (δ 42.0), four methine (δ 141.4,
109.7, 74.1, 56.7) and eleven quaternary carbons (δ 185.0, 169.1, 168.0,
167.2, 160.1, 158.0, 117.6, 106.0, 105.0, 80.0, 60.0) due to (DEPT, BB)
experiment. The structure was further confirmed through HMBC spectral
analysis (Figure 5.1) in which H-7 (δ 7.39) showed HMBC correlation with
carbonyl carbon at δ 169.1 to fix carboxylic function at C-6. The HMBC
interaction of H-13 (δ 5.67) with C-17 (δ 168.0) confirmed the location of
malonate moiety. Other diagnostic HMBC correlations are similar as
shown in figure 5.1. Stereochemistry at various chiral centers could be
defined through NOESY spectral information (Figure 5.4), deriding model
and due to comparable optical rotation value with that of 192.
Combination of the whole spectroscopic data and comparison with the
data (Table 5.1) of 192, compound 193 was identified as de-methyl
derivative of 192 and is named as cryptosporioptide A (193) (Tousif et al.,
2014).
Chapter 5 Endophytic Fungi
134
O
OOHO
HO
HN Me
OH
OOH
Me
O
O
H
H
193
Figure. 5.4: NOESY correlation observed in compound 193.
5.7.1.3. Characterization of cryptosporioptide B (194)
Compound 194 was also obtained as yellow amorphous powder.
The molecular formula C16H18O9 with
eight DBE was established through HR-
FABMS (m/z 377.0841, [M+Na]+. The 1H
and 13C-NMR spectrum displayed almost
similar signals to that of 192 and 193
that revealed the same skeleton of 194. The 1H-NMR spectrum of 194 was
missing the signals for a methylene group (Table 5.1), whereas, H-13
shifted upfiled (δ 4.09) by more than 1.0 ppm when compared to that of
194. This data indicated that compound 194 must be missing malonate
moiety. Molecular formula and 13C-NMR spectrum (Table 5.1) confirmed
this deduction. The 13C-NMR spectrum of 194 displayed total 16 signals,
which were attested for three methyl, four methine and nine quaternary
carbons. The above discussed data revealed that compound 194 is
missing malonate moiety and amide function, instead an additional
O
OOHO
H3CO
HO Me
OH
OHOH
Me
194
2
3 14
16
15
1312
H
1
4
56
7
89
1011
Chapter 5 Endophytic Fungi
135
hydroxyl group was found at C-2. This deduction was substantiated
through IR spectral analysis, which showed the presence of hydroxyl
(3470 cm-1), methine (3107, 2946 cm-1), carboxylate (1734 cm-1), ketonic
(1682 cm-1) and aromatic (1591, 1563 cm-1) systems, whereas, the
absorption band for amide function was missing (Tousif et al., 2014).
Table. 5.1: 1H and 13C-NMR data of 192 (CDCl3; 500 and 125 MHz) 193 and 194
(CD3OD; 500 and 125 MHz). Compound 192 Compound 193 Compound 194
Position δH (J in Hz) δc δH (J in Hz) δc δH (J in Hz) δc
2 - 104.2 - 106.0 - 105.6
3 3.42 (s) 55.8 3.57(s) 56.7 3.54 (s) 56.7
4 - 187.7 - 185.0 - 182.3
5 - 158.9 - 160.1 - 160.3
6 - 117.1 - 117.6 - 118.0
7 7.40 (d, J = 8.5) 140.4 7.39 (d, J = 8.3) 141.4 7.40 (d, J = 8.5) 141.8
8 6.43 (d, J = 8.5) 108.4 6.50 (d, J = 8.3) 109.7 6.41 (d, J = 8.5) 109.0
9 - 157.1 - 158.0 - 164.0
10 - 105.9 - 105.0 - 104.0
11 - 166.5 - 169.1 - 168.8
12 - 78.8 - 80.0 - 82.1
13 5.62 (s) 73.1 5.67 (s) 74.1 4.09 (s) 74.4
14 - 58.6 - 60.0 - 62.2
15 1.60 (s) 18.0 1.53 (s) 18.7 1.51(s) 18.0
16 1.70 (s) 28.4 1.62 (s) 28.4 1.59 (s) 28.4
17 - 165.3 - 168.0 - -
18 3.44 (d, J = 15.5)
3.39 (d, J = 15.5) 41.1 3.43 (d, J = 15.6)
3.40 (d, J = 15.6) 42.0
19 - 169.8 - 167.2 - -
OMe 3.66 (s) 52.5 - - 3.54 (s) 53.0
N-H 14.12 - - -
5-OH 11.78 - - -
Chapter 5 Endophytic Fungi
136
5.7.2. Computational Section for Compound 192
Conformational searches and DFT geometry optimizations were run
with Spartan’10 (Wave function, Inc., Irvine CA, 2010), using default
parameters, grids and convergence criteria. TDDFT calculations were run
with Gaussian’09 with default grids and convergence criteria. The
conformational search was run using MMFF force field. All structures
thus obtained within 10 kcal/mol were pre-optimized with B3LYP/6-
31G(d) and finally optimized with B3LYP/6-311+G(d,p). TDDFT
calculations were run using CAM-B3LYP and B3LYP functionals and TZVP
basis sets (www.gaussian.com/g_tech/g_ur/g09help.htm). ECD spectra
were generated by applying a Gaussian band shape with 0.38 eV
exponential half-width, using dipole-length rotational strengths; the
difference with dipole-velocity values was checked to be minimal for all
relevant transitions.
5.7. 4. Biological Activities of Compounds 192-194
Compounds 192-194 were tested against four enzymes
acetylcholinesterase, butyrylcholinesterase, -Glucosidase and
lipoxygenase and compound 192 was found significantly active against
the enzyme lipoxygenase with an IC50 value of 49.15±0.17 M and enzyme
-glucosidase with an IC50 value of 50.59±0.28 M, whereas it was
inactive against other two enzymes. Compounds 193 and 194 showed
moderate activity against -glucosidase (IC50 = 44.93±0.26 and
Chapter 5 Endophytic Fungi
137
41.42±0.14 respectively) and lipoxygenase (IC50 = 134.38±1.65 and
149.62±1.53 respectively), whereas, remained inactive against other two
enzymes. Cryptosporioptide (192) was also tested in an agar diffusion
assay against two bacterial test organimsms, Escherichia coli and Bacillus
megaterium, two fungi, Microbotyrum violaceum and Botrytis cinerea, and
the alga Chlorella fusca. At a concentration of 50 g per 9 mm paper disc,
it inhibited the growth of Bacillus megaterium moderately with a 9 mm
radius of the zone of inhibition. For comparison, the same concentration
of penicillin and streptomycin resulted in radii of inhibition of 26 and 13
mm, respectively. The metabolite was inactive against the other test
organisms. The comparison of the lipoxygenase inhibitory activity of
compounds 192-194, it is proposed that malonate moiety and
methylcarboxylate functions could equally be responsible to inhibit the
enzyme.
Table. 5.2: Enzyme inhibitory activity of compounds 192-194.
-Glucosidase activity Lipoxygenase activity
Compound Inhibition (%) at
0.25 mM
IC50 (µM) % Inhibition at 0.25
mM
IC50 (µM)
192 90.16±0.81 50.59±0.28 90.56 ± 1.19 49.15 ± 0.17
193 90.23±0.73 44.93±0.26 62.37±2.34 134.38±1.65
194 91.51±0.56 41.42±0.14 57.89±2.13 149.62±1.53
Acarbosea - 38.25±0.12 - -
Eserinea - - - 22.4 ± 1.3
Chapter 5 Endophytic Fungi
138
5.8. EXPERIMENTAL
5.8.1. General Experimental Procedures
Column chromatography (CC) was performed with silica gel (230-
400 mesh, E-Merck, Darmstadt, Germany) eluting with various organic
solvents, all commercial grades. Various fractions from CC were monitored
on the aluminium sheets pre-coated with silica gel 60 F254 (20×20 cm, 0.2
mm thick; E. Merck, Darmstadt, Germany), which were either visualized
under UV lamp (254 and 366 nm) or heating with ceric sulpahte spray.
The optical rotation was calculated on JASCO DIP-360 polarimeter. The
UV spectra were recorded on Jasco V-650 spectrophotometer, whereas, IR
spectra were recorded on Shimadzu 460 spectrometer (Duisburg,
Germany). The 1H-NMR spectra were scanned on Bruker NMR
spectrometer operating at 500 MHz, while 13C-NMR spectra were recorded
on the same instrument operating at 125 MHz. The 2D NMR (HMQC,
HMBC and COSY) spectra were also measured on the same machine
operating at 500 MHz. NOESY spectra were recorded with a Varian INOVA
600 (14.1 T) NMR spectrometer. FABMS and HR-FAB-MS were calculated
on Finnigan (Varian MAT)JMS H×110 with a data system and JMSA 500
mass spectrometers. CD spectra were measured on a J-715
spectropolarimeter using a 0.01 cm quartz cell.
Chapter 5 Endophytic Fungi
139
5.8.2 Culture, Extraction and Isolation
Cryptosporiopsis (internal strain no. 8999) endophytic fungal sp.
was isolated from the shoot tissues of Viburnum tinus, collected from
Gomera (Spain). The fungus strain was grown for four weeks at room
temperature on biomalt solid agar medium. The culture media was
repeatedly extracted with ethyl acetate. The extract was concentrated
under reduced pressure to get 4.0 g of the crude extract, which was
subjected to column chromatography over silica gel eluting with gradient
of ethyl acetate:n-hexane. As a result 05 fractions (A-E) were obtained
(Scheme 5.1).
The fractions A-C mainly contain a mixture of lipids, whereas,
fractions D and E with yellowish colored gummy mass showed several
spots on TLC. Therefore, these fractions were further purified on a silica
gel column eluting fraction D with 50% ethyl acetate in hexane that gave a
sub-fraction D2 which showed a major spot with fewer minor bands on
TLC. This sub-fraction on passing through sephadex LH-20 yielded
cryptosporioptide (192, 25mg). The fraction E was also purified on a silica
gel column eluting with an isocratic of 70% ethyl acetate in hexane to get
four sub-fractions (E1-E4). The sub-fractions E2 and E3 were separately
passed through sephadex LH-20 eluted with methanol that yielded
cryptosporioptide A (193, 15 mg), from fraction E2 and cryptosporioptide
B (194, 8 mg), from fraction E3.
Chapter 5 Endophytic Fungi
140
Crude Extract from culture media
Silica gel CC
Various Frcations on the basis of TLC profiles
Sephadax LH-20MeOH
Repeated Silica gel CC of Various Frcations on TLC basis Mostly free from Fats
EtOAc/ Hexane
192
D1-Fraction E2-Fraction
Sephadax LH-20MeOH
193 194
A B C D E
50% ethyl acetate in hexane isocratic of 70% ethyl acetate in hexane
E3-Fraction
Sephadax LH-20MeOH
Scheme 5.1: Extraction and isolation of secondary metabolites from Cryptosporiopsis (internal strain no. 8999) endophytic
fungal sp.
Chapter 5 Endophytic Fungi
141
5.8.3. Spectroscopic Data of Isolated Compounds
5.8.3.1. Spectroscopic data of cryptosporioptide (192): Light yellow
amorphous powder (25 mg). []D26 61.3 (c
0.001, CHCl3); UV max (max, 6.28 mM in
CH3CN) nm (104M–1cm–1): 374 (1.72), 269
(0.60), 243 (0.69), 195 (1.60); CD ext (ext,
6.28 mM in CH3CN) nm (M–1cm–1): 386.3
(+2.56), 341.0 (+5.0), 283.8 (0), 243.0 (–6.3),
192.5 (–7.3); IR max (KBr) cm-1: 3404, 2926, 1724, 1630; 1H-NMR (CDCl3,
500): δ 7.40 (1H, d, J = 8.5 Hz, H-7), 6.43 (1H, d, J = 8.5 Hz, H-8), 5.62
(1H, s, H-13), 3.42 (1H, s, H-3), 3.44 (1H, d, J =15.5 Hz, H-18), 3.39 (1H,
d, J =15.5 Hz, H-18), 3.66 (3H, s, OMe), 1.70 (1H, s, Me-16), 1.60 (1H, s,
Me-15), 14.12 (1H, s, N-H), 11.78 (1H, s, 5-OH); 13C-NMR (CDCl3, 125
MHz): δ 187.7 (C-4), 169.8 (C-19), 166.5 (C-11), 165.3 (C-17), 158.9 (C-5),
157.1 (C-9), 140.4 (C-7), 117.1 (C-6), 108.4 (C-8), 105.9 (C-10), 104.2 (C-
2), 78.8 (C-12), 73.1 (C-13), 58.6 (C-14), 55.8 (C-3), 41.1 (C-18), 28.4 (C-
16), 18.0 (C-15), 52.5 (OMe); HR-FAB-MS: m/z 444.0781 [M+Na]+ (calcd.
444.0907 for C19H19NNaO10, corresponding to the formula C19H19NO10).
O
OOHO
H3CO
HN Me
OH
OOH
Me
O
O
H
2
3
1918
14
56
7
11
16
17
15
1312
1
4
89
10
192
Chapter 5 Endophytic Fungi
142
5.8.3.2. Spectroscopic data of cryptosporioptide A (193): Light yellow
amorphous powder (15 mg). []D26 +81.1
(CH3OH, c 0.001); UV λmax (CH3OH) nm ():
372 (1.70), 268 (0.62), 242 (0.70), 194
(1.59); IR max (KBr) cm-1: 3402, 2924,
1722, 1629; 1H-NMR (CD3OD; 500 MHz): δ
7.39 (1H, d, J = 8.3 Hz, H-7), 6.50 (1H, d, J
= 8.3 Hz, H-8), 5.67 (1H, s, H-13), 3.57 (1H, s, H-3), 3.43 (1H, d, J =15.5
Hz, H-18), 3.40 (1H, d, J =15.5 Hz, H-18), 1.62 (1H, s, Me), 1.53 (1H, s,
Me); 13C-NMR (CD3OD, 125 MHz): δ 185.0 (C-4), 169.1 (C-11), 168.0 (C-
17), 167.2 (C-19), 160.0 (C-5), 158.0 (C-9), 141.4 (C-7), 117.6 (C-6), 109.7
(C-8), 105.0 (C-10), 106.0 (C-2), 80.0 (C-12), 74.1 (C-13), 60.0 (C-14), 56.7
(C-3), 42.0 (C-18), 28.4 (C-16), 18.7 (C-15); HR-FAB-MS: m/z 430.0721
[M+Na]+ (calcd. 430.0908 for C18H17NNaO10, corresponding to the formula
C18H17NO10).
5.8.3.3. Spectroscopic data of cryptosporioptide B (194): Light yellow
amorphous powder (8 mg). []D26 +22.1
(CH3OH, c 0.0008); UV λmax (CH3OH) nm
(): 376 (1.73), 270 (0.61), 244 (0.72), 196
(1.62); IR max (KBr) cm-1: 3405, 2928,
1720, 1628; 1H-NMR (CD3OD, 500 MHz): δ 7.40 (1H, d, J = 8.5 Hz, H-7),
6.41 (1H, d, J = 8.5 Hz, H-8), 4.09 (1H, s, H-13), 3.54 (1H, s, H-3), 3.54
O
OOHO
H3CO
HO Me
OH
OHOH
Me
194
2
3 14
16
15
1312
H
1
4
56
7
89
1011
O
OOHO
HO
HN Me
OH
OOH
Me
O
O
193
2
3
1918
14
16
17
15
1312
H
1
4
56
7
89
1011
Chapter 5 Endophytic Fungi
143
(3H, s, OMe), 1.59 (1H, s, Me), 1.51 (1H, s, Me); 13C-NMR (CD3OD, 125
MHz): δ 182.3 (C-4), 168.8 (C-11), 160.3 (C-5), 164.0 (C-9), 141.8 (C-7),
118.0 (C-6), 109.0 (C-8), 104.0 (C-10), 105.6 (C-2), 82.1 (C-12), 74.4 (C-
13), 62.2 (C-14), 56.7 (C-3), 28.4 (C-16), 18.0 (C-15), 53.0 (OMe); HR-FAB-
MS: m/z 377.0841 [M+Na]+ (calcd. 377.3201 for C16H19NaO8,
corresponding to the formula C16H19O8).
5.8.4. Enzyme Inhibitory Assays
The four enzyme inhibitory assays were performed according to the
pre-established protocols (Tappel 1953; Baylac and Racine 2003; Ellman
et al., 1961) with slight modifications, as described below.
5.8.4.1.-Glucosidase inhibition assay
The -glucosidase inhibition activity was performed according to the
method Reported by Pierre et al., (Pierre et al., 1978) with some
modification. Total volume of the reaction mixture of 100 µl contained 70
µl 50 mM phosphate buffer saline with pH 6.8, 10 µl (0.5 mM) test
compound, followed by the addition of 10 µl (0.057 units) enzyme. The
contents were mixed, preincubated for 10 min at 37ºC and pre-read at
400 nm. The reaction was initiated by the addition of 10 µl of 0.5 mM
substrate (p-nitrophenyl glucopyranoside). Acarbose was used as positive
control. After 30 min of incubation at 37ºC, absorbance was measured at
Chapter 5 Endophytic Fungi
144
400 nm using Synergy HT microplate reader. All experiments were carried
out in triplicates.
The percentage inhibition (%) of the enzyme was calculated using
the following formula:
Inhibition (%) = Control – Test × 100
Control
5.8.4.2. Acetylcholinesterase assay
The Acetylcholinesterase (AChE) inhibition activity was performed
according to the method described by Ellman et al., (Ellman et al., 1961)
with slight modifications. The 100 µL reaction mixture which contain 60
µL of buffer Na2H PO4 with pH 7.7, 10 µL (0.5 mM) test compound and 10
µL (0.005 unit well) enzyme was added and then pre-read at 405 nm. After
that it was incubated for 10 minutes at 37°C. The reaction was initiated
by the addition of 10 µL of 0.5 mM substrate (acetylthiocholine iodide),
followed by the addition of 10 µL DTNB (0.5 mM). Eserine (0.5 mM) was
used as a positive control. Then absorbance was measure at 405 nm on
Synergy HT (BioTek, USA) microplate reader.
The percentage inhibition (%) of the enzyme was calculated using the
following formula:
Inhibition (%) = Control – Test × 100
Control
Chapter 5 Endophytic Fungi
145
5.8.4.3. Butyrylcholinesterase assay
The butyrylcholinesterase inhibition activity was performed
according to same method as reported for Acetylchoinesterse with small
difference as butyrylcholinesterase enzyme was used here.
5.8.4.4. Lipoxygenase assay
The lipoxygenase (LOX)activity was perfirmed by same method as
dercribed for Acetylchoinesterse with small difference as lepoxygenase
enzyme used here (Evans et al., 1987; Baylac 2003) Baicalein (0.5 mM)
was used as a positive control.
The percentage inhibition (%) of the enzyme was calculated using the
following formula:
Inhibition (%) = Control – Test × 100
Control
Where Control = Total enzyme activity without inhibitor Test= Activity in the presence of test compound
In all enzyme inhibitory assays, the IC50 values were calculated
using EZ–Fit Enzyme Kinetics software (Perrella Scientific Inc. Amherst,
USA). All the measurements were done in triplicate and statistical analysis
was performed by Microsoft Excel 2003. Results are presented as mean ±
sem.
Chapter 5 Endophytic Fungi
146
5.8.5. Agar Diffusion Test for Antimicrobial Activity
Cryptosporioptide (192) was dissolved in acetone at a concentration
of 2 mg/mL. 25 μL of the solution (50 μg) were pipetted onto a sterile filter
disc, which was placed onto an appropriate agar growth medium for the
respective test organism and subsequently sprayed with a suspension of
the test organism. The test organisms were the gram-negative bacterium
Escherichia coli, the Gram-positive bacterium Bacillus megaterium (both
grown on NB medium), the fungi Microbotryum violaceum and Botrytis
cinerea, and the alga Chlorella fusca (fungi and alga were grown on MPY
medium). Reference substances were ketoconazole, penicillin and
streptomycin. Commencing at the outer edge of the filter disc, the radius
of zone of inhibition was measured in mm. These microorganisms were
chosen because they are non-pathogenic and had in the past proved to be
accurate initial test organisms for antibacterial, antifungal and
antialgal/herbicidal activities (Holler et al., 2000; Schulz et al., 1995).
compound 193 and 194 were not evaluated for antibacterial activity due
low amount.
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