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



4.3.2. 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



5.8.2 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. 2.3: 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


(CD3OD; 600 and 150 MHz)

52


MHz) 55


MHz) 56

Table. 2.10: 1H and 13C-NMR data of 86 (CDCl3 500 and 125 MHz) 58



Table. 2.13: Antioxidant and antiurease activity of isolated compounds 63

Table. 2.14: Antibacterial activity of isolated compounds 63




MHz)

100

Table. 4.4: 1H and 13C-NMR data of 140 (CDCl3, 400 and 100 MHz) 101


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



38



42



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-


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


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


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).


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


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,


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


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


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


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


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


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


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


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).


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


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


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.


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


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).


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


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).


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).


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


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).


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).


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


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).


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.


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


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.


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'


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



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


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.


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.


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


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


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.


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


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


Combination of the above spectroscopic data helped to establish

structure of 72 as 4-(4-methoxybenzyl) benzenamine, which is named

as vincetomine.


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


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


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


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


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


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


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]+).


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


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.


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


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


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


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


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).


(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


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-


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


55


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).


56

O

OH

HO

O

OH

85

OH

O

OH

OHHO

O

HO

O

OH

HO

O

OH

OOH

OHHO

O

OH84

OH


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


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.


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


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).


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


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).


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


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,


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.


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


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).


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


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).


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).


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


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


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


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).


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


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


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


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).


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


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


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


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


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-


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


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-



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.


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.


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


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).


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).


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


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).


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).


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).


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


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).


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


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.


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


95


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


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.


97

O

O

OCH3

OH

H3CO

H3CO

138


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


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.


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.


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).


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


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).


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


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.


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


104

4.3. EXPERIMENTAL


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.


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


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).


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.


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


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).


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).


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.


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).


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


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


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).


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


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).


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).


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).


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


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


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.


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

http://en.wikipedia.org/wiki/Fungus

http://en.wikipedia.org/wiki/Ascomycota

http://en.wikipedia.org/wiki/Leotiomycetes

http://en.wikipedia.org/wiki/Leotiomycetidae

http://en.wikipedia.org/wiki/Helotiales

http://en.wikipedia.org/wiki/Dermateaceae

http://en.wikipedia.org/w/index.php?title=Cryptosporiopsis&action=edit&redlink=1


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).


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).


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


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),


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


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).


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.


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


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).


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


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 - - -


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

http://www.gaussian.com/g_tech/g_ur/g09help.htm


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


138

5.8. EXPERIMENTAL


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.


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.


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.


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


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


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


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:


Control


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:


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.


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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