(E) Thesis, 1-09-15- HEC-Shabir-IUB.pdf

DISCOVERY OF NOVEL BIOACTIVE COMPOUNDS FROM AN

ENDOPYHTIC FUNGUS ASPERGILLUS SP. AND PHYTOCHEMICAL INVESTIGATION OF TECOMELLA UNDULATA

A Dissertation Submitted to The Islamia University of Bahawalpur

For

Fulfillment of the Requirement for the Award of Degree of Doctor of

Philosophy in Chemistry

By

Shabir Ahmad (M. Sc., M. Phil.)

Department of Chemistry

The Islamia University of Bahawalpur Bahawalpur-63100, Pakistan.

August 2014

The Islamia University of Bahawalpur Department of Chemistry

APPROVAL CERTIFICATE

It is certificated that the research work presented in this thesis,

entitled, “DISCOVERY OF NOVEL BIOACTIVE COMPOUNDS FROM AN

ENDOPYHTIC FUNGUS ASPERGILLUS SP. AND PHYTOCHEMICAL

INVESTIGATION OF TECOMELLA UNDULATA” has been carried out by

Shabir Ahmad s/o Zulfiqar Ali. He completed this research work under

my supervision for the fulfillment of the requirement for the award of

degree of doctor of philosophy (Ph.D.) in Chemistry during the session

2009-2013. The thesis is accepted in its present form and therefore,

recommended for submission and further processing.

Prof. Dr. Abdul Jabbar Supervisor

Prof. Dr. Faiz-ul-Hassan Nasim

Chairman

DECLARATION

I Shabir Ahmad, hereby declare that the research work entitled,

“DISCOVERY OF NOVEL BIOACTIVE COMPOUNDS FROM AN

ENDOPYHTIC FUNGUS ASPERGILLUS SP. AND PHYTOCHEMICAL

INVESTIGATION OF TECOMELLA UNDULATA” was carried out in the

Chemistry Department of The Islamia University of Bahawalpur, Pakistan.

Most of the work has been accepted for publication in peer reviewed

journals. I undertake that I have not submitted this work to any other

institution or university to earn the degree of Doctor of Philosophy (Ph.D.)

and will not, in future, submit for obtaining the similar degree to any

other university.

Shabir Ahmad

August 2014

Acknowledgement

I express my deepest gratitude from the insight of my heart

and all praises to the Almighty ALLAH the most merciful and the

most beneficent for the Blessings He had bestowed upon me to

complete this work.

All respects for the most perfect personality of the world

HAZRAT MUHAMMAD MUSTAFA (Peace Be upon Him) who

enlighten us to recognize our creator and showed us the right path

and gave a complete code of life.

It is like a hard nut to concentrate and to write a Ph.D. thesis

without the guidance and help of a sincere and learned supervisor.

For such a worthy contribution, I am immensely pleased to place on

record my profound gratitude and heartfelt thanks to my

supervisor Prof. Dr. Abdul Jabbar, Department of Chemistry, The

Islamia University of Bahawalpur, for his guidance during my

research, who extended all facilities and provided inspiring

guidance for the successful completion of my research work. I deem

it as my privilege to work under his able guidance. I whole

heartedly acknowledge his approach of work, guidance,

hardworking, skilled piece of advice and sincere efforts in turning

this work into success.

I am thankful to Dr. Muhammad Saleem for his precious

guidelines through my work. I am thankful for his scientific piece of

advice, knowledge and many insightful discussions and suggestions.

He is my primary resource for getting my scientific questions

answered and instrumental in helping me to crank up this thesis.

Infact he is one of the smartest people I know. The inspiration, help

and suggestions received from Dr. Naheed Riaz during research is

highly acknowledged. I am also whole heartedly thankful to Prof.

Dr. Faiz-ul-Hassan Nasim, Chairman Department of Chemistry, for

facilitating and guiding me in completion and submission of the

thesis. I also pay thanks to Prof. Dr. Muhammad Ashraf,

Department of Biochemistry and Biotechnology for screening my

samples in various bioassays.

I am thankful to Prof. Dr. Abdul JMalik and Prof. Dr. Muhammad

Shaiq Ali from HEJ Research Institute of Chemistry, University of

Karachi for providing some lab facilities.

I am also thankful to my laboratory fellows and senior

colleagues; Dr. Sara Musaddiq, Dr. Naseem Akhtar, Dr. Bushra

Jabeen, Dr. Nusrat Shafiq, Momina Zubair, Hafiza Mahwish Rafiq,

Jallat Khan, Imran Touseef, Bashart Ali, Akram Naveed ,Abdul

Ghaffar, Liaqat Ali, Mehreen Mukhtar and Saima Kanwal for their

support throughout the research work. I am very thankful to my

friends’ Prof. M. Ashraf Kamal, Imran Ghafoor and Rajab Ali who

encouraged me at every step during my work from research to

thesis submission. I pay sincere gratitude to my college principals

Prof. Mushtaq Ahmad Javed and Prof. Hamayoun Kabeer

Choudhary for their constant encouragement, pleasant association

and help.

I am thankful to all non-teaching staff, Department of

Chemistry, The Islamia University of Bahawalpur, for the help they

rendered me during my stay.

At this Juncture I think of my parents whose selfless sacrificial

life and their great efforts with pain and tears and unceasing

prayers have enabled me to reach the present position in life. I know

I cannot pay them back, thanks to Allah for blessing me with such

kind parents.

How can I ignore my soul mate, my wife Rabia Shabir and my

lovely daughter Youmnah Malik for their smile of love and

tolerance of partial depart which provided me an additional energy

for this completion. These past several years have not been an easy

ride, both academically and personally, I truly thank my wife for

sticking by my side even when I was irritable, I feel that what we

both learned a lot about life and strengthen our commitment and

determination to each other and to live life to the fullest.

Finally, I thank all those who have helped me directly or indirectly

in the successful completion of my thesis.

Shabir Ahmad

List of Abbreviations

LIST OF ABBREVIATIONS

Abbreviation Representation

AChE Acetylcholinesterase

ALP Alkaline Phosphatase

ALT Alanine amino Transferase

AST Aspartate amino Transferase

BChE Butyrylcholinesterase

CC50 Half maximal Cell-mediated Cytotoxicity

CD Circular Dichorism

CDCl3 Deuterated Chloroform

CDK Cyclin Dependent Kinase

CD3OD Deuterated Methanol

CIDS Cholistan Institute of Desert Studies

CNS Central Nervous System

COSY Correlation Spectroscopy

COX-1 Cytochrome Oxidase Subunit-1

COX-2 Cytochrome Oxidase Subunit-2

13C NMR Carbon Nuclear Magnetic Resonance

DEPT Distortionless Enhancement by Polarization Transfer

DBE Double Bond Equivalent

DNA Deoxyribonucleic Acid

DPPH 2,2-Diphenyl-1-Picrylhydrazyl

DMSO Dimethyl Sulfoxide

EC50 Half maximal Effective Concentration

EI-MS Electron Impact-Mass Spectrometry

ESI-MS Electron Spray Ionization-Mass Spectrometry

EtOAc Ethyl Acetate

FAB-MS Fast Atomic Bombardment-Mass Spectrometry

FPT Farnesyl Protein Transferase

GAE Gallic Acid Equivalent

Gr Greek

HCV Hepatitis C Virus

HDL High Density Lipoprotein

HIV Human Immunodeficiency Virus

HLE Human Leukocyte Elastase

List of Abbreviations

HMBC Heteronuclear Multiple Bond Correlation

HMQC Heteronuclear Multiple Quantum Correlation

HPTLC High Performance Thin Layer Chromatography

HR-EI-MS High Resolution-Electron Impact-Mass Spectrometry

HR-ESI-MS High Resolution Electron Spray Ionization Mass Spectrometry

HR-ESI High Resolution-Electron Spray Ionization

HSQC Heteronuclear Spin Quantum Coherence

HSV Herpes Simplex Virus

HPLC High Performance Liquid Chromatography

1H NMR Proton Nuclear Magnetic Resonance

IC50 Half maximal Inhibitory Concentration

IR Infrared

LDL Low Density Lipoprotein

LOX Lipoxygenase

g/ml Microgram per Milliliter

M Micro Mole

µl Micro Liter

MIC Minimum Inhibitory Concentration

MIQ Minimum Identifiable Quantity

MLR Mixed Lymphocyte Reaction

MRSA Methicillin Resistant Staphylococcus aureus

MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

ng Nanogram

NOESY Nuclear Overhauser Enhancement and Exchange Spectroscopy

QE Quericitin Equivalent

QRSA Quinolone Resistant Staphylococcus aureus

TP Thymocyte Proliferation

TLC Thin Layer Chromatography

TMS trimethylsilyl

TXB2 Thromboxane-B2

UK United Kingdom

US United State

UV Ultraviolet

VRE Vancomycin Resistant Enterococci

XTT 2,3-bis-(2-methoxy-4-nitro-5-sulfophenyl)-2H-tetrazolium-5-

carboxanilide

Table of Contents

TABLE OF CONTENTS

Title Page No.

Summary i-iii

CHAPTER 1

NATURAL PRODUCTS; A BLESSING TO MAN

1

1.1. What are Natural Products? 2

1.2. The Earliest Known Pharmaceuticals to Man 2

1.3. Natural Products under Clinical Trials 19

1.4. Aim of the Present Study (Research Question) 21

CHAPTER 2

LITERATURE REVIEW (PREVIOUS PHYTOCHEMICAL INVESTIGATION) OF TECOMELLA UNDULATA

22

2.1. Tecomella undulata 23

2.2. Scientific Classification of Tecomella undulata 23

2.3. Botanical Description of Tecomella undulata 24

2.4. Economic and Ecological Importance of Tecomella undulata 25

2.5. Medicinal Importance of Tecomella undulata 25

2.6. Pharmacological Studies of Techomella undulata 26

2.6.1. Analgesic activity of Techomella undulata 26

2.6.2. Antiacaricidal activity of Techomella undulata 26

2.6.3. Hepatoprotective activity of Techomella undulata 27

2.6.4. Anti-inflammatory activity of Techomella undulata 28

2.6.5. Antimicrobial activity of Techomella undulata 28

2.6.6. Anti-oxidant activity of Techomella undulata 29

2.6.7. Anticancer activity of Techomella undulata 29

2.6.8. Other activities of Techomella undulata 30

2.7. Previously Reported Phytoconstituents of Techomella undulata 31

CHAPTER 3

RESULTS AND DISCUSSION: STRUCTURE ELUCIDATION OF SECONDARY METABOLITES ISOLATED FROM TECOMELLA UNDULATA

37

3.1. Results and Discussion 38

3.1.1. Characterization of kaempferol-3-O-β-D-[3,4di-p-E-coumaroyl-α-L-rhamnosyl (1→6)] galactoside (142)

38

3.1.2. Characterization of the compound (6R,7Z)-9,10-dihydroxy-4,7-megastigmadien-3-one (143)

45

3.1.3. Characterization of (6E)-9,10-dihydroxy-4,6-megastigmadien-3-one (144)

48

Table of Contents

3.1.4. Characterization of (6R,9S)-9,10-dihydroxy-4-megastigmaen-3-one (145)

52

3.1.5. Characterization of 3S,5R,6R,9S-tetrahydroxy megastigmane (146)

52

3.1.6. Characterization of lapachol (118) 55

3.1.7. Characterization of 3-deoxyisolapachol (147) 57

3.1.8. Characterization of quercetin-3,4-dimethyl ether (148) 59

3.1.9. Characterization of 3,5,3,4-tetrahydroxy-6,7-dimethoxy flavone (149)

60

3.1.10. Characterization of quercetin-3-galactoside (150) 61

3.1.11. Characterization of ferulic acid (104) 65

3.1.12. Characterization of hexadecyl ferulate (151) 66

3.1.13. Characterization of betulinic acid (133) 68

3.1.14. Characterization of β-sitosterol (134) 69

3.1.15. Structure elucidation of β-sitosterol-3-O-β-D-glucopyranoside (152)

70

3.2. Antibacterial and Enzyme Inhibitory Evaluation of Compounds 118, 133-134, 142-152

72

3.2.1. Antibacterial activity of compound 142 72

3.2.2. Enzyme inbihitory activity of compounds 118, 133-134, 142-152

72

3.3. Experimental 76

3.3.1. General experimental procedures 76

3.3.1.1. Spectroscopic methods 76

3.3.1.2. Materials 76

3.3.2. Collection and identification of the plant material 77

3.3.3. Extraction of the plant material and isolation 77

3.4. Spectroscopic Data of Compounds Isolated from Tecomella Undulata

81

3.4.1. Spectroscopic data of kaempferol-3-O-β-D-[3,4di-p-E-coumaroyl-α-L-rhamnosyl (1→6)] galactoside (142)

81

3.4.2. Spectroscopic data of (6R,7Z,9S)-9,10-dihydroxy-4,7-megastigmadien-3-one (143)

82

3.4.3. Spectroscopic data of (6E,9S)-9,10-dihydroxy-4,6-megastigmadien-3-one (144)

83

3.4.4. Spectroscopic data of (6R,9S)-9,10-dihydroxy-4-megastigmaen-3-one (145)

83

3.4.5. Spectroscopic data of 3S,5R,6R,9S-tetrahydroxy megastigmane (146)

84

3.4.6. Spectroscopic data of lapachol (118) 84

3.4.7. Spectroscopic data of 3'-OH-deoxyisolapachol (147) 85

Table of Contents

3.4.8. Spectroscopic data of quercetin 3′,4′-dimethyl ether (148)

85

3.4.9. Spectroscopic data of 3,5,3′,4′-tetrahydroxy-6,7-dimethoxy flavone (149)

86

3.4.10. Spectroscopic data of quercetin-3-galactoside (150) 87

3.4.11. Spectroscopic data of ferulic acid (104) 87

3.4.12. Spectroscopic data of hexadecyl ferulate (151) 88

3.4.13. Spectroscopic Data of Betulinic Acid (133) 89

3.4.14. Spectroscopic data of β-sitosterol (134) 89

3.4.15. Spectroscopic data of β-sitosterol-3-O-β-D-glucopyranoside (152)

90

3.5. Acid Hydrolysis of Compound 142 91

3.6. Anti-bacterial Assay 91

CHAPTER 4

ENDOPHYTIC FUNGI AS POTENTIAL SOURCE OF BIOACTIVE ORGANIC COMPOUNDS WITH FASCINATING STRUTURES

93

4.1. Potential of Microorganisms with respect to Endophytes 94

4.2. What are Endophytes? 94

CHAPTER 5

LITERATURE SURVEY (SECONDARY METABOLITES PREVISOUSLY REPORTED) OF ASPERGILLUS SP.

115

5.1. Endophytes; A Natural Machinary to Synthesize Fascinating Bioactive Compounds

116

5.2. Secondary Metabolites Isolated from the Aspergillus sp 116

CHAPTER 6

STRUCTURE ELUCIDATION AND SPECTROSCOPIC DATA OF SECONDARY METABOLITES ISOLATED FROM ENDOPHYTIC FUNGUS ASPERGILLUS SP.

132

6.1. Results and Discussion 133

6.2. Characterization of Compounds Isolated from Aspergillus sp. 133

6.2.1. Characterization of dibenzofuran, Sch725421 (362) 133

6.2.2. Characterization of 4-acetyl-3,4-dihydro-6,8-dihydroxy-5-methyl-isocoumarin (302)

136

6.2.3. Characterization of 4-acetyl-6,8-dihydroxy-3-methoxy-5-methyl-3,4-dihydro isocoumarin (303)

138

6.2.4. Characterization of 2,3,4-trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran (304)

140

6.2.5. Characterization of 2,6-dihydroxy-4-(3-hydroxybutan-2-yl)-3-methyl benzoic acid (363)

142

6.2.6. Characterization of cytochalasin C (364) 145

6.2.7. Characterization of cytochalasin D (365) 148

Table of Contents

6.2.8. Characterization of ergosterol (244) 149

6.2.9. Characterization of 24-methylcholesta-7,22-E-diene-

3β,5-diol-6-one (366)

150

6.3. Enzyme Inhibitory Activites of the Compounds Isolated from Aspergillus sp.

153

6.4. Experimental 156

6.4.1. General experimental procedures 156

6.4.2. Culture, extraction and isolation 157

6.5. Spectroscopic Data of the Isolated Compounds 159

6.5.1. Spectroscopic data of dibenzofuran Sch725421 (362) 159

6.5.2. Spectroscopic data of 4-acetyl-3,4-dihydro-6,8-dihydroxy-5-methyl isocoumarin (302)

159

6.5.3. Spectroscopic data of 4-acetyl-6,8-dihydroxy-3-methoxy-5-methyl-3,4-dihydro isocoumarin (303)

160

6.5.4. Spectroscopic data of 2,3,4-trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran (304)

160

6.5.5. Spectroscopic data of 2,6-dihydroxy-4-(3-hydroxybutan-2-yl)-3-methyl benzoic acid (363)

161

6.5.6. Spectroscopic data of cytochalasin C (364) 161

6.5.7. Spectroscopic data of cytochalasin D (365) 162

6.5.8. Spectroscopic data of ergosterol (244) 163

6.5.9. Spectroscopic data of 24-methylcholesta-7,22-E-diene-

3β,5-diol-6-one (366)

164

References 166

Annexure-A 187

List of Tables

LIST OF TABLES

Table Page No.

1.1: Natural products under clinical trials 19

3.1: 1H and 13C NMR data of compound 142 (CD3OD+CDCl3, 400 and 100 MHz) and COSY correlations

43

3.2: 1H and 13C NMR data of compounds 143 and 144 (CD3OD, 400 and 100 MHz)

51

3.3: 1H and 13C NMR data of 145 and 146 (CD3OD, 400 and 100 MHz) 55

3.4: 1H and 13C NMR data of 118 and 147 (CDCl3, 500 and 125 MHz) 58

3.5: 1H and 13C NMR data of compounds (148-150) (CD3OD, 400 and 100 MHz)

64

3.6: 1H and 13C NMR data of 104 and 151 (CDCl3+CD3OD, 400 and 100 MHz)

67

3.7: 1H and 13C NMR data of 133, 134 and 152 (CDCl3+CD3OD, 400 and 100 MHz)

71

3.8: Enzyme inhibitory activities of the isolated compounds from Tecomella undulata

75

6.1: 1H and 13C NMR data of compound 362 (CD3OD, 400 and 100 MHz) 136

6.2: 1H and 13C NMR data of compound 302 (DMSO-d6, 500 and 125 MHz) and 303 (DMSO-d6, 400 and 100 MHz)

140

6.3: 1H and 13C NMR data of compound 304 (MeOD, 500 & 125 MHz) and 363 (DMSO-d6, 500 and 125 MHz)

144

6.4: 1H and 13C NMR data of compound 364 (DMSO-d6, 300 & 75 MHz) and 365 (DMSO-d6, 400 & 100 MHz)

149

6.5: 1H and 13C NMR data of compound 244 (CDCl3, 300 & 75 MHz) and 366 (CDCl3, 500 and 125 MHz)

152

6.6: Enzyme inhibitory activities of isolated compounds from Aspergillus sp. 155

List of Figures

LIST OF FIGURES

Caption of the Figure Page No.

3.1: Important HMBC correlations observed in the spectrum of 142 42

3.2: HMBC correlations observed in the spectrum of compound 143 47

3.3: HMBC correlations observed in the spectrum of 144 49




3.7: HMBC and COSY correlations observed in the spectrum of compound 150

63

6.1: Important HMBC and COSY correlations observed in the spectra of 362 135





6.6: Important COSY and HMBC correlations observed in the spectra of 364 147

Summary

i

Summary

With the dawn of civilization, plant-based natural products have

played a very important role in health care and prevention of diseases.

Since ancient times, plants are used by man to treat transferable

infections and some conventional medicines are still utilized for usual

treatment of many ailments. Plants produce secondary metabolites,

termed as “natural products” which provide a unique element of molecular

diversity and biological functionality. Many medicinal plants or their

extracts are used as medicine because of the biological action of secondary

metabolites that include steroids, terpenoids, alkaloids, flavonoids and

polyketides and or other numerous diverse structures etc.

Thousands of medicinal plants are growing in all regions of

Pakistan; unfortunately a little fraction of this number has been evaluated

for their medicinal values. Therefore there is a continuous need for

organized efforts to explore medicinal values of these plants and their

secondary metabolites. To address this issue, I selected a Pakistani

medicinal plant Tecomella undulata to investigate for its secondary

metabolites, besides, the culture extract of an endophytic fungus

Aspergillus sp. has also been studied for its chemicals.

The work embodied in this thesis has been divided into six chapters.

Chapter 1 offers information on the history of use of natural products and

provides a comprehensive review of important bioactive natural products.

Medicinal values, local uses and previous phytochemical work done

on Tecomella undulata has been incorporated in Chapter 2 as a review

article on related literature. During the course of this study, 15

metabolites (104, 118, 133, 134, 142-152), were isolated from the

extract of Tecomella undulata. Their structure elucidation has been

described in Chapter 3 as a part of results and discussion. The new

Summary

ii

compounds (142-144) were characterized with the help of 1D, 2D NMR

and HR-EI-MS techniques, whereas, the known compounds were

identified based on 1D-NMR (1H NMR and 13C NMR) studies, mass

spectrometry and comparison with the literature data. These compounds

are in process of publication in Journal of Chemical Society of Pakistan

(Annexure A). All the isolated compounds were evaluated for antibacterial

and enzyme inhibitory activities. The experimental techniques used in this

study and spectroscopic data of all the isolates have also been included in

the same chapter.

O

O

HO

OH

OH

O

O

HO

OH

OHO

O

HO

O

1

34a

8a

6

81'

3'

5'

1''

3''5''

O

OH

OO

OH142

O

OH

OH

R

Z

1

3

5

67

8

9

10

1112

13

143

O

144

OH

OH

E

[A], Shabir Ahmed, Sara Musaddiq, Muhammad Saleem, Naheed Riaz, Sundas Fatima, Asma Yaqoob, Abdul

Jabbar and Faiz-Ul-Hassan Nasim, Bioactive Secondary metabolites from Tecomella undulata, Journal of

Chemical Society of Pakistan, 2014. (In Press)

Chapter 4 deals with the introduction of endophytes and also describes

some important compounds previously isolated from various endophytic

fungi, whereas, previous studies on Aspergillus sp. has been described in

chapter 5 as a comprehensive literature review. Structure elucidation of

the metabolites isolated from our investigated Aspergillus sp. has been

Summary

iii

discussed in chapter 6. Spectroscopic analysis led to the identification of

nine known metabolites (244, 302-304, 362-366) from the ethyl acetate

extract of the culture of Aspergillus sp. All the metabolites were also

studied for their biological potential.

Chapter 1 Introduction

1

CHAPTER 1

NATURAL PRODUCTS; A BLESSING TO MAN


2

1.1. What are Natural Products?

The web definition of Natural Products is “the chemical compounds

found in nature that usually have a pharmacological or biological activity

for use in pharmaceutical drug discovery and drug design” or “the

compounds produced by living organisms including plants, animals and

microorganisms”. In the broadest sense, natural products include any

substance produced by life (Pearson et al., 2007; Samuelson and Gunnar,

1999). In terms of chemistry, the word natural products refer to organic

compounds produced by living organisms. Natural products are generally

either of prebiotic origin or originate from microbes, plants, or animal

sources (Nakanishi, 1999). Natural products include a large and diverse

group of substances from a variety of sources. They are produced by

marine organisms, bacteria, fungi, and plants. The term encompasses

complex extracts from these producers, but also the isolated compounds

derived from those extracts. It also includes vitamins, minerals and

probiotics.

1.2. The Earliest Known Pharmaceuticals to Man

The ancient man had to rely on its surroundings for his existence.

Plants had always been the major source that provided man the shelter,

clothing, food and health care products. Even with the dawn of

civilization, natural products have played a very important role in health

care and prevention of diseases. Several ancient civilizations provide


3

written evidence for the use of natural sources for curing various diseases

(Phillipson, 2001). The earliest known written document is a 4000 year old

Sumerian clay tablet that records remedies for various illnesses (Kong et

al., 2003). The document described the use of various herbs to cure

several diseases. For example the plants of genus Mandragora,

particularly the species Mandragora officinarum was prescribed for pain

relief. Turmeric possesses blood clotting properties, whereas, roots of the

endive plant were used for treatment of gall bladder disorders and raw

garlic was prescribed for circulatory disorders. These herbs or their

extracts are still being used as alternative medicines.

Biological organisms produce two distinctly different types of

chemical products. The first type, “primary metabolites” consists of the

compounds such as sugars, amino acids and proteins that are common to

all organisms and are essential for functional metabolism, reproduction

and growth. “Secondary metabolites” on the other hand are of limited

distribution in nature and are produced by a single species correlated

group of organisms. They are not crucially important for the life of

producer organism but serve in a wide variety of important roles, for

example they can function as communication tools, defense mechanism,

or sensory devices. The biological activity of these chemicals is beneficial

to the producer organism, but it can be harmful or beneficial to other

species, including humans (Howell and Passmore, 2012). Secondary

metabolites are derived from primary metabolites due to certain enzyme


4

activity. These chemicals are produced in response of the environmental

stress and or other factors, and usually play a role of defensive tools as

certain pharmacological activities are always associated to them.

Secondary metabolites include steroids, terpenoids, alkaloids, flavonoids

and polyketides and or other numerous diverse structures etc. Human

interest in natural sources to provide future drug candidates or

recreational use reaches back to the earliest points of history. Natural

products provide a unique element of molecular diversity and biological

functionality. The emergence of strategies to deliver drug leads from

natural products within the same time frame as synthetic chemical

screening has eliminated a major limitation of the past. Natural drugs may

be divided into three categories of compounds: i) those that are isolated

from biological organisms, ii) those that are derivatives of natural

products, and iii) those that are totally synthetic, but based on the models

of natural origin (Nisbet and Moore, 1997; Wrigley et al., 1997). In the

current scenario of pharmaceuticals, the natural products are responsible

for about half of the approved drugs (Tringali, 2004). This number is

rather higher in case of anticancer drugs. With the passage of time, the

field of natural product drug discovery has developed into the recent form

of science i.e. organic chemistry and pharmacognosy. The crude plant

drugs were subjected to the isolation of pure compounds, which became

the back bone of pharmaceutical industry today. The pure chemicals

isolated from plants and microorganisms were termed as secondary


5

metabolites. The early discoveries of such compounds include muscarine

(1), penicillin (2), nicotine (3), cocaine (4), quinine (5), morphine (6),

paclitaxel (7) and many others.

OCH3

OH

N

Me

Me

Me

1

N

SH

COOHO

HN

O

R

Me

Me

2

N

N

H

CH3

3O

O

NOMe

O

Me

4

N

HO

MeO

N

5

NMe

HO

O

H

HO

H

6

O NH

O

O

O OH

O

O

O

H

O

O

HO

O

O

OH

7

Secondary metabolites are a vast group of compounds, with no

distinct boundaries. Concepts of secondary metabolism include products

of certain enzymatic actions that may occur in response of nutrient

limitation and environmental stress. Perhaps the best defined theory of

secondary metabolism has been offered by Zahner, who described

secondary metabolism as evolutionary “elbow room” (Zahner and Drautz,

1992).

According to an estimate, out of 250,000 higher plants, not more

than 1% have been explored with respect to their pharmacological

properties (Krishnamurthi, 2007). However, several important compounds

have so far been isolated from fewer natural sources, such as vincristine


6

(8) and vinblastine (9) are phytochemicals which are used for acute

childhood leukemia and lymphoma types of cancer (Vita et al., 1970)

respectively. Then paclitaxel (7, taxol) and its analogue docetaxel (10,

taxotere) are known as microtubule-stabilizing anticancer drugs

(Rowinsky, 1997).

NH

N

OH

NR2

N

OAcOHH

CO2Me

HMeO

8: R1 = CO2Me, R2 = CHO9: R1=CO2Me, R2 = Me

R1

Ph O

NH

OH

O

O

OHHO O

O O

H

O

Ph O

O

HO

O

10

Epothilone A (11) and B (12) were discovered in 1993 (Gerth et al.,

1996; Hofle et al., 1996) and are supposed to have paclitaxel-like

mechanism of action (Bollag et al., 1995). Among various natural

products, the taxol (7) is currently the world’s best-selling anticancer drug

available for chemotherapy, obtained from plant sources. Other important

anticancer natural product based drugs are camptothecin (13),

flavopiridol (14), homoharringtonine (15) and podophyllotoxin (16) (Tang

and Eisenbrand, 1992).

A phytoflavonoid, flavopiridol (14) is known as the inhibitor of the

cyclin-dependent kinase (CDK) family (Rocha et al., 2001). Although, it


7

appears to be non-selective towards any particular CDK, yet it is creating

excitement due to its unique mode of action. Homoharringtonine (15) is

anti-leukemia constituent of the seeds of Cephalotaxus harringtonia

(Powell et al., 1970). Podophyllotoxin (16), an anti tumor phytochemical is

a dimeric lignan, isolated from the roots of plants belonging to the genera

Podophyllum and Juniperus (Kuo and King, 2001).

N

S

O

OOH

HO

O

O

H

N

S

O

OOH

HO

O

O

H3C

11 12

O

N

OOH

HO

Me

Cl

14 15

N

N

O

OHO

O

13

O

OO

OH

O

OMe

OMe

MeO

16

NO

O

OCH3

O

O

CH2COOCH3

HO

HO

Epipodophyllotoxin (17), an epimer of podophyllotoxin (16) has been

derivatized to synthesize etoposide (18) and teniposide (19) with more

activity and less toxicity (Angell et al., 1995; Newman et al., 2000).

18

19

R= CH3

R=S

OO

O

OMe

OMe

MeO

H

O H

OH

17

OO

O

O

O O

OMe

OMe

OH

O

HO

OH

R

O


8

Two anti-inflammatory tricyclic alkaloids, ascidiathiazone A (20) and

B (21) were isolated from Ascidian aplidium species. Both the compounds

affect superoxide production by human neutrophils in vitro with an IC50

value of 0.44-1.55 μM. Manzamine A (22), (-)-8-hydroxymanzamine A (23)

and hexahydro-8-hydroxymanzamine A (24) are known as potential

inhibitors of TXB2 generation in brain microglia (Mayer et al., 2005). A

low-toxic bis-prenylated quinone (25) is a constituent of brown alga

Perithalia capillaries. This compound has been reported to inhibit

superoxide anion production in human neutrophils with an IC50 value of

2.1 μM in vitro (Sansom et al., 2007).

N

S

NH

O

O

COOR

OO

20 R = H21 R =Me

NNH

R1

N

NH

H

OH

22 R = R1 = H23 R = H, R1 = OH

24

O

O

25

R

NNH

HN

N

H

O

H

The lignans 26-30 have been shown to be a valuable inhibitor of

both COX-1 and COX-2 (Ban et al., 2002; Bao-Ning et al., 2004) and

therefore, can be good candidates for anti-inflammatory drug

development.


9

O

OO

O

H

H

O

OO

OMe

MeO

OMe

OMe

26

O

OO

O

O

OO

27

O

OO

O

OO

28

OO

29

O

OO

OMe

OMe

OMe

30

O

H

H

H

H

A potent antimicrobial compound canthin-6-one (31) and its

derivatives 32-33 were isolated from Allium neapolitanum (Thouvenel et

al., 2003). These compounds showed potent antibacterial and antifungal

activity (Donnell and Gibbons, 2007).

NN

O

NN

OHO

31

NN

O

OMe32 33

Epidithiodioxopiperazines, bionectins A (34), B (35), C (36),

verticillin D (37) and G (38) were isolated from the fungus Bionectra

byssicola F120. Compounds 34, 35 and 37 exhibited antibacterial activity


10

against Staphylococcus aureus including methicillin-resistant

Staphylococcus aureus (MRSA) and quinolone-resistant Staphylococcus

aureus (QRSA), with MIC values of 10.0-30.0 µg/ml (Chaudhary et al.,

2006; Zheng et al., 2007; Zheng et al., 2006). Verticillin G (38) showed

high potential and inhibited the growth of Staphylococcus aureus

including methicillin-resistant and quinolone-resistant Staphylococcus

aureus with MIC of 3.0-10.0 µg/ml.

NH

N

N

O

O

S S

OH

H

CH(OH)CH3

HNN

NO

O

S S

CH(OH)CH3

HO

37

HN

NH

N

N

O

O

H

OH

R

S S

HN

NH

N

N

O

O

H

OH

SCH3

SCH3

34 R = H35 R = -CH(OH)CH3 36

NH

N

N

HN N

N

O

O

O

O

H

S

OH

OH

H

S S

HS

O

38

Six-acethylenic acids (39-45) were identified as very active anti

fungal agents (Li et al., 2008c) with no cytotoxicity, expressing their

specificity and importance to be developed as anti fungal drug.


11

OH

O

OH

O

OH

O

OH

O

OH

O

OH

O

OH

O

39 40

41 42

43

44

45

Chrysoeriol (46) is a natural product isolated from Newbouldia

laevis. It exhibited activity against four Gram-positive and ten Gram-

negative bacteria with MIC value 1.2-9.76 g/ml (Kuete et al., 2007).

Flavone glycoside (47) as a constituent of the leaves of Vitex negundo is

also identified as a potent antimicrobial agent (Sathiamoorthy et al.,

2007). Di-benzyloxy flavone (48) of the plant Helichrysum gymnocomum

possess high activity against Cryptococcus neoformans (Drewes and

Vuuren, 2008). Laburnetin (49) isolated from Ficus chlamydocarpa,

exhibited potential inhibition of Mycobacterium smegmatis and

Mycobacterium tuberculosis with MIC values of 0.61 and 4.88 g/ml,

respectively (Kuete et al., 2008).


12

46

OHO

OH O

OH

O

O

OHHO

HO

47

O

OO

O

48

O

OOH

HO

OH OH

49

O

MeO

O

OOH

HO

OH

OCH3

Phomolides, A (50) and B (51) are reported as the metabolites of the

fungus Phomopsis sp. Both the compounds displayed antimicrobial

activities against Escheria coli, Candida albicans and Saccharomyces

cerevisia with MIC value 5.0-10.0 g/ml. Both the compounds were

further found to be cytotoxic against the HeLa cell line at 10.0 g/ml (Du

et al., 2008). Another cytotoxic tetraene macrolide CE-108 (52), a

secondary metabolite of Streptomyces diastaticus also exhibited potent

antifungal properties (Perez-zuniga et al., 2004).


13

O

O

O

O

O

O

HO

50 51

O OH OH O

HOOH

HOO

O

O O

HO

H2NOH

52

Kanzonol C (53) (Mbaveng et al., 2008) and its analogues

isobavachalcone (54), stipulin (55) and 4-hydroxylonchocarpin (56) are

the natural products that showed broad spectrum high inhibitory

activities against Gram-positive, Gram-negative bacteria and fungi.

HO

O

OHHO

HO

O

OHHO HO

O

OHO

HO

O

OHHO

54

55 56

53

The polypeptides tripropeptins (TPPs) A-E (57-61) and Z (62)

produced by the bacterium (Lysobacter sp.) exhibited significant

antibacterial activities (MIC of 0.39-12.5 µg/ml) against a variety of

pathogens (Hashizume et al., 2001).


14

O

R

O

NH

OH

CO2H

O

N

OHN

HOO

NH

HO2C

HO

O

OH

57 R = (CH2)7CH(CH3)CH3

58 R = (CH2)8CH(CH3)CH3

59 R = (CH2)9CH(CH3)CH3

60 R = (CH2)10CH(CH3)CH3

61 R = (CH2)11CH(CH3)CH3

62 R = (CH2)6CH(CH3)CH3

NH

O

OH

N

ON

O

O

HH

H

NHNH

H2N

NH

Anthraquinones (63) and (64) are two phytochemicals, which were

isolated from the aerial part of Saprosma fragrans as antifungal agents with

MIC values of 12.5, 1.56, and 6.25 µg/ml against Sporitrichum schenckii,

Trichophyton mentagrophytes and Sporothrix schenckii respectively (Singh et

al., 2006a). Another anthraquinone: newbouldiaquinone A (65) was isolated

from Newbouldia laevis as antibacterial agent (Eyong et al., 2006).

O

O

OMe

OH

OH

O

H

O

O

OMe

OH

O

H

63 64

O

O

O

O

O

HO

65

A selective antibacterial drug platensimycin (66) is a novel natural

product isolated from various strains of Streptomyces platensis (Singh et

al., 2006b; Wang et al., 2007; Wang et al., 2006). This compound has

been reported to possess potent antimicrobial activity against Gram-


15

positive bacteria including methicillin-resistant Staphylococcus aureus

(MRSA) and vancomycin-resistant Enterococci (VRE).

66

OH

OH

HO

O

O

NH

O

O

Like other activities, the natural products also possess antioxidant

potential. For example a diarylbutane lignan, 2ʹ-hydroxy dihydroguaiaretic

acid (67) isolated from extracts of the underground parts of Saururus

chinensis, exhibited anti oxidative activity against low-density lipoprotein

(LDL) and radical scavenging activity for DPPH (1,1-diphenyl-2-

picrylhydrzyl) (Lee et al., 2004). Other compounds 68-70 isolated from the

stem and bark of Styrax japonica have been reported to possess

antioxidative properties (Min et al., 2004) as they inhibit the activity of

DPPH radical (Ono et al., 2004). Compounds 71-76 showed stronger anti

oxidative activity than that of -tocopherol (77), a standard drug.


16

H

O

R1

HO

R2

OH

OMe

OHR1 R2

71 H OMe

73 OMe H

X

OMeO

HO

OH

OMe

72 = X = NH74 = X = O

H

O

MeO

HO

OMe

OH

R

75 = R = H76 = R = OH

R3O

R4O

O

O

OR1

OR2

H

H

R1 R2 R3 R4

68 = CH3 Glc H CH3

69 = CH3 Glc CH3 H

70 = CH3 H CH3 GlcMeO

OH

OMe

OH

OHMe

Me

67

O

HO

77

Quercetin (78), quercetin-3-O-β-glucopyranoside (79) and morin

(80) are well known phytochemicals with potent antioxidant activity and

are important factors of our food (Tachakittirungrod et al., 2007).

8079

OHO

OH O

OH

OHO

OH O

OH

OHO

OH O

O

OH

OH

OH

OH

O

OH OH

OH

OH

78

HO OH


17

Polypeptide, cyclosporin A (81) is a natural immunosuppressant

drug isolated from a fungus Tolypocladium inflatum (Dreyfuss et al., 1976).

The current worldwide use of this compound has placed it on the list of

the 25 top overall drugs and is mostly used among all immunosuppressive

drugs (Grabley and Thiericke, 1999). Nemerosin (82) and isochaihulactone

(83), isolated from Bupleurum scorzonerifolium, were also found to possess

immunosuppressive activity (Chang et al., 2003).

Me

N

O

NNH

O

OMeO

N

Me

N

Me

HO

O

ON Me

N

Me

O

O

HN

O

HN

NH

OO N Me

Ala (Me)Leu Val

(Me)Leu

Bar(Me)Leu

(Me)Leu (Me)Val (Me)Bmt Abu

D-Ala

81

OO

OMe

OMe

OMe

O

O

82

MeO

MeO

OMe

O

O

OO

83

Talarico et al (Talarico et al., 2007) isolated a low toxic compound;

DL-galactan hybrid C2S-3 (84) from the marine alga Cryptonemia

crenulata. This compound exhibited potent antiviral activity against

dengue virus sero-type 2 (IC50 = 0.8–16 μg/ml). Another low sulfated


18

xylomannan (85) (Mandal et al., 2008) was found to be a potential

inhibitor of HSV-1 and HSV-2 with an IC50 value of 0.5–1.4 μg/ml.

Similarly a non-toxic phlorglucinol derivative 6,6′-bieckol (86) isolated

from an edible marine brown alga species Ecklonia cava inhibited HIV-

induced syncytia formation with an IC50 value of 1.72 μM (Artan et al.,

2008).

O

O

OOO O O

OHOH

OSO3-

HO

OSO3-

OH OH OH

OSO3-

-O3SO

OOH

n84

O

O

O

O

O

O

OHHO

OH

OH

OH

HOOH

OH

OHHO

HO

OH

86

O

O

HO OH

HOO

HOOH

O

n

85


19

1.3. Natural Products under Clinical Trials

More than 100 natural products are under clinical trials for new drug discovery, whereas, more

than 100 compounds are in the preclinical studies (Table 1.1) for drug development process (Butler,

2008). Majority of these are developed from plants and microbial sources (Harvey, 2008).

Table 1.1: Natural products under clinical trials

Natural Product Class Clinical trial Phase Disease Area Biological Source Reference

(-)-Gossypol (87) Phenolic aldehyde derivative

II (b) Brain and lung cancer

genus Gossypium (Kim et al., 2009; Polsky et

al., 1989) Trodusquemine (88)

Cholesterol derivative I & II

Type 2 Diabetes Squalus acanthias (Ahima et al., 2002; Rao et

al., 2000)

Rostafuroxin (89) Digitoxygenin derivative

II chronic arterial hypertension

(Schoner and Bobis, 2007)

Pyridoxamine (90) VitaminB6

analogue II (b) Type 2 Diabetes (Roje, 2007)

Tetrameprocol (91) Lignin I & II leukemia, solid tumors and

glioma

Larrea tridentata (Khanna et al., 2007)

Silybin (92) Flavonolignin II Cancer chemotherapy

Silybum marianum (Lee and Yanze, 2003)

(Gazak et al., 2007)

Combretastatin A-4 phosphate (93)

III anaplastic thyroid cancer

(ATC).

Combretum caffrum

(Escalona-Benz et al., 2005)


20

O

OH

OH

OHHO

H

H

89

NH

OS

O

O OH

OHH

NH

HNH2N

88

HO

HO

O

OH

OH

OH

OHO

87

N

OH

NH2

HO

H3C

90

MeO

OMe

OMe

OMe

91

MeO

HO

O

O

O OH

OHO

HO

OH

92

MeO

MeO

OMe

OMe

OP

OH

OOH

93

Currently the projects for the natural product drug discovery are

mostly focused on infectious diseases, anticancer and many other

therapeutic areas. Careful literature search revealed that mostly the lead

compounds, in clinical trials, are either from plants or from microbial

sources (Butler, 2005).

If a secondary metabolite has no adverse effect on the producing

organism at any of the levels of concentration, it may be potential

substitute of a synthetic drug, which have several side effects. Therefore,

natural products are blessing for man to serve as life saving drugs.


21

1.4. Aim of the Present Study (Research Question)

More than 80% of Pakistani population is dependent on traditional

medicines for their primary healthcare needs. Allopathic drugs are so

expensive that they are out of reach of most of the people of Pakistan;

therefore, they prefer to use medicinal herbs or their extracts to cure their

diseases. Unfortunately, no scientific or systematic study has been done on

the local flora of Pakistan that may play significant role in maintaining the

health and other economical matters of the community. More than 90% of

the country’s medicinal herbs are imported, although Pakistan is rich in

medicinal flora, the only need is to investigate them for their biological

potential.

To address a part of this issue, I selected a medicinal plant Tecomella

undulata growing in Pakistan, to investigate it for its bioactive secondary

metabolites. Although the same plant has been investigated from other

region of the world, but never been explored in Pakistan. Additionally, the

drug discovery from endophytic fungi has never been initiated in Pakistan,

therefore, to setup this important area of drug discovery, a part of my work

deals with the search of secondary metabolite from an endophytic fungus.

Chapter 2 Literatire Review on Tecomella undulata

22

CHAPTER 2

LITERATURE REVIEW (PREVIOUS PHYTOCHEMICAL INVESTIGATION) OF

TECOMELLA UNDULATA


23

2.1. Tecomella undulata

Tecomella undulata is a monotypic genus and an important large

ornamental shrub or small deciduous tree of arid and semi arid regions

(Chopra et al., 1956; Rohilla and Garg, 2014). It belongs to family

Bignoniaceae and mostly distributed in western parts of India and South

Eastern parts of Pakistan. In Pakistan it is found in Attock, Kalachita

Mountain where it is named as Haroora. In Baluchistan it is found in

Musakhel and Khuzdar, also it grows in some parts of sindh. This plant is

usually grown as a source of timber due to its quality wood. The plant is

known by various names. It is called Rohida and locally is known as honey

tree, white cedar, desert teak (Ammora) in English, rohira in Punjabi, lohira

in Sindhi and rakhtroda in Marathi (Sharma et al., 2001).

2.2. Scientific Classification of Tecomella undulata

Kingdom: Plantae

Order: Lamiales

Family: Bignoniaceae

Genus: Tecomella

Species: T. Undulata


24

2.3. Botanical Description of Tecomella undulata

It is a shrub or small tree having drooping branches and attaining a

height of about 2.5-5.0 m.

Leaves are greyish green in

colour and are simple,

narrowly oblong and entire

with undulate margins. Size of

the leaves varies in length from

5.0 to 12.5 cm and in width

ranges between 1.0-3.2 cm.

Large beautifully coloured,

odourless flowers are present

in corymbose; usually orange,

red and yellow coloured flowers

are present and flowering season is December-February. Orange yellow

corolla is 3.8-6.3 cm long campanulate, 5 lobes are sub equal and rounded.

Seeds have narrow wings round the apex of the seed while absent at base.

Outer surface of bark is dull brown or grey in color while inner surface is

dark brown in colour also it is tasteless and odorless. In one locality three

different types of flower bearing trees can be seen near to each other (Jindal

et al., 1985).


25

2.4. Economic and Ecological Importance of Tecomella undulata

Tecomella undulata is economically valuable as it is a good source of

strong, durable and very good quality wood. Due to softness, fine finishing

and being capable to take good polish, this timber is precious and valuable

for toys, carvings and engraved furniture. Also it is excellent firewood and

charcoal. All these factors make the species economically important by

helping local people in livelihood. Besides its importance for human, it

provides shelter for birds and desert wild life. Cattle and goats consume

leaves while flowers are consumed by camels, goats and sheep. This tree

also plays an important role in ecology of this region. The roots and lateral

roots of the tree act as soil binder by creating a network spread on the

upper soil surface. Mycorrhizal nature of this plant is found to increase the

carbon and nitrogen content of soil significantly (Rao et al., 1986). Mathur

et al. (2010) suggested its use in phytoremediation of soil contaminated

with crude petroleum oil (Mathur et al., 2010).

2.5. Medicinal Importance of Tecomella undulata

Traditionally, people extensively use the plant for the treatment of

leucoderma, leucorrhoea, spleen enlargement and to treat urinary

discharge (Saxena, 2000). The traditional healers also use it in the

treatment of gynaecological troubles and digestive system. The paste of its

bark is applied on traumatic wounds while powdered bark is used to treat

piles, worm infestations, anorexia and flatulence (Dhir and Shekhawat,


26

2012). Pulp of roots of Tecomella undulata mixed with rice water is used for

leucorrhea treatment (Katewa and Galav, 2005). Tribes of Rajasthan found

roheda useful against allergies (Meena and Rao, 2010), Garasia tribes used

as a remedy for syphilis (Meena and Yadav, 2010) and in treatment of old

wounds and snakebite in the tribal (Bhil, Meena, Garasia, Sahariya,

Damor, Kathodia) dominated areas of Rajasthan (Jain et al., 2011). The

paste of fresh leaves is applied on the head for migraine, Flower extract is

used to reduce thirst and also found beneficial for sterile women (Tareen et

al., 2010).

2.6. Pharmacological Studies of Techomella undulata

2.6.1. Analgesic activity of Techomella undulata

Methanolic extract of whole plant of T. undulata was evaluated for

analgesic potential; hot water tail immersion test in mice was used for this

purpose. The results obtained in the experiment at dose level of 300, 500 or

1000 mg/kg were comparable to that of acetylsalicylic acid, a standard

analgesic drug (Ahmad et al., 1994).

2.6.2. Antiacaricidal activity of Techomella undulata

Methanolic extract of T. undulata was found effective against

Sarcoptes scabiei where 45, 65, and 80% mortality of the Sarcoptes scabiei

mites was observed for 10, 20 and 30% concentrations of extract,

respectively (Khan et al., 2013).


27

2.6.3. Hepatoprotective activity of Techomella undulata

Jaina et al., concluded that betulinic acid plays important role in

hepatoprotective potential of T.undulata stem bark (Jain et al., 2012).

Effectiveness of Rohitaka ghrita, an ayurvedic formulation, was reported

against paracetamol induced toxicity. This formulation is a combination of

Tecomella undulata and four other plants (Goyal et al., 2012). Patel et al

validated the hepatoprotective potential of bark of T. undulata against

paracetamol induced hepatic damage using acetone, chloroform, methanol

soluble fractions and methanol insoluble fraction of ethanolic bark extract.

It was evaluated that methanol soluble fraction was most potent among

others by reducing the elevated levels of alkaline phosphatase (ALP),

alanine amino transferase (ALT), aspartate amino transferase (AST) and

total bilirubin (TBil). It was also found effective to normalize decreased level

of total protein (TP), increased wet liver weight and volume and abnormal

histopathology (Patel et al., 2011). In 2011 methanolic extract of Tecomella

undulata leaves was studied against paracetamol and alcohol induced

hepatic damage in rats and found effective at dose of 100-200 mg/kg for 15

days (Singh and Gupta, 2011). Rana et al., in 2008 evaluated

hepatoprotective potential of Tecomella undulata stem bark methanolic

extract inducing hepatotoxicity (liver damage) in rats using carbon

tetrachloride, while in 2009 Khatri et al. studied the same effect with

Tecomella undulata stem bark ethanolic extracts using thioacetamide for

inducing hepatotoxicity in rats. Both studies depicted significant reduction


28

in serum aspartate aminotransferase (AST), gamma-glutamyl

transpeptidase (GGT), alanine aminotransferase (ALT), alkaline phosphatise

(ALP), total protein, albumin, bilirubin and cholesterol levels (Khatri et al.,

2009; Rana et al., 2008).

2.6.4. Anti-inflammatory activity of Techomella undulata

Significant anti-inflammatory activity was observed for butanolic

extract of T.undulata bark against carrageenan induced paw edema and

cotton pellet induced granuloma in wistar albino rat. Methanolic extract of

T.undulata whole plant has analgesic potential, oral administration of 300,

500 or 1000 mg/kg bodyweight of extract significantly reduced paw edema

in rat induced by carrageenan (Ahmad et al., 1994).

2.6.5. Antimicrobial activity of Techomella undulata

Leaves of T. undulata, and leaf extracts in hexane, chloroform and

methanol have antibacterial activity against Bacillus subtilis, Escherichia

coli, Enterococcus faecalis, Pseudomonas aeruginosa, Klebsiella pneumonia,

Proteus vulgaris and Micrococcus luteus. Methanolic extract was found

effective against Klebsiella pneumonia (MIC: 0.01 mg/ml) and Micrococcus

luteus (MIC: 0.1 mg/ml) (Abhishek et al., 2013). Both the alcoholic and

aqueous leaf extracts of Tecomella undulata were reported to have

antibacterial activity against the pathogens including Salmonella typhi

(Gehlot and Bohra, 2000). Antimicrobial studies of acetone, alcoholic,


29

petroleum ether and water extracts of Tecomella undulata were carried out

where alcoholic and petroleum ether extract showed 9 and 10 mm zone of

inhibition respectivly against Escherichia coli, while acetone extract showed

activity against Bacillus subtilis and Staphylococcus aurens with zone of

inhibition of 17 and 10 mm respectively (Thanawala and Jolly, 1993).

Methanolic extract of the specie was active for inhibition against five

medicinally important bacterial strains Bacillus subtilis, Salmonella

typhimurium, Staphylococcus epidermidis, Proteus vulgaris and

Pseudomonas pseudoalcaligenes, although the extract was found more

active against susceptible bacteria Bacillus subtilis and Staphylococcus

epidermidis (Parekh et al., 2005).

2.6.6. Anti-oxidant activity of Techomella undulata

The methanolic extract of leaves, stem, bark and root of Tecomella

undulata was found rich in total phenolics as gallic acid equivalent per

gram dry weight (13.75±0.125 mgGAE/gdw) and total flavonoid as

quericitin equivalent per gram dry weight (71.875±18.393 mgQE/gdw).

Stem extract showed highest radical scavenging activity with IC50 value

92.29±7.693 µg/ml (Sharma et al., 2013).

2.6.7. Anticancer activity of Techomella undulata

Traditionally genus Tecomella is believed to have anticancer

properties, this traditional claim was confirmed scientifically and an anti


30

cancerous polyherbal formulation (SJT ONC) was evaluated in 2012 by

Savjiyani in which stem bark extracts of Tecomella undulata was as an

important component. Other species used in this formulation are Oroxylum

indicum, Bauhinia variegata and Indigofera tinctoria. 1000 µg/ml of this

formulation showed cytotoxicity against MCF-7 and Caco-2 cell lines.

Further, when 300 mg/kg was fed to Dimethylbenz anthracene treated

(high fat diet fed) rats for 12 weeks, marked reduction was observed in

mammary tumor volume (Savjiyani et al., 2012). Stem bark chloroform

extract of the plant showed considerable anti-tumor potential against

chronic myeloid leukemia cell line (K562) (Ravi et al., 2011).

2.6.8. Other activities of Techomella undulata

Rohitakarista, Ayurvedic drug used for treatment of pliha (disease of

spleen), udara (disease of abdomen), gulma (localized abdominal swelling or

tumour), major component of which is Tecomella undulata stem and bark

with some other medicinal plants in small amount. Oral administration of

rohitakarista caused a decrease in triglycerides and a significant increase

in the total cholesterol, VLDL (very low density lipoprotein) and HDL (high

density lipoprotein) (Ullah et al., 2010). The ethanolic leaves extract of

Tecomella undulata has in-vivo anti-hyperglycemic and anti-oxidant

potential. The extract lowered blood glucose level, maintained cholesterol,

glycogen contents, glycosylated haemoglobin and antioxidant parameters in


31

intraperitoneal treated animals. Tecomella undulata can be used as new

candidate for antihyperglycemic and antioxidant (Kumar et al., 2012).

2.7. Previously Reported Phytoconstituents of Techomella undulata

Phytochemical studies have been carried out on different parts of the

plant, leading to isolation and identification of compounds with great

structural diversity such as flowers of Tecomella undulata yielded alkaloids

named as 2-pyrrolidinemethanol (94), 2-methyl-6-propylpiperidine (95), 3-

amino-4-pyrazolecarbonitrile (96), 3-(1-methyl-2-pyrrolidinyl) pyridine (97),

1-piperidineethanol (98), 5-acetylpyrimidine-2,4,6 (1H,3H,5H)-trione (99),

4-formyl-1,3-dihydro-1,3-dimethyl-2H-imidazole-2-thione (100), 1-(1-

cyclohexen-1-yl) pyrrolidine (101), 2,4-dihydro-5-methyl-2-phenyl-3H-

pyrazol-3-one (102) and decahydroquinoline (103) (Laghari et al., 2014).

HN

OH N

HN

NH2

N

N

N

NH

N

OH

N N

S

ONH

NH

O

OO

ON H

N

NN O

94 96 9795 98

10099 101 103102


32

Ferulic acid (104) having anti-obesity activity and its ester cluytyl

ferulate (105) is isolated from the bark of Tecomella undulata (Alvala et al.,

2013; Singh et al., 2008). Rutin (106), tiliroside (107), genistein-4',7-O-

diglucosidemethyl malonylated (108), luteone-4',7-O-diglucoside (109),

luteolin-3',4'-dimethylether-7-O-β-D-glucoside (110) and 5,6-dimethyoxy-

3',4'-dioxymethylene-7-O-(6''-β-D-glucopyranosyl-β-D-lucopyranosyl)

flavanone (111) were identified from the extracts of leaves and flowers of T.

undulata (Laghari et al., 2013).

O

O

HO

OH

OH

O OHO OH

O

O

OH

OH

O

O

HO

OH

OH

O

O

HO

OH

OH

O

O

HO

HO

HO

OH

106

107

OHO

HOOH

OH

O

O

O

OHO

OOH

OHHO

O

108

O

OO

OOMe

MeO

O

OHO

HOOH

O

OHO

HOOH

OH

111

OHO

HOOH

OH

O

O

O

OHO

O

OHOH

OH

HO

OH

CH2

CH

(H3C)2C

OHO

HOOH

OH

O

O

O

OH

OMe

OMe

109

110

OH

OMe

O O

26

105

OH

OMe

O OH

104

MeO

OO

Rutin (106), quercetin (78), luteolin-7-glucoside (112) are isolated

from flowers of Tecomella undulata (Taneja et al., 1975). The concentrated


33

petroleum ether leaves extract of Tecomella undulata yielded cirsimaritin

(113) and cirsilineol (114) (Azam and Ghanim, 2000). Chromone glycoside

undulatoside-A (115) has been isolated from the bark of this species (Gujral

et al., 1979). Petroleum ether extract of the heartwood of this plant afforded

radermachol (116), techomaquinone-I (117), lapachol (118), 2-isopropenyl

naphthol [2,3-b] furan-4,9-quinone (119), dehydro-α-lapachone (120), α-

lapachone (121) and β-lapachone (122) (Singh et al., 2008; Singh et al.,

1972).

O

O

MeO

MeO

OH

OH

113

O

O

MeO

MeO

OH

OH

OMe

114

112

O

OOH

OH

OH

OHO

HOOH

O

OH

O

OOH

O

OHO

HOOH

OH

115

O

O

OHO116

O

O

OH

118

O

O

O

O

117

O

O

O119

O

O

O120

O

O

O121

O

O

O

122


34

Lapachol (118) is also reported along with dehydrotectol (123) and

dehydro-α-lapachone (120) from this species by another group of

researchers (Joshi et al., 1977). Tectoquinone (124) and deoxylapachol

(125) are reported from heartwood extract of this species (Verma et al.,

1986).

O

O

O123

O

O

125

O

O

124

The compound iso-β-lapachol (126) is an isomer of lapachol (118).

Two analogs of 118; 2-(3ʹ,7ʹ-dimethyl-2ʹ,6ʹ-octadienyl)-3-hydroxy-1,4-

naphthoquinone (127) and 2-(3ʹ,3ʹ-dibromo-2ʹ-propenyl)-3-hydroxy-1,4-

naphthoquinone (128) were also purified from T. undulata, highly active

against the Walker 256 tumor cell line.

O

O

OH

126

O

O

OH

127

O

O

OH

Br

Br

128

Another analog of lapachol (118); 2-(3ʹ-methyl-2ʹ-buteny1)-3-

(tetraacetyl-β-D-glucopyranosyloxy)-1,4-naphthoquinone (129) was found


35

effective in increasing the life span in mice inoculated with leukemic cells

(Hussain et al., 2007a). Nagpal et al., reported quantitiative estimation of

techomin (130) (Nagpal et al., 2013).

131

OHO

OMe

MeO

OMe

OMe

O O

OOH

OHHO

OH

130

O

O

O

129

O

OAc

OAc

OAc

HAcO

Iridoids are reported from Tecomella undulata e.g., 6-O-veratryl

catalposide, undulatin has been isolated from the acetone extract of the

heartwood (Joshi et al., 1975; Verma et al., 1986). Veratric acid (131) and

steroids; stigmasterol (132) and β-sitosterol (134) have been isolated from

this species (Joshi et al., 1977; Singh et al., 2008). Betulinic acid (133),

oleanolic acid (135) and ursolic acid (136) have also been isolated from

leaves of T. undulata (Mukerji, 1977). Other reported compounds include

triacontanol (137) (Joshi et al., 1977), linoleic acid (138), oleic acid (139),

stearic acid (140) and palmitic acid (141) from seeds of this plant (Khare,

2004).


36

HO

H H

134132 133

135 136

HO

O

OH

OH

O

HO

HO

O

OH

HO

H

H

HO

O

OH

O

OH

139

O

HO

140

O

HO

141

137

138

Chapter 3 Results and Discussion: Characterization of Metabolites from Tecomella undulata

37

CHAPTER 3

RESULTS AND DISCUSSION: STRUCTURE

ELUCIDATION OF SECONDARY METABOLITES

ISOLATED FROM TECOMELLA UNDULATA


38

3.1. Results and Discussion

Methanolic extract of Techomella undulata was partitioned between

water and n-hexane, chloroform, ethyl acetate and n-butanol. The ethyl

acetate fraction showed antibacterial activity and thus was subjected to

purification to get several secondary metabolites which were characterized

with the help of various spectroscopic techniques.

3.1.1. Characterization of kaempferol-3-O-β-D-[3,4di-p-E-coumaroyl-

-L-rhamnosyl (1→6)] galactoside (142)

Compound 142 was isolated as yellow amorphous powder, which

showed a strong fluorescence under

UV lamp at 254 nm, whereas, it also

remained yellow on TLC after heating

with ceric sulphate reagent. This

observation indicated flavonoid

nature of 142, which was further

supported by the UV analysis, which

exhibited absorption maxima at 258,

265, 313 and 356 nm. The similar UV

data has been reported for acylated

kaempferol glycosides (Mabry et al., 1970; Saleh et al., 1990) therefore, it

was assumed that compound 142 could also have the same structural

features.

O

O

HO

OH

OH

O

O

HO

OH

OH

O

O

HO

O

1

34a

8a

6

81'

3'

5'

1''

3''5''

O

OH

OO

OH

142

3´´´

1´´´

5´´´

6´´´

1´´´´

3´´´´5´´´´

6´´´´´

1´´´´´

3´´´´´


39

The IR spectrum displayed absorption bands for hydroxyl function

(3380 cm-1), conjugated ester (1715 cm-1), conjugated ketone (1685 cm-1)

and aromatic system (1600, 1545, 1525 cm-1). The positive FAB-MS

analysis revealed its molecular weight as m/z 886 amu, whereas, the

positive HR-FAB-MS (m/z 887.2320) depicted the molecular formula as

C45H42O19 with 25 double bond equivalence (DBE).

The aromatic region of the 1H NMR spectrum of 142 (Table 3.1)

displayed three pairs of o-coupled doublets [δ 8.06 (2H, d, J = 7.6 Hz) and

6.86 (2H, d, J = 7.6 Hz), 7.36 (2H, d, J = 8.4 Hz) and 6.76 (2H, d, J = 8.4

Hz), and 7.29 (2H, d, J = 8.8 Hz) and 6.73 (2H, d, J = 8.8 Hz)] attributed to

three p-substituted benzene rings. The first pair among these doublets

could be attested for ring B of kaempferol moiety, whereas the other two

pairs were attributed to two E-p-coumaroyl units in the molecule. The four

more protons resonated in the downfield region at δ 7.66 (1H, d, J = 16.0

Hz), 6.32 (1H, d, J = 16.0 Hz), 7.55 (1H, d, J = 16.0 Hz) and 6.31 (1H, d, J =

16.0 Hz). Due to their coupling constants (J = 16.0 Hz), they were

attributed to two coumaroyl moieties in compound 142. The aromatic

region of the same spectrum further displayed resonances of two m-coupled

doublets at δ 6.42 (1H, d, J = 1.2 Hz) and 6.19 (1H, d, J = 1.2 Hz). Due to

their chemical shifts and J values, these two protons were identified as H-6

and H-8, respectively of ring A of kaempferol nucleus. In addition the 1H

NMR spectrum displayed signals for two anomeric protons δ 5.30 (1H, d, J


40

= 7.2 Hz) and 4.47 (1H, br s) due to β- and -hexoses respectively. The

remaining sugar proton displayed their positions between δ 4.81-3.31

(Table 3.1). With reference to anomeric protons, the other sugar protons

could be differentiated due to mutual COSY correlations. The coupling

constant (J = 2.8 Hz) of an oxymethine of β-sugar at 3.58 due to H-4

indicated it to be a galactose unit, whereas the resonance of a doublet (J =

6.5 Hz) methyl at 0.92 indicated that the -sugar could be rhamnose. The

most downfield oxymethines resonated at δ 4.81 and 4.78 were attested for

H-3 and H-4 of rhamnose due to COSY correlations. Relatively downfield

shift of these two oxymethines were attributed to the acylation on these two

centers.

The 13C NMR spectrum of compound 142 (Table 3.1) fully supported

the mass and 1H NMR data as it displayed signals for kaempferol nucleus (

177.4, 164.2, 161.1, 156.6, 156.4, 133.2, 103.8, 98.7, 93.7), two p-

coumarates ( 166.3, 159.7, 144.5, 130.2, 125.1, 114.8, 114.3) and (

165.4, 158.8, 143.3, 132.6, 125.4, 115.7, 115.4), and two sugar moieties (

102.1, 100.1, 73.4, 73.3, 72.9, 71.0, 70.5, 68.2, 68.0, 66.0, 65.9, 17.1).

The C-H connectivities were established due to HSQC and COSY

spectral analysis, whereas, the sugar attachment was determined through

HMBC analysis (Figure 3.1) in which the anomeric proton (δ 5.30) of the

galactose moiety was correlated with a quaternary carbon at δ 133.2 (C-3),

whereas, the HMBC correlation of anomeric proton (δ 4.47) of rhamnose


41

suggested its attachment to C-6 of the galactose which was also evident

from downfield chemical shift (δ 65.9, C-6) of this carbon. The HMBC

interaction of H-4 (δ 4.78) of rhamnose with the carbonyl carbon at δ

166.3 confirmed the connectivity of one coumaroyl moiety on C-4 of

rhamnose, whereas other coumaroyl connectivity was substantiated due to

HMBC correlation of H-3 of rhamonse with another carbonyl carbon at δ

165.4.

The nature of the glycosidic part was finally confirmed through acidic

medium hydrolysis of compound 142, which provided three products. The

hydrolysed products were separated by solvent extraction. Kaempferol and

coumaric acid moieties were detected in the ethyl acetate layer and were

not processed further, whereas, the aqueous layer containing sugar units

was concentrated and the sugars were purified through preparative TLC

developed in EtOAc-MeOH-H2O-AcOH; 4:2:2:2. Both the sugars were

identified as D-galactose and L-rhamnose through their optical rotation

values [α]20D +78.5 (c, 0.1 in H2O) (Kardosova et al., 1969) and

[α]23D +7.7 (c, 0.2 in H2O) (Liocharova et al., 1989) and comparison of the

retention time of their trimethylsilyl (TMS) ethers with those of the

standards in gas chromatography (GC). The above discussed data was

further compared with the related published compounds (Saleh et al.,

1990; Soliman et al., 2002).


42

O

O

HO

OH

OH

O

O

HO

OH

OH

O

O

HO

OO

OH

OO

OH

142

Figure 3.1: Important HMBC correlations observed in the spectrum of 142

Based on all above discussed evidences, compound 142 was

identified as kaempferol-3-O-β-D-[3,4di-p-E-coumaroyl-α-L-rhamnosyl

(1→6)] galactoside, which we have reported as a new natural product and is

named as tecomeside (142) (Ahmed et al., 2014).


43

Table 3.1: 1H and 13C NMR data of compound 142 (CD3OD+CDCl3, 400 and 100 MHz) and COSY correlations

Position H (J in Hz) C COSY (H→H)

2 _ 156.6 (C) _ 3 _ 133.2 (C) _ 4 _ 177.4 (C) _ 4a _ 103.8 (C) _ 5 _ 161.1 (C) _ 6 6.19, (d, 1.2) 98.7 (CH) H-6/H-8 7 _ 164.2 (C) _ 8 6.42, (d, 1.2) 93.7 (CH) H-8/H-6 8a _ 156.4 (C) _

1 _ 120.8 (C) _

2,6 8.06, (d, 7.6) 130.9 (CH) H-2,6/H-3,5

3,5 6.86, (d, 7.6) 115.0 (CH) H-3,5/H-2,6

4 _ 159.9 (C) _

1 5.30, (d, 7.2) 102.1 (CH) H-1/H-2

2 3.52, (t, 7.6) 71.0 (CH) H-2/H-1,3

3 3.50, (dd, 7.6, 2.8) 70.5 (CH) H-3/H-2,4

4 3.58, (d, 2.8) 68.2 (CH) H-4/H-3,5

5 3.41, (m) 72.9 (CH) H-5/H-4,6

6 3.59, (Ha, dd, 10.4, 4.9) 3.31, (Hb, dd, 10.4, 2.8)

65.9 (CH2) H-6/H-5

1 4.47, (br s) 100.1 (CH) H-1/H-2

2 3.49, (H, br d, 6.5) 68.0 (CH) H-2/H-1,3

3 4.81, (dd, 9.0, 6.5) 73.3 (CH) H-3/H-2,4

4 4.78, (t, 9.5) 73.4 (CH) H-4/H-3,5

5 3.60, (m) 66.0 (CH) H-5/H-4,6

6 0.92, (d, 6.5) 17.1 (CH3) H-6/H-5

1 _ 125.1 (C) _

2,6 7.36, (d, 8.4) 130.2 (CH) H-2,6/H-3,5

3,5 6.76, (d, 8.4) 114.8 (CH) H-3,5/H-2,6

4 _ 159.7 (C) _

7 7.66, (d, 16.0) 144.5 (CH) H-7/H-8


44

8 6.32, (d, 16.0) 114.3 (CH) H-8/H-7

9 _ 166.3 (C) _

1 _ 125.4 (C) _

2,6 7.29, (d, 8.8) 132.6 (CH) H-2,6/H-3,5

3,5 6.73, (d, 8.8) 115.7 (CH) H-3,5/H-2,6

4 _ 158.8 (CH) _

7 7.55, (d, 16.0) 143.3 (CH) H-7/H-8

8 6.31, (d, 16.0) 115.4 (CH) H-8/H-7

9 _ 165.4 (C) _


45

3.1.2. Characterization of the compound (6R,7Z)-9,10-dihydroxy-4,7-megastigmadien-3-one (143)

The EIMS spectrum of compound 143 showed molecular ion at m/z

224, whereas, the molecular formula C13H20O3

with four DBE was deduced with the help of HR-

EI-MS. The IR spectrum showed diagnostic

absorption bands for hydroxy functional (3465

cm-1), conjugated ketone (1650 cm-1) and olefinic system (1630 cm-1). The

UV analysis revealed a substituted cyclohexenone system as it displayed

absorption maxima at 243 nm.

The 1H NMR spectrum of 143 displayed signal for an olefinic proton

at 5.92 (br s), which was correlated in COSY spectrum with a doublet

methyl at 1.96 (J = 1.2 Hz). The chemical shift and amount of coupling

constant of this doublet methyl revealed its allylic nature. Other doublet of

two olefinic protons displayed its position at 5.70 (J = 6.3 Hz), which was

correlated in COSY spectrum with two different methines in 1H NMR

spectrum at 4.16 (m) and 2.69 (d, J = 7.8 Hz). This information revealed

that compound 143 must have a Z-olefinic system. The oxymethine ( 4.16)

was further correlated in COSY spectrum with an oxymethylene at 3.49.

In addition, the same spectrum displayed resonances of a methylene at

2.46 (Ha, d, J = 16.8 Hz) and 2.08 (Hb, d, J = 16.8 Hz), and two geminal

methyl at 1.03 (s) and 0.99 (s).

O

OH

OH

R

Z

1

3

5

67

8

9

10

1112

13

143


46

The above-discussed data revealed a megastigmane skeleton of 143,

which was further substantiated due to 13C NMR spectrum of the

compound (Table 3.2). The spectrum showed total 13 carbon signals,

which, on the basis of DEPT experiment, were differentiated as three

methyl, two methylene, five methine and three quaternary carbons. This

data was in full agreement with the obtained molecular formula. The

resonance of three carbon atoms at 201.3 (C), 165.2 (C) and 125.4 (CH)

substantiated the presence of a substituted enone system that

accommodated two DBE, whereas, the other olefinic methines [ 135.0 (CH)

and 129.0 (CH) (Table 3.2)] justified the third DBE, therefore, the remaining

fourth DBE could be attributed to a six membered cyclic system. This data

was comparable with the reported data of ionols and ionones (D'Abrosca et

al., 2004; Goda et al., 1999; Greger et al., 2001). Greger et al., has also

reported 9,10-dihydroxy-4,7-megastigmadien-3-one with E-configuration of

the double bond between carbon 7 and 8 (D'Abrosca et al., 2004) whereas,

our isolated compound was found to be the same but with (Z) configuration.

Complete C-H connectivities were identified due to COSY and HSQC

spectral analysis, whereas, the structure was finally confirmed through

HMBC experiment (Figure 3.2) in which the olefinic proton ( 5.92) was

correlated with a carbonyl carbon ( 201.3), a methine ( 56.1), a methylene

( 47.4) and a methyl carbon ( 23.0). In addition to this, the HMBC

interaction of two geminal methyls ( 1.03 and 0.99) with the carbons at


47

36.4 (C), 47.4 (CH2) and 56.1 (CH) substantiated the cyclohexenone moiety

in compound 143. The HMBC correlations of olefinic methine ( 5.70) with

the carbons at 165.2 (C), 56.1 (CH) and 36.4 (C) revealed further

substitution pattern on cyclohexenone nucleus, whereas other important

long-range interactions are shown in (Figure 3.2).

O

OH

OH

143

Figure 3.2: HMBC correlations observed in the spectrum of compound 143

The Circular Dichorism (CD) analysis showed positive maxima (87.0)

at 245.6 nm, however this information could not be found sufficient to

establish the absolute stereochemistry at C-9. Only a comparison was

made with the reported 13C NMR shift of C-9. In the published NMR data of

similar compounds, the 13C NMR chemical shifts for C-9 of the -D-

glucopyranoside of 9R and 9S-3-oxo--ionol have been reported to be 77.0

and 74.7, respectively (Lee et al., 2003; Pabst et al., 1992). The 13C NMR

spectrum (Table 3.2) of our isolate 143 displayed C-9 at 73.0, similar to

the center with S-configuration of the reported compound. The absolute

stereochemistry at C-6 was established as R on the basis of CD data

leading to the structure of compound 143 as (6R,7Z)-9,10-dihydroxy-4,7-


48

megastigmadien-3-one, which is a new phytochemical and is named as

undustigmene A (143) (Ahmed et al., 2014).

3.1.3. Characterization of (6E)-9,10-dihydroxy-4,6-megastigmadien-3-one (144)

Compound 144 displayed the same mass and formula (C13H20O3) in

HR-EI-MS spectrum as was found in the spectra of

143. In addition, the IR spectrum displayed identical

absorption bands indicating the similar nature of

functional groups in 144. The UV spectrum (243 nm)

was indicative of a cyclohexenone moiety with an extended conjugation.

The NMR data of compound 144 was nearly similar to the data of

143 with few differences. The 1H NMR signals for two geminal methyl (H-11

& H-12), allelic methyl (H-13) and oxygenated methylene (H-10) displayed

their positions nearly at the same shifts, whereas, methylene (H-2)

appeared as singlet ( 2.35) instead of two doublets. The main difference

was found in the downfield region of the 1H NMR spectrum where the

resonances [( 6.28 (1H, t, J = 6.6 Hz) and 5.90 (1H, s)] for only two

olefinic protons were observed instead of three. Further, in aliphatic region,

two changes were observed; the signal for H-6 disappeared, in addition two

resonances for a methylene were observed at 2.74 (dd, J = 4.5, 15.0 Hz)

and 2.45 (br t, J = 15.0 Hz). The COSY relation of this methylene with the

olefinic triplet at 6.28 and H-9 at 3.75 (m) suggested an exo-cyclic

double bond between carbon 6 & 7 instead of an isolated double bond

2

3

6

7

89

12 11

13

101

O

144

OH

OH

E


49

between carbon 7 & 8 as was observed in compound 143. This deduction

was further supported by the 13C NMR spectrum, which afforded altogether

13 carbon signals (Table 3.2) out of which the carbons resonated at 157.9

(C) and 124.9 (CH) were attributed to the exo-cyclic double bond. D’Abrosca

et al. have reported (6E,9S)-9-hydroxy-4,6-megastigmadien-3-one

(D'Abrosca et al., 2004) and compound 144 was found to be its 10-hydroxy

derivative. In HMBC spectrum, H-7 ( 6.28) was correlated with the carbons

at 38.6 (C-1) and 157.9 (C-6) and 143.1 (C-5) whereas, aliphatic H-8 (

2.74 & 2.45) was found to interact with the carbon at 124.9 (C-7) to

substantiate the position of exocyclic double bond between C-6 and C-7.

The other important HMBC correlations are shown in (Figure 3.3).

Like compound 143, the absolute stereochemistry at C-9 of the

compound 144 could not be determined, however only a comparison of 13C

NMR shift of C-9 could be made (D'Abrosca et al., 2004; Lee et al., 2003;

Pabst et al., 1992).

OOH

OH

144

Figure 3.3: HMBC correlations observed in the spectrum of 144

The 6-E geometry of exo-cyclic double bond was established on the

basis of chemical shifts of H-7 ( 6.28) which is reported for Z isomer as


50

5.66 and for E isomer as 6.11 (D'Abrosca et al., 2004; Ito et al., 1997).

The above-discussed information when coupled with HR-EIMS data led to

the structure of 144 as (6E)-9,10-dihydroxy-4,6-megastigmadien-3-one,

which is also a new natural product and is named as undustigmene B

(144) (Ahmed et al., 2014).


51

Table 3.2: 1H and 13C NMR data of compounds 143 and 144 (CD3OD, 400 and 100 MHz)

143 144

Position H (J in Hz) C H (J in Hz) C

1 _ 36.4 (C) _ 38.6 (C) 2 2.46, (Ha, d, 16.8)

2.08, (Hb, d, 16.8)

47.4 (CH2) 2.35, (s) 53.9(CH2)

3 _ 201.3 (C) _ 201.3 (C) 4 5.92, (br s) 125.4 (CH) 5.90, (br s) 133.7 (CH) 5 _ 165.2 (C) _ 143.1 (C) 6 2.69, (d, 7.8) 56.1 (CH) _ 157.9 (C) 7 5.70, (d, 6.3) 129.0 (CH) 6.28, (t, 6.6) 124.9 (CH) 8 5.70, (d, 6.3) 135.0 (CH) 2.74, (Ha, dd,

4.5, 15.0) 2.45, (Hb, br t, 15.0)

34.4 (CH2)

9 4.16, (m) 73.0 (CH) 3.75, (m) 72.6 (CH) 10 3.49, (m) 66.6 (CH2) 3.54, (d, 5.5) 66.2 (CH2) 11 1.03, (s) 26.6 (CH3) 1.32, (s) 28.5 (CH3) 12 0.99, (s) 27.3 (CH3) 1.32, (s) 28.5 (CH3) 13 1.96, (d, 1.2) 23.0 (CH3) 2.13, (d, 0.78) 21.8 (CH3)


52

3.1.4. Characterization of (6R,9S)-9,10-dihydroxy-4-megastigmaen-3-

one (145)

The HR-EI-MS spectrum of compound 145 exhibited the molecular

formula as C13H22O3 with DBE one less than that

of compound 144. This change was supported by

the NMR spectral data (Table 3.3) in which signals

for an exo-cyclic double bond were missing, rather

an additional methine ( H 2.01, C 50.5) and a methylene ( H 2.06 and

1.47, C 27.5) were found in the spectra. However, other signals were

found nearly at the same positions as were observed in the spectra of 144.

This observation revealed that the exo-cyclic double bond between C-6 and

7 has been reduced. Therefore, compound 145 was characterized as

(6R,9S)-9,10-dihydroxy-4-megastigmaen-3-one, which is also a known

metabolite, thus the whole data was compared with the reported values

(Pan et al., 2013).

3.1.5. Characterization of 3S,5R,6R,9S-tetrahydroxy megastigmane (146)

Compound 146 was purified as white amorphous powder, which was

also found to be a megastigmane derivative. It exhibited characteristic IR

absorption bands at 3505 cm-1 and 1660 cm-1 attested for hydroxyl and

olefinic systems respectively. The EIMS spectrum afforded molecular ion at

m/z 244, whereas negative HR-FAB-MS m/z: 243.1584 [M-H]-1(Calcd for

C13H23O4, 243.1596) depicted the molecular formula as C13H24O4.

O

OH

12

7

10

OH

145


53

The 1H NMR spectrum of 146 displayed resonance for four methyls at

1.26 (d, J = 6.4 Hz), 1.24 (s), 1.07 (s) and 0.86 (s), and two methylenes at

2.02 (Ha, dd, J = 12.4, 4.8 Hz), 1.92 (Hb, dd, J

= 12.4, 2.5 Hz) and 1.67 (Ha, dd, J = 13.4, 4.6

Hz) and 1.51 (Hb, dd, J = 13.4, 5.6 Hz). Two

oxymethine protons displayed their positions at

4.31 (H-9) and 4.09 (H-3). The first oxymethine

signal 4.31 (1H, m, H-9) was correlated in COSY spectrum with the

doublet methyl ( 1.26) and an olefinic proton resonating at 5.75 (1H, dd,

J = 16.0, 6.4 Hz), which in turn was correlated with another olefinic proton

at 6.17 (1H, dd, J = 16.0, 2.0 Hz). Other oxymethine ( 4.09) was

correlated in the COSY spectrum with the two methylenes. The smaller

coupling constant (J = 2.0 Hz) of olefinic methine ( 6.17) was attributed to

the allylic coupling with oxymethine (H-9), whereas, larger J value (16.0 Hz)

could be attributed to the E geometry of the double bond. This data was

comparable to the previously discussed data of megastigmanes 143-145,

therefore compound 146 was also found to have the same nature.

The 13C NMR spectrum displayed altogether 13 carbon signals (Table

3.3), which were identified as four methyls ( 27.3, 25.2, 25.1 and 24.3),

two metylenes ( 46.1 and 45.2), four methines ( 130.0, 129.3, 70.2 and

66.3) and three quaternary carbons ( 80.1, 78.5 and 40.0). This data

indicated that the carbonyl carbon in compounds 143-145 at C-3 must

12

4

7

10

HO

OH

OH

OH11 12

13

146


54

have been reduced in compound 146 to a secondary hydroxyl function,

whereas, the double bond between C-4 and 5 must have been hydrated to

add a hydroxyl group at C-5.

This deduction was substantiated through HMBC analysis (Figure

3.4), in which singlet methyl at 1.07 exhibited correlations with the two

oxygenated quaternary carbons ( 80.1 and 78.5) and a methylene ( 45.2).

This is how one hydroxyl group was fixed at C-6, which was further

confirmed due to HMBC correlations of the two olefinic protons ( 6.17 and

5.75) with the oxygenated quaternary carbon at 80.1. Other important

HMBC correlations are shown in (Figure 3.4). The relative stereochemistry

at all stereogenic centers could only be established due to comparison of

the NMR data and optical rotation values with the reported data. Further,

the whole data was identical to the reported data for 3S,5R,6R,9S-

tetrahydroxy megastigmane, and thus compound 146 was also found to be

the same, which is a known phytochemical (Takeda et al., 2000) and has

been isolated for the first time from T. undulata.

HO

OH

OH

OH

146



55

Table 3.3: 1H and 13C NMR data of 145 and 146 (CD3OD, 400 and 100

MHz)

145 146


1 _ 36.2 (C) _ 40.0 (C) 2 2.06, (Ha, d, 17.1)

2.39, (Hb, d, 17.1) 45.5 (CH2) 1.67, (Ha dd, 13.4,

4.6) 1.51, (Hb, dd, 13.4, 5.6)

46.1 (CH2)

3 _ 202.0 (C) 4.09, (m) 66.3 (CH) 4 5.80, (br s) 123.7 (CH) 2.02, (Ha, dd, 12.4,

4.8) 1.92, (Hb, dd, 12.4, 2.5)

45.2 (CH2)

5 _ 167.7 (C) _ 78.5 (C) 6 2.01, (m) 50.5 (CH) _ 80.1 (C) 7 2.06, (Ha, m)

1.47, (Hb, m) 27.5 (CH2) 6.17, (dd, 16.0, 2.0) 129.3 (CH)

8 1.61, (Ha, m) 1.43, (Hb m)

33.4 (CH2) 5.75, (dd, 16.0, 6.4) 130.0(CH)

9 3.54, (m) 73.8 (CH) 4.31, (m) 70.2 (CH) 10 3.45, (d, 5.2) 64.3 (CH2) 1.26, (d, 6.4) 25.1 (CH3) 11 1.10, (s) 26.5 (CH3) 1.24, (s) 24.3 (CH3) 12 1.03, (s) 28.0 (CH3) 0.86, (s) 25.2 (CH3) 13 2.01, (s) 22.8 (CH3) 1.07, (s) 27.3 (CH3)

3.1.6. Characterization of lapachol (118)

Compound 118 was obtained as yellow needle like crystals, which

exhibited characteristic IR absorption bands at 3350, 1655 and 1645, 1605

and 1545 cm-1 due to hydroxyl, conjugated ketone, olefinic system and

aromatic moiety, respectively. The UV analysis (272, 280, 289 nm) also

substantiated a substituted aromatic system. The EI-MS spectrum of 118

afforded the molecular ion at m/z 242, whereas, the molecular formula

C15H14O3 with nine DBE was determined through HR-EI-MS, which

displayed the molecular ion at m/z 242.0961.


56

The 1H NMR spectrum (Table 3.4) of 118 showed signals for a 1,2-

disubstitiuted benzene ring at 8.12 (1H, d, J = 7.5 Hz), 8.05 (1H, d, J =

7.5 Hz), 7.68 (1H, t, J = 7.5 Hz) and 7.62 (1H, t, J = 7.5 Hz). In addition, the

same spectrum displayed the resonances of an isoprene unit at 5.21 (1H,

t, J = 6.8 Hz), 3.27 (2H, d, J = 6.8 Hz), 1.78 (3H, s)

and 1.69 (3H, s). The isoprenoid nature of these

signals could be established due to allelic COSY

correlation of the two singlet methyls ( 1.78 and

1.69) with olefinic system ( 5.21) and that of

olefinic proton with the methylene ( 3.27) protons. 13C NMR spectrum

(Table 3.4) substantiated the isoprenoid moiety in 118 due to the carbon

signals at 135.1 (C), 119.2 (C-H), 26.7 (CH3), 24.5 (CH2) and 18.1 (CH3).

The signals for benzene ring were observed at 131.3, 130.1, 129.6, 129.3,

127.2 and 126.3. In addition, the same spectrum afforded four more

signals at 182.5, 181.1, 153.6 and 121.0. This information revealed that,

in addition to benzene ring and isoprenoid moiety, compound 118 must

have two ketonic functions and one additional double bond. This data when

coupled with the molecular formula and DBE, it was established that 118

has a quinone moiety condensed with the benzene ring.

The attachment of the isoprenoid group with the quinone moiety

could be determined due to HMBC experiment (figure 3.5), in which

methylene protons ( 3.27) exhibited correlation with the carbons at 182.5

O

O

OH

118

1

35

7 9

10

1'

2'

3'

4'

5'


57

(C-4), 153.6 (C-2), 121.0 (C-3) and 135.1 (C-3). The olefinic proton showed

HMBC interaction with the carbons at 121.0 (C3), 26.7 (C-5) and 18.1 (C-

4). These determinations helped to fix the position of hydroxyl function at

C-2 of quinone moiety (Figure 3.5). This data was when matched with the

literature values, it fully superimposed with reported data for lapachol

(Oliveira et al., 2002) and therefore, compound 118 was also found to be

the same i e; lapachol.

OH

O

O

118

1

3

5

79

10

1'

3'

4'

5'


3.1.7. Characterization of 3-deoxyisolapachol (147)

The IR, UV and mass data of compound 147 was super imposable to

that of 118 indicating it to be the derivative of 118. On the other hand, the

same molecular formula C15H14O3 and DBE raised

confusion that 118 and 147 could be the same

compounds. However, the difference was observed

in 1H NMR data, which displayed a tarns-olefin

system due to the resonances at 6.81 (1H, d, J =

16.4 Hz) and 6.76 (1H, d, J = 16.4 Hz). The chemical shift of these two

O

O

147

1

35

79

101'

2'

3'

4'

5'

OH


58

methine protons could be attributed to an extended conjugation with

another olefin at 6.96 (1H, s) and then with carbonyl system. This data

suggested that 2-OH in 147 is absent, rather it is a methine group,

whereas, the double bond between 2 and 3 must have been shifted to the

carbons 1 and 2. This deduction was substantiated due to relatively up

field chemical shift of methyls 4 and 5 collectively at 1.45. Further, 13C-

NMR shifts of these two methyls ( 30.1) and that of C-1 ( 116.1) and C-3

( 72.0). It was thus characterized that 2-OH has been shifted as 3-OH.

Other NMR data was comparable with that of compound 118. Therefore,

the structure of compound 147 was established as 3-deoxyisolapachol,

which is also a known metabolite (Sumthong et al., 2006; Sumthong et al.,

2008), but has been isolated for the first time from Techomella undulata.

Table 3.4: 1H and 13C NMR data of 118 and 147 (CDCl3, 500 and 125 MHz)

118 147


1 _ 181.1 (C) _ 183.6 (C)

2 _ 153.6 (C) 6.96, (s) 130.9 (CH)

3 _ 121.0 (C) _ 142.1 (C)

4 _ 182.5(C) _ 184.3 (C)

5 8.12, (d, 7.5) 127.2 (CH) 8.11, (d, 8.0) 127.1 (CH)

6 7.68, (t, 7.5) 131.3 (CH) 7.76, (m, 7.8) 133.5 (CH)

7 7.62, (t, 7.5) 130.1 (CH) 7.37, (m, 7.8) 133.8 (CH)

8 8.05, (d, 7.5) 126.3 (CH) 8.06, (d, 8.06) 124.1 (CH)

9 _ 129.3 (C) _ 133.1 (C)

10 _ 129.6 (C) _ 133.6 (C)

1 3.27, (d, 6.8) 24.5 (CH2) 6.76, (d, 16.4) 116.1 (CH0

2 5.21, (t, 6.8) 119.2 (CH) 6.81, (d, 16.4) 148.2 (CH)

3 _ 135.1 (C) _ 72.0 (C)

4 1.78, (s) 18.1 (CH3) 1.45, (s) 30.1 (CH3)

5 1.69, (s) 26.7 (CH3) 1.45, (s) 30.1 (CH3)


59

3.1.8. Characterization of quercetin-3,4-dimethyl ether (148)

Compound 148 was purified as yellow amorphous powder, which

also showed a yellow spot on TLC complexation with ceric sulphate, as an

indication of a flavonoid nucleus. This idea was

substantiated due to UV spectral analysis, in

which the absorption maxima were observed at

257, 287 and 351 nm for a substituted

flavonoid. The EIMS of 148 showed molecular ion at m/z 330, while

HREIMS displayed molecular ion at m/z 330.0762 corresponded to the

molecular formula as C17H14O7.

The aromatic region of the 1H NMR spectrum of compound 148

resembled closely to the spectrum of compound (150) with small

differences. In the hydrogen spectrum of 148, five signals were seen at

7.60 (1H, dd, J = 8.4, 2.0 Hz), 7.32 (1H, d, J = 2.0 Hz), 6.89 (1H, d, J = 8.4

Hz), 6.37 (1H, d, J = 2.0 Hz) and 6.17 (1H, d, J = 2.0 Hz). The first three

signals splitted at ABX pattern were attributed to ring B of flavonoid

nucleus, whereas, the last two signals were attested for ring A of the

flavonoid skeleton (Table 3.5). Two methoxyl signals were also observed in

the same spectrum at 3.84 and 3.80. The 13C NMR spectrum of 148

afforded 17 carbon signals (Table 3.5). The absence of signal for H-3 and

resonance of a quaternary carbon at 133.3 revealed a flavonol nature of

148, whereas, two methoxyl groups were placed on ring B due to their

7

3

5

8

9

10

13´1´

5´

OHO

OH O

OMe

OMe

OH

148


60

HMBC correlations with the aromatic carbons at 149.1 and 148.4

respectively. The whole data was comparable with the reported data of

quercetin-3,4-dimethyl ether (Pavanasasivam and Sultanbawa, 1975) and

therefore, compound 148 was also found to be the same.

3.1.9. Characterization of 3,5,3,4-tetrahydroxy-6,7-dimethoxy flavone

(149)

TLC analysis and the UV data of compound 149 also indicated its

flavonoid nature. The EI-MS exhibited the heaviest ion at m/z 346, whose

HR-EI-MS analysis (m/z 346.0681) depicted the molecular formula as

C17H14O8. The 1H NMR data (Table 3.5) was nearly similar to that of

compound 148, with the difference that the

resonance of only one proton at 6.54 (1H, s)

was observed for ring A of flavonoid nucleus,

whereas, other three signals resonating at

7.44 (1H, dd, J = 8.4, 1.8 Hz), 7.32 (1H, d, J = 1.8 Hz) and 7.08 (1H, d, J =

8.4 Hz) were attributed to ring B. Two methoxyl moieties were determined

due to the resonances at 3.83 and 3.81.

The 13C NMR data (Table 3.5) substantiated the above determinations

as it displayed 17 signals, which were differentiated as two methoxyl, four

methine and eleven quaternary carbons. The methoxyl groups were placed

at C-6 and C-7 due to their HMBC correlations (Figure 3.6) with the carbon

signals at 159.5 (C-7) and 133.6 (C-6). HMBC correlation of H-8 ( 6.54)

7

3

5

8

9

10

13´1´

5´

OMeO

OH O

OH

OH

OH

149

MeO


61

with C-7 and C-6 substantiated the above evidence. Based on these

evidences and comparison with the literature (Voirin et al., 1985)

compound 149 could be identified as 3,5,3,4-tetrahydroxy-6,7-dimethoxy

flavone.

O

O

OH

OH

MeO

OH

OH

MeO149


3.1.10. Characterization of quercetin-3-galactoside (150)

Compound 150 was isolated as yellow amorphous powder, which

showed a flavonoid nature on TLC when

heated with ceric sulphate. The UV data

(254, 276, 320 and 335 nm) depicted a

substituted flavonoid skeleton. The HR-

FABMS in positive mode exhibited pseudo

molecular ion [M+H]+ at m/z 465.3869

corresponding to the formula as C21H20O12.

The molecular formula indicated that compound 150 could be a flavonoid

glycoside. Aromatic region of the 1H NMR spectrum of compound 150 was

identical to that of compound 148 with five signals appeared at δ 7.68 (1H,

7

5

8

9

10

5´

1

3

1'

3'

1''

3''

5''

6´´

O

O

O

OH

HO

OH

OH

O

HO

HO

OHHO

150


62

d, J = 2.0 Hz), 7.54 (1H, dd, J = 8.4, 2.0 Hz), 6.84 (1H, d, J = 8.4 Hz), 6.34

(1H, d, J = 1.8 Hz) and 6.16 (1H, d, J = 1.8 Hz). This data indicated the

aglycon part to be the quercetin. The 1H NMR spectrum further displayed

signals for a hexose moiety at 5.26 (1H, d, J = 7.6 Hz), 3.69 (1H, dd, J =

12.0, 2.6 Hz), 3.58 (1H, dd, J = 12.0, 5.6 Hz), 3.51 (1H, dd, J = 8.0, 2.8 Hz),

3.48 (1H, t, J = 8.4 Hz) and 3.30 (1H, d, J = 2.8 Hz). The coupling constant

of anomeric proton (J = 7.6 Hz) and that of H-4 ( 3.30, d, J = 2.8 Hz)

depicted the β-galactose nature of the hexose moiety (Guvenalp and

Demirezer, 2005).

The 13C NMR spectrum (Table 3.5) of compound 150 displayed total

21 carbon signals, in which 15 were attested for quercetin part, whereas,

the six signals resonating at 103.5, 74.1, 72.5, 71.6, 69.0 and 62.6 were

attributed to the galactose moiety. The connectivity of the galactose moiety

at C-3 could be determined through HMBC spectrum (Figure 3.7) in which

anomeric proton ( 5.26) exhibited correlation with the carbon at 133.5

(C-3). The above whole discussed data was identical to the reported data of

quercetin-3-O-β-D-galactoside (Guvenalp and Demirezer, 2005) which has

been isolated for the first time from our investigated source.


63

O

OH

OH

OOH

HO

O

HOOH

OH

O

HO

HMBC and COSY( ) )(

150

Figure 3.7: HMBC and COSY correlations observed in the spectrum of

compound 150


64

Table 3.5: 1H and 13C NMR data of compounds (148-150) (CD3OD, 400 and 100 MHz)

148 149 150

Position H (J in Hz) C H (J in Hz) C H (J in Hz) C

1 _ _ _ _ _ _ 2 _ 159.0 (C) _ 158.9 (C) _ 158.2 (C) 3 _ 133.3 (C) _ 132.8 (C) _ 133.5 (C) 4 _ 179.5 (C) _ 178.0 (C) _ 178.4 (C) 5 _ 163.0 (C) _ 153.5 (C) _ 162.5 (C) 6 6.17, (d, 2.0) 99.8 (CH) _ 133.6 (C) 6.16, (d, 1.8) 99.8 (CH) 7 _ 166.1 (C) _ 159.5 (C) _ 166.0 (C) 8 6.37, (d, 2.0) 95.0 (CH) 6.54, (s) 90.5 (CH) 6.34, (d, 1.8) 94.1 (CH) 9 158.8 (C) _ 156.3 (C) _ 158.3 (C) 10 _ 105.0 (C) _ 104.6 (C) _ 104.0 (C)

1 _ 123.1 (C) _ 121.0 (C) _ 121.0 (C)

2 7.32, (d, 2.0) 109.8 (CH) 7.32, (d, 1.8) 111.9 (CH) 7.68, (d, 2.0) 116.1 (CH)

3 _ 148.4 (C) _ 147.4 (C) _ 147.6 (C)

4 _ 149.1 (C) _ 148.5 (C) _ 149.8 (C)

5 6.89, (d, 8.4) 115.9 (CH) 7.08, (d, 8.4) 115.5 (CH) 6.84, (d, 8.4) 114.8 (CH)

6 7.60, (dd, 8.4, 2.0)

122.9 (CH) 7.44, (dd, 8.4, 1.8)

129.0 (CH) 7.54, (dd, 8.4, 2.0)

123.1 (CH)

1 5.26, (d, 7.6) 103.5 (CH)

2 3.48, (t, 8.4) 71.6 (CH)

3 3.51, (dd, 8.0, 2.8) 72.5 (CH)

4 3.30, (d, 2.8) 69.0 (CH)

5 3.21, (m) 74.1 (CH)

6 3.69, (Ha, dd, 12.0, 2.6) 3.58, (Hb dd, 12.0, 5.6)

62.6 (CH2)

6 -OMe 3.81, (s) 62.7 (CH3) 7 -OMe 3.83, (s) 55.6 (CH3)

3-OMe 3.80, (s) 55.3

4-OMe 3.84, (s) 56.0


65

3.1.11. Characterization of ferulic acid (104)

Compound 104 was obtained as white crystalline solid, which

exhibited IR absorption bands for carboxylic

function (3420 cm-1 for chelated hydroxyl group

and 1710 cm-1 for carbonyl group). IR spectrum

further displayed absorption bands due to double

bond (1625 cm-1), aromatic system (1600 and 1545

cm-1) and methoxyl moiety (1305 cm-1). The EI-MS spectrum displayed the

molecular ion peak at m/z 194, whereas, The HR-EI-MS analysis of this ion

depicted the molecular formula C10H10O4.

Aromatic region of the 1H NMR spectrum of compound 104 showed

resonances of five hydrogen atoms at 7.52 (1H, d, J = 15.6 Hz), 6.96 (1H,

d, J = 1.8 Hz), 6.86 (1H, dd, J = 8.0, 1.8 Hz), 6.79 (1H, d, J = 8.0 Hz) and

6.20 (1H, d, J = 15.6 Hz). The first and the last signals were attested for a

conjugated trans olefinic system whereas the rest of aromatic protons

splitted at ABX pattern were attributed to a tri-substituted aromatic

system. The same spectrum further afforded a signal of three protons at

3.89 due to a methoxyl moiety. This data indicated that compound 104

could be methyl ether of ferulic acid, which was further confirmed due to

13C NMR analysis (Table 3.6). The 13C NMR spectrum afforded total ten

resonances with the most downfield quaternary carbon resonated at

172.0, was attested for carboxylic acid function, whereas, the olefinic

OMe

HO

OH

O

104

1

3

5 9


66

methines displayed their positions at 144.1 and 115.0. The resonances

for aromatic carbons were observed at 146.0, 145.5, 124.6, 120.2, 114.9

and 110.6. This data was identical to the data reported for methyl ether of

ferulic acid (Scalbert et al., 1985) therefore, compound 104 was also found

to be the same.

3.1.12. Characterization of hexadecyl ferulate (151)

IR spectrum of compound 151 displayed diagnostic absorption bands

at 3415, 1715, 1635 and 1550 cm-1 for

hydroxyl function, ester moiety, double

bond and aromatic system, whereas, the

molecular formula could be determined

through HR-EI-MS (m/z 418.3080) as C26H42O4. Aromatic region of the 1H

NMR spectrum (Table 3.6) of compound 151 was nearly super imposable to

that of compound 104 as it displayed five resonances at 7.50 (1H, d, J =

16.0 Hz), 6.97 (1H, d, J = 2.0 Hz), 6.93 (1H, dd, J = 7.6, 2.0 Hz), 6.83 (1H,

d, J = 7.6 Hz) and 6.18 (1H, d, J = 16.0 Hz). This data indicated a ferulic

acid derived molecular nature of compound 151. In addition to the

methoxyl signal, the 1H NMR spectrum showed a triplet oxymethylene at

4.22 (J = 7.2), which showed COSY correlation with another methylene at

1.69 (m), which in turn was correlated with a broad singlet of several

methylenes at 1.54-1.23. A triplet methyl ( 0.81, J = 6.5 Hz) was also

found to show correlation in COSY spectrum with the same broad singlet at

7

8

91

3

6

1'

16'

OMe

HO

O

O

12

151


67

1.54-1.23. This information revealed a long chain hydrocarbon part in

151. The chemical shift of oxymethylene ( 4.22) indicated its attachment

with the carboxylate function. These observations revealed 151 to be an

alkyl ferulate derived molecule.

The 13C NMR data (Table 3.6) was also in agreement with the 1H NMR

data as it displayed signals for ferulate moiety and a hydrocarbon chain.

The length of the hydrocarbon chain could be fixed due to mass spectrum

analysis and finally compound 151 was identified as hexadecyl ferulate

(Bernards and Lewis, 1992; Mussadiq et al., 2013).

Table 3.6: 1H and 13C NMR data of 104 and 151 (CDCl3+CD3OD, 400 and

100 MHz)

104 151)

Position H (J = Hz) C H (J = Hz) C

1 _ 124.6 (C) _ 125.1 (C)

2 6.96, (d, 1.8) 110.6(CH) 6.97, (d, 2.0) 110.1 (CH)

3 _ 146.0 (C) _ 145.1 (C)

4 _ 145.5 (C) _ 146.2(C)

5 6.79, (d, 8.0) 114.9 (CH) 6.83, (d, 7.6) 116.0 (CH)

6 6.86, (dd, 8.0,1.8) 120.2 (CH) 6.93, (dd, 7.6, 2.0) 121.2 (CH)

7 7.52, (d, 15.6) 144.1 (CH) 7.50, (d, 16.0) 145.2 (CH)

8 6.20, (d, 15.6) 115.0 (CH) 6.18, (d, 16.0) 115.1 (CH)

9 _ 172 0(C) _ 169.1 (C)

3-OMe 3.89. (s) 61.2 (CH3) 3.84, (s) 60.3 (CH3)

1 4.22, (t, 7.2) 64.1 (CH2)

2 1.69, (m) 29.8 (CH2)

3-15 1.54-1.23, (br s) 30.5-29.6

(CH2)13

16 0.81, (t 6.5) 14.5 (CH3)


68

3.1.13. Characterization of betulinic acid (133)

Compound 133 was isolated as white amorphous solid, which

exhibited IR absorption bands at 3405, 2420, 1710 and 1685 cm-1 due to

carboxylic acid group and olefinic function,

respectively. The EI-MS of compound 133

displayed molecular ion at m/z 456 whereas,

HR-EI-MS depicted the molecular formula as

C30H48O3 with seven DBE. The fragmentation

pattern in EI-MS (m/z 440, 426, 208, 79 and

41) and the formula revealed a pentacyclic triterpenoid of betulane series

(Sharma et al., 2010).

The 1H NMR spectrum (Table 3.7) of 133 displayed resonances of six

tertiary methyls at 1.63, 0.97, 0.95, 0.93, 0.81 and 0.79. A terminal

olefinic methylene displayed its position at 5.01 (Hb, s) and 4.76 (Ha, s),

whereas, the resonance of a methine, characteristic of betulane series

appeared at 2.32 (m), which showed allelic correlation in the COSY

spectrum with the olefinic methylene. The 1H NMR spectrum displayed an

oxymethine signal at 3.33 (m).

The 13C NMR spectrum (Table 3.7) showed total 30 carbon signals,

which were separated as six methyl, eleven methylene, six methine and

seven quaternary carbons due to DEPT experiment. The most downfield

quaternary carbon at 178.3 was attested for carboxylic acid function.

HO

OH

O

133

29

30

20

28

2324

2526

27


69

The olefinic carbon resonances appeared at 151.2 and 108.7, while the

oxymethine carbon resonated at 79.0. This data was identical to that

published for betulinic acid (Sharma et al., 2010), therefore, compound

133 was also found to be the same, which is a known phytochemical.

3.1.14. Characterization of β-sitosterol (134)

The EI-MS of compound 134 displayed molecular ion at m/z 414

with characteristic fragment ions at m/z

399, 396, 381, 329, 275, 273 and 255 for

a 5-sitosterol. The molecular formula was

established as C29H50O with five DBE due

to HR-EI-MS. The IR spectrum of

compound 134 showed the absorption bands for hydroxyl group at 3450

cm-1 along with 1645, 816 cm-1 for C=C functionality. The 1H NMR

spectrum of compound 134 displayed two tertiary methyls at 1.02 and

0.68, three secondary methyls at 0.95 (J = 6.2), 0.86 (J = 6.5) and 0.82 (J

= 6.5) and one primary methyl at 0.86 (J = 7.0). The same spectrum

displayed a signal for an olefinic methine at 5.12 (m) and an oxymethine

at 3.38 (m). This data revealed a β-sitosterol nucleus, which was

subantiated due to the 13C NMR spectrum (Table 3.7) of compound 134.

The 13C NMR spectrum afforded 29 carbon signals, which were separated

due to DEPT experiment, as six methyls, eleven methylenes, nine methines

and three quaternary carbons. The most downfield resonances at 141.9,

HO 134

1

2

3 5 7

9

1113

15

17

18

19

2021

23 25

26

27

2829


70

122.9 and 76.9 were attested for double bond and oxymethine respectively.

Other 13C NMR shift are shown in (Table 3.7) Based on this information,

and comparison of TLC profile with standard sample, compound 134 was

identified as β-sitosterol, which is a common phytochemical (Ahmad et al.,

2009; Kamboj and Saluja, 2011).

3.1.15. Structure elucidation of β-sitosterol-3-O-β-D-glucopyranoside

(152)

The FAB-MS of compound 152 showed molecular ion peak at m/z

576.4386 corresponding to molecular formula C35H60O6 (calcd. for

C35H60O6, 576.4336). Like

compound 134, the mass

fragmentation pattern of

compound 152 was

characteristic for sterol

with double bond at C-5

(Ageta and Ageta, 1984; Mizushina et al., 2006b; Wyllie et al., 1977).

The 1H NMR spectrum of 152 displayed an olefinic proton at δ 5.30

(distorted t, H-6), a carbinylic proton at δ 3.98 (m, H-3), two singlet methyl

signals at δ 0.96 and 0.69, three doublet methyl signals at δ 0.86 (J = 6.5

Hz), 0.88 (J = 6.7 Hz) and 0.92 (J = 6.5 Hz), and one triplet methyl signal at

δ 0.83 (t, J = 6.6 Hz). In addition, an anomeric proton signal at δ 4.95 (d, J

= 7.0 Hz, H-1′), four oxymethines and an oxymethylene were observed

between δ 4.28-3.93 in the same spectrum.

O

152

O

HO

HOOH

OH1

3 5 7

9

11 13

15

17

1824

19

20

21

28

29

25

26

27

1''3''

5''6''


71

The 13C NMR spectrum (Table 3.7) of compound 152 showed 35

carbon signals, out of which 29 were attributed to the aglycone moiety and

six to the sugar moiety. The carbon signals of the sugar appeared at δ

103.0, 76.4, 75.6, 73.6, 70.1 and 62.4 giving the idea of glucose. On the

basis of above data and comparison with literature, 152, was identified as

β-sitosterol-3-O-β-D-glucopyranoside (Haque et al., 2008; Iribarren and

Pomilio, 1983; Mizushina et al., 2006a), which is also a common

phytochemical.

Table 3.7: 1H and 13C NMR data of 133, 134 and 152 (CDCl3+CD3OD, 400

and 100 MHz)

133 134 152

Position H (J = Hz) C H (J = Hz) C H (J = Hz) C

1 39.1 (CH2) 37.3 (CH2) 37.3 (CH2)

2 26.8 (CH2) 31.8 (CH2) 29.4 (CH2)

3 3.33, (m) 79.0 (CH) 3.38, (m) 76.9 (CH) 3.98, (m) 79.3 (CH)

4 _ 39.2 (C) 39.6 (CH2) 38.2 (CH2)

5 54.6 (CH) _ 141.9 (C) _ 141.1 (C)

6 17.9 (CH2) 5.12, (m) 122.9 (CH) 5.30, (m) 121.7 (CH)

7 35.0 (CH2) 32.1 (CH2) 31.6 (CH2)

8 40.9 (C) 36.9 (CH) 31.8 (CH)

9 50.6 (CH) 45.6 (CH) 46.9 (CH)

10 _ 38.0 (C) _ 37.0 (C) _ 36.3 (C)

11 21.3 (CH2) 20.3 (CH2) 21.5 (CH2)

12 24.6 (CH2) 23.7 (CH2) 38.3 (CH2)

13 38.0 (CH) _ 36.8 (C) _ 42.1 (C)

14 _ 43.1 (C) 45.1 (CH) 45.2 (CH)

15 29.8 (CH2) 28.8 (CH2) 24.2 (CH2)

16 33.4 (CH2) 30.4 (CH2) 28.2 (CH2)

17 49.0 (C) 51.2 (CH) 56.0 (CH)

18 47.6 (CH) 0.68, (s) 13.9 (CH3) 0.69, (s) 12.0 (CH3)

19 2.32 (H, m) 49.9 (CH) 1.02, (s) 19.4 (CH3) 0.96, (s) 19.4 (CH3)

20 _ 151.2 (C) 36.3 (CH) 35.9 (CH)

21 31.0 (CH2) 0.95, (d,

6.2)

19.1 (CH3) 0.92, (d,

6.5)

18.8 (CH3)

22 37.6 (CH2) 34.0 (CH2) 36.5 (CH2)

23 0.93, (s) 27.9 (CH3) 29.3 (CH2) 26.4 (CH2)

24 0.78, (s) 15.5 (CH3) 45.4 (CH) 45.7 (CH)


72

25 0.81, (s) 16.8 (CH3) 26.2 (CH) 29.2 (CH)

26 0.95, (s) 17.1 (CH3) 0.95, (d,

6.5)

18.8 (CH3) 0.88, (d,

6.7)

19.4 (CH3)

27 0.97, (s) 15.5 (CH3) 0.82, (d,

6.5)

19.8 (CH3) 0.86, (d,

6.5)

19.0 (CH3)

28 _ 178.3 (C) 23.1 (CH2) 23.0 (CH2)

29 4.76, (Ha, s)

5.01, (Hb, s)

108.7 (CH2) 0.86, (t

7.0)

13.9 (CH3) 0.83, (t, 6.6) 11.6 (CH3)

30 1.63, (s) 21.1 (CH3)

1' 4.95, (d,

7.0)

103.0 (CH)

2' 4.08, (m) 73.6 (CH)

3' 4.02, (m) 76.4 (CH)

4' 4.02. (m) 70.1 (CH)

5' 3.93, (m) 75.6 (CH)

6' 4.28, (Ha,

dd,

11.2,2.1)

4.14, (Hb,

dd,

11.2,4.9)

62.4 (CH2)

3.2. Antibacterial and Enzyme Inhibitory Evaluation of Compounds

118, 133-134, 142-152

3.2.1. Antibacterial activity of compound 142

Compound 142 was tested in vitro against four Gram negative

(Escherichia coli, Salmonella typhi, Shigella dysenteriae and Psedumonas

aeruginosa) and two Gram positive (Staphylococcus aureus and Bacillus

subtilis) bacterial species. The test compound 142 only inhibited the growth

of E. coli and S. typhi with MIC values 96.0 and 91.8 µg/ml, respectively.

These results are in consistence with the reported antibacterial activity

(Abhishek et al., 2013) of leaves extract of Tecomella undulata.


73

3.2.2. Enzyme inbihitory activity of compounds 118, 133-134, 142-

152

The purified compounds 118, 133-134, 142-152 were evaluated for

enzyme inhibitory potential against the enzymes Acetylcholinesterase,

Butyrylcholinesterase and Lipoxygenase (Table 3.8). All the tested

compounds showed 35-82% inhibition of the enzymes at a concentration of

0.5 mM, however, the IC50 values were quite high which means the tested

compounds are either weakly active or inactive at tested concentration.

Unfortunately no other biological studies could be performed for the

isolates due to lack of lab facilities but literature survey revealed that some

of the isolated compounds were potent in some other activities e.g., ferulic

acid (104) exhibits antioxidant activity (Graf, 1992), inhibits melanin

formation and strong absorption of harmful UV-wavelength has also been

reported (Murray et al., 2008; Tournas et al., 2006).

Lapachol (118) is medicinally an important compound and a wide

spectrum of therapeutic activities have been attributed to the compound

118 and its derivatives e.g, anti-ulcer, antiseptic, antiviral, antimalarial,

antileishmanial, anticarcinomic, antiedemic, anti-inflammatory, antitumor,

fungicidal and bactericidal etc (Hussain et al., 2007b). Betulinic acid (133)

is reported as potent anti-HIV (Hashimoto et al., 1997) and

hepatoprotective agent (Jain et al., 2012). β-sitosterol (134) is used against

heart disease, modulating the immune system, hypercholesterolemia, to


74

prevent cancer and against rheumatoid arthritis. It is also found useful

against tuberculosis, cervical cancer and hair loss (Saeidnia et al., 2014).

Some megastegmanes and their glycosides are reported to show lipid

accumulation inhibitory and 2,2-diphenyl-picrylhydrazyl (DPPH) radical-

scavenging activities respectively (Matsunami et al., 2006; Muraoka et al.,

2009).


75

Table 3.8: Enzyme inhibitory activities* of the isolated compounds from Tecomella undulata

No.of compound AChE (%)

AChE (IC50) M BChE (%)

BChE (IC50) M

LOX (%)

LOX (IC50) M

118 29.23±0.24 NIL 40.13±0.15 NIL 47.81±0.14 NIL

133 35.02±0.19 NIL 37.62±0.25 NIL 60.72±0.14 NIL

134 41.16±0.11 NIL 29.72±0.22 NIL 61.02±0.14 NIL

142 67.82±0.14 199.01±0.21 40.51±0.14 <700 35.65±0.21 <700

143 37.62±0.26 NIL 57.80±0.14 NIL 59.03±0.14 NIL

144 46.90±0.13 NIL 69.02±0.14 NIL 48.9±0.10 NIL

145 31.51±0.10 NIL 70.12±0.14 NIL 51.79±0.15 NIL

146 56.9±0.10 <400 53.4±0.4 <300 5.4±0.13 NIL

147 82.26±0.10 <700 71.2±0.2 <700 45.6±0.18 NIL

148 62.36±0.19 <700 65.8±0.2 <700 55.7±0.16 NIL

149 57.82±0.13 <900 38.80±0.14 <800 58.12±0.14 NIL

150 28.12±0.19 <700 67.82±0.14 <700 69.2±0.11 NIL

151 30.25±0.83 NIL 65.1±0.4 263.9±0.22 73.2±0.4 134.1±0.2

152 39.29±0.77 NIL 35.66±0.5 NIL 63.69±0.6 NIL

Eserine** 91.29±1.17 0.04±0.001 82.82±1.09 0.85±0.001 - -

Baicalein** - - - - 93.79±1.2 22.4±1.3

* All experiments were performed in triplicate

** Standard drugs

Chapter 3 Experimental

76

3.3. Experimental

3.3.1. General experimental procedures

3.3.1.1. Spectroscopic methods

The EI-MS, HR-EI-MS, FAB-MS and HR-FAB-MS were recorded on

Finnigan (Varian MAT, Waldbronn, Germany) JMS H×110 with a data

system and JMSA 500 mass spectrometers respectively. The UV spectra

were recorded on Schimadzu UV-240 or U-3200 HITACHI

spectrophotometer (Duisburg, Germany), whereas, the IR spectra were

recorded as KBr pellets on Shimadzu 460 spectrometer/JASCO 320-A

infrared spectrometer (Duisburg, Germany). The optical rotations were

measured on a JASCO DIP-360 polarimeter (Tokyo, Japan). The 1H NMR

spectra were recorded on Bruker AM-300, 400, 500 and 600 MHz in

deuterated solvents, while the 13C NMR spectra were recorded at 75, 100,

125 or 150 MHz, on the same instruments. The 2D-NMR spectra were also

measured on the same instruments operating at 400, 500 and 600 MHz.

The chemical shift values (δ) are reported in ppm and the coupling

constant (J) in Hz.

3.3.1.2. Materials

Commercially available solvents were used after distillation at their

respective boiling points for extraction of plant material and

chromatographic techniques. Column chromatography was performed

using silica gel (Keiselgel-230-400 mesh, Darmstadt, Germany and

Keiselgel-70-230 mesh, E-Merck) as stationary phase packed in glass


77

column. Chromatographic separations were monitored using aluminium

sheets precoated with silica gel 60 F254 (20×20 cm, 0.2 mm thick; E-Merck;

Darmstadt, Germany). UV light (254 and 366 nm) was used to see

fluorescence of chromatogram, and ceric sulphate solution followed by

heating was used to locate UV inactive spots on chromatogram.

3.3.2. Collection and identification of the plant material

The whole plant material was collected from National Park, Lal Sohanra,

Bahawalpur in June 2008 and was identified by Dr. Muhammad Arshad

(Late), Ex-Director, CIDS, The Islamia University of Bahawalpur, Pakistan.

3.3.3. Extraction of the plant material and isolation

The shade dried plant of Tecomella undulata (5.0 kg) was extracted

thrice with MeOH (15.0 L) at room temperature. The combined MeOH

extract was concentrated and the residue (205.0 g) was divided into n-

hexane (50.0 g), chloroform (40.0 g), ethylacetate (55.0 g), n-butanol (25.0

g) and water-soluble (20.0 g) fractions (Scheme 3.1). The ethylacetate

fraction (55.0 g) was subjected to column chromatography over silica gel

eluting with n-hexane, n-hexane-CHCl3, CHCl3 and CHCl3-MeOH in

increasing order of polarity, and five fractions (A, C-F) were obtained.

The fraction F (1.1 g) obtained from main column with 5% MeOH in

ethyl acetate was processed further to get three sub fractions (1-3). Sub

fraction 3 (0.6 g) showed one major and fewer minor components on TLC


78

(CHCl3:MeOH:H2O in ratio7.5:2.3:0.2) and thus was re-chromatographed

on silica gel column eluting with isocratic of 5% MeOH in ethyl acetate. The

resulting fraction was then passed through sephadex LH-20 eluting with

methanol to get kaempferol-3-O-β-D-[3,4di-p-E-coumaroyl--L-

rhamnosyl (1→6)] galactoside (142, 21.0 mg). Other components obtained

from the same sub fraction were too minute to be processed further.

Sub fraction 2 (0.4 g) obtained with 50% ethyl acetate in hexane

afforded a semi-pure compound, which was passed through silica gel

coloumn eluting with EtOAc:hexane (6:4) to get quercetin-3-galactoside

(150, 25.0 mg), 3,5,3′,4′-tetrahydroxy-6,7-dimethoxy flavone (149, 22.0 mg)

as yellow amorphous powders and quercetin-3′,4′-dimethyl ether (148, 35.0

mg), when eluted with n-hexane:ethyl acetate (6:4). Fraction E (1.7 g)

obtained with pure chloroform from main coloumn was further purified by

coloumn chromatography eluting with CHCl3:hexane (4:6) yielding two sub

fractions E1 and E2; deoxyisolapachol (147, 19.0 mg) was isolated with

solvent system CHCl3:hexane (4:6) and betulinic acid (133, 32.0 mg) was

isolated using pure CHCl3 from E1, whereas coloumn chromatography of E2

afforded lapachol (118, 25.0 mg) at CHCl3:hexane (5:5).

Fraction D (1.5 g) obtained from the main column with 90% CHCl3 in

n-hexane, on silica gel column chromatography yielded β-sitosterol 3-O-β-

D-glucopyranoside (152, 76.0 mg) at CHCl3:CH3OH (9.8:0.2).


79

Fraction C (450.0 mg) from the main column obtained with 80%

CHCl3 in n-hexane was chromatographed on a silica gel column eluted with

100% methylene chloride (CH2Cl2) to get 5 sub-fractions (1-5). Fraction 3

was further purified on RP-18 silica packed column eluted with isocratic of

70% aqueous methanol to get two sub-fractions (2a and 2b). The fraction

2a was purified by reversed phase HPLC eluting with 25% aqueous

methanol to yield compounds: (6R,7Z,9S)-9,10-dihydroxy-4,7-

megastigmadien-3-one (143, 12.0 mg, Rt = 31.2 min), (6E,9S)-9,10-

dihydroxy-4,6-megastigmadien-3-one (144, 7.5 mg, Rt = 25 min), (6R,9S)-

9,10-dihydroxy-4-megastigmaen-3-one (145, 16.0 mg, Rt = 33.8 min).

Fraction 2b was passed over Sephadex LH-20 eluted with methanol to get

3S,5R,6R,9S-tetrahydroxy megastigmane (146, 21.0 mg).

The fraction A (2.5 g) obtained from main column with n-

hexane:CHCl3 (8:2) was further subjected to silica gel column

chromatography to get two sub-fractions (A1 and A2). Sub fraction A1 on

repeated coloumn chromatography yielded β-sitosterol (134, 112.0 mg) at

n-hexane:CHCl3 (9:1). The sub fraction A2 on further purification by silica

gel column eluting with n-hexane:CHCl3 (6:4) afforded ferulic acid (104,

37.0 mg) and with n-hexane:CHCl3 (6.5:3.5) hexadecyl ferulate (151, 20.0

mg).


80

Dry powdered T. undulata (5 kg)

Hexane

Hexane part(50 g)

Water

EtOAc part(55 g)

Water part(20 g)

CC over flash silica using varying polarity of solvents

Fract. A Fract. C Fract. D Fract. E Fract. F

EtOAc

CHCl3 part(40 g)

Butanol part(25 g)

104, 134, 151 143-146 152 118, 133, 147 142, 148-150

MeOH7 days

Methanolic extract (205 g)

BuOHCHCl3

Scheme 3.1: Isolation protocol of the secondary metabolites of T. undulata


81

3.4. Spectroscopic Data of Compounds Isolated from Tecomella

Undulata

3.4.1. Spectroscopic data of kaempferol-3-O-β-D-[3,4di-p-E-

coumaroyl--L-rhamnosyl (1→6)] galactoside (142)

Yellow amorphous powder (21.0 mg); [α]26D: -43.0 (c 0.10, MeOH); UV

(MeOH) max nm: 258, 265, 313, 356; IR

(KBr) max cm-1: 3380, 1715, 1685, 1600,

1545, 1525; 1H NMR (CD3OD+CDCl3, 400

MHz): δ 8.06 (2H, d, J = 7.6 Hz, H-2´, 6´),

7.66 (1H, d, J = 16.0 Hz, H-7´´´´), 7.55

(1H, d, J = 16.0 Hz, H-7´´´´´), 7.36 (H, d, J

= 8.4 Hz, H-2´´´´, 6´´´´), 7.29 (H, d, J = 8.8

Hz, H-2´´´´´, 6´´´´´), 6.86 (H, d, J = 7.6 Hz,

H-3´, 5´), 6.76 (H, d, J = 8.4 Hz, H-3´´´´,

5´´´´), 6.73 (H, d, J = 8.8 Hz, H-3´´´´´,

5´´´´´), 6.42 (1H, d, J = 1.2 Hz, H-8), 6.19 (1H, d, J = 1.2 Hz, H-6), 6.32 (1H,

d, J = 16.0 Hz, H-8´´´´), 6.31 (1H, d, J = 16.0 Hz, H-8´´´´´), 5.30 (1H, d, J =

7.2 Hz, H-1´´), 4.81 (1H, dd, J = 9.0, 6.5 Hz, H-3´´´), 4.78 (1H, t, J = 9.5 Hz,

H-4´´´), 4.47 (1H, br s, H-1´´´), 3.60 (1H, m, H-5´´´), 3.59 (1H, dd, J = 10.4,

4.9 Hz, Ha-6´´), 3.58 (1H, d, J = 2.8 Hz, H-4´´), 3.52 (1H, t, J = 7.6 Hz, H-

2´´), 3.50 (1H, dd, J = 7.6, 2.8 Hz, H-3´´), 3.49 (1H, br d, J = 6.5 Hz, H-2´´´),

3.41 (1H, m, H-5´´), 3.31 (1H, dd, J = 10.4, 2.8 Hz, Hb-6´´), 0.92 (3H, d, J =

6.5 Hz, H-6´´´); 13C NMR (CD3OD+CDCl3, 100 MHz): δ 177.4 (C-4), 166.3 (C-

9´´´´), 165.4 (C-9´´´´´), 164.2 (C-7), 161.1 (C-5), 159.9 (C-4´), 159.7 (C-4 ´´´´),

O

O

HO

OH

OH

O

O

HO

OH

OH

O

O

HO

O

1

34a

8a

6

81'

3'

5'

1''

3''5''

O

OH

OO

OH142


82

158.8 (C-4´´´´´), 156.6 (C-2), 156.4 (C-8a), 144.5 (C-7´´´´), 143.3 (C-7´´´´´),

133.2 (C-3), 132.6 (C-2´´´´´, 6´´´´´), 130.9 (C-2´, 6´), 130.2 (2´´´´, 6´´´´), 125.4

(C-1´´´´´), 125.1 (C-1´´´´), 120.8 (C-1´), 115.7 (C-3´´´´´, 5´´´´´), 115.4 (C-8´´´´´),

115.0 (C-3´, 5´), 114.8 (3´´´´, 5´´´´), 114.3 (C-8´´´´), 103.8 (C-4a), 102.1 (C-

1´´), 100.1 (C-1´´´), 98.7 (C-6), 93.7 (C-8), 73.4 (C-4´´´), 73.3 (C-3´´´), 72.9

(C-5´´), 71.0 (C-2´´), 70.5 (C-3´´), 68.2 (C-4´´), 68.0 (C-2´´´), 66.0 (C-5´´´),

65.9 (C-6´´), 17.1 (C-6´´´); HR-FAB-MS m/z: 887.2312 (calcd. for C45H43O19,

887.2320).

3.4.2. Spectroscopic data of (6R,7Z)-9,10-dihydroxy-4,7-

megastigmadien-3-one (143)

Colorless oil (12.0 mg); []20D: + 113.2 (c 0.004, MeOH); UV (MeOH) max nm

(log Ɛ): 243.0 (0.81); CD (MeOH) nm (): 245.6

(+87.0); 1H NMR (CD3OD, 400 MHz): δ 5.92 (1H, br

s, H-4), 5.70 (1H, d, J = 6.3 Hz, H-7), 5.70 (1H, d, J

= 6.3 Hz, H-8), 4.16 (1H, m, H-9), 3.49 (2H, m, H-

10), 2.69 (1H, d, J = 7.8 Hz, H-6), 2.46 (1H, d, J = 16.8 Hz, Ha-2), 2.08 (1H,

d, J = 16.8 Hz, Hb-2), 1.96 (3H, d, J = 1.2 Hz, H-13), 1.03 (3H, s, H-11),

0.99 (3H, s, H-12); 13C NMR (CD3OD, 100 MHz): δ 201.3 (C-3), 165.2 (C-5),

135.0 (C-8), 129.0 (C-7), 125.4 (C-4), 73.0 (C-9), 66.6 (C-10), 56.1 (C-6),

47.4 (C-2), 36.4 (C-1), 27.3 (C-12), 26.6 (C-11), 23.0 (C-13). EI-MS m/z:

224.0; HR-EI-MS m/z: 224.1407 (calcd. for C13H20O3, 224.1412).

O

OH

OH

R

Z

1

3

5

67

8

9

10

1112

13

143


83

3.4.3. Spectroscopic data of (6E,9S)-9,10-dihydroxy-4,6-

megastigmadien-3-one (144)

Colorless oil (7.5 mg); []20D: +11.0 (c 0.00145, MeOH); UV (MeOH) max nm

(log Ɛ): 243.0 (0.49), 295.0 (0.15); 1H NMR (CD3OD,

400 MHz): δ 6.28 (1H, t, J = 6.6 Hz, H-7), 5.90 (1H,

br s, H-4), 2.35 (2H, s, H-2), 3.75 (1H, m, H-9), 3.54

(2H, d, J = 5.5 Hz H-10), 2.74 (1H, ddd, J = 15.0, 6.9,

4.5 Hz, Ha-8), 2.45 (1H, br t, J = 15.0 Hz, Hb-8), 2.13 (3H, d, J = 0.78 Hz,

H-13),1.32 (6H, s, H-11, H-12); 13C NMR (CD3OD, 100 MHz): δ 201.3 (C-3),

157.9 (C-6), 143.1 (C-5), 133.7 (C-4), 124.9 (C-7), 72.6 (C-9), 66.2 (C-10),

53.9 (C-2), 38.6 (C-1), 34.4 (C-8), 28.5 (C-11), 28.5 (C-12), 21.8 (C-13). HR-

EI-MS m/z: 224.1405 (calcd. for C13H20O3, 224.1412).

3.4.4. Spectroscopic data of (6R,9S)-9,10-dihydroxy-4-megastigmaen-3-one (145)

Colorless oil (16.0 mg); []20D: +58.0 (c 0.001, MeOH); UV (MeOH) λmax

(logε): 241 (3.81) nm; IR (film) ν max: 3410, 2955,

2875, 1645, 1440 cm−1; 1H NMR (CD3OD, 400

MHz): δ 5.80 (1H, br s, H-4), 3.54 (1H, m, H-9),

3.45 (2H, d, J = 5.2 Hz, H-10), 2.39 (d, J = 17.1 Hz,

Hb-2), 2.06 (d, J = 17.1 Hz, Ha-2), 2.06 (m, Ha-7), 2.01 (3H, s, H-13), 2.01

(1H, m, H-6), 1.61 (m, Ha-8), 1.47 (m, Hb-7), 1.43 (m, Hb-8), 1.10 (3H, s, H-

11), 1.03 (3H, s, H-12); 13C NMR (CD3OD, 100 MHz): δ 202.0 (C-3), 167.7

(C-5), 123.7 (C-4), 73.8 (C-9), 64.3 (C-10), 50.5 (C-6), 45.5 (C-2), 36.2 (C-1),

O

OH

12

7

10

OH

145

7

5

9

13

11

101

3

12

O

144

OH

OH

E


84

33.4 (C-8), 28.0 (C-12), 27.5 (C-7), 26.5 (C-11), 22.8 (C-13); EI-MS m/z:

226.0; HR-EI-MS m/z: 226.1671 (calcd. for C13H22O3, 226.1675).

3.4.5. Spectroscopic data of 3S,5R,6R,9S-tetrahydroxy megastigmane (146)

An amorphous powder (21.0 mg); []20D: -27.1 (c=0.002, MeOH); IR (film) ν

max: 3505, 1660 cm-1; 1H NMR (CD3OD, 400

MHz): δ 6.17 (1H, dd, J = 16.0, 2.0 Hz, H-7), 5.75

(1H, dd, J = 16.0, 6.4 Hz, H-8), 4.31 (1H, m, H-9),

4.09 (1H, m, H-3), 2.02, (Ha, dd, J = 12.4, 4.8 Hz,

H-4), 1.92, (Hb, dd, J = 12.4, 2.5 Hz, H-4), 1.67

(Ha, dd, J = 13.4, 4.6 Hz, H-2), 1.51 (Hb, dd, J = 13.4, 5.6.Hz, H-2), 1.26

(3H, d, J = 6.4 Hz, H-10), 1.24 (3H, s, H-11), 1.07 (3H, s, H-13), 0.86 (3H, s,

H-12); 13C NMR (CD3OD, 100 MHz): 130.0 (C-8), 129.3 (C-7), 80.1 (C-6),

78.5 (C-5), 70.2 (C-9), 66.3 (C-3), 46.1 (C-2), 45.2 (C-4), 40.0 (C-1), 27.3 (C-

13), 25.2 (C-12), 25.1 (C-10), 24.3 (C-11); HR-FAB-MS m/z: 243.1584 [M-

H]-1(Calcd for C13H23O4, 243.1596)

3.4.6. Spectroscopic data of lapachol (118)

Yellow needle like crystals (25.0 mg); melting point: 139-140˚C; UV λmax:

272, 280, 289 and 341 nm, IR (KBr) ν max: 3350,

1655, 1645, 1605 and 1545 cm-1; 1H NMR (CDCl3,

500 MHz): 8.12 (1H, d, J = 7.5 Hz, H-5), 8.05 (1H,

d, J = 7.5 Hz, H-8), 7.68 (1H, t, J = 7.5 Hz, H-6), 7.62

12

4

7

10

HO

OH

OH

OH11 12

13

146

O

O

OH

118

1

357

9

10

1'

2'

3'

4'

5'


85

(1H, t, J = 7.5 Hz, H-7), 5.21 (1H, t, J = 6.8 Hz, H-2′), 3.27 (2H, d, J = 6.8 H-

1′), 1.78 (3H, s, H-4′), 1.69 (3H, s, H-5′); 13C NMR (CDCl3, 125 MHz):

182.5 (C-4), 181.1 (C-1), 153.6 (C-2), 135.1 (C-3′), 131.3 (C-6), 130.1 (C-7),

129.6 (C-10), 129.3 (C-9), 127.2 (C-5), 126.3 (C-8), 121.0 (C-3), 119.2 (C-

2′), 26.7 (C-5′). 24.5 (C-1′), 18.1 (C-4′); EIMS: m/z 242 [M]+; HR-EI-MS: m/z

242.0939 [M]+(242.0943 calcd. for C15H14O3).

3.4.7. Spectroscopic data of 3'-OH-deoxyisolapachol (147)

Yellow powder (19.0 mg); UV λmax: 210, 251, 300 and 343 nm; IR (KBr):

3495, 1655, 1645, 1605 and 1545 cm-1; 1H NMR

(CDCl3, 500 MHz): 8.11 (1H, t, J = 8.0 Hz, H-5),

8.06 (1H, d, J = 8.0 Hz, H-8), 7.76 (2H, m, H-6),

7.35 (2H, m, H-7), 6.96 (1H, s, H-2), 6.81 (1H, d, J

= 16.4 H-2′), 6.76 (1H, d, J = 16.4 Hz, H-1′), 1.45 (6H, s, H-4′, 5′); 13C NMR

(CDCl3, 125 MHz): 184.3 (C-4), 183.6 (C-1), 148.2 (C-2′), 142.1 (C-3),

133.8 (C-7), 133.6 (C-10), 133.5 (C-6), 133.1 (C-9), 130.9 (C-2), 127.1 (C-5),

124.1 (C-8), 116.1 (C-1′), 72.0 (C-3′), 30.1 (C-4′, 5′); EI-MS: m/z 242 [M]+;

HR-EI-MS: m/z 242.0939 [M]+ (242.0943 calcd. for C15H14O3)

3.4.8. Spectroscopic data of quercetin 3′,4′-dimethyl ether (148)

Yellow amorphous powder (35.0 mg); U V λmax: 257, 287, 351 nm; IR (KBr):

3417, 1745, 1654, 1617, 1307 cm-1; 1H NMR (CD3OD, 400 MHz): 7.60

(1H, dd, J = 8.4, 2.0 Hz, H-6′), 7.32 (1H, d, J = 2.0 Hz, H-2′), 6.89 (1H, d, J

O

O

143

1

357

9

10

1'

2'

3'

4'

5'

OH


86

= 8.4 Hz, H-5′), 6.37 (1H, d, J = 2.0 Hz, H-8), 6.17 (1H, d, J = 2.0 Hz, H-6),

3.84 (3H, s, 4′-OMe), 3.80 (3H, s, 3′-OMe); 13C

NMR (CD3OD, 100 MHz): 179.5 (C-4), 166.1

(C-7), 163.0 (C-5), 159.0 (C-2), 158.8 (C-9),

149.1 (C-4′), 148.4 (C-3′), 133.3 (C-3), 123.1

(C-1′), 122.9 (C-6′), 115.9 (C-5′), 109.8 (C- 2′), 105.0 (C-10), 99.8 (C-6), 95.0

(C-8), 56.0 (4′-OMe), 55.3 (3′-OMe); EI-MS: m/z 330 [M]+; HR-EI-MS:

330.0735 (330.0740 calcd. for C17H14O7).

3.4.9. Spectroscopic data of 3,5,3′,4′-tetrahydroxy-6,7-dimethoxy

flavone (149)

Yellow powder (22.0 mg); UV λmax: 215, 271, 278, 345, 351 nm; IR (KBr):

3452, 1671, 1656, 1502, 1462, 1314 cm-1; 1H

NMR (CD3OD, 400 MHz): 7.44 (1H, dd, J =

1.8, 8.4 Hz, H-6′), 7.32 (1H, d, J = 1.8 Hz, H-

2′), 7.08 (1H, d, J = 8.4 Hz, H-5′), 6.54 (1H, s,

H-8), 3.83 (3H, s, 7-OMe), 3.81 (3H, s, 6-OMe); 13C NMR (CD3OD, 100

MHz): 178.0 (C-4), 159.5 (C-7), 158.9 (C-2), 156.3 (C-9), 153.5 (C-5),

148.5 (C-4′), 147.4 (C-3′), 133.6 (C-6), 132.8 (C-3), 129.0 (C-6′), 121.0 (C-

1′), 115.5 (C-5′), 111.9 (C-2′), 104.6 (C-10), 90.5 (C-8), 62.7 (6-OCH3), 55.6

(7-OCH3); EI-MS: m/z 346 [M]+; HR-EI-MS: m/z 346.0681 [M]+(346.0689

calcd. for C17H14O8).

7

3

5

8

9

10

13´1´

5´

OMeO

MeO

OH O

OH

OH

OH

149

7

3

5

8

9

10

13´1´

5´

OHO

OH O

OMe

OMe

OH

148


87

3.4.10. Spectroscopic data of quercetin-3-galactoside (150)

Yellow amorphous powder (25.0 mg); UV λmax: 254, 276, 320 and 335 nm;

IR (KBr): 3235, 1660, 2915, 1607, 1515 cm-1;

1H NMR (CD3OD, 400 MHz): 7.68 (1H, d, J =

2.0 Hz, H-2′), 7.54 (1H, dd, J = 2.0, 8.4 Hz, H-

6′), 6.84 (1H, d, J = 8.4 Hz, H-5′), 6.34 (1H, d,

J = 1.8 Hz, H-8), 6.16 (1H, d, J = 1.8 Hz, H-6),

5.26 (1H, d, J = 7.2 Hz, H-1″), 3.69 (Ha, dd, J

=2.6, 12.0 Hz, H-6″), 3.58 (Hb, dd, J = 5.6, 12.0 Hz, H-6″), 3.51 (1H, dd, J

=2.8, 8.0 Hz, H-3″), 3.48 (1H, t, J = 8.4 Hz, H-2″), 3.30 (1H, d, J =2.8 Hz, H-

4″), 3.21 (1H, m, H-5″); 13C NMR (CD3OD, 100 MHz): 178.4 (C-4), 166.0 (C-

7), 162.5 (C-5), 158.3 (C-9), 158.2 (C-2), 149.8 (C-4′), 147.6 (C-3′), 133.5 (C-

3), 123.1 (C-6′), 121.0 (C-1′), 116.1 (C-2′), 114.8 (C-5′), 104.0 (C-10), 103.5

(C-1″), 99.8 (C-6), 94.1 (C-8), 74.1 (C-5″), 72.5 (C-3″), 71.6 (C-2″), 69.0 (C-

4″), 62.6 (C-6″); FAB-MS: m/z 465.0 [M+H]+; HR-FAB-MS: 465.3869 [M+H]+

(465.3842 calcd. for (C21H20O12).

3.4.11. Spectroscopic data of ferulic acid (104)

White crystalline solid (37.0 mg); melting point: 169-171˚C; UV λmax (EtOAc)

nm: 235, 290, 326; IR (KBr): max: 3417, 2910,

1715, 1625, 1540, 1460, 1265 cm-1; 1H NMR

(CDCl3+CD3OD, 400 MHz): δ 7.52 (1H, d, J = 15.6

Hz, H-7), 6.96 (1H, d, J = 1.8 Hz, H-2), 6.86 (1H, dd, J = 8.0, 1.8 Hz, H-6),

2

6

7

89 OH

O

HO

MeO

104

1

4

1''

3''6''

2

36

89

10

1

O

OH

OH

OOH

HO

O

HOOH

OH

O

HO

150

3´1´

5´


88

6.79 (1H, d, J = 8.0 Hz, H-5), 6.20 (1H, d, J = 15.6 Hz, H-8), 3.83, (3H, s,

OMe-3); 13C NMR (CDCl3+CD3OD, 100 MHz): δ 172.0 (C-9), 146.0 (C-3),

145.5 (C-4), 144.1 (C-7), 124.6 (C-1), 120.2 (C-6), 115.0 (C-8), 114.9 (C-5),

110.6 (C-2), 61.2 (3-OMe); EI-MS: m/z 194.1 [M]+; HR-EI-MS: m/z

194.0573 (194.0579 calcd. for, C10H10O4).

3.4.12. Spectroscopic data of hexadecyl ferulate (151)

White crystalline powder (20.0 mg); UV λmax (EtOAc) nm: 234, 290, 325; IR

(KBr) max: 3415, 2985, 1715, 1635,

1550, 1462, 1260 cm-1; 1H NMR

(CDCl3+CD3OD, 400 MHz): δ 7.50

(1H, d, J = 16.0 Hz, H-7), 6.97 (1H, d,

J = 2.0 Hz, H-2), 6.93 (1H, dd, J = 7.6, 2.0 Hz, H-6), 6.83 (1H, d, J = 7.6 Hz,

H-5), 6.18 (1H, d, J = 16.0 Hz, H-8), 4.22 (2H, t, J = 7.2 Hz, H-1´), 3.84,

(3H, s, -OMe), 1.69 (2H, m, H-2´), 1.23-1.54 (26H, br s, H-3´-15´), 0.81 (3H,

t, J = 6.5 Hz, H-16´); 13C NMR (CDCl3+CD3OD, 100 MHz): δ 169.1 (C-9),

146.2 (C-4), 145.2 (C-7), 145.1 (C-3), 125.1 (C-1), 121.2 (C-6), 116.0 (C-5),

115.1 (C-8), 110.1 (C-2), 64.1 (C-1´), 60.3 (3-OMe), 30.5-29.6 (C-3´-15´),

29.8 (C-2´) and 14.5 (C-16´); EI-MS: m/z 418.4 [M]+; HR-EI-MS: m/z

418.3080 (418.3083 calcd. for C26H42O4).

O

O

HO

OMe

1'

2'1

3

4

6 7

9

151

1116'


89

3.4.13. Spectroscopic Data of Betulinic Acid (133)

White solid (32.0 mg); [α]25D: +37˚ (c 0.2, CHCl3 ); IR (KBr) max : 3405, 2420,

1710 and 1685 cm-1; 1H NMR (CDCl3, 400

MHz): δ 5.01 (Hb, br s, H-29), 4.76 (Ha, br s,

H-29), 3.33 (1H, m, H-3), 2.32 (1H,.m, H-19),

1.63 (3H, s, H-30), 0.97 (3H, s, H-27), 0.95

(3H, s, H-26), 0.93 (3H, s, H-23), 0.81(3H, s,

H-25), 0.78 (3H, s, H-24); 13C NMR (CDCl3,

100 MHz): δ 178.3 (C-28), 151.2 (C-20), 108.7 (C-29), 79.0 (C-3), 54.6 (C-5),

50.6 (C-9), 49.9 (C-19), 49.0 (C-17), 47.6 (C-18), 43.1 (C-14), 40.9 (C-8),

39.2 (C-4), 39.1 (C-1), 38.0 (C-10), 38.0 (C-13), 37.6 (C-22), 35.0 (C-7), 33.4

(C-16), 31.0 (C-21), 29.8 (C-15), 27.9 (C-23), 26.8 (C-2), 24.6 (C-12), 21.3

(C-11), 21.1 (C-30), 17.9 (C-6), 17.1 (C-26), 16.8 (C-25), 15.5 (C-27), 15.5

(C-24); EI-MS: m/z 456); HR-EI-MS: m/z 426.3858 [M]+ (calcd. 426.3862

for C30H48O3).

3.4.14. Spectroscopic data of β-sitosterol (134)

White crystalline solid (112.0 mg); MP: 135 Cº; IR(KBr): 3450 (O-H), 3050

(C-H), 1645, 816 (C=C) cm-1; 1H NMR

(CDCl3, 400 MHz): 5.12 (1H, m, H-6),

3.38 (1H, m, H-3), 1.02 (3H, s, C-19), 0.95

(3H, d, J = 6.2 Hz, H-21), 0.95 (3H, d, J =

6.5 Hz, H-26), 0.86 (3H, t, J = 7.0 Hz, H-

HO

OH

O

133

29

30

20

28

2324

2526

27

17

21

29

25

1

3

57

9

11

14

20

23

28

HO

HH

134

10


90

29), 0.82 (3H, d, J = 6.5 Hz, H-27), 0.68 (3H, s, H-18); 13C NMR (CDCl3, 100

MHz): 141.9 (C-5), 122.9 (C-6), 76.9 (C-3), 51.2 (C-17), 45.6 (C-9), 45.4

(C-24), 45.1 (C-14), 39.6 (C-4), 36.8 (C-13), 37.3 (C-1), 37.0 (C-10), 36.9 (C-

8), 36.3 (C-20), 34.0 (C-22), 32.1 (C-7), 31.8 (C-2), 30.4 (C-16), 29.3 (C-23),

28.8 (C-15), 26.2 (C-25), 23.7 (C-12), 23.1 (C-28), 20.3 (C-11), 19.8 (C-27),

19.4 (C-19), 19.1 (C-21), 18.8 (C-26), 13.9 (C-29) and 13.9 (C-18); EI-MS:

m/z 414, 399, 396, 381, 329, 275, 273 and 255; HR-EI-MS: m/z

414.38459 (calcd. for C29H50O, 414.38617).

3.4.15. Spectroscopic data of β-sitosterol-3-O-β-D-glucopyranoside (152)

Colorless amorphous solid (76.0 mg); M.p: 283-286 Cº; []D20: +62.4o (c =

0.2, CHCl3); IR (KBr)

νmax: 3460, 1655, 1618,

1021 and 800 (C=C) cm-

1; 1H NMR (CDCl3, 400

MHz): δ 5.30 (1H, m, H-

6), 4.95 (1H, d, J = 7.0

Hz, H-1′), 4.28 (Ha, dd, J = 11.2, 2.1 Hz, H-6′), 4.14 (Hb, dd, J = 11.2, 4.9

Hz, H-6′), 4.08 (1H, m, H-2′), 4.02 (2H, m, H-3′, 4′), 3.98 (1H, m, H-3), 3.93

(1H, m, H-5′), 0.96 (3H, s, H-19), 0.92 (3H, d, J = 6.5 Hz, H-21), 0.88 (3H, d,

J = 6.7 Hz, H-26), 0.86 (3H, d, J = 6.5 Hz, H-27), 0.83 (3H, t, J = 6.6 Hz, H-

29) and 0.69 (3H, s, H-18); 13C NMR (CDCl3, 100 MHz): δ 141.1 (C-5), 121.7

(C-6), 103.0 (C-1′), 79.3 (C-3), 76.4 (C-3′), 75.6 (C-5′), 73.6 (C-2′), 70.1 (C-

O

152

OHO

HOOH

OH1

3 5 7

9

11 13

15

17

1824

19

20

21

28

29

25

26

27

1''3''

5''6''


91

4′), 62.4 (C-6′), 56.0 (C-17), 46.9 (C-9), 45.7 (C-24), 45.2 (C-14), 42.1 (C-13),

38.3 (C-12), 38.2 (C-4), 37.3 (C-1), 36.5 (C-22), 36.3 (C-10), 35.9 (C-20),

31.8 (C-8), 31.6 (C-7), 29.4 (C-2), 29.2 (C-25), 28.2 (C-16), 26.4 (C-23), 24.2

(C-15), 23.0 (C-28), 21.5 (C-11), 19.4 (C-26), 19.4 (C-19), 19.0 (C-27), 18.8

(C-21), 12.0 (C-18) and 11.6 (C-29); m/z 576.4386 corresponding to

molecular formula C35H60O6 (calcd. for C35H60O6, 576.4336).

3.5. Acid Hydrolysis of Compound 142

A solution of compound (142) (8.0 mg) in MeOH (5.0 ml) containing 1N

HCl (4.0 ml) was refluxed for 4 hrs, concentrated under reduced pressure,

and diluted with H2O (8.0 ml). The aglycones were extracted with EtOAc (3

× 15 ml). The aqueous phase was concentrated under reduced pressure

and purified on preparative thin layer chromatography using solvent

system (EtOAc-MeOH-H2O-HOAc; 4:2:2:2) and identified as D-galactose and

L-rhamnose by the sign of their optical rotation ([α]D20 +78.5 ) and ([α]D20

+7.7 ), respectively. These sugars were also confirmed by the retention time

of their TMS ether with the standards and D-galactose (retention time of -

anomer, 3.0 and β-anomer, 5.2 min).

3.6. Anti-bacterial Assay

The antibacterial activity was performed in sterile 96-wells

microplates under aseptic environments. The method is based on the

principle that microbial cell number increases as the microbial growth


92

proceeds in the log phase of growth which results in increased absorbance

of broth medium (Kaspady et al., 2009; Patel et al., 2009).

The microorganisms were kindly provided by the Department of

Biological and Biomedical Sciences, Aga Khan University, Karachi,

Pakistan, and were maintained on stock culture agar. The test sample

prepared in DMSO was pipetted into wells (20.0 µg/well). After suitable

dilution with fresh nutrient broth, the overnight maintained fresh bacterial

culture was poured into wells (180 µl). The initial absorbance of the culture

was maintained between 0.12-0.19 at 540 nm. The total volume in each

well was kept to 200.0 µl. The incubation was done at 37 0C for 16-24

hours with lid on the microplate. The absorbance was measured at 540 nm

using Synergy HT BioTek® USA microplate reader, before and after

incubation and the difference was noted as an index of bacterial growth.

Ciprofloxacin, gentamycin and ampicillin were taken as standard.

Minimum inhibitory concentration (MIC) was measured with suitable

dilutions (5.0-30.0 µg/well) and results were calculated using EZ-Fit5

Perrella Scientific Inc. Amherst USA software, and data expressed as IC50.

Chapter 4 Introduction of Endophytes

93

CHAPTER 4

ENDOPHYTIC FUNGI AS POTENTIAL SOURCE OF

BIOACTIVE ORGANIC COMPOUNDS WITH

FASCINATING STRUTURES


94

4.1. Potential of Microorganisms with respect to Endophytes

Among the natural sources, the potential of microorganisms in drug

discovery is recently exploited. Most of the drugs especially the antibiotics

currently in market have been reported from microorganisms. After the

discovery by Nobel laureate Alexander Fleming in 1928 and the clinical use

of penicillin in 1940s opened a new area of drug discovery, followed by the

isolation of a large number of antibiotics from microbes. Latter, the

derivatization of many antibiotics, which were discovered until the early

1970s, established a new generations of clinically useful antibiotics (Agusta

et al., 2006; Overbye and Barrett, 2005). The first systematic study of

fungal metabolites was initiated soon after World War I by Harold Raistrick,

who in the course of the following four decades made a seminal

contribution to the recognition of fungi as a major source of natural

products (Petrini, 1986; Saleem et al., 2007).

4.2. What are Endophytes?

The literal meaning of the word endophyte is “in the plant”, which is

derived as (endo, Gr. = within, phyton = plant). Endophytes most often are

fungi and bacteria that live within plant cells without creating any apparent

disease. More than 100 year of research suggests that most, if not all,

plants in natural ecosystems are symbiotic with mycorrhizal fungi and/or

fungal endophytes (Petrini, 1986). These fungal symbionts play a vital role

in plant ecology, fitness, and evolution (Schulz et al., 2006). However,


95

unlike mycorrhizal fungi that colonize plant roots and grow into the

rhizosphere, endophytes live entirely within plant tissues and grow within

nearly all parts of plant (Carroll, 1988; Sherwood and Carroll, 1974; Stone

et al., 2004). According to several reports, among 300,000 plant species

existing on the earth, each individual plant hosts one or more endophytes

(Ryan et al., 2008; Strobel and Daisy, 2003; Strobel et al., 2004a; Strobel,

2006; Zhang et al., 2006).

The discovery of anticancer compound, paclitaxel (taxol) (7) from the

endophytic fungus Pestalotiopsis microspora has increased the interest of

researchers in investigating the endophytes for their bioactive metabolites

(Bacon and White, 2000). Till now, large number of organic compounds

have been reported from few endophytic fungal species, and these

compounds may have significant impact to the life of their host plants, e.g.

they may provide protection and survival value to the plants as

antimicrobials, antivirals and insecticidals (Miller et al., 2008; Schulz et al.,

2002). Therefore, these compounds may also offer their services to the

ecological community by serving as plant growth regulators (Tudzynski and

Sharon, 2002).

Endophytic fungi use multiple metabolic pathways to synthesize

countless natural chemicals of diverse structural classes (Strobel et al.,

2004b; Zhang et al., 2006) and have considerable potential as sources of

novel natural products with agrochemical, pharmaceutical and industrial


96

potential (Gunatilaka, 2006; Strobel, 2002; Strobel, 2006; Wiyakrutta et al.,

2004). Some of the endophytes can be pathogenic to the host. After the

isolation of taxol as a potent microtubule stabilizer (Bacon and White,

2000; Stierle et al., 1993), researchers have reported the identification of

several other important secondary metabolites, for example, camptothecin

(13), vincristine (8) and podophyllotoxin (16) or their analogues (Kharwar et

al., 2011).

Another example is the pestalone (153), a chlorinated benzophenone,

an antibiotic which was produced by a co-cultured endophytic algal marine

fungus/unicellular marine bacterium, strain CNJ-328. Pestalone exhibits

moderate in vitro cytotoxicity and shows potent antibiotic activity against

methicillin-resistant Staphylococcus aureus and Enterococcus faecium with

MIC values of 37.0 and 78.0 ng/ml, respectively (Cueto et al., 2001).

Pestalachloride (154), a chlorinated benzophenone derivative was

discovered in culture of an isolate of the plant endophytic fungus

Pestalotiopsis adusta. Compound 154 was obtained as a mixture of two

inseparable atropisomers which displayed significant antifungal activities

against plant pathogens (Li et al., 2008a; Slavov et al., 2010).


97

CHO

OHHO

O

H3CO

Cl

CH3

Cl

OH

OHHO

H3CO

Cl

CH3

Cl

OHNH

O

H

153 154

Epoxphomalins, A (155) and B (156) were isolated from the marine-

derived fungus Paraconiothyrium sp. and they are two new prenylated

polyketides with unusual structural features (Mohamed et al., 2010).

Compound 155 showed superior cytotoxicity at nanomolar concentrations

toward 12 of a panel of 36 human tumor cell lines. In comparative

analyses, the observed cytotoxic selectivity pattern of epoxyphomalin A

(155) did not correlate with those of reference anticancer agents with

known mechanisms of action. Their mechanism of action was studied

through the potent inhibition of the 20S proteasome (Mohamed et al.,

2010).

HO

OH

R

O

O

H

R=CH2OH 155

R=CH3 156


98

The most prominent feature of endophytic fungi is that these organisms

produce secondary metabolites of nearly all classes. For example an

aliphatic dicarboxylic acid, 2-hexyl-3-methylbutanedioic acid (157) was

isolated from the endophytic fungus Xylaria sp. which showed antifungal

activity against Cladosporium cladosporioides and Cladosporium

sphaerospermum with a limit of 25.0 and 10.0 μg/ml, respectively (in

bioautographic assay) (Cafeu et al., 2005). The tricarboxylated alkylsulfate

(158) is another carboxylic acid, isolated from endophytic fungus residing

in the leaves of Berberis oregana, is a potent (IC50 = 14.0 nM) and specific

inhibitor of FPTase (farnesyl-protein transferase) (Jayasuriya et al., 1996).

COOH

H3C

COOH

CH3HO

O COOH

COOH

OSO3H

157 158

The acetonitrile soluble fraction of the Penicillium sp. 1 cultivated in

potato-dextrose-broth afforded two different compounds, cyclo-(L-Pro-L-Val)

(159) and uracil (160). Compounds 159 and 160 showed

acetylcholinesterase (AChE) inhibitory activity with a detection limit of 10.0

and 60.0 µg respectively (Oliveira et al., 2009). Penicidones, A-C (161-163)

were isolated from the culture of Penicillium sp. IFB-E022, separated from

the stem tissues of Quercus variabilis. Penicidones, A-C (161-163) were the

first group of natural products possessing a penicidone framework which

exhibited moderate cytotoxicity against four cancer cell lines SW1116,


99

K562, KB and Hela, indicating that 161-163 were cytotoxic with their IC50

values between 21.1 and 90.8 µM (Ge et al., 2008).

NH

NH

O

O

N

NH

O

O

CH3

CH3

NH

O

CH3

O

O

RO

OCH3OCH3 N

H

OO OCH3

H3CO

O

CH3

159 160 161 R = H

162 R = CH3

163

Pyrrolo [1,4] benzodiazepines named, limazepine B1 (164) and B2 (165),

C-E (166-168) were isolated from the culture broth of Micrococcus sp.

strain ICBB 8177. Limazepines, B1/B2 (164, 165), C (166) and E (168)

were active against the Gram-positive bacterium Staphylococcus aureus and

the Gram-negative bacterium Escherichia coli. Limazepine D (167) was also

active against Staphylococcus aureus but found inactive against Escherichia

coli. Only the limazepines B1/B2 (164, 165) mixture and D (167) were

active against Pseudomonas aeruginosa (Fotso et al., 2009). Terpeptin A

(169) and B (170) and enamide derivative (171) were identified from a

strain of Aspergillus sp. (w-6), an endophytic fungus associated with

Acanthus ilicifolius. Compounds 169-171 showed moderate cytotoxicities

against A-549 cell lines with IC50 values of 23.3, 28.0, and 15.0 µM,

respectively (Lin et al., 2008).


100

N

HN

O

OH

MeOH

N

N

O

MeO

N

N

O

OH

MeOH

164 166

NH

O

NH

CH3

H3CO

N

O

NH

O

H3C

H3C

CH3

H3C CH3

CH3

169

170

NH

O

NH

CH3

H3CO

N

O

NH

O

H3C

H3C

CH3

H3C CH3

CH3

NH

NH

O

N

O

NH

O

H3C

H3C

CH3

H3C CH3

CH3

171

CH3

CH3

OH

N

HN

O

OH

MeOH

165

OH

167

N

N

O

MeO

168

H

OH

Pyrrospirones, A and B (172, 173) and pyrrocidine, A (174) were

isolated from unpolished rice cultures of the endophytic fungus Neonectria

ramulariae Wollenw KS-246. Compounds 172-174 showed cytotoxicity

against HL-60 (human promyelocytic leukemia), K562 (human chronic

myelogenous leukemia) and LNCaP (human prostate carcinoma) cell lines

with IC50 values ranging from 0.12 to 20.24 µM. pyrrocidine A (174) was

found to be more cytotoxic than the others against HL-60 (IC50:0.12 µM)

(Shiono et al., 2008).Peniprequinolone (175) and gliovictin (176) were

produced by an endophytic fungi Penicillium janczewskii, isolated from

Chilean gymnosperm Prumnopitys andina. The cytotoxicity (IC50) of

peniprequinolone (175) towards fibroblasts cell line derived from human


101

lung was 116.0 µM while the compound 175 presented an IC50 of 89.0 µM

against AGS cells (gastric adenocarcinoma cells). (Schmeda-Hirschmann et

al., 2005).

NHHCH3

H3C

H

H

O

H

H3C

H3CR2

R1

O

OHO

H

NHHCH3

H3C

O

H

H3C

H3C

O

OHO

H

CH2

H

NH

H3C

CH3 OH

OCH3

O

OCH3

OH

N

N

O

O

CH3

SOH

CH3

H3C

S

H3C

172 R1 = H, R2 = OH

173 R1 = OH, R2 = H

175 176

174

Pyrrole alkaloids, N-[4-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-butyl]-

acetamide (177) and N-[5-(2-formyl-5-hydroxymethyl-pyrrol-1-yl)-pentyl]-

acetamide (178) were isolated from an endophytic ascomycetous fungus,

Fusarium incarnatum (HKI00504) extracted from the mangrove plant

Aegiceras corniculatum. Both the compounds (177, 178) showed weak

activity against tumor cell lines HeLa, K-562 and L-929 (IC50 ranges from

6.3 to 11.6 µg/ml) and were almost inactive against Bacillus subtilis,

Staphylococcus aureus, Escherichia coli, and Candida albicans (Li et al.,

2008b). Cytochalasin H (179) was separated from the mangrove endophytic


102

fungus Phomopsis sp. (ZZF08) obtained from the South China Sea coast.

Compound 179 exhibited strong cytotoxicity toward KB cells and KBv200

cells with IC50 less than 1.25 µg/ml. (Tao et al., 2008).

OHCOH

NH

CH3

O

OHCOH

HN

H3C

O

177 178

HN

H3C

O

OH

H

O

H3C OH

CH3

O

OH3C

H

179

More cytochalasins, Z17 (180) and rosellichalasin (181) were isolated

from Aspergillus flavipes, an endophytic fungus associated with Acanthus

ilicifolius. Cytochalasin Z17 (180) and rosellichalasin (181) showed cytotoxic

activities against A-549 cell lines with IC50 values of 5.6 and 7.9 µM,

respectively (Lin et al., 2009).

HN

H

CH3

CH3

OO

O O

HN

H3C

HCH3

CH3

OO

O O

OH3C

180 181

OHH3CCH3

An anthraquinone, (+)-3,3',7,7',8,8'-hexahydroxy-5,5'-dimethyl-

bisanthraquinone (182) was isolated from the culture media of mangrove

endophytic fungus # 2240 collected from a mangrove Castaniopsic Fissa

from South China Sea coast. The DNA hTopo I isomerase inhibition test

showed that 182 possessed strong activity (Nia et al., 2009). A novel


103

metabolite xylopyridine, A (183) was isolated from mangrove endophytic

fungus Xylaria sp. (#2508) collected from the South China Sea coast. The

compound expressed strong DNA-binding capacity, thus it is exploitable as

strong DNA-binder (Xu et al., 2009a). Bisanthraquinones named (+)-

epicytoskyrin (184) and (+)-1,1ʹ-bislunatin (185) were produced by the

endophytic fungus from a tea plant, which is a species closely related to

Diaporthe phaseolorum strain sw-93-13. Both the compounds showed

moderate cytotoxic activity against KB cells at IC50 value 0.5 µg/ml and 3.5

µg/ml, respectively (Agusta et al., 2006). Many important compounds such

as 7-methoxy-2-methyl-3,4,5-trihydroxyanthraquinone (186), physcion

(187), deoxybostrycin (188), altersolanol B (189), dactylariol (190) and

pleospdione (191) were isolated from the culture of Pleospora sp. IFB-E006,

an endophytic fungus residing in the normal stem of Imperata cylindrical.

The Compounds 188-190 exhibited significant cytotoxic activity against

human colon cancer (SW1116; IC50 5.8, 3.3, 0.8 µg/ml) and leukemia

(K562, IC50 3.1, 3.3, 1.3 µg/ml) (Ge et al., 2005).


104

O

O

H3C

OH OH

OH

CH3

OHO

OOH

OH

OH

HO

O

O

OCH3

OH

OH

OCH3

O

O

HO

OHOH

O

O

OH

H3CO

OH

OCH3

O

O

HO

OH

OH

CH3

R1

R2O

O

H3CO

OH OH

O

O

H3CO

OH

R1 R2

OH

CH3

OH

OHO

O

H3CO

OH

OH

CH3

OHOH

OH

182184

185

186 R1, R2 = OH

187 R1 = H, R2 =

OH188 R1 = OH, R2 = H

189 R1, R2 = H

190 R1 = H, R2 = OH

191

O

N

O

N

183

Microsphaeropsones, A and B (192, 193) with a unique oxepino [2,3-b]

chromen-6-one (ring-enlarged xanthone) skeleton were isolated from the

endophytic fungus Microsphaeropsis sp. Both the compounds exhibited

antibacterial, fungicidal and algicidal properties (Krohn et al., 2009).

Pestaloficiols, J and L (194, 195), isoprenylated chromone derivatives

including one heterodimer, have been isolated from the plant endophytic

fungus Pestalotiopsis fici. Pestaloficiols J (194) showed an inhibitory effect

on HIV-1 replication in C8166 cells, with an EC50 value of 8.0 µM (the CC50

value is greater than 100 µM, the positive control indinavir sulfate showed

an EC50 value of 8.2 nM), whereas pestaloficiol L (195) displayed cytotoxic

activity against the HeLa and MCF7 cells with IC50 values of 8.7 and 17.4

µM, respectively (the positive control fluoro uracil showed IC50 values of

10.0 and 15.0 µM, respectively) (Liu et al., 2009).


105

O O

OH O

CH3

R

OOCH3

O

O

H3C

H3C

H3C CH3

OH

OH3C

H3C

H3C CH3

OH

O

H3C

OH COOCH3

OCH3

OH OH

192 R = OH193 R = H

194195

OH

Alternariol-4-methyl-10-acethyl ester 2240B (196), together with

alternariol (197), alternariol-4,10-dimethyl ether (198) and alternariol-4-

methyl ether (199) were isolated from the ethyl acetate extract of the

mangrove endophytic fungus. The anticancer tests showed that 197 and

199 had strong activities against KB and KBv200 cells with IC50 values of

3.17, 3.12, and 4.82, 4.94 µg/ml, while compounds 196, 198 exhibited

weak activities against the KB and KBv200 cell lines with IC50 values >50

µg/ml (Tan et al., 2008). Two compounds identified as alternariol

monomethyl ether (200) and (4S)-α,β-dehydrocurvularin (201) were

isolated from Alternaria tenuissima and Alternaria sp. extracted from the

leaves of Acacia mangium, displaying the activity with IC50 value 51.0 and

10.6 µg/ml, respectively (Jeon et al., 2010). Monocerin (202) and

isocoumarin derivative (203) were isolated from Microdochium bolleyi, an

endophytic fungus associated with a plant Fagonia cretica, of the semiarid

coastal regions of Gomera. The Compounds 202 and 203 showed good

antifungal, antibacterial and antialgal activities against Microbotryum


106

violaceum, Escherichia coli, Bacillus megaterium, and Chlorella fusca (Zhang

et al., 2008).

O

O

OCH3

OH

H3C CH3

O

HO

OH O

O OCH3

O

CH3

R2

R1

OH

O

O

O

O

H

H

CH3

R

OH

H3CO

H3CO

196 R1 = OCH3, R2 = Ac

197 R1, R2 = OH

198 R1, R2 = OCH3

199 R1 = OCH3, R2 = OH

202 R = H203 R = -OH200 201

Three depsidones (204-206) were isolated from the endophytic fungus

BCC 8616. Compound 204 exhibited weak cytotoxic activity against KB

and BC cell lines with IC50 values 6.5 and 4.1 µg/ml, respectively whereas,

compounds 205 and 206 were inactive against the KB and BC cell lines

(IC50 > 5.0 µg/ml) (Pittayakhajonwut et al., 2006). Phomosines, A-C (207-

209) three biaryl ethers have been isolated from the endophytic fungus

Phomopsis sp. These compounds displayed moderate antibacterial,

antifungal and algicidal activities in vitro (Krohn et al., 1995).


107

O

O

O

CH3

OHHO

CHO

CH3H3C

CH3

CH3

O

O

O

CH3

OHHO

CH3H3C

CH3

CH3

RO

O

OH

HO CH3

H O

OH

CH3

H3C

OCH3O

O

OH

HO CH3

OH

CH3

H3C

OCH3O

OCH3

O

OH

HO CH3

OH

CH3OCH3O

OH

204 205 R = H

206 R = CH3

207

208 209

Ergoflavin (210), a pigment from an endophytic fungus, growing on the

leaves of an Indian medicinal plant Mimosops elengi (bakul), inhibited

significantly human TNF-α and IL-6 with IC50 values of 1.9 and 1.2 µM. It

also induced cytotoxicity in ACHN, H460, Panc1, HCT116 and Calu1 cancer

cell lines with IC50 values 1.2, 4.0, 2.4, 8.0 and 1.5 µM, respectively

(Deshmukh et al., 2009).

O

OHO

H3C

OH

OH

O

OH

OH

CH3O

O

O

OO

OH 210

Five antileishmanial compounds, Preussomerin EG1 (211),

palmarumycin CP2 (212), palmarumycin CP17 (213), palmarumycin CP18

(214) and CJ-12371 (215) were isolated from the mycelium of the fungus

Edenia sp. These metabolites displayed significant inhibition of the growth


108

of Leishmania donovani in the amastigote form with IC50 values 0.12, 3.93,

1.34, 0.62, and 8.40 µM, respectively. They also showed weak cytotoxicity

to Vero cells (IC50 of 9.0, 162.0, 174.0, 152.0 and 150.0 µM, respectively)

(Martinez-Luis et al., 2008). Phomoeuphorbins A and C (216, 217) were

purified from the cultures of Phomopsis euphorbiae, an endophytic fungus

isolated from Trewia nudiflora. Both the isolates exerted minimal

cytotoxicity against C8166 cells (CC50 > 200.0 µg/ml) and each showed

anti-HIV activity with EC50 value 79.0 and 71.0 µg/ml, respectively (Yu et

al., 2008).

OHO

O

O

O O

O

R

OH

O O

OH

O

O

O O

OH OH

O O

O

COOH

O

HO

HO

H3CO

CH3

O

HO

HO

H3C

OH

OH

211

212 R = H

213 R = OH214 215

216217

Eutypellin A (218), a -lactone, was isolated from the endophytic fungus

Eutypella sp. BCC 13199. Compound 218 exhibited weak cytotoxic

activities against cell lines NCI-H187, MCF-7, KB and Vero with IC50 values

12.0, 84.0, 38.0 and 88.0 µM, respectively (Isaka et al., 2009). Bicyclic

fusidilactone D (219) and E (220) along with fusidilactone B (221) were


109

separated from the fungal endophyte Fusidium sp. These metabolites

exhibited moderate antifungal, antibacterial and antialgal activities (Qin et

al., 2009).

Macrolides; lasiodiplodin (222), de-O-methyllasiodiplodin (223) and 5-

hydroxy-de-O-methyllasiodiplodin (224) are known as the constituents of

the mycelium extracts of a brown algal endophytic fungus (No. ZZF36)

obtained from the South China Sea. Compound de-O-methyllasiodiplodin

(223) exhibited the inhibiting activities to Staphylococcus aureus with MIC

value 6.25 µg/ml. Lasiodiplodin (222) inhibited the in vitro growth of S.

aureus, Bacillus subtilis and Fusarium oxysporum with MIC value 25.0,

50.0 and 100.0 µg/ml, respectively, whereas compound 224 inhibited the

growth of S. aureus at MIC value 100.0 µg/ml (Yang et al., 2006).

O

O

HO

H

OOH

O

O

O

CH3 OH

OH

H3C OH

H3C

H3C

O

O

O

CH3 OH

OH

H3C

H3C

CH3

H

H

O

O

O

CH3 OH

OH

H3C OH

H3C

H3C

H

H

218

219220

221

O

O CH3OR1

HO R2

222 R1 = CH3, R2 = H

223 R1, R2 = H

224 R1 = H, R2 = OH

Cyclic depsipeptide 1962A (225) was isolated from the fermentation broth


110

of the mangrove endophytic fungus obtained from leaf of plant Kandelia

candel. In the MTT bioassay, 1962A (225) showed activity against human

breast cancer MCF-7 cells with an IC50 value of 100.0 µg/ml (Huang et al.,

2007). Another cyclohexadepsipeptide, pullularin A (226) was separated

from the endophytic fungus Pullularia sp. BCC 8613. Pullularin A (226)

exhibited activities against the malarial parasite Plasmodium falciparum K1

(IC50 value 3.6 µg/ml) and herpes simplex virus type 1 (HSV-1; IC50 value

3.3 µg/ml). Compound 226 also showed weak cytotoxicity to Vero cells

(IC50 value 36.0 µg/ml) (Isaka et al., 2007).

NH

NH

NH

HN

O

HN

OH3C

H3C

O

O

OHO

H3C

CH3O

O

CH3

H3C

N

OO

O

HN

O

N

O

NHNH

O O

OH

CH3

CH3

CH3

O CH3

CH3

H3C

226225

The endophytic fungus Stemphylium globuliferum, isolated from the

stem tissues of the Moroccan medicinal plant Mentha pulegium, produces

polyketides, alterporriol G (227) and its atropisomer alterporriol H (228),

altersolanol K (229), altersolanol L (230) and stemphypyrone (231). Among

the alterporriol-type anthranoid dimers, the mixture of alterporriols G and

H (227, 228) exhibited considerable cytotoxicity against L5178Y cells with

an EC50 value 2.7 µg/ml while Compounds altersolanol L and


111

stemphypyrone (230-231) have also been reported as potent inhibitors of

kinases displaying EC50 value 1.4 µg/ml (Debbab et al., 2009). Javanicin

(232) is another polyketide produced by an endophytic fungus of

Chloridium sp. This compound exhibits strong antibacterial activity against

Pseudomonas fluorescens (Kharwar et al., 2009).

O

O

OH

OH

CH3

OH

OH

H3CO

O

O

H3CO OH

OH

OH

CH3O

OH

OH

OH

CH3

OH

OH

H3CO

OH

H

H

OH

O

OH

OH

OH

H3CO

OH

H

H

O O

OCH3

CH3

CH3

H3C

OHCH3

O

CH3

OH

OHO

O

H3CO

227, 228 Atropisomers

229 230

231232

Endophytic fungi particularly from higher plants are reported to

produce terpenoids. For example, tuberculariols, A-C (233-235) have been

isolated from the mutant strain M-741 of Tubercularia sp. TF5, an

endophytic fungus of Taxus mairei. Antitumor and antibacterial properties

of these compounds were evaluated and were found weakly active (Xu et al.,

2009b). Tauranin (236) was isolated from Phyllosticta spinarum, a fungal

strain, endophytic in plant Platycladus orientalis. When tested in a flow

cytometry-based assay, tauranin (236) induced apoptosis in PC-3M and

NIH 3T3 cell lines at cytotoxic concentrations (2.5-10.0 µM) resulted in a

significant and dose-dependent accumulation of PC-3M cells in the sub-G1

phase (Wijeratne et al., 2008).


112

CH3CH3OH

OH

OH

CH3CH3 CH2

OH

OH

HO

CH3 CH2

OH

OH

OH

OH

OH

CH2

O

O

H3C

H3C CH3

H

OH

OH

233 234 235

236

Higher terpenoids and terpenoid glycosides are also produced by

endophytic fungi. Among them a diterpene glycoside, 16-O-α-D-

tetraacetylglucopyranosyl hymatoxin C (237) was isolated from the culture

of the endophytic fungal strain Tubercularia sp. TF5. The inhibiting rates of

hymatoxin C (237) at 10.0 µg/ml concentration, against Hela and HepG2

lines were 5.67 and 15.84% respectively and at 50.0 µg/ml concentration

against bacteria (Escherichia coli, Bacillus Subtilis and Staphylococcus

aureus) and yeasts (Saccharomyces cerevisiae and Candida albicaus) were

21.63, 22.27, 42.05, 13.75 and 12.69% respectively (Li et al., 2009).

Geniculol (238) has been purified from the culture of an endophytic fungus

of the genus Geniculosporium associated with Teucrium scorodonia. The

compound exhibited algacidal properties at a concentration of 50.0 µg/ml

and caused 2-mm radius inhibition zones in this agar diffusion test (Konig

et al., 1999). Another fungal strain of Fusarium subglutinans, isolated from

a Chinese medicinal plant Tripterygium wilfordii, produced the

immunosuppressive but noncytotxic diterpene pyrones, subglutinol, A

(239) and B (240). Both the compounds are equipotent in the mixed


113

lymphocyte reaction (MLR) assay and thymocyte proliferation (TP) assay

(IC50 value 0.1 µM) (Lee et al., 1995).

CH3

CH3 O

H3C

H

HO

AcO

OAc

OAc

OAc

COOH

237

H3C H

O

O

H

H

H3CHO

H3C

OH

O

CH3

CH3

H2C

O

OH

CH3

H3C

O

H3C

CH3

H

12

239 12=S240 12=R

238

O

O

Steroids are also among common constituents of endophytic fungi. A

sterol ergosta-8(9)-22-diene-3,5,6,7-tetraol(3β,5α,6β,7α,22E) (241) was

isolated from the mycelia of an unidentified endophytic fungus separated

from Castaniopsis fissa (chestnut tree). This compound 241 exhibited

potent selective cytotoxicity against Bel-7402, NCI4460 and L-02 cell lines

with IC50 values 8.45, 5.03, 13.621 µg/ml, respectively (Li et al., 2004).

Other two antimicrobial sterols, 3β,5-dihydroxy-6β-acetoxy-ergosta-7,22-

diene (242) and 3β,5α-dihydroxy-6β-phenylacetyloxy-ergosta-7,22-diene

(243) were characterized from the culture of Colletotrichum sp., an

endophyte isolated from inside the stem of Artemisia annua. Both the


114

compounds 242 and 243 inhibited the growth of all the tested bacteria

(Bacillus subtilis, Staphylococcus aureus, Sarcina lutea and Pseudomonas

sp.) with minimal inhibitory concentrations (MICs) ranging from 25.0 to

75.0 µg/ml. Moreover, metabolites 242 and 243, showed inhibitory

potential against the fungi Candida albicans and Aspergillus niger with MIC

value between 50.0 and 100.0 µg/ml. At the concentration level 200.0

µg/ml, the compounds 242, 243 were shown to be fungistatic to the crop

pathogenic fungi Gaeumannomyces graminis (var. tritici), Rhizoctonia

cerealis, Helminthosporium sativum and Phytophthora capisici (Lu et al.,

2000).

H3C

CH3

CH3

CH3

CH3

H3C

HO

OHOH

OH

H3C

CH3

CH3

CH3

H3C

H3C

HOOH

OR

H H

241 242 R = COCH3

243 R = COCH2C6H5

Chapter 5 Literature Survey of Aspergillus sp

115

CHAPTER 5

LITERATURE SURVEY (SECONDARY

METABOLITES PREVISOUSLY REPORTED) OF ASPERGILLUS SP.


116

5.1. Endophytes; A Natural Machinary to Synthesize Fascinating Bioactive Compounds

In recent years, chemical studies of culturable microorganisms

specially endophytes have led to the discovery of numerous, structurally

novel, biologically active secondary metabolites, most of which are isolated

from marine bacteria, filamentous marine fungi collected from diverse

marine sources including fish, algae, invertebrates and sediments.

Although distributions and ecological roles of endophytic fungi and bacteria

are not well explored, yet the fungus Aspergillus sp. has been proved a good

source of new secondary metabolites.

5.2. Secondary Metabolites Isolated from the Aspergillus sp

In 2013 Pinheiro et al. isolated ergosterol (244), ergosterol peroxide

(245), trypacidin A (246), monomethylsulochrin (247) and mevalolactone

(248) from Aspergillus sp. EJC08 separated from the tissues of Bauhinia

guianensis. The Compounds 246, 247 and 248 showed considerable

activity against Bacillus subtilis, Escherichia coli, Staphylococcus aureus and

Pseudomonas aeruginosa (Pinheiro et al., 2013).


117

HO

244HO

OO

245

O

O

HO

248

O

MeOMe

OHOMe

O OMe

247

OMe

OMe O

OMeO

MeO

O

246

O

O

O

MeO

HO Me

249

O

O OH

OMe

OH

250

OH

HO COOH

251

O

O

O

HO

253

O O

HO

MeO

252

N

N

NH

O

O

254

O

OH

HO

O

255

OHHO

256

OH

Spiro compound aspergispiroketal (249) along with semivioxanthin

(250), 2,3-dihydroxybenzoic acid (251), dihydropenicillic acid (252), (R)-2-

acetonyl-7-hydroxy-5-methylchromone (253), circumadatin F (254), 2-(2´-

hydroxypropyl)-5-methyl-7-hydroxychromone (255) and orcinol (256) were

isolated from EtOAc extract of fermentation broth of the endophytic fungus

Asperilligus sp. HS-05 (Chen et al., 2013). Leporizines, A-C (257−259) were


118

isolated from an Aspergillus sp. strain. Leporizines A (257) and B (258)

were highly correlated with cell toxicity while Leporizine C (259) was not

cytotoxic to cells (Reategui et al., 2013).

NH

N

NO

OH

OHO

O

Sn

OH

HOO

257 n=3258 n=2

NH

N

NO

OH

OHO

OOH

HOO

S

S

259

Aflaquinolones, A & B (260, 261) were obtained from a Hawaiian

fungicolous isolate of Aspergillus sp. while Aflaquinolones, C-G (262-266)

were obtained from a marine Aspergillus isolate (SF-5044) Aflaquinolones

(260-266) except aflaquinolone-D (263),were tested for growth inhibitory

activity against chronic myelogenous leukemia cells (K562), hepatocellular

carcinoma cells (Hep3B), human acute promyelocytic leukemia cells (HL-

60), human breast cancer adenocarcinoma cells (MDA-MB-231) and murine

melanoma cells (B16F10) using in vitro cell viability assays, the compounds

(260-262, 264-266) were effective at high concentrations values > 80μM,

although extract of the Hawaiian isolate was more effective due to the

presence of compounds 260 and 261 (Neff et al., 2012).


119

NH

O

OOH

OMeOH

260NH

O

OH

OMeOH

261

HO

NH

O

OH

OMeOH

262

NH

O

OH

OMeOH

263

O

O

NH

O

R2

R3 OH

R1

264 R1 = OMe, R2 =H, R3 = OH265 R1 = OH, R2 =H, R3 = H266 R1 = H, R2 =OH, R3 = H

Three phenolic bisabolane sesquiterpenoid dimers, disydonols, A–C

(267-269) along with (S)-(+)-sydonol (270) were isolated from the

fermentation broth of a marine-derived fungus Aspergillus sp. isolated from

Xestospongia testudinaria, a sponge collected from South China Sea.

Among them, compounds 267, 269 showed cytotoxicity against the two cell

lines (HepG-2 and Caski human tumour cell lines) (Sun et al., 2012). Some

more bisabolane sesquiterpenoids were reported from the fermentation

broth of a marine-derived fungus Aspergillus sp. associated with

Xestospongia testudinaria. These compounds included aspergiterpenoid A

(271), (-)-sydonol (272), (-)-sydonic acid (273), (-)-5-(hydroxymethyl)-2-

(2′,6′,6′-trimethyltetrahydro-2H-pyran-2-yl) phenol (274) and (Z)-5-

(Hydroxymethyl)-2-(6′-methylhept-2′-en-2′-yl) phenol (275) fungal

metabolites. Compounds 271-275 showed antibacterial activity against

eight bacterial strains: Staphylococcus albus, Bacillus subtilis, Bacillus


120

cereus, Sarcina lutea, Escherichia coli, Micrococcus tetragenus, Vibrio

Parahaemolyticus and Vibrio anguillarum with the MIC values between 1.25

and 20.0 µM (Li et al., 2012).

HO

OH

271

HO

OH

272

OH

HOOC

OH

273

OH

HO

OH

O

274

OH

HO

275

OH

HOO

HO

OH

267

OH

HO

269

OH

HO

OH

HOO

OH

OH

OH

268

HOOH

OH

270

Aspergilone A (276) and its symmetrical dimer aspergilone B (277)

were isolated from the culture broth of a marine-derived fungus Aspergillus

sp. Aspergilone A (276) exhibited in vitro selective cytotoxicity against

human promyelocytic leukemia HL-60 and potent antifouling activity with

an EC50 value 7.68 µg/ml (Shao et al., 2011).

The Mixed cultured mycelia of two different mangrove-derived epiphytic

fungi belonging to the same genus Aspergillus, yielded aspergicin (278),


121

neoaspergillic acid (279) and ergosterol (244). MIC value for Aspergicin

(278) was 15.62 µg/ml against B. subtilis whereas neoaspergillic acid (279)

showed the MIC value in the range of 0.49–15.62 µg/ml against Gram-

positive bacteria, Staphylococcus epidermidis, Staphylococcus aureus,

Bacillus dysenteriae, Bacillus subtilis and Bacillus proteus and Gram-

negative bacteria Escherichia coli (Zhu et al., 2011).

Two compounds, emodin (280) and chrysophanol (281) were isolated

from marine fungus Aspergillus sp. and were tested for their protective

effects against ethanol-induced toxicity. Emodin was found a potential

candidate against ethanol induced HepG2/CYP2E1 liver cells damage (Qian

et al., 2011).

O

O

O

O

O

O O

O

O

276 277

N

N

O

O

O

NH

O

278

279

OHOH O

O

OH

280

OHOH O

O

Me

281

N

N

O

OH H3C


122

EtOAc extract of the fermentation broth of the endophytic fungus

Aspergillus sp. HS-05 obtained from the leaves of Huperzia serrata yielded

N-[4'-hydroxy-(E)-cinnamoyl]-L-tyrosine methyl ester (282) and methyl 4-

methoxy-3-(3'-hydroxy-2'-methyl) propionyloxy-benzoate (283). Compounds

282, 283 showed no anticancer activities against HL-60 cell lines in vitro

(Zhang et al., 2011). Teadenol A (284) and teadenol B (285), were isolated

from tea (Camellia sinensis L.) leaves fermented with Aspergillus sp.

Compounds 284, 285 were prepared biosynthetically from (-)-

epigallocatechin-3-O-gallate (EGCG) and (-)-gallocatechin-3-O-gallate (GCG)

respectively, by enzymatic reaction during the fermentation process of tea

leaves (Wulandari et al., 2011). Extract of the strain Aspergillus sp. (2P-22),

isolated from a marine sponge, Cliona chilensis afforded four compounds,

286-289. The compound namely butylrolactone-VI (286) and 287 were

active against Clavibacter michiganensis. The MIQ value of butyrolactone VI

(286) was 50.0 μg (San-Martin et al., 2011).


123

O

HO

NH

OMeO OH

282 O OMe

O

O

OH

OMe

283

O

O

OH

HO

COOH

284

O

OH

HO

COOH

285

OOH

O

HO

HO

OH

OHCOOMe

OOH

O

MeO

HO

COOH

287

O

O

OH

O

O

O

OMe

O

288

O

O

OH

O

O

O

OMe

O

289

OH

286

Yaming Zhou screened the extract of Aspergillus sp. obtained from

the Mediterranean sponge Tethya aurantium and isolated alkaloids

tryptoquivaline K (290) and fumiquinazolines K–P (291-296). Compounds

290-296 were evaluated for their cytotoxicity against the murine lymphoma

cancer cell-line L517Y.and showed weak activity up to a dose of 10.0

μg/ml. (Zhou et al., 2011). Austalides, M–Q (297-301) were also obtained

from Aspergillus sp. extracted from Mediterranean sponge Tethya

aurantium. Compounds 297-301 were evaluated for their effect on human

alveolar basal epithelial cells (A549) viability by XTT viability assay and

none of them showed potential bioactivity (Zhou, 2012).


124

N

N

OO

O

N

N

O

O

290

N

N

O

N

O

N

N

HO

O

291

N

NNH

O

O

N

NH

O

HHO

292

N

NNH

O

O

N

NH

O

HHO

O

OH

HO

HO

O

293

N

NN

O

O

N

N

O

HHO

294

HO

O

N

NN

O

O

N

N

O

HHO

O

O

295

Other compounds obtained from the same source were, 4-acetyl-3,4-

dihydro-6,8-dihydroxy-5-methylisocoumarin (302), 4-acetyl-3,4-dihydro-

6,8-dihydroxy-3-methoxy-5-methylisocoumarin (303), 2,3,4-tri-methyl-5,7-

dihydroxy-2,3-dihydrobenzofuran (304), citrinin (305), butyrolactone II

(306), methyl (3,4,5-trimethoxy-2-[2 (nicotinamido) benzamido] benzoate

(307), phenol A acid (308), dicitrinin A (309), fumiquinazoline J (310), 3ʹ-O-

acetylthymidine (311), 6,8-dihydroxy-3,4,5-tri-methylisocoumarin (312),

dihydrocitrinone (313), dihydrocitrinin (314) and pretrichodermamide A

(315) (Zhou et al., 2013).


125

O

O

O

O HOMe

HO

OMe

OMe O

297

O

O

O

O H

HO

OMe

OMe O

298

O

O

O

O

O HOH

HO

OMe

OMe O

299

O

O

HOH

HO

OMe O

300

MeO O

HO

O

O

HOH

HO

OMe O

301MeO O

HO

N

NN

O

O

N

N

O

HHOO

296

OO

HO

OMe

O

OH

OH

306

O

O

O

OH

O

309

O

O

HO

OH O

OMe

303

O

HO

OH

304

O

HO

OH O

O

302

O

O

HO

O

OH

305

HO OH

OH

OHO

308

N

NH

O

O

NH

MeO

MeO

OMe

OMe

O

307


126

O

HO

OH O

312

O

NH

O

OH

OH

OHO

S S

OH

OMe

OMe315

N

N NH

O

HN

O 310

N

NH

O

O

O

HO

OO

311

O

HO

HOOC

OH

313

O

HO

HOOC

OH O

314

Compounds tryptoquivaline K (290), Cytochalasin Z17 (180),

Dihydroisoflavipucine (316), Austalide R (317), 3-((1-Hydroxy-3-(2-

methylbut-3-en-2-yl)-2-oxoindolin-3-yl)methyl)-1-methyl-3,4-dihydro-1H-

benzo[e] [1,4] diazepine-2,5-dione (318) and 3-(4-(4-(2-Carboxyvinyl)-2-

methoxyphenoxy)-3-methoxyphenyl)-2-hydroxyacrylic acid (319) obtained

from Aspergillus sp. extract were tested against cell line human

Philadelphia chromosome-positive chronic myelogenous leukaemia, showed

no activity in the assay (Zhou, 2012).


127

NH

OO

O

HO H

316

O

O

O

OOMeOH

HO

HO

O

317

O

OMe

O

MeO

OH

O

HO

319

NH

NO

NOH

O

O

318

Column chromatography of fermentation extract of seaweed fungus

Aspergillus sp. AF044 yielded, 6-hydroxy-5-methoxy-3-methyl-3,6-dihydro-

2H-pyran-4-carboxylic acid (320), 8,9-dihydroxy-8,9-deoxyaspyrone (321)

and penicillic acid (322). Penicillic acid (322) showed activity against

Ruegeria atlantica (Zhuo et al., 2010). Compounds, JBIR-74 (323) and

JBIR-75 (324) have been isolated from the Aspergillus sp. fS14 derived from

sponge (Takagi et al., 2010). Aspergillus sp. isolated from the inner tissue of

soft coral Sarcophyton tortuosum afforded compounds, 3,6-Diisobutyl-

2(1H)-pyrazinone (325), 3-Isobutyl-6-(1-hydroxy-2-methylpropyl)-2(1H)-

pyrazinone (326), 3-Methoxy-4-methyl-2,4-dien-pentanoic acid (327) and

Penicillic acid (322). These compounds show potent cytotoxicities toward

several cancer cell lines (Hou-jin et al., 2010).


128

OHO

MeO

OHOO

O

OH

HO

OH

OO

OH

OMe

320321 322

NH

HNO

O NH

N

323

NH

HNO

O NH

N

324

HN

N

O

325

HN

N

O

326OH

O OH

O

327

A difuranxanthone, asperxanthone (328), and a biphenyl,

asperbiphenyl (329) were isolated from Aspergillus sp. (MF-93), a marine

fungus. Compounds, 328,329 showed moderate activity (inhibitory rates

62.9 and 35.5% respectively) in inhibiting multiplication of TMV but much

lower than that of the extract at the identical concentration of 0.2 mg/ml

(Wu et al., 2009).

Prenylated indole alkaloids notoamides A-D (330-333) (Kato et al., 2007a)

and notoamides F-K (334-339) along with sclerotiamide (340),

deoxybrevianamide E (341), deoxybrevianmide J (342), 3-epi-notamide C

(343) and stephacidin A (344) were isolated from a marine-derived

Aspergillus sp. Notoamide I (337) showed cytotoxicity against HeLa cells


129

with an IC50 value 21.0 μg/ml (Kato et al., 2007b).

NH

NH

N

O

O

O

HO

H

338

O

HO

OHO

OH

OH

329

O N

R1

N

O

HO

R2

330 (R1 = OH, R2 = H)

331 (R1 = R2 = H)

336 (R1 = R2 = OH)

340 (R1 = H, R2 = OH)

O NH

HN

N

O

H

O

O

H

332

O NH

N

NHO

O

OH

R

333 (R = )339 (R = )

NH

HN

N

O

OH

H

341

NH

O

O N

R1

N

O

H

NH

OR3

R2

344 (R1 = R2 =R3 = H)

334 (R1 = R2 = H, R3 = OMe)

335 (R1 = OH, R2 = H, R3 = OMe)

337 (R1 = H, R2, R3 = O)

NH

HN

N

O

OHO

H

342

NH

HN

N

O

O

HO

H

343

O

O

OO

O

OMe

328

Antifungal macrolide, Sch 725674 (345) was isolated from an

Aspergillus sp. culture SPRI-0836. Compound 345 showed inhibitory

activity against Saccharomyces cerevisiae (PM503) and Candida albicans

(C43) with MICs of 8.0 and 32.0 mg/ml, respectively (Yang et al., 2005).

β,γ-dehydrocurvularin (346), ,β-dehydrocurvularin (347), 8-β-hydroxy-7-

oxo curvularin (348) and oxocurvularin (349) have been reported from

mycelial mats and cultur filterate of Aspergillus sp. Compound 346

accelerated the primary root growth in proportion to its concentration from

3.0 mg/l to 300.0 mg/l, and compound 349 accelerated the primary root


130

growth to 167% of control at the concentration of 300.0 mg/l. Compound

346 was non toxic to plant growth, making it a potentially useful

nematicide (Kusano et al., 2003). The extract of cultured marine-derived

fungus of the genus Aspergillus (strain CNM-713) afforded a sesterterpene

epoxide-diol and aspergilloxide (350). Compound 350 showed little in vitro

cytotoxicity toward HCT-116 human colon carcinoma (Cueto et al., 2002).

OH

O

OH

OH

O

345

OHOHH

O

H H

350

OO

O

OO

O

OO

O

O

OH

OO

O

O

346347

348 349

OH

HO HO

OHOH

HOHO

Mycelium of a cultured marine fungus of the genus Aspergillus (CNC-

120) yielded two isomeric linear peptides, aspergillamide A (351) and B

(352). A poorly defined metabolite L-phenylalaninamide (353) was also

isolated in low yield from CNC120 along with aspergillamides (351, 352).

Compounds 351,353 showed in vitro mild cytotoxicity toward the human

colon carcinoma cell line HCT-116 with an IC50 value 16.0 µg/ml (Toske et


131

al., 1998). Aspergillimide VM55598 (354), 16-keto aspergillimide SB202327

(355), paraherquamides VM54159 (356), SB203105 (357) and SB200437

(358) have been isolated from the Aspergillus strain IMI 337664. The

compound (355) showed evidence of activity in vitro but not in vivo against

adult Trichostrongylus colubriformis infections in gerbils (Banks et al.,

1997). Cycloaspeptides, A (359), B (360) and C (361) were isolated along

with ergosterol (244) from the mycelial chloroform extract of Aspergillus sp.

NE-45. The compound 359 has antifungal and antibacterial activity at

concentrations more than 100.0 µg/disc (Kobayashi et al., 1987).

NH

NH

O

NH

CH3

O

NH

O

351

H

NH

NH

O

NH

CH3

352

O

NH

OH

NH

NH

O

NH

CH3

O

NH

OH

353

N

N

N

H Me

RO

O

Me

O

Me

354 R=H355 R=O

O

O

R'

N

N

N

H Me

O

H

O

R

356 R=Me R'=H 357 R=Me R'=OH 358 R=H R'=H

N

N

NN

N

O

R1O

O

R1

OR2

OR4

O

R1

R3

359 R1=R4=H, R2=R3=CH3

360 R1=R2=R4=H, R3=CH3

361 R1=R3=R4=H, R2=CH3

H

Chapter 6 Characterization of Metabolites of Aspergillus sp

132

CHAPTER 6

STRUCTURE ELUCIDATION AND SPECTROSCOPIC

DATA OF SECONDARY METABOLITES ISOLATED

FROM ENDOPHYTIC FUNGUS ASPERGILLUS SP.


133

6.1. Results and Discussion

The culture extract of an Aspergillus sp. internal strain No. 9297 B,

was chromatographed to purify its metabolites, filtration through Sephadex

LH-20 followed by HPLC purification of the culture extract, eight

compounds were obtained and identified as: dibenzofuran Sch725421

(362), 4-acetyl-3,4-dihydro-6,8-dihydroxy-5-methyl-isocoumarin (302), 4-

acetyl-6,8-dihydroxy-3-methoxy-5-methyl-3,4-dihydroisocoumarin (303),

2,3,4-Trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran (304), 2,6-

dihydroxy-4-(3-hydroxybutan-2-yl)-3-methylbenzoic acid (363),

cytochalasin C (364), cytochalasin D (365), ergosterol (244) and 24-

methylcholesta-7,22-E-diene-3β,5-diol-6-one (366).

6.2. Characterization of Compounds Isolated from Aspergillus sp.

6.2.1. Characterization of dibenzofuran, Sch725421 (362)

Compound 362 was isolated as yellowish amorphous powder, which

exhibited characteristic absorption bands at

3460, 1655, 1510, 1470 and 1240 cm-1 due

to hydroxyl function, olefinic system,

aromatic moiety and ether group

respectively. The EI-MS of 362 exhibited molecular ion peak at m/z 296.

The high resolution (HR-EI-MS) analysis of the same peak (m/z 296.1407)

depicted the molecular formula C19H20O3 with ten DBE.

7

4b5

8a5´1

3

1'

3'

O

OH

Me

Me

OH

Me

Me

362

4a

8b

4´

7´

6´


134

The 1H NMR spectrum (Table 6.1) of 362 displayed resonances for

three aromatic protons at 6.74 (1H, d, J = 1.8 Hz), 6.72 (1H, s) and 6.48

(1H, d, J = 1.8 Hz). This observation revealed that compound 362 must

have at least two aromatic ring systems. In addition, the same spectrum

displayed signals for an olefinic methine at 5.09 (t, J = 7.1 Hz), which

showed COSY correlation with a methylene resonating at 3.45 (d, J = 7.1

Hz). Four tertiary methyl resonances were observed at 2.87, 2.34, 1.80

and 1.67. The chemical shifts of first two methyls revealed their attachment

with the aromatic system, while other two were found to be allelic in

nature.

The 13C NMR spectrum (Table 6.1) of compound 362 fully supported

the mass and 1H NMR data as it displayed total 19 carbon atoms, which

were identified due to DEPT experiment as four methyl ( 25.9, 20.5, 18.1

and 18.0), one methylene ( 27.0), four methine ( 125.0, 110.9, 104.0 and

95.7) and ten quaternary carbon atoms ( 159.4, 155.9, 155.3, 153.0,

137.3, 133.3, 131.1, 123.8, 117.2 and 111.9). Four downfield aromatic

carbon resonances at 159.4, 155.9, 155.3, 153.0 indicated that these

carbons must have oxygen atom attached to them, whereas, the molecular

formula afforded only three oxygen atoms. This analysis indicated that one

oxygen atom must have been shared by two aromatic moieties. This

deduction was substantiated due to the DBE. The above information


135

accommodated nine DBE, therefore, the remaining one DBE could be

attributed to a furan ring system condensed between two benzene rings.

The mutual HMBC correlations (Figure 6.1) of allelic methyls ( 1.80

and 1.67) and with the carbons at 131.0 and 125.4 revealed an isopropyl

group and therefore, led to the elucidation of an isoprene unit in 362.

Further, the HMBC correlation of olefinic methine with the aromatic carbon

at ( 123.8) and that of methylene ( 3.45) with three aromatic carbons at

123.8, 133.3 and 155.3 determined the position of isopropyl moiety. Other

methyl groups ( 2.87 and 2.34) were also fixed on two different aromatic

rings due to their HMBC correlations as are shown in (figure. 6.1).

O

OH

Me

Me

OH

Me

COSY correlations

HMBC correlations

Me

362

Figure 6.1: Important HMBC and COSY correlations observed in the

spectra of 362

The combination of the whole data and careful analysis revealed the

structure of compound 362 as dibenzofuran Sch725421, which is a known

fungal metabolite. Therefore, observed spectroscopic data was compared


136

with the published data (Yang et al., 2004) for final confirmation of the

structure as dibenzofuran Sch725421 (362).

Table 6.1: 1H and 13C NMR data of compound 362 (CD3OD, 400 and 100

MHz)

Position H (J in Hz) C

1 133.3 (C)

2 _ 123.8(C)

3 _ 155.3(C)

4 6.72, (s) 95.7 (CH)

4a _ 155.9 (C)

4b _ 159.4 (C)

5 6.74, (d, 1.8) 104.0 (CH)

6 _ 137.3 (C)

7 6.48, (d, 1.8) 110.9 (CH)

8 _ 153.0 (C)

8a _ 111.9 (C)

8b _ 117.2 (C)

1 3.45, (d, 7.1) 27.0 (CH2)

2 5.09, (t, 7.1) 125.0 (CH)

3 _ 131.1 (C)

4 *1.80, (s) 18.1 (CH3)

5 *1.67, (s) 25.9 (CH3)

6 2.87, (s) 18.0 (CH3)

7 2.34, (s) 20.5 (CH3)

*Values interchangeable

6.2.2. Characterization of 4-acetyl-3,4-dihydro-6,8-dihydroxy-5-

methyl-isocoumarin (302)

Compound 302 was purified from the fungal culture as yellow

amorphous powder. The EI-MS of 302 displayed molecular ion peak at m/z

236, while the molecular formula was established due to HR-EI-MS (m/z

236.0681) as C12H12O5 with seven DBE. The IR spectrum displayed

absorption bands (3415, 1712, 1735, 1510, 1456 cm-1) for hydroxyl,


137

aromatic system and ketonic function and UV data (214, 272 and 313 nm)

was indicative of an isocoumarin skeleton of 302.

The 1H NMR spectrum (Table 6.2) of compound 302 showed the most

downfield signals at 12.0 (1H, s) and 10.9 (1H, s), which were attested for

two hydroxyl functions, whereas, the aromatic region afforded resonance of

only one proton at 6.34 (1H, s), which was attributed

to a benzene ring. The same spectrum also showed

resonances of an oxymethylene at 4.93 (Ha, d, J =

12.4 Hz) and 4.60 (Hb, dd, J = 12.4 and 4.0 Hz), which

was correlated in COSY spectrum with a methine

proton at 4.30 (1H, d, J = 4.0 Hz). This proton signal was correlated in

HSQC spectrum with a carbon resonated at 48.1, indicated its non-

oxygenated nature, and rather depicted its presence between two sp2

hybridized carbon centers. Relatively downfield shift of oxymethylene

revealed its connectivity with a carboxylate function. An acetyl function was

elucidated due to the resonance of a singlet methyl at 2.26 which showed

HMBC correlations with a ketonic carbon resonating at 202.5 and with

the methine carbon at 48.1 in 13C NMR spectrum. (Table 6.2) This

observation additionally helped to identify the location of acetyl moiety.

Another methyl proton displayed its resonance in 1H NMR spectrum at

2.03, which was correlated with the carbon at 11.5 and showed HMBC

correlation (Figure 6.2) with the aromatic carbons at 115.1, 139.4 and

O

OMe

HO

OH O

Me

302

9

10

11

12

13


138

163.0 indicating its attachment with the benzene ring. The resonances for

other carbon nuclei were observed in 13C NMR spectrum (Table 6.2) at

168.8 (C=O), 159.8 (C), 103.1 (C), 100.7 (CH), 68.6 (CH2) and 27.6 (CH3).

COSY correlation

HMBC correlations

O

OMe

HO

OH O

Me

302


spectra of 302

The above data was identical to the published data for an isocoumarin,

(Krohn et al., 2001) which was further confirmed through HMBC

experiment (Figure 6.2) in which oxymethylene displayed its correlation

with the carbonyl carbon at 168.8, therefore, compound 302 was finally

established as 4-acetyl-3,4-dihydro-6,8-dihydroxy-5-methyl-isocoumarin

(302) which has previously been reported from the endophytic fungi (Krohn

et al., 2001; Krohn et al., 2004)

6.2.3. Characterization of 4-acetyl-6,8-dihydroxy-3-methoxy-5-methyl-

3,4-dihydro isocoumarin (303)

Compound 303 was also found to be isocoumarin as it displayed

nearly similar IR and UV data as was observed for compound 302. The EI-


139

MS showed molecular ion peak at m/z 266, whereas, HR-EI-MS displayed

molecular ion at m/z 266.0785 corresponding to the molecular formula as

C13H14O6. This formula afforded one carbon and one oxygen atom more but

the same DBE when compared to that of 302. This

information gave the idea that compound 303 could

have an additional methoxyl group. This idea was

substantiated through 1H NMR data of compound

303 (Table 6.2) which was nearly identical to that of

compound 302 with the only difference of the absence of signals for

oymethylene, and in lieu thereof, the spectrum of compound 303 displayed

resonance of a methine proton at 5.81 (d, J = 1.4 Hz) and a methoxyl

proton at 3.46. The methine proton ( 5.81) was correlated in HSQC

spectrum with a carbon resonating at 103.0. This information clearly

established that compound 303 has a methoxyl moiety at C-3 ( 103.0),

which was substantiated due to HMBC spectral analysis (Figure 6.3), in

which methoxyl proton showed correlation with C-3. Other 1H and 13C NMR

data (Table 6.2) was identical to that of 302. Based on these analyses,

compound 303 was identified as 4-acetyl-6,8-dihydroxy-3-methoxy-5-

methyl-3,4-dihydro isocoumarin, which is also a known fungal metabolite

(Krohn et al., 2004).

O

OMe

HO

OH O

Me

303

OMe

9

10

11

12

13

1

3


140

COSY correlation

HMBC correlations

O

OMe

HO

OH O

Me

OMe

303


spectra of 303

Table 6.2: 1H and 13C NMR data of compound 302 (DMSO-d6, 500 and 125

MHz) and 303 (DMSO-d6, 400 and 100 MHz)

302 303

Position

H (J in Hz) C H (J in Hz) C

1 _ 168.8 (C) _ 168.2 (C) 3 4.93, (Ha, d, 12.4)

4.60, (Hb dd, 12.4, 4.0) 68.6 (CH2) 5.81, (d, 1.4) 103.0 (CH)

4 4.30, (d, 4.0) 48.1 (CH) 4.49, (d, 1.4) 53.1 (CH) 5 _ 115.1 (C) _ 117.9 (C) 6 _ 163.0 (C) _ 162.9 (C) 7 6.34, (s) 100.7 (CH) 6.37, (s) 101.2 (CH) 8 _ 159.8 (C) _ 160.1 (C) 9 _ 103.1 (C) _ 99.1 (C) 10 _ 139.4 (C) _ 136.3 (C) 11 _ 202.5 (C) _ 203.6 (C) 12 2.26, (s) 27.6 (CH3) 2.30, (s) 30.0 (CH3) 13 2.03, (s) 11.5 (CH3) 1.97, (s) 11.7 (CH3) 3-OMe _ _ 3.46, (s) 56.5 (CH3) 6-OH 10.90, (s) _ 10.90, (s) _ 8-OH 12.0, (s) _ 10.94, (s) _

6.2.4. Characterization of 2,3,4-trimethyl-5,7-dihydroxy-2,3-

dihydrobenzofuran (304)

Compound 304 was found to be benzofuran in nature with molecular

formula C11H14O3 and five DBE, determined through HR-EI-MS (m/z

194.0941). The IR absorption bands were observed for hydroxyl function


141

and aromatic moiety (3433, 1605, 1525, 1458 cm-1). The 1H NMR of 304

exhibited only one signal in aromatic region at 6.18 (1H, s), however, the

up-field region of the spectrum displayed signals for methyl groups at

2.02 (s), 1.31 (d, J = 6.4 Hz) and 1.27 (d, J = 6.4 Hz), and two methine

protons at 4.35 (m) and 2.97 (m). These two methine protons were found

correlated with each other in COSY spectrum (Figure 6.4), whereas, the

oxymethine proton ( 4.35) was further correlated with a doublet methyl at

1.31 and the other aliphatic methine ( 2.97) was correlated in COSY

spectrum with the doublet methyl at 1.27.

The 13C NMR spectrum (Table 6.3) of 304 supported the above data

as it displayed signals for aromatic carbons at 154.3 (C), 139.0 (C), 138.5

(C), 133.6 (C), 112.3 (C), 100.1 (CH), an oxymethine

carbon at 87.1, an aliphatic methine carbon at

45.0, three methyl carbons at 20.2 (CH3), 19.0

(CH3) and 11.2 (CH3). This data accommodated four

DBE, therefore, the remaining DBE could be attributed to another possible

ring system, which could be found as a furan ring condensed with the

aromatic ring. This deduction was substantiated due to HMBC correlations

(Figure 6.4) of oxymethine proton ( 4.35) with the carbon at 138.5 and

133.6. The aliphatic methine also exhibited HMBC correlations with

aromatic carbons at 133.6, 138.5 and 112.3. Other important HMBC

correlations have been described in figure 6.4. This data was

5

68

10

9

11

12

O

Me

HO

OH

Me

Me

304

2

3

4


142

superimposable with the reported data for the compound 2,3,4-trimethyl-

5,7-dihydroxy-2,3-dihydrobenzofuran (Chen et al., 2002), therefore,

compound 304 was found to be the same. However, this compound has

been isolated for the first time from our investigated source.

COSY correlations

HMBC correlations

O

Me

HO

OH

Me

Me

304


spectra of 304

6.2.5. Characterization of 2,6-dihydroxy-4-(3-hydroxybutan-2-yl)-3-

methyl benzoic acid (363)

The UV data (214, 253 and 315 nm) of 363 indicated the presence of

a substituted benzoic acid, whereas, IR spectrum

displayed characteristic absorption bands for carboxylic

acid group, hydroxyl function and aromatic system (3420,

3375, 1708, 1515, 1452 cm-1). The molecular formula

C12H16O5 was established through HR-EI-MS which

displayed molecular ion peak at m/z 240.0995. The most downfield signals

at 13.60 (s), 12.91 (s) and 11.1 (s) observed in the 1H NMR of 363 were

1

4

5

6

OH

Me

OHO

OHHO

Me

363

Me

2

3

12

7

9

8 10

11


143

attested for hydroxyl protons, whereas, in aromatic region, only one

resonance was observed at 6.03 (s) due to a penta-substituted benzene

ring. Further, the same spectrum showed the resonance of an oxymethine

proton at 3.71 (m), which was correlated in COSY spectrum with a

doublet methyl resonating at 1.12 (J = 6.5 Hz) and a methine proton at

2.93 (m). The latter methine ( 2.93) was further correlated in COSY

spectrum with another doublet methyl at 1.04 (J = 6.0 Hz). This

information revealed that a benzoic acid derived molecule must have 2-

hydroxy-3-methylpropyl moiety attached to benzene ring. The 1H NMR

spectrum (Table 6.3) also showed signal for a singlet methyl at 2.01 (s).

The chemical shift of this methyl protons revealed that it must be

connected to benzene ring. Therefore, from the above data, it was deduced

that the remaining two attachments with the benzene ring could be

hydroxyl functions.

The 13C NMR spectrum was in full agreement with mass and 1H NMR

data as it displayed twelve carbon signals at 174.8 (C), 160.1 (C), 159.7

(C), 147.0 (C), 111.0 (C), 108.7 (CH), 102.1 (C), 70.0 (CH), 42.3 (CH), 20.1

(CH3), 16.7 (CH3) and 10.9 (CH3). The two alkyl moieties were placed on

benzene ring at ortho to each other due to HMBC correlation of singlet

methyl with the aromatic carbons at 159.7, 147.0 and 111.0, and that of

doublet methyl ( 1.04) with the carbons at 147.0, 42.3 and 70.0. Further

the methine proton at 2.93 exhibited HMBC correlation with the aromatic


144

carbons at 147.0, 111.0 and 108.7 (Figure 6.5). Based on the above data,

compound 363 was characterized as 2,6-dihydroxy-4-(3-hydroxybutan-2-

yl)-3-methylbenzoic acid, which has already been isolated from fungal

sources (Brown et al., 1949; Rodel and Gerlach, 1995).

COSY correlations

HMBC correlations

OH

Me

OHO

OHHO

Me

363


spectra of 363

Table 6.3: 1H and 13C NMR data of compound 304 (MeOD, 500 & 125 MHz)

and 363 (DMSO-d6, 500 and 125 MHz)

Compound 304 Compound 363


1 _ _ _ 102.1 (C) 2 4.35, (m) 87.1 (CH) _ 159.7 (C) 3 2.97, (m) 45.0 (CH) _ 111.0 (C) 4 _ 112.3 (C) _ 147.0 (C) 5 _ 154.3 (C) 6.03, (s) 108.7 (C) 6 6.18, (s) 100.1 (CH) _ 160.1 (C) 7 _ 139.0 (C) 2.93, (m) 42.3 (CH) 8 _ 138.5 (C) 3.71, (m) 70.0 (CH) 9 _ 133.6 (C) 1.12, (d, 6.5) 20.1 (CH3) 10 1.31, (d, 6.4) 20.2 (CH3) 1.04, (d, 6.0) 16.7 (CH3) 11 1.27, (d, 6.4) 19.0 (CH3) 2.01, (s) 10.9 (CH3) 12 2.02, (s) 11.2 (CH3) 174.8 (C) 2-OH _ _ 13.60, (s) _ 6-OH _ _ 12.91, (s) _ 8-OH _ _ 11.1, (s) _


145

6.2.6. Characterization of cytochalasin C (364)

Compound 364 was obtained as white amorphous powder, which

exhibited characteristic absorption

bands for hydroxyl function (3410 cm-1),

ester carbonyl (1730 cm-1), ketonic and

amide carbonyl (1695 cm-1), olefinic

function (1648 cm-1) and for aromatic

moiety (1590-1455 cm-1). The HR-ESI-

MS analysis in +ve mode depicted the molecular formula as C30H37NO6 with

13 DBE due to a pseudo molecular ion at 530.2513 [M+Na]+.

Aromatic region of the 1H NMR spectrum (Table 6.4) of 364 exhibited

three signals at 7.31 (2H, t, J = 8.8 Hz, H-3, 5), 7.27 (1H, d, J = 8.8 Hz,

H-4) and 7.21 (2H, d, J = 8.8 Hz, H-2, 6) attested for a mono-substituted

benzene ring. In the olefinic region of the same spectrum, four resonances

due to two double bonds were observed at 5.83 (1H, d, J = 16.0 Hz, H-20),

5.15 (1H, d, J = 16.0 Hz, H-19), 5.58 (1H, dd, J = 15.6, 10.0 Hz, H-13)

and 5.07 (1H, m, H-14). The bigger J values revealed E-geometry of the

double bonds. 1H NMR spectrum further displayed signals for two

oxymethine protons at 5.61 (1H, br, s) and 3.50 (1H, d, J = 8.9 Hz), three

aliphatic methine protons at 2.40 (1H, m), 2.32 (1H, m) and 3.08 (1H, d, J

= 1.1 Hz), five methyl signals at 2.23 (s), 1.61 (s), 1.49 (s), 1.06 (d, J = 6.4

Hz) and 1.35 (s) and two methylenes at 3.01 (Ha, dd, J = 13.1 and 8.9 Hz),

15

1721

1''

2''

2

3

5

9

12

13

11

19

22

23

1

5´

1'3'

710

HN

MeOH

OOO

Me

O

MeHO

E

E Me

Me

364


146

2.76 (Hb, dd, J = 13.1 and 5.5 Hz) and 2.19 (Ha, m) and 1.92 (Hb, m). The

first two methylene signals exhibited COSY correlation (Figure 6.6) with a

methine proton resonating at 3.14 (1H, m), which was correlated in HSQC

spectrum with a carbon resonating at 59.3, therefore, was identified as

azamethine. The above data closely resembled with the data reported for

cytochalasins, which are known as microbial metabolites (Aldridge and

Turner, 1969). The 13C NMR spectrum (Table 6.4) supported the above

data, as it afforded signals for a mono-substituted benzene ring ( 136.5

,129.1, 127.9, 127.1), four olefinic methines ( 128.1, 130.4, 132.5, 131.8),

two olefin quaternary carbons (133.0 and 125.3), The most downfield

signals resonated at 208.7, 174.6 and 170.1 were attributed to a ketonic,

an amide and an acetyl function respectively.

The above discussed data justified ten DBE, therefore, the remaining

three DBE was attributed to three ring systems in compound 364.

Relatively downfield shift of oxygenated methine proton at 5.61 (H-21) and

its HMBC correlation (Figure 6.6) with the carbonyl carbon at 170.1

helped to fix acetyl group at C-21. The COSY correlation of azamethine (

3.14, H-3) with H-4 ( 3.08) and amide hydrogen at 8.20 (s), and its

HMBC correlation (Figure 6.6) with a quaternary carbon at 51.2 (C-9) and

carbonyl at 174.6 (C-1) revealed a five membered cyclic amide moiety in

364. Further COSY correlation of H-3 with the methylene at 3.01 (Ha-10)

and 2.76 (Hb-10), and its HMBC interaction with the aromatic carbon at


147

136.5 (C-1) and that of methylene C-10 confirmed a benzyl moiety

connected with cyclic amide system.

The COSY coupling of the proton at 2.32 (H-8) with oxymethine at

3.50 (H-7) and its HMBC correlations with C-9 ( 51.2), C-4 ( 49.7) and C-

6 ( 133.0), and the HMBC interaction of H-4 with the carbons at 125.3

(C-5), 133.0 (C-6) and 48.5 (C-8) confirmed a condensed cyclohexene ring

with the amide system. These observations accommodated two more DBE.

Both the 1H and 13C NMR spectra afforded five methyls, therefore, the

remaining eight carbons in the molecule could be assumed to be present as

part of a macrocyclic system, thus elucidated an 11 membered ring.

HN

MeMe

OH

O

OO

Me

O

Me

HO

COSY correlations

HMBC correlations

364

Figure 6.6: Important COSY and HMBC correlations observed in the

spectra of 364

Finally the whole spectroscopic data was found to be identical to the

reported data for cytochalasin C (Aldridge and Turner, 1969) which has

already been reported as fungal metabolite. Therefore, compound 364 was

confirmed as cytochalasin C.


148

6.2.7. Characterization of cytochalasin D (365)

The mass and IR data of 365 was super imposable to the data of 365

with the same molecular formula

C30H37NO6 and 13 DBE. The main

difference was seen in the NMR data

which showed signals for an exo-cyclic

double bond in 365. Signals for two

allelic methyls in 365 disappeared, and

an additional doublet methyl displayed its position at 1.15 (J = 6.5 Hz),

besides an additional olefinic methylene resonated in 1H NMR spectrum at

5.31 (Ha, s) and 5.09 (Hb, s). This information attested that the endo-

cyclic double bond at C-5 and C-6 in 365 is reduced and rather one allelic

methyl has been oxidized to an exo-cyclic in 365. The 13C NMR spectral

data (Table 6.4) substantiated the presence of exo-cyclic double bond as it

displayed signals due to a quaternary carbon at 148.7 (C-6) and

methylene at 114.0 (C-12). The rest of the NMR data of compound 365

was super imposable to that of 364 and was identical to the reported data

for cytochalasin D (Aldridge and Turner, 1969). Therefore, compound 365

was characterized as cytochalasine D, which is also a known fungal

compound but has been reported first time from Aspergillus sp.

15

1721

1''

2''

2

3

5

9

12

13

11

19

22

23

1

5´

1'3'

710

HN

MeOH

OOO

Me

O

MeHO

E

E Me

365


149

Table 6.4: 1H and 13C NMR data of compound 364 (DMSO-d6, 300 & 75

MHz) and 365 (DMSO-d6, 400 & 100 MHz)


Position

H (J in Hz) C H (J in Hz) C

1 _ 174.6 (C) _ 172.8 (C) 2-NH

8.20, (s) _ 8.12, (s) _

3 3.14, (m) 59.3 (CH) 3.15, (m) 60.1 (CH) 4 3.08, (d, 1.1) 49.7 (CH) 3.20, (d, 0.9) 49.0 (CH) 5 _ 125.3 (C) 2.96, (m) 36.7 (C) 6 _ 133.0 (C) _ 148.7 (C) 7 3.50, (d, 8.9) 68.1 (CH) 3.88, (d, 9.8) 68.8 (CH) 8 2.32, (m) 48.5 (CH) 2.32, (t, 9.8) 48.1 (CH) 9 _ 51.2 (C) _ 51.6 (C) 10 3.01, (Ha, dd, 13.1, 8.9)

2.76, (Hb, dd, 13.1, 5.5) 43.8 (CH2) 2.92, (Ha, dd, 13.4,

9.0) 2.65, (Hb, dd, 13.4, 5.4)

42.5 (CH2)

11 1.61, (s) 16.8 (CH3) 1.15, (d, 6.5) 14.0 (CH3) 12 1.49, (s) 14.1 (CH3) 5.31, (Ha, s)

5.09, (Hb, s) 114.0 (CH2)

13 5.58, (dd, 15.6, 10.0) 131.8 (CH) 5.51, (dd, 16.0, 9.8) 131.0 (CH) 14 5.07, (m) 132.5 (CH) 5.01, (m) 130.8 (CH) 15 2.19, (Ha, m)

1.92, (Hb, m) 37.8 (CH2) 2.18, (Ha, m)

1.87, (Hb, m) 38.0 (CH2)

16 2.40, (m) 42.3 (CH) 2.41, (m) 40.9 (CH) 17 _ 208.7 (C) _ 207.9 (C) 18 _ 78.0 (C) _ 76.4 (C) 19 5.15, (d, 16.0) 128.1 (CH) 5.16, (d, 15.6) 128.6 (CH) 20 5.83, (d, 16.0) 130.4 (CH) 5.79, (d, 15.6) 130.4 (CH) 21 5.61, (br, s) 75.2 (CH) 5.48, (s) 73.8 (CH) 22 1.06, (d, 6.4) 19.7 (CH3) 1.01, (d, 6.4) 19.4 (CH3) 23 1.35, (s) 25.1 (CH3) 1.35, (s) 23.8 (CH3) 1' _ 136.5 (C) _ 136.5 (C) 2', 6' 7.21, (2H, d, 8.8) 129.1 (CH) 7.16, (2H, d (8.8) 128.7 (CH) 3', 5' 7.31, (2H, t, 8.8) 127.9 (CH) 7.30, (2H, t, 8.8) 127.1 (CH) 4' 7.27, (1H, d, 8.8) 127.1 (CH) 7.21,(1H, d, 8.8) 125.8 (CH) 1'' _ 170.1 (C) _ 169.8 (C) 2'' 2.23, (s) 20.8 (CH3) 2.21, (s) 21.0 (CH3)

6.2.8. Characterization of ergosterol (244)

The shining crystals of compound 244 melted at 155-157.5 C, and

displayed IR absorption bands due to hydroxyl group (3350 cm-1) and


150

olefinic system (1645 cm-1). The EIMS of compound 244 displayed

molecular ion peak at m/z 396, and the high resolution analysis of the

same peak depicted the molecular

formula C28H44O with seven DBE. The 1H

NMR spectrum (Table 6.5) exhibited

resonances for four olefinic methines at

5.53 (1H, d, J = 8.1 Hz), 5.47 (1H, d, J =

8.1 Hz), 5.27 (1H, dd, J = 15.5, 5.1 Hz) and 5.10 (1H, dd, J = 15.5, 5.9 Hz)

attributed to ergosterol nucleus. A resonance due to an oxymethine was

seen at 3.50 (1H, m, H-3), whereas, six methyls displayed their positions

at 1.05 (3H, d, J = 6.5 Hz, H-21), 0.93 (3H, s, H-19), 0.91 (3H, d, J = 6.5

Hz, H-27), 0.86 (3H, d, J = 6.5 Hz, H-28), 0.82 (3H, d, J = 6.6 Hz, H-26) and

0.65 (3H, s, H-18). The 13C NMR data (Table 6.5) fully supported the mass

and NMR, which was identical to the reported data of ergosterol (Soubias et

al., 2005). Therefore, compound 244 was identified as ergosterol, which

was further confirmed through comparative TLC with authentic sample.

6.2.9. Characterization of 24-methylcholesta-7,22-E-diene-3β,5-diol-

6-one (366)

Compound 366 was found to be an ergosterol derived molecule, which

showed molecular ion peak in EI-MS, spectrum at m/z 428. The molecular

formula C28H44O3 showed two more oxygen atom but the same DBE when

compared to that of 244. The IR spectrum exhibited characteristic

absorption bands for a hydroxyl function at 3440 cm-1 and for an enone

HO

Me

Me

Me Me

Me

Me

1

3 5

6

7

89

10

11 13

14 15

161719

18 20

2122

2425

26

27

28

244


151

system at 1690 and 1615 cm-1. This information gave the idea that one

double bond in ergosterol might have

oxidized to a ketone, thus justifying one

additional oxygen atom, whereas, the third

oxygen atom could be attributed to an

addition hydroxyl function in 366.The 1H

MR spectral data (Table 6.5) of 366 was nearly identical to the spectral data

of 244 with fewer differences. The 1H NMR spectrum of 366 was missing

two doublet olefinic signals, rather displayed a singlet at 5.75 (H-7), along

with usual signals for a trans-olefin 5.18 (1H, dd, J = 15.0, 5.3 Hz, H-22)

and 5.10 (1H, dd, J = 15.3, 5.8 Hz, H-23). The 13C NMR spectrum (Table

6.5) substantiated the above evidence as it displayed signals for an enone

moiety at 199.9 (C-6), 164.0 (C-8) and 120.7 (C-7). Similarly an additional

oxygenated quaternary carbon resonance was observed at 77.1 (C-5).

Combination of the whole data finally led to the structure of 366 as 24-

methylcholesta-7,22-E-diene-3β,5-diol-6-one (366), which is also a known

fungal metabolite and thus the whole data was also compared with the

reported data (Aiello et al., 1991).

HO

Me

Me

O

Me Me

Me

Me

HO

1

3 56 7

89

10

11 13

14 15

161719

18 20

2122

2425

26

27

28

366


152

Table 6.5: 1H and 13C NMR data of compound 244 (CDCl3, 300 & 75 MHz)

and 366 (CDCl3, 500 and 125 MHz)



1 38.7 (CH2) 32.1 (CH2) 2 32.6 (CH2) 29.9 (CH2) 3 3.50, (m) 71.4 (CH) 3.97, (m) 67.6 (CH) 4 2.46, (Ha, dd, 12.3, 1.2)

2.28, (Hb, t, 12.0) 40.9 (CH2) 2.39, (Ha, m)

2.08, (Hb, dd, 12.0, 3.0)

38.8 (CH2)

5 _ 142.3 (C) _ 77.1 (CH) 6 5.53, (d, 8.1) 119.9 (CH) _ 199.9 (C) 7 5.47, (d, 8.1) 115.9 (CH) 5.75, (s) 120.7 (CH) 8 _ 139.8 (C) _ 164.0 (C) 9 39.7 (CH) 43.8 (CH) 10 36.5 (C) _ 40.1 (C) 11 20.7 (CH2) 20.8 (CH2) 12 32.6 (CH2) 36.9 (CH2) 13 _ 42.9 (C) 45.0 (C) 14 54.2 (CH) 2.60, (dd, 12.0, 5.6) 54.0 (CH) 15 23.0 (CH2) 23.8 (CH2) 16 34.1 (CH2) 27.6 (CH2) 17 54.6 (CH) 55.9 (CH) 18 0.65, (s) 12.3 (CH3) 0.64, (s) 14.2 (CH3) 19 0.93, (s) 20.0 (CH3) 0.96, (s) 21.1 (CH3) 20 40.5 (CH) 2.05 (1H, m) 40.6 (CH) 21 1.05, (d, 6.5) 19.1 (CH3) 1.01, (d, 6.4) 21.0 (CH3) 22 5.27, (dd, 15.5, 5.1) 133.1 (CH) 5.18, (dd, 15.0, 5.3) 131.7 (CH) 23 5.10, (dd, 15.5, 5.9) 135.8 (CH) 5.10, (dd, 15.3, 5.8) 133.9 (CH) 24 42.9 (CH) 42.6 (CH) 25 33.7 (CH) 34.6 (CH) 26 0.82, (d, 6.4) 21.5 (CH3) 0.87, (d, 6.0) 19.8 (CH3) 27 0.91, (d, 6.5) 16.1 (CH3) 0.91, (d, 6.0) 20.3 (CH3) 28 0.86, (d, 6.5) 22.6 (CH3) 0.83, (d, 6.3) 17.6 (CH3)


153

6.3. Enzyme Inhibitory Activites of the Compounds Isolated from

Aspergillus sp.

The compounds 244, 302-304, 362-366 isolated from our

investigsted Aspergillus sp. were also evaluated for their enzyme inhibitory

potential against the enzymes Acetylcholinesterase, Butyrylcholinesterase

and Lipoxygenase (Table 6.6). The tested compounds were weakly active

against the three enzymes as they showed 45-62% inhibition at a

concentration of 0.5 mM. The higher IC50 values revealed that the natural

isolates were inactive at tested concentration. However, the literature

search revealed that some of these compounds have been reported as

potential bioactive molecules.For example derivatives of ergosterol (244) are

reported to inhibit human neutrophil elastase (Lee et al., 2012). Similarly

prenylted dibenzofuran Sch725421 (362) is reported to show antibacterial

and antifungal properties (Yang et al., 2004).

Cytochalasins are a group of common fungal constituents that bind

to actin and alter its polymerization (Cooper, 1987). Cytochalasins are

known to possess several biological potentials, like inhibition of the division

of cytoplasm, reversible inhibition of cell movement, nuclear extrusion

(Carter, 1967; Krishan, 1972), platelet aggregation and clot retraction

(Shepro et al., 1970; Thorsen et al., 1972), glucose transport (Estensen and

Plagemann, 1972; Kletzien et al., 1972) and thyroid secretions (Williams

and Wolff, 1971). Our isolated compound 365 has been reported to exhibit

antimicrobial (Betina and Micekova, 1972; Betina et al., 1972; Fu et al.,


154

2011) and anticancer activity (Katagiri and Matsuura, 1971). Cytochalasins

received great attention as subject of cytological research, therefore they are

now commercially available, and more cytochalsin derivatives are being

discovered.


155

Table 6.6: Enzyme inhibitory activities* of isolated compounds from Aspergillus sp.

No. AChE (%) AChE (IC50) M

BChE (%) BChE (IC50) M

LOX (%) LOX (IC50) M

244 51.51±0.12 <400 38.14±0.34 <700 40.12±0.54 <700

362 55.63±0.18 <700 47.62±0.25 NIL 62.72±0.14 <500

302 49.26±0.19 NIL 31.72±0.22 NIL 56.09±0.30 NIL

303 56.30±0.24 NIL 41.23±0.18 NIL 37.81±0.44 NIL

304 47.62±0.27 NIL 51.80±0.22 NIL 60.03±0.49 NIL

363 46.90±0.13 NIL 69.02±0.14 NIL 48.19±0.14 NIL

364 45.51±0.10 NIL 60.12±0.14 NIL 51.79±0.14 NIL

365 56.91±0.10 <400 53.48±0.34 <300 50.44±0.1 NIL

366 42.39±0.10 <700 71.29±0.22 <700 45.61±0.1 NIL

Eserine** 91.29±1.17 0.04±0.001 82.82±1.09 0.85±0.001 - -

Baicalein** - - - - 93.79±1.2 22.4±1.3

* All experiments were performed in triplicate

** Standard drugs


156

6.4. Experimental

6.4.1. General experimental procedures

Open column chromatography was carried out using silica gel

(Keiselgel-230-400 mesh, Darmstadt, Germany and Keiselgel-70-230 mesh,

E-Merck) as a stationary phase packed in glass columns with organic

solvents as the mobile phase. Commercially available solvents were used

after distillation at their respective boiling points, for extraction of fungal

material and chromatographic techniques. Chromatographic separations

were monitored by using aluminium sheets precoated with silica gel 60 F254

(20×20 cm, 0.2 mm thick; E-Merck; Darmstadt, Germany). UV light (254

and 366 nm) was used to see fluorescence of UV active compounds on

chromatogram. Ceric sulphate solution was prepared in 65% sulphuric acid

and used as the spraying agent with subsequent heating to locate UV

inactive spots on chromatogram. IR spectra were recorded as KBr pellets on

a Shimadzu 460 infrared spectrophotometer /JASCO 320-A infrared

spectrometer (Duisburg, Germany). The EI-MS, HR-EI-MS, FAB-MS and

HR-FAB-MS were recorded on Finnigan (Varian MAT, Waldbronn, Germany)

JMS H×110 with a data system and JMSA 500 mass spectrometers,

respectively. The 1H NMR and 13C NMR spectra were recorded on a Bruker

(300, 400, 500 and 600 MHz in deuterated solvents for 1H NMR and 75,

100 and 125 MHz for 13C NMR) spectrometer. The chemical shift values (δ)

are reported in ppm and the coupling constant (J) in Hz.


157

6.4.2. Culture, extraction and isolation

The endophytic fungus Aspergillus sp. internal strain no. 9297 B was

isolated from the tissues of the Sea Sponge Geodia lydonium of Spanish Sea

in Adria. 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 2.5 g of the crude extract, which was subjected to column

chromatography over silica gel eluting with gradient of n-hexane:EtOAc and

EtOAc:MeOH to get 5 fractions (A1-A5).

Fraction A5 was purified on silica gel coloumn using n-hexane:EtOAc

(4:6) where 4-acetyl-3,4-dihydro-6,8-dihydroxy-5-methylisocoumarin (302,

17.0 mg) was isolated at same solvent system along with a semi pure

fraction which on further purification using Sephadex LH-20 eluting with

MeOH afforded 2,6-dihydroxy-4-(3-hydroxybutan-2-yl)-3-methylbenzoic

acid (363, 12.0 mg) as a colourless solid and Cytochalasin C (364, 25.0

mg). Silica gel coloumn chromatography of Fraction A4 at n-hexane:EtOAc

(6:4) afforded 2,3,4-Trimethyl-5,7-dihydroxy-2,3-dihydrobenzofuran (304,

18.0mg) and two subfractions (A4a & A4b), Where A4a when purified on

Sephadex LH-20 eluting with MeOH, yielded Cytochalasin D (365, 22.0 mg)

and dibenzofuran Sch725421 (362, 28.0 mg). Fraction A3 yielded 4-acetyl-

6,8-dihydroxy-3-methoxy-5-methyl-3,4-dihydroisocoumarin (303, 21.0mg)

by using Sephadex LH-20 coloumn at pure methanol.


158

Fraction A2 showed three major with few minor spots on TLC, this

fraction was purified on silica gel column eluting with a gradient of n-

hexane:EtOAc (9:1 to 6:4) to get compound ergosterol (244, 32.0 mg) along

with a semi-pure fraction which on passing through Sephadex LH-20

eluting with MeOH yielded 24-methylcholesta-7,22-E-diene-3β,5-diol-6-

one (366, 22.0 mg) (Scheme 6.1).

Aspergillus sp. EtOAc Extract

2.5 g

CC over flash silica using varying polarity of solvents

Fract. A2Fract. A3 Fract. A4 Fract. A5

302, 363, 364

304, 362, 365

244, 366 303

Scheme 6.1: Isolation Scheme of Metabolites from Aspergillus sp.


159

6.5. Spectroscopic Data of the Isolated Compounds

6.5.1. Spectroscopic data of dibenzofuran Sch725421 (362)

Yellow amorphous powder (28.0 mg), UV λmax (MeOH): 214, 272, 313 nm; IR

(KBr): 3460, 1655, 1510, 1470 and 1240

cm-1; 1H NMR (CD3OD, 400 MHz): δ 6.74

(1H, d, J = 1.8, H-5), 6.72 (1H, s, H-4), 6.48

(1H, d, J = 1.8, H-7), 5.09 (1H, d, J = 7.1

Hz, H-2), 3.45 (2H, d, J = 7.1 H-1), 2.87 (3H, s, H-6), 2.34 (3H, s, H-7),

1.80 (3H, s, H-4), 1.67 (3H, s, H-5); 13C NMR (CD3OD, 100 MHz): 159.4

(4b), 155.9 (C-4a), 155.3 (C-3), 153.0 (C-8), 137.3 (C-6), 133.3 (C-1), 131.1

(C-3), 125.0 (C-2), 123.8 (C-2), 117.2 (C-8b), 111.9 (C-8a), 110.9 (C-7),

104.0 (C-5), 95.7 (C-4), 27.0 (C-1), 25.9 (C-5), 20.5 (C-7), 18.1 (C-4), 18.0

(C-6); EI-MS: m/z 296 [M]+; HR-EI-MS: m/z 296.1407 [M]+( 296.1412

calcd. for C19H20O3).

6.5.2. Spectroscopic data of 4-acetyl-3,4-dihydro-6,8-dihydroxy-5-

methyl isocoumarin (302)

Yellow amorphous solid (17.0 mg); UV λmax (MeOH): 214, 272, 313 nm; IR

(KBr); 3415, 1712, 1735, 1510, 1456 cm-1; 1H NMR

(DMSO-d6, 500 MHz): δ 12.0 (1H, s, 8-OH), 10.90

(1H, s, 6-OH), 6.34 (1H, s, H-7), 4.93 (d, J = 12.4 Hz,

Ha-3), 4.60 (dd, J = 12.4, 4.0 Hz, Hb-3), 4.30 (1H, d,

J = 4.0 Hz, H-4), 2.26 (3H, s, H-12), 2.03 (3H, s, H-

13); 13C NMR (DMSO-d6, 125 MHz): 202.5 (C-11), 168.8 (C-1), 163.0 (C-6),

O

OH

Me

Me

OH

362

1

2

454a4b

6

88a 8b

1'

2'

3'

4'

5'

7'

6'

1

45

6

8

O

OMe

HO

OH O

Me

10

9

11

12

13

302


160

159.8 (C-8), 139.4 (C-10), 115.1 (C-5), 103.1 (C-9), 100.7 (C-7), 68.6 (C-3),

48.1 (C-4), 27.6 (C-12), 11.5 (C-13); EI-MS: m/z 236 [M]+; HR-EI-MS: m/z

236.0681 [M]+ (236.0685 calcd. for C12H12O5).

6.5.3. Spectroscopic data of 4-acetyl-6,8-dihydroxy-3-methoxy-5-

methyl-3,4-dihydro isocoumarin (303)

Yellow amorphous solid (21.0 mg), UV λmax (MeOH): 231, 275.1, 315 nm; IR

(KBr); 3425, 1715, 1738, 1512, 1455, 1030 cm-1; 1H

NMR (DMSO-d6, 400 MHz): δ 10.94 (1H, s, 8-OH),

10.90 (1H, brs, 6-OH), 6.37 (1H, s, H-7), 5.81 (1H,

d, J = 1.4, H-3), 4.49 (1H, d, J = 1.4, H-4), 3.46 (s,

3-OMe), 2.30 (3H, s, H-12), 1.97 (3H, s, H-13); 13C

NMR (DMSO-d6, 100 MHz): 203.6 (C-11), 168.2 (C-1), 162.9 (C-6), 160.1

(C-8), 136.3 (C-10), 117.9 (C-5), 103.0 (C-3), 101.2 (C-7), 99.1 (C-9), 56.5

(OMe, C-3), 53.1 (C-4), 30.0 (C-12), 11.7 (C-13). EI-MS: m/z 266 [M]+; HR-

EI-MS: m/z 266.0785 [M]+ (266.0790 calcd. for C13H14O6).

6.5.4. Spectroscopic data of 2,3,4-trimethyl-5,7-dihydroxy-2,3-

dihydrobenzofuran (304)

Brown solid (18.0 mg); UV λmax (MeOH): 222.6, 294.0 nm; IR (KBr); 3433,

1605, 1525, 1458 cm-1; 1H NMR (MeOH, 500 MHz):

δ 6.18 (1H, s, H-6), 4.35 (1H, m, H-2), 2.97 (1H, s,

H-3), 2.02 (3H, s, H-12), 1.31 (3H, d, J = 6.4 Hz, H-

10), 1.27 (3H, d, J = 6.4 Hz, H-11); 13C NMR (DMSO-

d6, 125 MHz): 154.3 (C-5), 139.0 (C-7), 138.5 (C-8), 133.6 (C-9), 112.3 (C-

1

45

6

8

O

OMe

HO

OH O

Me

303

OMe10

9

11

12

13

5

68

10

9

11

12

O

Me

HO

OH

Me

Me

304


161

4), 100.1 (C-6), 87.1 (C-2), 45.0 (C-3), 20.2 (C-10), 19.0 (C-11), 11.2 (C-12).

EI-MS: m/z 194.0 [M]+; HR-EI-MS: m/z 194.0941 [M]+ (194.0943 calcd. for

C11H14O3).

6.5.5. Spectroscopic data of 2,6-dihydroxy-4-(3-hydroxybutan-2-yl)-3-

methyl benzoic acid (363)

Colourless Solid (12.0 mg); UV λmax: 214, 253, 315 nm; IR (KBr): 3420,

3375, 1708, 1515, 1452; 1H NMR (DMSO-d6, 500

MHz): 13.60 (1H, s, 2-OH), 12.91 (1H, s, 6-OH), 11.1

(1H, s, 8-OH), 6.03 (1H, s, H-5), 3.71 (1H, m, H-8), 2.93

(1H, m, H-7), 2.01 (3H, s, H-11), 1.12 (3H, d, J = 6.5 Hz

H-9), 1.04 (3H, d, J = 6.0 Hz, H-10); 13C NMR (DMSO-

d6, 125 MHz): 174.8 (C-12), 160.1 (C-6), 159.7 (C-2), 147.0 (C-4), 111.0

(C-3), 108.7 (C-5), 102.1 (C-1), 70.0 (C-8), 42.3 (C-7), 20.1 (C-9), 16.7 (C-

10), 10.9 (C-11). EI-MS: m/z 240.0 [M]+; HR-EI-MS: m/z 240.0995 [M]+

(240.0998 calcd. for C12H16O5).

6.5.6. Spectroscopic data of cytochalasin C (364)

White amorphous powder (25.0 mg); IR (KBr): 3410, 1730, 1695, 1648,

1590-1455, 1230 and 1030 cm-1; 1H NMR (DMSO-d6, 300 MHz): 8.20 (1H,

s, N-H), 7.31 (2H, t, J = 8.8 Hz, H-3, 5), 7.27 (1H, d, J = 8.8 Hz, H-4), 7.21

(2H, d, J = 8.8 Hz, H-2, 6), 5.83 (1H, d, J = 16.0 Hz, H-20), 5.61 (1H, s, H-

21), 5.58 (1H, dd, J = 15.6, 10.0 Hz, H-13), 5.15 (1H, d, J = 16.0 Hz, H-19),

5.07 (1H, m, H-14), 3.50 (1H, d, J = 8.9 Hz, H-7), 3.14 (1H, m, H-3), 3.08

MeMe

OH

Me

HO OH

OHO

1

2

4

6

363

78

9

10

11

12


162

(1H, d, J = 1.1 Hz, H-4), 3.01 (1H, dd, J = 13.1, 8.9 Hz, Ha-10), 2.76 (1H,

dd, J = 13.1, 5.5 Hz, Hb-10), 2.40 (1H, m,

H-16), 2.32 (1H, m, H-8), 2.23 (3H, s, H-

2), 2.19 (1H, m, Ha-15), 1.92 (1H, m,

Hb-15), 1.61 (3H, s, H-11)1.49 (3H, s, H-

12), 1.35 (3H, s, H-23), and 1.06 (3H, d, J

= 6.4 Hz, H-22); 13C NMR (DMSO-d6, 75

MHz): 208.7 (C-17), 174.6 (C-1), 170.1 (C-1), 136.5 (C-1), 133.0 (C-6),

132.5 (C-14), 131.8 (C-13), 130.4 (C-20), 129.1 (C-2,6), 128.1 (C-19),

127.9 (C-3,5), 127.1 (C-4), 125.3 (C-5), 78.0 (C-18), 75.2 (C-21), 68.1 (C-

7), 59.3 (C-3), 51.2 (C-9), 49.7 (C-4), 48.5 (C-8), 43.8 (C-10), 42.3 (C-16),

37.8 (C-15), 25.1 (C-23), 20.8 (C-2), 19.7 (C-22), 16.8 (C-11) and 14.1 (C-

12); ESI-MS: m/z 530 [M+Na]+; HR-ES-IMS: m/z 530.2513 (calcd. 530.2519

for C30H37NO6Na corresponding to the formula C30H37NO6).

6.5.7. Spectroscopic data of cytochalasin D (365)

White amorphous powder (22.0 mg); IR (KBr): 3440, 1732, 1690, 1650-

1525, 1235 cm-1; 1H NMR (DMSO-d6, 400

MHz): δ 8.12 (1H, s, N-H), 7.30 (2H, t, J=

8.8 Hz, H-3′,5′), 7.21 (1H, d, J= 8.8 Hz, H-

4′), 7.16 (2H, d, J= 8.8 Hz, H-2′, 6′), 5.79

(1H, d, J= 15.6 Hz, H-20), 5.51 (1H, dd,

15

1721

1''

2''

2

3

5

9

12

13

11

19

22

23

1

5´

1'3'

710

HN

MeOH

OOO

Me

O

MeHO

E

E Me

365

15

1721

1''

2''

2

3

5

9

12

13

11

19

22

23

1

5´

1'3'

710

HN

MeOH

OOO

Me

O

MeHO

E

E Me

Me

364


163

J= 16.0, 9.8 Hz, H-13), 5.48 (1H, s, H-21), 5.31 (Ha, s, H-12), 5.16 (1H, d,

J= 15.6 Hz, H-19), 5.09 (Hb, s, H-12), 5.01 (1H, m, H-14), 3.88 (1H, d, J=

9.8 Hz, H-7), 3.20 (1H, d, J= 0.9 Hz, H-4), 3.15 (1H, m, H-3), 2.96 (1H, m,

H-5), 2.92 (Ha, dd, J= 13.4, 9.0 Hz, H-10), 2.65 (Hb, dd, J= 13.4, 5.4 Hz, H-

10), 2.41 (1H, m, H-16), 2.32 (1H, t, J = 9.8, H-8), 2.21 (3H, s, H-2′′), 2.18

(Ha, m, H-15), 1.87 (Hb, m, H-15), 1.35 (3H, s, H-23), 1.15 (3H, d, J= 6.4

Hz, H-11), 1.01 (3H, d, J= 6.4 Hz, H-22); 13C NMR (DMSO-d6, 100 MHz): δ

207.9 (C-17), 172.8 (C-1), 169.8 (C-1′′), 148.7 (C-6), 136.5 (C-1′), 131.0 (C-

13), 130.8 (C-14), 130.4 (C-20), 128.7 (C-2′,6′), 128.6 (C-19), 127.1 (C-3′,5′),

125.8 (C-4′), 76.4 (C-18), 73.8 (C-21), 68.8 (C-7), 60.1 (C-3), 51.6 (C-9),

49.0 (C-4), 48.1 (C-8), 42.5 (C-10), 40.9 (C-16), 38.0 (C-15), 36.7 (C-5), 23.8

(C-23), 21.0 (C-2′′), 19.4 (C-22), 14.0 (C-11); ESI-MS: m/z 530 [M+Na]+; HR-

ESI-MS: m/z 530.2513 (calcd. 530.2519 for C30H37NO6Na corresponding to

the formula C30H37NO6).

6.5.8. Spectroscopic data of ergosterol (244)

White solid, melting point 155-157.5C (32.0 mg); IR (KBr): 3350 (O-H),

3110, 2940 (C-H), 1645 (C=C) cm-1; 1H NMR

(CDCl3, 300 MHz): 5.53 (1H, d, J = 8.1 Hz,

H-6), 5.47 (1H, d, J = 8.1 Hz, H-7), 5.27

(1H, dd, J = 15.5, 5.1 Hz, H-22), 5.10 (1H,

dd, J = 15.5, 5.9 Hz, H-23), 3.50 (1H, m, H-

3), 2.46 (Ha, dd, J = 12.3, 1.2 Hz, H-4), 2.28 (Hb, t, J = 12.0 Hz, H-4), 1.90-

HO

Me

Me

Me Me

Me

Me

1

3 5

6

7

89

10

11 13

14 15

161719

18 20

2122

2425

26

27

28

244


164

1.35 (various methylenes), 1.05 (3H, d, J = 6.5 Hz, H-21), 0.93 (3H, s, H-

19), 0.91 (3H, d, J = 6.5 Hz, H-27), 0.86 (3H, d, J = 6.5 Hz, H-28), 0.82 (3H,

d, J = 6.6 Hz, H-26) and 0.65 (3H, s, H-18); 13C NMR (CDCl3, 75 MHz):

142.3 (C-5), 139.8 (C-8), 135.8 (C-23), 133.1 (C-22), 119.9 (C-6), 115.9 (C-

7), 71.4 (C-3), 54.6 (C-17), 54.2 (C-14), 42.9 (C-13), 42.9 (C-24), 40.9 (C-4),

40.5 (C-20), 39.7 (C-9), 38.7 (C-1), 36.5 (C-10), 34.1 (C-16), 33.7 (C-25),

32.6 (C-2), 32.6 (C-12), 23.0 (C-15), 22.6 (C-28), 21.5 (C-26), 20.7 (C-11),

20.0 (C-19), 19.1 (C-21), 16.1 (C-27), 12.3 (C-18); EI-MS: m/z 396 [M]+,

381, 377, 366, 325, 299, 271; HR-EI-MS: m/z 396.3379 (calcd. 396.3392

for C28H44O).

6.5.9. Spectroscopic data of 24-methylcholesta-7,22-E-diene-3β,5-

diol-6-one (366)

IR (KBr): 3440, 3065, 2940, 1690 and 1615 cm-1; 1H NMR (CDCl3, 500

MHz): δ 5.75 (1H, br, s, H-7), 5.18 (1H, dd,

J = 15.0, 5.3 Hz, H-22), 5.10 (1H, dd, J =

15.3, 5.8 Hz, H-23), 3.97 (1H, m, H-3), 2.60

(1H, dd, J = 12.0, 5.6, Hz, H-14), 2.39 (Ha,

m, H-4), 2.08 (Hb, dd, J = 12.0, 3.0 Hz, H-

4), 2.05 (1H, m, H-20), 1.84-1.28 (various methylenes), 1.01 (3H, d, J = 6.4

Hz, H-21), 0.96 (3H, s, H-19), 0.91 (3H, d, J = 6.0 Hz, H-27), 0.87 (3H, d, J

= 6.0 Hz, H-26), 0.83 (3H, d, J = 6.4 Hz, H-28) and 0.64 (3H, s, H-18); 13C

NMR (CDCl3, 125 MHz): 199.9 (C-6), 164.0 (C-8), 133.9 (C-23), 131.7 (C-

22), 120.7 (C-7), 77.1 (C-5), 67.6 (C-3), 55.9 (C-17), 54.0 (C-14), 45.0 (C-

HO

Me

Me

O

Me Me

Me

Me

HO

1

3 56 7

89

10

11 13

14 15

161718

19 20

2122

2425

26

27

28

366


165

13), 43.8 (C-9), 42.6 (C-24), 40.6 (C-20), 40.1 (C-10), 38.8 (C-4), 36.9 (C-

12), 34.6 (C-25), 32.1 (C-1), 29.9 (C-2), 27.6 (C-16), 23.8 (C-15), 21.1 (C-

19), 21.0 (C-21), 20.8 (C-11), 20.3 (C-27), 19.8 (C-26), 17.6 (C-28), 14.2 (C-

18); EI-MS: m/z 428 [M]+, 409, 357, 331 and 302; HR-EI-MS: m/z

428.3268 (calcd. 428.3290 for C28H44O3).

References

166

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