PHYTOCHEMICAL AND BIOLOGICAL STUDIES OF EUPHORBIA …

PHYTOCHEMICAL AND BIOLOGICAL STUDIES OF EUPHORBIA SERPENS, EUPHORBIA GRANULATA (EUPHORBIACEAE) AND

VERNONIA CINERASCENS (COMPOSITAE)

Thesis submitted For the Fulfillment of the Degree of

DOCTOR OF PHILOSOPHY Pharmacy

By

Irshad Ahmad

FACULTY OF PHARMACY BAHAUDDIN ZAKARIYA UNIVERSITY

MULTAN, PAKISTAN 2009

ACKNOWLEDGMENTS

I

Acknowledgments

First of all, I bow my head to the omnipotent, omnipresent and omniscient Al-Mighty

Allah. As a result of His clemency my success was finally consummated.

Words would barely express my feeling of gratitude for the kind support and valuable

guidance I received from my supervisors, Prof. Dr. Bashir Ahmad Chaudhary,

Dean, Faculty of Pharmacy, Bahauddin Zakariya University, Multan and Prof. Dr.

Khalid Hussain Janbaz, Chairman, Department of Pharmacy, Bahauddin Zakariya

University, Multan.

I am much obliged to Prof. Dr. Anwar-ul-Hassan Gillani, Agha Khan Medical

University, Karachi for providing me excellent facilities in his research laboratory

during my course of work.

I also feel pleasure and honour to express my gratitude to Prof. Dr. M. Iqbal

Chaudhary (S.I., T.I.) and Prof. Dr. Atta-ur-Rehman (N.I., H.I., S.I., T.I.) H.E.J.

Research Institute of Chemistry, University of Karachi, for their kind help regarding

to the technical support.

My humble thanks are extended to Dr. Naheed Riaz Chaudhary, Assistant Professor,

Department of Chemistry, The Islamia University of Bahawalpur for his

encouragement and co-operation throughout my research work.

I am much obliged to Mr. Muhammad Uzair, Assistant Professor, Department of

Pharmacy Bahauddin Zakariya University Multan; without his dedicated help this

study would not have been possible.

I am grateful to all the laboratory staff for their cooperation during all this period.

I am forever grateful to my father and mother, whose foresight and values paved the

way for a privileged education and unconditional support at each turn of my life, my

ACKNOWLEDGMENTS

II

sisters and brothers for their prayers, constant encouragement, love and care, without

which this was quite impossible.

Last but not the least, I wish to thank my family members, very particularly to my

wife, for their well understanding and support throughout the entire period of my

studies. Without their support, this work would not have been possible.

Irshad Ahmad

Dedicated to

my

Parents, Wife and Kids

LIST OF CONTENTS

List of Contents

No. Contents Page

Acknowledgments I

Abstract III

CHAPTER 1: INTRODUCTION

1 Introduction 1 1.1 Importance of natural products 1

1.2 Botanical aspects of the family Asteraceae 7

1.2.1 Botanical aspects of the genus Vernonia 8

1.2.1.1 Botanical classification of Vernonia cinerascens 9

1.3 Botanical aspects of the family Euphorbiaceae 9

1.3.1 Botanical aspects of the genus Euphorbia 10

1.3.1.1 Botanical aspects of Euphorbia granulata 11

1.3.1.2 Botanical classification of Euphorbia granulata 11

1.3.1.3 Botanical aspects of Euphorbia serpens 12

1.3.1.4 Botanical classification of Euphorbia serpens 12

1.4 Important classes of secondary metabolites 12

1.4.1 Steroids 12

1.4.2 Terpenoids 13

1.4.3 Saponins 13

1.4.4 Alkaloids 14

1.4.5 Tannins 14

1.4.6 Glycosides 14

1.4.7 Fatty acids 14

1.4.8 Coumarins 14

1.5 Flavonoids and their biosynthesis 15

1.5.1 Introduction 15

1.5.2 General aspects of Flavonoids biosynthesis 19

1.5.3 Biosynthesis of flavanone 23

1.5.4 Biosynthesis of isoflavone 24

1.5.5 Biosynthesis of flavone 26

1.5.6 Biosynthesis of flavonol 27

1.5.7 Glycosylation 28

LIST OF CONTENTS

1.5.8 Methylation 29

CHAPTER 2: LITERATURE REVIEW 2 Literature review 31

2.1 Literature survey on the biological activities of the genera

Vernonia and Euphorbia

31

2.1.1 Literature survey on the biological activities of the genus

Vernonia

31

2.1.1.1 Antimicrobial activity 31

2.1.1.2 Antimalarial activity 31

2.1.1.3 Immunomodulating properties 32

2.1.1.4 Anticancer activity 32

2.1.1.5 Lipid lowering effect 32

2.1.1.6 Antiulcer activity 32

2.1.1.7 Antidiabetic activity 33

2.1.1.8 Antioxidant and hepatoprotective activities 33

2.1.1.9 Antiarthritis activity 33

2.1.1.10 Analgesic and anti-inflammatory activities 33

2.1.1.11 Cathartic effect 33

2.1.1.12 Anti-leishmanial activity 34

2.1.1.13 Muscle relaxant activity 34

2.1.1.14 Wound healing effect 34

2.1.1.15 Anthelmintic properties 34

2.1.1.16 Mutagenecity, insecticidal and tripanocidal activities 34

2.1.2 Literature survey on the biological activities of the genus

Euphorbia

35

2.1.2.1 Antimicrbial activity 35

2.1.2.2 Antipyretic and analgesic activities 36

2.1.2.3 Antidiarrheal activity 36

2.1.2.4 Molluscicidal and antifeedant activities 37

2.1.2.5 Inhibition of allergic reactions 37

2.1.2.6 Cytotoxicity 38

2.1.2.7 Effect on the cell division 38

2.1.2.8 DNA damaging activity 38

LIST OF CONTENTS

2.1.2.9 Modulatority of multidrug resistant 38

2.1.2.10 Tumor promoting activity 38

2.1.2.11 Proinflamatory activity 39

2.1.2.12 Inhibition action on the mammalian mitochondrial

respiratory chain

39

2.1.2.13 Antidipsogenic activity 39

2.1.2.14 Survival effect on fibroblasts PGE2 inhibition activity 39

2.1.2.15 PEP inhibitory activity 39

2.1.2.16 Urease inhibitory activity 40

2.1.2.17 Angiotensin converting enzyme inhibitig activity 40

2.1.2.18 Other activities 40

2.2 Literature survey on the Phytochemical studies of the genera

Vernonia and Euphorbia

40

2.2.1 Literature survey on the Phytochemical studies of the genus

Vernonia

40

2.2.1.1 Steroids 40

2.2.1.2 Sesquiterpenes 46

2.2.1.3 Diterpenes 50

2.2.1.4 Coumarins 51

2.2.1.5 Flavonoids 51

2.2.2 Literature survey on the Phytochemical studies of the genus

Euphorbia

53

2.2.2.1 Sesquiterpenoids 53

2.2.2.2 Higher diterpenoids 53

2.2.2.2.1 ent-Abietanes 54

2.2.2.2.2 ent-Atisanes 54

2.2.2.2.3 ent-Kauranes 55

2.2.2.2.4 ent-Isopimaranes and ent-Pimaranes 55

2.2.2.2.5 Other diterpenoids 56

2.2.2.3 Lower diterpenoids 56

2.2.2.3.1 Casbanes 57

2.2.2.3.2 Jatrophanes 57

2.2.2.3.3 Lathyranes 59

2.2.2.3.4 Myrsinanes, Cyclomyrsinanes and Premyrsinanes 60

LIST OF CONTENTS

2.2.2.3.5 Jatropholanes 62

2.2.2.3.6 Daphnanes 62

2.2.2.3.7 Tiglianes 63

2.2.2.3.8 Ingenanes 63

2.2.2.3.9 Segetanes 64

2.2.2.4 Triterpenoids 65

2.2.2.4.1 Cycloartanes 66

2.2.2.5 Steroids 66

2.2.2.6 Phenolics 67

2.2.2.7 Flavonoids 68

2.2.2.8 Miscellaneous compounds 68

CHAPTER 3: MATERIALS AND METHODS

3 Materials and Methods 70

3.1 Plant materials 70

3.2 Extraction 70

3.3 Chromatographic studies 70

3.3.1 Analytical 70

3.3.2 Visualization of components on TLC plates 70

3.3.3 High performance liquid chromatography (HPLC) 71

3.4 Isolation 71

3.4.1 Column chromatography 71

3.4.2 Gel chromatography 71

3.4.3 Solvents and chemicals 73

3.5 Preparation of reagents 74

3.5.1 Wagner,s reagent 74

3.5.2 Mayer,s reagent 74

3.5.3 Hager,s reagent 74

3.5.4 Dragendorff,s reagent (solution of Potassium Bismuth Iodide 74

3.5.5 Godine reagent 75

3.6 Preparation of solutions 75

3.6.1 Preparation of dilute HCl 75

3.6.2 Preparation of dilute ammonia solution 75

3.6.3 Preparation of 70 % alcohol 75

3.6.4 Preparation of lead subacetate solution 75

LIST OF CONTENTS

3.6.5 10 M NaOH 75

3.6.6 10 % Ferric chloride solution 75

3.6.7 3.5 % Ferric chloride in glacial acetic acid 76

3.6.8 1 % gelatin solution in 10 % Sodium chloride 76

3.6.9 10 % sulfuric acid 76

3.7 Detection of various classes of secondary metabolites 76

3.7.1 Detection of alkaloids 76

3.7.2 Detection of anthraquinones glycosides 76

3.7.3 Detection of cardioactive glycosides 77

3.7.4 Detection of tannins 77

3.7.4.1 Ferric chloride test 77

3.7.4.2 Gelatin test 77

3.7.4.3 Catechin test 78

3.7.5 Detection of Flavonoids 78

3.8 Biological activities 78

3.8.1 Spasmolytic activity 78

3.8.2 Antifungal assay 79

3.8.3 Antibacterial assay 79

3.8.4 Brine-Shrimp Toxicity Assay 80

3.8.5 Phototoxic Assay 80

3.8.6 Antioxidant activity 80

3.8.7 Actylcholinestrase inhibitory assay 81 3.9 Spectroscopy 81

3.10 Physical and spectroscopical data of the isolated compounds

(A-J)

83

3.10.1 Physical and spectroscopical data of the isolated compound

A (Vernonione)

83


B (Cinerascenone)

84


C (2-Hydroxy-3-methoxy-5-(2′-propenyl)-phenol)

85


D (Vanillic acid)

86


E (Isoferulic acid)

87

LIST OF CONTENTS


F (Caffeic acid)

88


G (Methyl gallate)

89


H (Uridine)

90

3.10.9 Physical and spectroscopical data of the isolated compound I

(3′ -Methylquercetin)

91


J (Quercetin)

92

CHAPTER 4: RESULTS

4 Results 93

4.1 Extraction 93

4.2 Phytochemical Analysis 93

4.3 Biological screening 94

4.4 Isolation of compounds A-J 100

4.4.1 Isolation of compound A 100

4.4.2 Isolation of compounds B-J 100

4.5 Structure elucidation of the isolated compounds 104

4.5.1 Compound A (Vernonione) 104

4.5.2 Compound B (Cinerascenone) 111

4.5.3 Compound C [2-Hydroy-3-methoxy-5-(2′-propenyl)-phenol] 118

4.5.4 Compound D (Vanillic acid) 119

4.5.5 Compound E (Isoferulic acid) 120

4.5.6 Compound F (Caffeic acid) 121

4.5.7 Compound G (Methyl gallate) 122

4.5.8 Compound H (Uridine) 123

4.5.9 Compound I (3′ Methylquercetin) 124

4.5.10 Compound J (Quercetin) 125

CHAPTER 5: DISCUSSION

5. Discussion 126

CHAPTER 6: CONCLUSION

6. Conclusion 129

LIST OF CONTENTS

CHAPTER 7: REFERENCES

7. References 132

LIST OF TABLES

List of Tables

No. Tables Page

1.1 A list of glycosyltransferases and their sources 30

3.1 Solvent systems used for the analysis of dichloromethane extracts of

V. cinerascens

72

3.2 Solvent systems used for the analysis of methanol extracts of V.

cinerascens

73

4.1 Results of the extraction of plants Vernonia cinerascens, Euphorbia

granulata and Euphorbia serpens with different solvents

93

4.2 Result of chemical tests for identification of constituents of V.

cinerascens, E. granulata and E. serpens

94

4.3 Results of spasmolytic activities of dichloromethane and methanol

extracts of V. cinerascens, E. granulata and E. serpens

95

4.4a Results of antifungal activities of dichloromethane and methanol

extracts of V. cinerascens

95

4.4b Results of antifungal activities of dichloromethane and methanol

extracts of E. granulata and E. serpens

96

4.5a Results of antibacterial activities of dichloromethane and methanol


96

4.5b Results of antibacterial activities of dichloromethane and methanol

extracts of E. granulata and E. serpens

97

4.6 Results of brine shrimp lethality bioassay of the different extracts of V. cinerascens, E. granulata and E. serpens

98

4.7 Result of phytotoxicity bioassay of the different extracts of V.


99

4.8 Result of antioxidant bioassay of the different extracts of V.


100

4.9 1H-NMR chemical shift, 1H→13C, HMQC direct correlation (1J) and 1H→13C HMBC long range correlations (2J and 3J) in compound A

107

LIST OF TABLES

4.10 13C-NMR chemical shift assignments of compound A 108

4.11 1H-NMR chemical shift, 1H→13C, HMQC direct correlation (1J) and 1H→13C HMBC long range correlations (2J and 3J) in compound B

114

4.12 13C-NMR chemical shift assignments of compound B 115

LIST OF FIGURES

List of Figures No. Title Page 1.1 Flavonoids having 1,3-diphenylpropane skeleton 15

1.2 Flavonoids having 1,2-diphenylpropane skeleton 16

1.3 Basic skeleton and numbering pattern in flavonoids 17-18

1.4 Basic skeleton and numbering pattern in homoflavonoids 19

1.5 Biosynthesis of p-coumaric acid 20-21

1.6 Biosynthesis of chalcone 22

1.7 Biosynthesis of flavanone 23

1.8 Biosynthesis of isoflavone 25

1.9 Biosynthesis of flavone 27

1.10 Biosynthesis of flavonol 28

4.1 Isolation scheme of compound A from the dichloromethane

extract of the roots of Vernonia cinerascens

102

4.2 Isolation scheme of compound B-J from the dichloromethane

extract of the roots of Vernonia cinerascens

103

4.3 EI-MS Fragmentation pattern of compound A 105

4.4 UV spectrum of compound A 109

4.5 IR spectrum of compound A 109

4.6 1H-NMR spectrum of compound A 110

4.7 13C-NMR spectrum of compound A 110

4.8 EI-MS Fragmentation pattern of compound B 112

4.9 UV spectrum of compound B 116

4.10 IR spectrum of compound B 116

4.11 1H-NMR spectrum of compound B 117

4.12 13C-NMR spectrum of compound B 117

LIST OF PUBLICATIONS

LIST OF PUBLICATIONS

1. Irshad Ahmad, Bashir Ahmad Chaudhary and Khalid Hussain Janbaz.

Cinerascenone, A New Flavonoid from Vernonia cinerascens. J. Chem. Soc. Pak.,

32, 101 (2010).

2. Irshad Ahmad, Bashir Ahmad Chaudhary, Khalid Hussain Janbaz, Muhammad

Uzair and Muhammad Ashraf. Urease Inhibitors and Antioxidants from Vernonia

cinerascens. J. Chem. Soc. Pak., 33, 114 (2011).

3. Irshad Ahmad, Arif-Ullah Khan, Bashir Ahmad Chaudhary, Khalid Hussain

Janbaz, Muhammad Uzair, Muhammad Akhtar, Muhammad Ashraf and Anwarul-

Hassan Gilani. Antifungal and Antispasmodic activities of the extracts of

Euphorbia granulata. J. Med. Plants Res., (in press).

4. Irshad Ahmad, Bashir Ahmad Chaudhary, Muhammad Uzair, Khalid Hussain

Janbaz and Muhammad Ashraf. Vernonione, A New Carvotacetone derivative

from Vernonia cinerascens J. Chem. Soc. Pak., (submitted).

ABSTRACT

III

Abstract The present research work emphasis on the biological and Phytochemical studies of

the medicinal plants Vernonia cinerascens (Compositae), Euphorbia granulata and

Euphorbia serpens (Euphorbiaceae). Dichloromethane and methanol extracts of the

different parts of Vernonia cinerascens, Euphorbia granulata and Euphorbia serpens

were prepared. These extracts were subjected to a battery of biological screening and

it was observed that dichloromethane extracts of Vernonia cinerascens roots and that

of Euphorbia granulata whole plant exhibited significant antifungal activity against

Microsporum canis. Microsporum canis is responsible for tinea capitis and tinea

corporis. Methanol extract of Vernonia cinerascens also showed significant antifungal

activity against Fusarium solani. Fusarium solani is a pathogen responsible for fungal

infections in plant and human. Dichloromethane and methanol extracts of Euphorbia

serpens exhibited moderate antifungal activity against Aspergillus flavus. Methanol

extracts of Vernonia cinerascens and Euphorbia serpens showed antioxidant activity.

All the extracts have dose dependent spasmolytic activity. Dichloromethane extract

of Vernonia cinerascens root was subjected for Phytochemical investigations and

afforded a new monoterpene, (3β-acetoxy-5α-angeloyloxy-7-deoxy-carvotacetone).

Methanol extract of the same plant when subjected for isolation offered a new flavone

(5,4'-dihydroxy-7-(4-hydroxybenzoyl)-3'-methoxyflavone) and eight known

compounds, namely, 2-hydroxy-3-methoxy-5-(2′-propenyl)-phenol, vanillic acid,

isoferulic acid, caffeic acid, methyl gallate, uridine, 3'-methylquercetin and quercetin.

CHAPTER # 1 INTRODUCTION

1

1. INTRODUCTION

1.1 Importance of Natural Products

The use of natural products to cure ailments of mankind is as old as human civilization

and for thousands of year, mineral, plant and animal products were the main sources of

drugs (De Pasquale, 1984). As 20th century, the achievements in advanced technology

and the development of organic chemistry resulted in a preference for synthetic products

as therapeutic agents. This was due to the easy availability of pure compounds and

chances of their structure modifications to produce more potent and safer drugs.

Moreover, during the development of human civilization, the use of plants as drug has

had magical-religious importance and different ideas regarding the concepts of health and

disease existed within each civilization. Obviously, this was totally against the ideas of

developed western civilization. They considered that drugs from natural resources were

for poorly educated or low income people or simply as religious superstition of no

therapeutic importance. However, the importance of natural products is clearly

understood from the discovery of the penicillin as an anti-infective therapy. Plants

contribute about 25% of the drugs prescribed worldwide, 121 such active molecules being

in current use. According to World Health Organization (WHO) list of essential 252

drugs, 11% are from plant origin and a significant number of synthetic drugs are obtained

from natural precursors. A few examples of important drugs obtained from plants are

digoxin (1) from dried leaves of Digitalis lanata (Fam. Scrophulariaceae), quinine (2) and

quinidine (3) from the bark of Cinchona species (Fam. Rubiaceae), Vincristine and

vinblastine (4) from the dried whole plant Catharanthus roseus (Fam. Apocynaceae),

atropine (5) from dried leaves and flowering or fruiting tops of Atropa belladonna (Fam.

Solanaceae) and morphine (6) and codeine (7) from the air dried milky exudates obtained

by incising the unripe capsules of Papaver somniferum (Fam. Papaveraceae). It is

estimated that 60% of anti-tumor and anti-infectious drugs already in the market or under

clinical trial are of natural origin (Yue-Zhong Shu, 1998).


2

O

CH3

CH3

OH

OH

O

O

HO

OO

OO

OH

CH3

OH

CH3

CH3

OHOH

N

N

CH2

H3CO

H

HO

N

N

CH2

H3CO

HO

H

NH

N

OH

CH3

HAcO

N

N

H

H3CO

CH3

H

CH3

AcOOH

OAc

O

O OH

NCH3

O

CH3

N

HO

HO

O

CH3

N

H3CO

HO

1

2

3 4

5 6 7

The economical synthetic procedures for the most of these phytoconstituents can not yet

be developed and are still obtained from wild or cultivated plants. Natural compounds can

be lead compounds, allowing the design and rational planning of new drugs (Hamburger

and Hostettmann 1991). In addition, secondary metabolites played an important role in

pharmacological, physiological and biochemical studies. A few examples of such

secondary metabolites are, muscarine (8) from Amanita muscaria (Fam. Amanitaceae),

physostigmine (9) from dried ripe seed of Physostigma venenosum (Fam. Fabaceae),

yohimbine (10) from the bark of Pausinystalia yohimbe (Fam. Rubiaceae), forskolin (11)

from the roots of Coleus forskohlii (Fam. Lamiaceae), colchicine (12) from dried corm of


3

Colchicum autumnale (Fam. Liliaceae) and phorbol ester (13) from Croton tiglium (Fam.

Euphorbiaceae) (Williamson et al. 1996).

O N

H3C

CH3

CH3

H3C

HO

NN

CH3CH3

CH3

O

O

HN

H3C

H

NH

N

H

H

H3COOC

OH

H O

OH

H3C CH3 OH

OAc

OCH3

CH2

OH

CH3 CH3

NH

H3CO

H3CO

O

H3C

O

OCH3

H3CO

OH

H

H3C

OH

O

H3C

OH

CH3

CH3

HO

89

10 11

1213

OH

H

In recent years, there has been growing interest in alternative medicines and the

pharmacological use of natural products, especially those derived from plants (Goldfrank

et al. 1982, Vulto and Smet 1988, Mentz and Schenkel 1989). This interest in plant drugs

is due to some important reasons. Most of the synthetic medicines are either inefficient or

their incorrect use resulted into side effects. On the other hand, natural products have no

side effects and are harmless. It is estimated that, in 1997, the world market for Over-The-

Counter phytomedicinal products was US$ 10 billions, with an annual growth of 6.5%

(Soldati 1997).


4

The WHO considers phytotherapy in its health programs and suggests basic procedures

for the validation of drugs from plant origin in developing countries. Eastern countries,

like China and India, have a well-established herbal medicines industry and Latin

American countries have been investing in research programs in medicinal plants and the

standardization and regulation of phytomedicinal products, following the example of

European countries, such as France and Germany. In Germany, 50% of phytomedicinal

products are sold on medical prescription, the cost being refunded by health insurance

(Gruenwald 1997). In America, where phytomedicinal products are sold as “health foods”

(Brevoort 1997, Calixto 2000), consumers and professionals have struggled to change this

by gathering information about the efficacy and safety of these products (Israelsen 1997).

The modern social context and economic view of health services, the needs of the

pharmaceutical market and the recognition that research on medicinal plants used in folk

medicine represents a suitable approach for the development of new drugs (Elisabetsky

1987, Calixto 1996). The NCI (National Cancer Institute, USA) has tested more than

50,000 plant samples for anti-HIV activity and 33,000 samples for anti-tumor activity.

However, the potential use of higher plants as a source of new drugs is still poorly

explored. Of the estimated 250,000–500,000 plant species, only a small percentage has

been investigated phytochemically and even a smaller percentage has been properly

studied in terms of their pharmacological properties; in most cases, only pharmacological

screening or preliminary studies have been carried out. It is estimated that 5000 species

have been studied for medical use (Payne et al. 1991). Between the years 1957 and 1981,

the NCI screened around 20,000 plant species from Latin America and Asia for anti-

tumor activity, but even these were not screened for other pharmacological activities

(Hamburger and Hostettman 1991).

Research into, and development of, therapeutic materials from plant origin is a hard and

expensive task. Each new drug requires an investment of around US$ 100–360 millions

and a minimum of 10 years of work, with only 1 in 10,000 tested compounds being

considered promising and only 1 in 4 of these being approved as a new drug. The NCI

had only found 3 plant extracts active against HIV out of 50,000 tested, and only 3 out of

33,000 plant extracts tested were found to have anti-tumor activity (Borris 1996, Turner

1996, Williamson et al. 1996). Quantitative considerations regarding the average yield of

active compounds and the amount of starting crude plant material required for the

discovery, development and launch of a new drug in the market were presented by

McChesney (1995), 50 Kg of raw material are necessary to provide 500 mg of pure


5

compound for bioassays, toxicology, and “in vivo” evaluation; full pre-clinical and

clinical studies can require 2 Kg of pure compounds obtained from 200 ton of raw

material. The process is multi-disciplinary (De Pasquale 1984, Verpoorte, 1989). The

basic sciences involved are botany, chemistry and pharmacology, including toxicology.

Any research into pharmacologically active natural compounds depends on the

integration of these sciences. In any case, a particular discipline should not be seen as

secondary to another. Other fields of knowledge may also be involved if the long path

from plant to medicine is taken into account. Anthropology, agronomy, biotechnology

and organic chemistry can play very important roles. In addition, pharmaceutical

technology is fundamental to the development of any drug, including drugs of plant origin

(Petrovick et al. 1997, Sharapin 1997). Plants can be used as drugs in several ways. They

can be used as herbal decoctions or other home made remedies. When plants are

considered as phytopharmaceutical preparations, they can be used as crude extracts in

pharmaceutical preparations, such as tinctures, fluid extracts, powders, pills and capsules.

For isolation of active constituents, plants can be subjected to successive extraction and

purification procedures. These active compounds can be used directly as a drug; for

example quinine, digoxin and ergotamine, or they can be used as precursors e.g.

diosgenin in semisynthetic processes or as models for total synthesis, e.g. morphine.

The approach for drug development from plant resources depends on the objective.

Different strategies will result in a herbal medicine or in an isolated active compound.

However, apart from this consideration, the selection of a suitable plant for a

pharmacological study is a very important and decisive step. There are several ways in

which this can be done, including traditional use, chemical content, toxicity, randomized

selection or a combination of several criteria (Ferry and Baltassat-Millet 1977, Soejarto

1996, Williamson et al. 1996). The most common strategy is careful observation of the

use of natural resources in folk medicine in different cultures; this is known as

ethnobotany or ethnopharmacology. Information on how the plant is used by an ethnic

group is extremely important. The preparation procedure may give an indication of the

best extraction method. The formulation used will provide information about

pharmacological activity, oral versus non-oral intake and the doses to be tested. However,

certain considerations must be taken into account when the ethnopharmacological

approach of plant selection is chosen. For instance, each ethnic group has its own

concepts of health or illness, as well as different healthcare systems (Elisabetsky and

Posey 1986). The signs and symptoms should be translated, interpreted and related to


6

western biomedical concepts, thus allowing a focused study of a particular

pharmacological property. Selection based on chemical composition uses phylogenetic or

chemotaxonomic information in the search, mainly in certain genera and families, for

compounds from a defined chemical class with known pharmacological activity (Gottlieb

and Kaplan 1993, Souza Brito 1996). The search for highly specific potent drugs for

therapeutic use and, more precisely, as an investigation tool in biological research has

been quite productive in toxic plants. A number of important compounds now used in

research came from toxic plants (Williamson et al. 1996). Observation of the plant’s

environment has led to the isolation of active compounds, mainly anti-bacterial and anti-

insect drugs (Harmburger and Hostettman 1991). Another method of selecting a plant is

that the investigator decides on a well-defined pharmacological activity and performs a

randomized search, resulting in active species to be considered for further study. The

search for anti-tumor drugs is a good example of the use of this strategy. The present

situation of exploitation of the world’s vegetation may lead to the extinction of some

species, which results in the loss of interesting chemical compounds as potential drugs. It

is, therefore, crucial for the developing countries which have the largest natural resources,

preservation of environment while searching for new drugs (Soejarto 1996, Brito and

Nunes 1997, Rouhi 1997). Sensible use of these resources must be based on the amounts

available, ease of access, the possibility of preservation and replanting and the

establishment of priorities in relation to a desirable pharmacological activity. If possible,

consideration should be given to the use of cultivated plants, which allows the production

of homogeneous material, thus guaranteeing chemical homogeneity, and the use of plants

from genetic enhancement projects, which preserve species threatened with extinction

(Labadie 1986). The search for drugs active against tumors, viruses and cardiovascular

and tropical diseases is a priority. The largest research fields, as defined by the number of

publications describing bioactive plant-derived compounds in the last few years, are anti-

tumor drugs, antibiotics, drugs active against tropical diseases, contraceptive drugs, anti-

inflammatory drugs, immunomodulators, kidney protectors and drugs for psychiatric use

(Hamburger and Hostettman 1991). Taxol (14) is isolated from T. brevifolia (Fam.

Taxaceae) and is the most important natural product-derived diterpene with anti-tumor

activity. It is both an example of the importance of natural products and of the complexity

and necessity of finding alternative routes by which it can be obtained.


7

H3C

CH3

CH3

AcO O

CH3

OH

OAcO

OO

HO

O

O

OH

NH

O

14

However, the biggest obstacle to its clinical use is obtaining the material. In order to

produce 2.5 Kg of taxol, 27,000 tons of T. brevifolia bark is required and 12,000 trees

must be cut down. Due to the high demand, this species of Taxus will soon be extinct if

no alternative source of taxol can be developed. An economically possible and technically

realistic alternative is its partial synthesis, in considerable yield, from an analogue as well

as the production of other semi-synthetic analogues (Hamburger and Hostettman 1991,

Wall and Wani 1996).

1.2 Botanical aspects of the family Asteraceae

The family “Asteraceae” is also known by name “Compositae” is a largest angiosperm

family consists of about 1300 genera and 25,000 species distributed all over the world

and in almost all habitats. Most of the members of this family are annual or perennial;

xerophytes, succulents or normal mesophytes; herbs, shrubs, climbers and occasionally

trees. Roots are mostly branched tap that may grow deep into the soil. Sometimes

tuberous adventitious roots are known. The taps of roots are thickened like carrot. Stem

are aerial, tuberous, erect or weak and climbing, cylindrical or angular, woody. Many

species, the stem contain latex. Leaves are radical cauline and remal, exstipulate, petiolate

or sessile, alternate or opposite. In some plants the leaves contains latex (Ali et al. 2008).

Flowers having small flower-shaped head mean capitulum surrounded by an involurce of

one or more whorls of free or connate bracts, unisexual and epigymous. Calyx is 2-3 scale

like outgrowth and hairy it is reduced or even absent in many cases. Corolla is five,

gamopetalous, tubular. Androcium are five, filaments are mostly free, slightly connate;


8

anthers usually long are fused to form a tube around the style. Gynacium are bicarpellay

syncarpus, inferior, unilocular, placentation basal, ovule one, anatropous. Fruits are

cypsela. Seeds are non-endospermic or has a scenty endospermic. The embryo is straight

(Bhattacharyya 2005). The two subfamilies and their genera are as follows:

1) Tubuliflorae: In this subfamily latex vessels are absent, but schizogenous oil ducts are

common. The corollas of the disc-florets are nonligulate. Genera include Senecio (1300

spp.), Xanthium (30 spp.), Ambrosia (30-40 spp.), Zinnia (20 spp.), Helianthus (110

spp.), Dahlia (27 spp.), Helenium (40 spp.), Tagetes (50 spp.), Solidago (100 spp.), Bellis

(15 spp.), Aster (500 spp.), Erigeron (200 spp.), Achillea (200 spp.), Anthemis (200 spp.),

Chrysanthemum (c. 200 spp.), Matricaria (40 spp.), Tanacetum (50-60 spp.), Artemisia

(400 spp.), Blumea (50 spp.), Inula (200 spp.), Doronicum (35 spp.), Calendula (20-30

spp.), Eupatorium (1200 spp.), Arnica (32 spp.), Vernonia (600 spp.), Echinops (100

spp.), Carlina (20 spp.), Arctium (5 spp.), Carduus (100 spp.), Centaurea (600 spp.),

Carthamus (13 spp.), and Gerbera (70 spp.).

2) Liguliflorae: In this subfamily latex vessels are present but volatile oil is rare. All the

flowers have ligulate corollas. Genera include Cichorium (9 spp.), Crepis (200 spp.),

Hieracium (over 1000 spp.), Taraxacum (60 spp.), Lactuca (100 spp.), Scorzonera (150

spp.), and Sonchus (50 spp.) (Evans 2002).

1.2.1 Botanical aspects of the genus Vernonia

The genus vernonia is one of major genus of family Asteraceae which consist of about

600-650 species. Most of these are herbs or shrubs. Leaves are simple, alternate, entire or

toothed. Heads are terminal or axillary, homogamous, cymose or panicled. Involucre

ovoid, globose or hemispheric, equaling or shorter than the flowers; Bracts in many

series, the inner longest. Recepticle naked or pitted, sometime shortly hairy. Corollas all

equal, regular, tubular slender; lobes five, narrow. Anther-bases obtuse. Style-arms

subulate, hairy. Pappus usually two seriate of many hairs, often girt with a row of outer

short hairs or flattened bristles. Achenes striate, ribbed or angled, rarely terete (Mhaskar

et al. 2000).


9

1.2.1.1 Botanical Classification of Vernonia cinerascens

Kingdom Plantae

Sub-kingdom Vascular plants

Division Flowering plant

Supper division Seed plants

Class Dicotyledons

Family Asteraceae

Subfamily Tubuliflorae

Genus Vernonia

Species cinerascens

1.3 Botanical aspects of the family Euphorbiaceae

Dioecious or monoecious often poisonous, prostrate, erect or scandent annual,

biennial or perennial herbs, shrubs or trees, succulent or not, spiny or unarmed,

sometimes with phylloclades, with or without a milky latex or coloured sap.

Indumentum 0 or of simple, branched or stellate hairs or peltate scales, the hairs

sometimes urticating. Leaves usually alternate, sometimes opposite or whorled,

occasionally all 3, green or scarious and squamiform, petiolate or sessile, stipulate or

exstipulate, simple, lobed or compound, entire or toothed, peltate or not, palminerved

or penninerved, glandular or eglandular. Stipules free or connate, sometimes

spathaceous, membranaceous, capilliform, glandular or spiny, subpersistent to readily

caducous. Inflorescences terminal, axillary, lateral or leaf-opposed, cymose, paniculate,

racemose, spicatc or cyathial, or with the flowers fasciculate or solitary. Flowers

unisexual, usually actinomorphic and small to minute. Calyx in both sexes usually of

3-6 imbricate, valvate or open equal or unequal lobes or free sepals, often dissimilar

between the sexes, rarely the 9 calyx turbinate or spathaceous, sometimes accrescent,

minute or 0. Corolla in one or both sexes of 3-6 free (rarely united), subvalvate or

imbricate petals, or petals minute or 0, Disc in the o* flowers of 5-6, occasionally more,

free or united glands, or disc annular, cupular or 0; in the 9 flowers hypogynous, usually


10

annular or cupular, entire or lobed, rarely glands free, sometimes 0. Stamens (1~) 3-

100 (-1000), free or connate, simple, rarely branched, anthers usually 2-locular and

longitudinally dehiscent, erect or inflexed in bud, the cells usually parallel and adnate

to the connective, sometimes free, variously positioned. Pistillode present or 0. Ovary

superior, usually sessile, usually 3-celled; placenta-tion axile, the ovules solitary or

paired in each loculus. Styles usually 3, free or united, erect or spreading, entire, bifid

or laciniate, the inner faces stigmatic. Staminodes sometimes present. Fruit usually

schizocarpic, often dehiscing into 3 (occasionally less or more) bivalved cocci leaving a

persistent columella, or else fruit indehiscent and drupaceous. Seeds 1 or 2 per cell,

or by abortion 1 per fruit, carunculate or not, smooth or variously ornamented and

sculptured, concolorous or variously patterned; endosperm usually copious and

fleshy; embryo straight, radicle superior, cotyledons usually broad and flat (Nasir and

Ali 1986).

A very large family, the sixth largest amongst the Anthophyta, with 300 genera and

6000 or more species, subcosmopolitan but with the strongest representation in the

humid tropics and subtropics of both hemispheres. This family is represented in

Pakistan by 24 genera of which 11 are not native. Genera include Euphorbia (about

2000 spp.), Phyllanthus (about 500 spp.), Mallotus (2 spp.), Ricinus (1 spp.), Croton

(750 spp.), Heva (12 spp.), Jatropha (175 spp.), Manihot (170 spp.), Sapium (120

spp.), Poranthera (10 spp.), Securinega (25 spp.), Aleurites (2 spp.), and Hippomane

(5 spp.) (Evans 2002).

1.3.1 Botanical aspects of the genus Euphorbia

Monoecious herbs, shrubs or trees, often succulent, with milky latex and with a

simple indumentum, when present. Leaves often of 3 types, lower, median and

upper; lower or stem-leaves usually alternate, median or pseudumbel-leaves whorled,

upper or ray-leaves whorld or opposite, free or connate; all or most leaves usually

sessile, rarely shortly petiolate, stipulate or not, simple, entire or toothed, penni or

palminerved. Stipules, when present, minute and subulate, interpetiolar and chaffy,

glandular and sessile, or spiny. Inflorescence a cyathium, wkh 1-9 flower and several

bracteate <3 flowers enclosed in a gland-bearing involucre; cyathia axillary or arranged

pseudodichasially, often in a pseudumbel of radiating 'dichasia'. Involucre usually 5-

lobed, with 1-5 glands alternating with them. Male flowers each consisting of a single

stamen borne directly on its own pedicel; anther-cells subglobose, longitudinally


11

dehiscent. Female flower consisting of a trilocular ovary on a pedicel which usually

elongates in fruit; ovules 1 per cell; styles 3, free or connate at the base, stigmas often

bifid. Fruits 3-celled, dehiscent into bivalved cocci; endocarp woody or cartilaginous.

Seeds often carunculate; testa thin, crustaceous, smooth, ornamented or sculptured;

albumen thick; embryo straight; cotyledons broad, flat. One of the six largest genera

of flowering plants (the others being Astragalus, Qirex, Piper. Senecio and

Solatium), having some 2000 species. It is ± cosmopolitan, but chiefly restricted to

tropical, subtropical and warm temperate regions (Nasir and Ali 1986).

1.3.1.1 Botanical aspects of Euphorbia granulata

E. granulata is a glabrous much-branched prostrate annual or perennial herb with

stems to 20 cm long. Petioles 0.5 mm long. Leaf-blades obo vale-oblong, 1-8 × 0.5-5

mm, rounded or emarginate at the apex, obliquely rounded at the base, entire or almost

so, thick and slightly fleshy, rugulose when dry, glaucous. Stipules subulate, 0.5 mm

long. Cyathia axillary, solitary. Glands transversely ovate, yellowish or ochreous,

sometimes reddish, with unequal, subentire white or pink appendages. Fruits

trigonous, keels carinate, 1.1 × 1.1 mm, smooth, cither pubescent all over or else on

the keels only, with simple hairs. Seeds narrowly ovoid-cylindric, quadrangular, 0.8

× 0.4 mm, irregularly foveolate-rugulose, pinkish-grey (Nasir and Ali 1986).

1.3.1.2 Botanical Classification of Euphorbia granulata

Kingdom Plantae

Sub-kingdom Vascular plants



Class Dicotyledons

Family Euphorbiaceae

Genus Euphorbia

Species granulata


12

1.3.1.3 Botanical aspects of Euphorbia serpens

A completely glabrous much-branched prostrate annual herb with stems to c. 25 cm long,

rooting at the nodes. Petioles 0.3-1 mm long. Leaf-blades subor-bicular-ovate, 1-5 × 1-4.5

mm, rounded to emarginate at the apex, obliquely shallowly cordate at the base, entire,

pale green. Stipules interpetiolar, fused above and beneath to form a triangular laciniate or

fimbriate white scale 0.5 mm long. Cyathia axillary, solitary, in the uppermost axils;

glands transversely ovate or oblong, purplish, with narrow, subentire white appendages.

Fruits trigonous, keels carinate, 1.2 × 1.5 mm, smooth. Seeds ovoid, quadrangular, 0.8 ×

0.5 mm, smooth, pinkish -brown.

Distribution: Native of the Americas, introduced into the Mediterranean, S. W. Asia and

Africa. On sandy clay in moist situations; near sea-level to 1640/500m (Nasir and Ali

1986).

1.3.1.4 Botanical Classification of Euphorbia serpens

Kingdom Plantae

Sub kingdom Vascular plants



Class Dicotyledons

Family Euphorbiaceae

Genus Euphorbia

Species serpens

1.4 Important classes of secondary metabolites

1.4.1 Steroids

Steroids (Greek stereos = solid) are compounds possessing a characteristic tetracyclic

carbon skeleton, the perhydrocyclopentano phenanthrene nucleus. They include a


13

wide range of naturally occurring compounds like sterols, the bile acids, the sex

hormones, the adrenocortical hormones, the cardiac glycosides, the sapogenins and

some alkaloids. The sterols, the bile acids, the sex hormones and the adrenocortical

hormones have a number of functions in human physiology and are of immense

biological importance.

1.4.2 Terpenoids

The terpenoids are defined as natural products whose structures may be devided into

isoprene units; hence, these compounds are also called isoprenoids. During the

formation of terpenoids, the isoprene units are usually linked in a head to tail manner,

and the number of units incorporated into a particular unsaturated hydrocarbon

terpenoid serves as a basis for the classification of these compounds.

Hemiterpenoids are made of one isoprene unit, have the formula C5H8 and are the

simplest of all terpenoids.

Monoterpenoids are composed of two isoprene units, have the molecular formula

C10H16, and are the major component of many essential oils and, as such, have

economic importance as flavors and perfumes.

Sesquiterpenoids contain three isoprenes units, have the molecular formula C15 H24 and

exist in aliphatic, bicyclic, and tricyclic framework.

Diterpenoids are a widely varied group of compounds based on four isoprene units and

have the molecular formula, C20H32.

Triterpenoids are based on six isoprene units and have the formula C30H48. They are

biosynthetically derived from squalene, often high-melting colorless solids and are widely

distributed among plant resins, cork, and cutin.

1.4.3 Saponins

Saponins are high molecular weight triterpene glycosides, containing a sugar group

attached to either a sterol or other triterpenes. They are widely distributed in the plant

kingdom. Typically, they have detergent properties, readily form foams in water, have a

bitter taste, and are piscicidal.


14

1.4.4 Alkaloids

Alkaloids are classically defined as being plant-derived, pharmacologically active,

basic compounds derived from amino acids that contain one or more heterocyclic

nitrogen atoms.

1.4.5 Tannins

Tannins are water-soluble oligomers, rich in phenolic groups, capable of binding or

precipitating water-soluble proteins. Tannins may be divided into two groups: either

condensed tannins or hydrolysable tannins. Condensed tannins are formed

biosynthetically by the condensation of flavanols to form polymeric networks.

Hydrolyzable tannins are esters of a sugar (usually glucose) with one or more

trihydroxybenzenecarboxylic acids (gallic acid).

1.4.6 Glycosides

Glycosides are compounds that yield one or more sugars among the products of

hydrolysis. The nonsugar component of a glycoside is known as the aglycone; the

sugar component is called the glycone.

1.4.7 Fatty acids

Fatty acids are carboxylic acids having hydrocarbons chain of 4 to 36 carbons. In

some acids chains are fully saturated; while others contain one or more double bonds.

A few of them contain hydroxyl groups. In higher plants and animals the predominant

fatty acids residues are palmatic, oleic, linoleic and stearic acids. Fatty acids are

obtained by the hydrolysis of triacylglycerols.

1.4.8 Coumarins

Coumarin is 5,6-benzo-2-pyrone and it along with its various derivatives occurs

abundantly in plants of different families. The name coumarin is derived from the

Caribbean word “Coumarou” for the tonka tree from which coumarin with the

characteristic aroma of new-mown hay was isolated. Unsubstituted coumarin is

widespread in nature. Most of these naturally occurring lactones are oxygenated at C-7

position and hence 7-hydroxy coumarin, umbeliferone is regarded as the parent

compound.


15

1.5 Flavonoids and their Biosynthesis

1.5.1 Introduction

The flavonoids are one of the most diverse and widespread group of natural products,

occupy a prominent position among the natural phenols commonly present in plants. The

name “flavonoid” is obtained from Greek word “flavus” means yellow. These are

important biologically active compounds and about 2% of all the carbon

photosynthesized is converted into Flavonoids (Harbone 1988). Flavonoids are the

coloring co-pigment of the plants (Zechmeister 1957). The wide range of shades and

colors in flowers are generally due to the presence of flavonoids. These usually occur as

pigments in the cells of flower tissues (Manitto et al. 1981). Flavonoids occur in a variety

of structural forms. All contain fifteen carbon atoms in their parent nucleus and share the

common structural feature of two phenyl rings linked by a three-carbon chain (diphenyl

propane derivatives). The compounds possessing a 1,3-diphenylpropane skeletons are

regarded as chalconoids.

O

Chalconoids

O

A C

B

Flavan

(a)

O

O

A

B

C

Auronoids

O

A C

B

O

Flavonoids

Figure 1.1: Flavonoids having 1,3-diphenylpropane skeleton

The three-carbon chain may be formed into a third five or six membered ring through

oxygen on one of these phenyl rings producing a tricycle system. The tricycle compounds

possessing a five membered heterocyclic ring are referred as auronoids, whereas those

possessing a six membered heterocyclic ring are designated as Flavonoids (Figure 1.1).


16

The tricycle compounds derived from 1,2-diphenylpropane system are known as

isoflavonoids (Figure 1.2).

O

A B

C

O

Isoflavan Isoflavonoid

Figure 1.2: Flavonoids having 1,2-diphenylpropane skeleton

O

__________________________________________________

In tricycle compounds of the flavonoid, auronoid and isoflavonoid types, rings are labeled

A, B, C and the individual carbon atoms are referred by a numbering system which

utilizes ordinary numerals for the A and C rings and primed numerals for the B ring

(Figure 1.3). Some authors refer to carbon 9 and 10 in flavonoids as 8a and 4a,

respectively. In chalcones, the A ring normally written to the left, is given primed

numbers, while the B ring carbons are given ordinary numbers (Figure 1.1).

Natural flavonoinds and isoflavonoids are usually oxygenated and bear hydroxyl and/or

methoxy substituents. The structure and numbering of some common naturally occurring

classes of monomeric Flavonoids (Figure 1.3).


17

O

O

O

OH

O

OH

O

O

O

O

O

O

O

O

OH

O

O

O

O

Flavanone

1 2

34

5

6

7

89

10

1'

2'

3'

4'

5'

6'

Flavan-3-ol

Flavanol

Anthocynanidin

Flavone

Isoflavanone

Flavonol

Isoflavone

IsoflavanFlavan

Figure 1.3: Basic skeleton and numbering pattern in flavonoids


18

1

2

3

4

5

6

1'

2'

3'

4'

5'

6'

a

b

b'-Chalcanol

OH

b'-Chalcanone

O

a-Chalcanol

OH

b-Chalcanol

O

HO

O

O

Chalcan-1,3-dione

O

O

H1 2

34

5

6

7

89

10 1'

2'

3'

4'

5'

6'

Aurone

O

O

H

OH

Aurononol

O

O

H

Isoaurone

O

b'

Chalcone

Figure 1.3: Basic skeleton and numbring pattern in flavonoids (continued)


19

Homoflavonoids are the class of flavonoids which contain an additional carbon in their

skeleton (Figure 1.4). This additional carbon is designated as C-11. A large number of

flavonoids occur as O-glycosides in which one or more of the hydroxyl groups of the

flavonoid are bound to a sugar or sugars via an acid labile hemiacetal bond. In flavonoid

with C-glycosides (Harbone et al. 1975, Harbone and Mabry 1982), the sugar is C-linked

and this linkage is acid resistant. The effect of glycosylation renders the flavonoid less

reactive and more water soluble.

O

O

1 2

2'

345

6

7

89 1'

3'

4'

5'

6'10

11

Homoflavone

O

O

Homoisoflavone

O

O

Homoisoflavanone

O

Homoisoflavan

OO

O

Rotenoid

12

34

5

6

7

89

10

11

1'2'

3'

4'

5'

6'

OO

O

Dehydrorotenoid

Figure 1.4: Basic skeleton and numbering pattern in homoflavonoids

1.5.2 General aspects of flavonoids biosynthesis

The biosynthesis of all flavonoids involve the formation of a C-15 intermediate known as

chalcone. The central role of chalcones in flavonoid biosynthesis has been attested in a

number of investigations (Grisebach et al. 1965). The formation of chalcone is catalyzed

by chalcone synthase, which is the key enzyme of flavonoid biosynthesis. The precursors

for chalcone formation are malonyl-CoA and 4-coumaroyl-CoA (hydroxycinnamic acid

CoA ester) (Figure 3.7). Both flavonoid precursors are derived from carbohydrates.

Malonyl-CoA is synthesized from the glycolysis intermediate acetyl-CoA and carbon

dioxide, the reaction being catalyzed by acetyl-CoA carboxylase. The synthesis of 4-

coumaroyl-CoA is more complex and involves the shikimate pathway, which is the main


20

route to aromatic amino acid, phenylalanine and tyrosine in higher plants (Figure 1.5).

The biosynthesis of shikimic acid begins with the condensation of D-erythrose-4-

phosphate and phosphoenolpyruvic acid (Figure 1.5) (Manitto et al. 1981).

O

OH

OH

CO2

H2O

O

OH

OH

HO CO2

H2O

H

H

O2C

O P

NADPH

PO

CO2

O

HO

OH

OH

NADP

HO H

CHPOH2C

H

OH

O

H

CO2

O

HO

OH

OH

HO

OH

OH

CO2

H

D-Erythrose-4-phosphate Phosphoenol pyruvic acid (PEP)

..

Pi

..

Syn elimination

Shikimate

D-Glucose + CO2

Pentose phosphate cycle

Glycoslysis

Figure 1.5: Biosynthesis of p-coumaric acid


21

OH

O

CO2

CO2

PO

OH

O

CO2

CO2

HPO CO2

PO

OH

OH

CO2

CH2COCO2

OH

CO2

OH

H2CCO2

NH3

NH3

OH

H2CCO2H

NH2

NH2

NADH

NAD

H

OH

O2C CH2COCO2

CH2COCO2

O

O

O

ATP

OH

HCCO2H

H

HO

OH

OH

CO2

..

PEP

Chorismate

p-coumaric acid

Pi

PiShikimate

Figure 1.5: Biosynthesis of p-coumaric acid (continued)


22

CH3 C

O

SCoA

OH

HO

O

CoASHH2OHOOC

SCoA

O

OH

CoAS

O

O

OO

O SCoA

O

CO2

O

O

O

SCoA

OH

O

OH

HO

O

OH

OH

Chalcone Synthase

Chalcone

GLYCOLYSIS

Acetyl-CoA

3

Malonyl-CoA

p-coumaric acid

Figure 1.6: Biosynthesis of chalcone


23

1.5.3 Biosynthesis of Flavanone

Flavanones are formed from chalcones by isomerization. There is a good evidence for the

in vitro and in vivo existence of equilibrium between flavanones and the corresponding

chalcones (Manitto et al. 1981). The interconversion between chalcones and flavanones is

catalyzed in vivo by chalcone isomerase. The stereospecificity of this enzymatic reaction

is apparent in the (S) chirality of C-2 in flavanone derivative.

O

OOH

HO

OHH

H A

B..

Chalcone

BH

OH

HO

OH O

H

H OH*

A..

OH

O

H

HO

OH O

OH

*

Flavanone

Figure 1.7: Biosynthesis of flavanone

Therefore, it is not accidental that all the flavanones found in nature have the (S)

configuration at C-2 and are levorotatory. The chalcones having two free hydroxyl groups

at C-2 and C-6, the equilibrium is shifted completely in an aqueous solution and rapidly

to the flavanone (Figure 1.7). The stabilization energy of the strong hydrogen bond

between the carbonyl group and the ortho-phenolic hydroxyl group greatly influence the

position of equilibrium and the interconversion rate.


24

1.5.4 Biosynthesis of Isoflavone

The key step in isoflavone formation is the 2,3-migration of the aryl side chain of a

flavanone chalcone intermediate. An enzyme catalyzing this transformation was recently

found in microsomal preparations from elicitor-challenged soybean cell suspension

cultures. It transforms (2S)-naringenin into genistein (Figure 1.8).

It was found that two enzymatic steps are involved in this transformation. The first step

comprises of oxidation and rearrangement of naringenin to 2-hydroxy-2,3-

dihydrogenistein. This step is strictly dependent on NADPH and molecular oxygen. The

second enzyme which catalyses the elimination of water from the 2-hydroxyisoflavanone

is identified but has not yet been characterized (Kochs et al. 1986).

Due to the following reasons, (2S)-flavanones and not the chalcones are very probably the

actual substrates: (a) a stereospecific incorporation of (2S)-naringenin into biochanin A

(5,7-dihydroxy-4′-methoxyisoflavone) (Patschke 1966). (b) Only the (2S) but only the

(2R) enantiomer acts as a substrate in vitro” (Kochs et al. 1986). (c) “The equilibrium of

4,2′,4′,6′-tetrahydroxychalcone is at least 1000:1 in favor of the flavanone (Boland et al.

1975). The equilibrium of 4,2′,4′,6′-tetrahydroxychalcone is flavanone to isoflavone

(Figure 1.8) is consistent with the participation of NADPH and molecular oxygen.


25

O

OH

HO

OH

O

OHO

HO O

O

H2O

HB Enz

O

O

HO

OH

OH

OH

H2O

-H2O

H

O

HO

HO

O

H

HOO

O

O

HO

OHOH

O

OH

HO

OH

OH

NADPH

[O]B

EnzNaringenin

..

..

2-Hydroxydihydrogenistein Genistein(Isoflavone)

Figure 1.8: Biosynthesis of isoflavone

The sequence of events may be initiated by an epoxidation. Protonation and subsequent

cleavage of the epoxide would render a positive charge to the B-ring. Keto-enol

tautomerism, as indicated by proton exchange at C-3 in flavanones allows homoallylic

interaction between C-3 and C-1′, and rearrangement of the structure then takes place

(Grisebach and Zilg 1968). Addition of hydroxyl ion to C-2 leads to the 2-

hydroxyisoflavanone intermediate, which is transformed to the isoflavone by elimination

of water molecule.


26

1.5.5 Biosynthesis of Flavone

The conversion of flavanones to flavones was first observed in parsley plants. The

reaction has been studied in more detail in parsley cell suspension culture and in

Antirrhinum flowers (Britsch et al. 1981, Stotz and Forkmann 1981).

The parsley enzyme requires 2-oxoglutarate, Fe2+ and possibly ascorbate as co-factors.

Ascorbate stimulates this and other 2-oxoglutarate dependent dioxygenases involved in

the flavonoid pathway (Britsch and. Grisebach 1986). In Antirrhinum (Stotz. and

Forkmann 1981) and in flavone producing flowers including Verbena (Stotz et al. 1975),

Dahlia, Streptocarpus and Zinnia (Forkmann and Sartz 1984), reduction of flavanone to

flavone is catalyzed by a microsomal enzyme requiring NADPH as co-factor. Although

mutants recessive with respect to falvone formation are not known, yet, evidence for the

enzyme being involved in flavone formation is provided by the fact that flowers of plants

that naturally lack flavones are also devoid of this NADPH dependent microsomal

enzyme activity.

Both the parsley and the flower enzyme catalyzed the reaction from (2S)-naringenin to

apigenin (Figure 1.9). The mechanism of double bond formation is still unclear. It has

been suggested that 2-hydroxyflavanone is formed in the first step, and water is then

eliminated via a dehydratase (Britsch et al. 1981, Stotz and Forkmann 1981). However,

no such 2-hydroxy intermediate has yet been observed even with a nearly homogenous

enzyme protein (Britsch and. Grisebach 1986). 2-Hydroxyflavones certainly exist as plant

metabolites and they are indeed, the substrates in C-glycosylflavones formation (Kerscher

et al. 1987).


27

O

OHO

HO

OH

[O]

O

OHO

HO

OH

H2 H2O

O

OHO

HO

OH

OH

Naringenin

Apigenin (Flavone)

Figure 1.9: Biosynthesis of flavone

1.5.6 Biosynthesis of Flavonol

Enzymatic conversion of dihydroflavonols to flavonols was first observed with enzyme

preparations from parsley cell suspension cultures (Britsch et al. 1981). Synthesis of

flavonols was found to be catalyzed by a soluble 2-oxoglutarate dependent oxygenase.

Flavonol synthesis, most probably, proceeds via a 2-hydroxy intermediate such as 2-

hydroxydihydrokaempferol with subsequent dehydration, giving rise to the respective

flavonols (Figure 1.10). Flavonol synthase has also been demonstrated in flower extract

of Matthiola (Spribille and Forkmann 1984) and Petunia (Forkmann and Sartz 1984). As

in parsley, flavonol formation in these flowers is catalyzed by 2-oxoglutarate dependent

dioxygenase.


28

O

OHO

HO

OH

Naringenin

O

OHO

HO

OH

OH

Dihydrokaempferol

[O]

[O]

O

OHO

HO

OH

OH

OH

2-Hydroxydihydrokaempferol

-H2OO

OHO

HO

OH

OH

Kaempferol (Flavonol)

Figure 1.10: Biosynthesis of flavonol

1.5.7 Glycosylation

The vast number of flavonoid glycosides found in nature suggests the occurance of a

great range of glycosyltransferases with varying substrate specificities (Harborne et al.

1975, 1982, Stumpf and Conn 1981).

Novel Flavonol O-glycosyltransferases were demonstrated in enzyme preparations from

Tulip anthers (Kleinehollenhorst et al. 1982), Pisum flowers, Chrysosplenium shoots

(Bajaj et al. 1983, Khouri et al. 1986) and Anethum cell cultures (Mohle et al. 1985).

These enzymes exhibit a pronounced specificity with regard to the substrate, the position

and the sugar transferred. The isoflavone 7-O-glucosyltransferase isolated from Cicer

shows a similar high specificity. Two enzymes isolated from Chrysosplenium extracts

glycosylate the B-ring of highly methoxylated flavonols in the 2′ and 5′ positions. A

particular interesting situation has been found during extensive genetic and biochemical

studies of the glycosylation of isovitexin (6-C-glucosylapigenin) in Silene paratensis and

Silene dioica (Steyns et al. 1984). Eleven functional alleles, spread over six loci, have

now been identified. These codes for different glycosyltransferases which catalyzed


29

glycosylation, of the 7-hydroxyl group or glycosylation of the 2-hydroxyl group of the

carbon bound glucose of isovitexin. Different glycosyltransferases and their sources are

listed in (Table 1.1).

1.5.8 Methylation

Many enzymes catalyzing a methyl transfer from S-adenosylmethionine to the various

hydroxyl groups of flavonoid substrates (Harborne and Mabry. 1982). Some of them

require Mg+2 as an obligatory co-factor. S-andenosylhomocysteine formed in the reaction

is an inhibitor of these enzymes. An 8-hydroxyflavonol-8-O-methyltransferase was

reported from Lotus corniculatus flowers. Lotus flowers also contain 3- and 3′-O-

methyltransferases, which have been shown to methylate the flavone and flavonols in the

relevant positions (Jay et al. 1985).


30

Table 1.1. A list of glycosyltransferases and their sources

S. No. Source S. No. Enzyme

1 Silene paratensis

(Petals, green parts)

a

b

c

d

2′′-O-Xylosyltransferase

7-O-Galactosyltranferase

7-O-Glucosyltranferase

2′′-O-Rhamnoside glucosyltransferase

2 Cicer arietinum

(Roots)

a 7-O-Glucosyltransferase

3 Chrysosplenium

americanum (shoots)

a Flavonol 2′ and

5′-O-Glucosyltransferase

4 Anethum graveolens

(Cell cultures)

a 3-O-Glucuronosyltranseferase

5 Pisum sativum

(Flowers)

a

b

c

3-O-Glucosyltranferase

3-O-Glucoside glucosyltransferase

3-O-Diglucoside glucosyltransferase

6 Tulipa (Anthers) a

b

c

3-O-Glucosyltransferase

3-O-Glucoside rhamnosyltransferase

3-O-Glucoside xylosyltransferase

7 Matthiola incana

(Flowers)

a

b

c

3-O-Glucosyltransferase

3-O-Glucoside xylosyltransferase

3-O-Glycoside 5-O-glucosyltransferase

CHAPTER # 2 LITERATURE REVIEW

31

2. LITERATURE REVIEW

2.1 Literature survey on the biological activities of the genera Vernonia and

Euphorbia

2.1.1 Literature survey on the biological activities of the genus Vernonia

The genus Vernonia is enriched with pharmacological properties. Various species of

genus Vernonia are used for the treatment of schistomiasis, amoebic dysentery,

gastrointestinal problems, malaria, venereal diseases, wounds, hepatitis, diabetes,

colic, fever, stomachache, toothache, cough, nasal and bronchial pain. The previous

report regarding pharmacological properties of the genus Vernonia are given here

with title.

2.1.1.1 Antimicrobial activity

Sesquiterpenes vernolide and vernodalol isolated from V. amygdalina reported to

exhibit significant activity against five gram positive bacteria. In antifungal test,

vernolides exhibited potent activity with LC50 values of 0.2, 0.3 and 0.4 mg/ml

against Penicillium notatum, Aspergillus flavus, Aspergillus niger and Mucor

hiemalis, respectively. Vernodalol showed moderate inhibition against Aspergillus

flavus, Penicillium notatum and Aspergillus niger with LC50 values of 0.3, 0.4 and 0.5

mg/ml, respectively (Erasto et al. 2006). Vernodalin and vernolepin isolated from the

same plant are used as antibacterial and antifungal agent (Al Magboul et al. 1997).

The sesquiterpens isolated from V. colorata and V. fastigata have also been reported

as antibacterial agent (Rabe et al. 2002, Roos et al. 1998). Zaluzanin D isolated from

the aerial parts of V. arborea showed 100% inhibition in mycelial growth of

Rhizoctonia solani. The effect being ca. 75% with Curvularia lunata and Botrytis

cinerea at 200 ppm concentration. The moderate activity (60%) was observed at 200

ppm with Colletotrichum lindemuthianum, Fusarium equisetii, and F. oxysporum at

lower concentrations (Kumari et al. 2003).

2.1.1.2 Antimalarial activity

Sesquiterpene lactones isolated from V. cinerea and methanolic extract of V. lasiopus

are used for the treatment of malaria. The aerial parts of V. chlorata extracts showed

significant antiplasmodial activity with IC50 < than 5 µg/ml (Kaou et al. 2008). The

leaves of V. staehelinoides showed in vitro activity against the chloroquine sensitive


32

and the chloroquine resistant strains of Plasmodium falciparum with IC50 value 3

µg/ml (Pillay et al. 2007). The extracts of V. lasiopus, singly combined with

chloroquine and tested against the multi-drug resistant P. falciparum isolate, exhibited

IC50 values 5 mg/ml (Muregi et al. 2003).

2.1.1.3 Immunomodulating properties

The ethanol extract of leaves of V. amygdalina showed significant

immunomodulating activity with screening doses of 25, 50 and 100 µg/ml using

luminal/lucigenin-based chemiluminescence assay (Kokoa et al. 2008).

2.1.1.4 Anticancer activity

Vernolide A and B isolated from V. cinerea demonstrated potent cytotoxicity against

human and Hela tumor cell lines while vernolide B has marginal cytoxicity (Kuo et

al. 2003). Vernobockolides B, piptocarphin C, piptocarphin F, piptocarphin A and

hirsutolide isolated from V. bockiana showed strong cytotoxicity against mouse

lymphoid tumor cell line with IC50 values of 1.81, 1.32, 0.77 and 0.73 μm,

respectively (Huo et al. 2008). Glaucolides M isolated from leaf extract of V.

pachyclada showed significant activity in the human ovarian cancer cell line, with the

IC50 value of 3.3 μm (Williams et al. 2005). The dichloromethane fraction of V.

scorpioides totally inhibited tumor development in direct contact with tumor cells

(Pagno et al. 2006). Water-soluble extract of leafs of V. amygdalina potently inhibited

DNA synthesis in a concentration-dependent fashion both in the absence and presence

of serum (Izevbigie 2003).

2.1.1.5 Lipid lowering effect

The methanol extract of V. amygdalina showed lipid-lowering effects in rats

compared with a standard hypolipidemic drug, questran. The treatment with extract of

V. amygdalina at doses of 100 and 200 mg/kg caused a dose dependent reduction in

the plasma and post mitochondrial fraction cholesterol by 20%, 23% and 23%, 29%,

respectively similar reduction in cholesterol levels in questran-treated rats

(Adaramoye et al. 2008).

2.1.1.6 Antiulcer activity

Methanol and chloroform extracts of the aerial parts of V. polyanthes showed

significant antiulcer activty. The methanol (250 mg/kg) and chloroform extracts (50


33

mg/kg) significantly inhibited the gastric mucosa damage 64% and 90%, respectively

(Barbastefano et al. 2007).

2.1.1.7 Antidiabetic activity

In Nigeria V. amygdalina is the most traditionally used antidiabetic herb (Gbolade

2009). The acetone extract of the leaves of V. colorata induced significant decrease of

blood glucose in normoglycaemic rats with dosage of 100 mg/kg (Sy et al. 2005).

2.1.1.8 Antioxidant and Hepatoprotective activities

An aqueous extract of the leaves of V. amygdalina resulted in a dose-dependent (50-

100 mg/kg) reversal of acetaminophen-induced alterations of all the liver function

parameters by 51.9-84.9%. Suppression of acetaminophen-induced lipid peroxidation

and oxidative stress by the extract was also dose-dependent (50-100 mg/kg). This

plant elicits hepatoprotectivity through antioxidant activity on acetaminophen-induced

hepatic damage in mice (Iwalokun et al. 2006).

2.1.1.9 Antiarthritis activity

The flower extract of V. cinerea showed that the adverse physical, biochemical and

histopathological changes in arthritic animals (Latha et al. 1998).

2.1.1.10 Analgesic and anti-inflammatory activities

V. cinerea showed a significant analgesic effect in acetic acid-induced writhing

response and mechanical-induced pains. The analgesic effects of the extracts were

higher than that of acetylsalicylic acid, but lower when compared to morphine. The

methanol and ether extracts showed more potent analgesic activity than that of the

chloroform extract. The aqueous extract of the leaves has peripheral and central

analgesic properties (Njan et al. 2008). The chloroform, methanol and ether extracts

of leaves showed a potent and significant suppressant activity on acute inflammatory

model of carragenin-induced paw oedema in rats (Iwalewa et al. 2003). In the chronic

model the methanolic extract exhibited significant anti-inflammatory activity

(Mazumder et al. 2000).

2.1.1.11 Cathartic effect

The methanol extract of the leaves of V. amygdalina exhibited significant promotion

of intestinal motility on charcoal meal test in mice. Frequency of defecation of faeces

was markedly increased following administration of the extract which also promoted


34

gastric emptying in rats. The studies on the isolated rat fundus strip showed a

contractile effect, which was blocked by atropine (Awe et al. 1999). The aqueous

extract of V. amygdalina leaves stimulates gastric acid secretion and increases

intestinal motility. These results support the use of V. amygdalina in stomach upset

and constipation (Owu et al. 2008).

2.1.1.12 Anti-leishmanial activity

The methanol extract of V. polyanthes showed promising activity against Leishmania

amazonensis and L. chagasi (Braga et al. 2007). The chloroform and methanol

extracts of the plant, has been assessed in vitro on L. aethiopica. Amastigotes were

more sensitive to V. amygdalina than promastigotes. The chloroform extract of V.

amygdalina showed parasiticidal activity with effective doses (ED50) of 18.5µg/ml

and 13.3 µg/ml for promastigotes (Tadesse et al. 1993).

2.1.1.13 Muscle relaxant activity

Glaucolides E, the sesquiterpene lactone isolated from V. liatroides showed potent

relaxing high KCl or noradrenaline-induced contractions in aorta and to relax the high

KCl-contraction in uterus (Campos et al. 2003).

2.1.1.14 Wound healing effect

The ethanol leaves extract of V. scorpioides showed improved regeneration and

organization of the new tissue in guinea pigs (Leite et al. 2002).

2.1.1.15 Anthelmintic properties

The ethanol extract of the seeds of V. anthelmintica diplayed efficient anthelmintic

efficacy of up to 93%, relative to pyrantel tartrate tested against exsheathed infective

larvae of Haemonchus contortus using a modified methyl-thiazolyltetrazolium (MTT)

reduction assay (Hordegen et al. 2006).

2.1.1.16 Mutagenicity, isecticidal and tripanocidal activities

The ethanol extract of V. brasiliana showed insecticidal and trypanocidal effects

evaluated on Triatoma infestans and bloodstream forms of Trypanosoma cruzi,

respectively. Both mutagenicity and toxicity were evaluated by sister chromatid

exchange in human peripheral lymphocyte culture and by the lethality test of Artemia

salina (Arias et al. 1995).


35

2.1.2 Literature survey on the biological activities of the genus Euphorbia

2.1.2.1 Antimicrobial activity

Ent-11β-hydroxyabieta-8(14),13(15)-dien-16,12α-olide obtained from E. seguieriana

showed moderate to strong growth inhibition against Bacillus cereus, B. subtilis,

Micrococcus flavas, Moraxella catarrhalis, Neisseria sicca, and Candida albicans at

12.5 µg/ml concentration. Jolkinolide A isolated from E. fischeriana moderately

inhibited the growth of M. catarrhalis at 50 µg/ml concentration (Sutthivaiyakit et al.

2000). Heliscopinolide A and Heliscopinolide B isolated from E. helioscopia showed

significant activity against Staphylococcus aureus (2.5 µg/spot) (Valente et al. 2004).

In vitro bioassays showed that yuexiandajisu A from E. ebracteolata exhibited anti-

bacterial activity (Xu et al. 1998). A mixture of three cerebrosides from E. peplis

showed a synergistic antifungal activity against candida spp. and Cryptococcus

neoformans strain. Moreover, only a single compound (n=3) showed an intresting

antitubercular activity with MIC of 40 µg/ml against clinical strain (Cateni et al.

2003). The antibacterial activity of E. fusiformis was investigated against pathogenic

strains of Gram positive (Bacillus subtilis and S. aureus) and Gram negative bacteria

(Escherichia coli, Klebsiella pneumonieae, Proteus vulgaris, Pseudomonas

aeruginosa, Salmonella typhii A, and S. typhii B. The different extracts differed

significantly in their antibacterial properties with the methanolic extract being very

effective followed by acetone and chloroform extracts. Aqueous and ethanolic

extracts showed the very least activity. Root extracts had better antibacterial

properties than the leaf extracts. The results of this study supported the use of this

plant in traditional medicine to treat the fever, wound infections and intestinal

disorders (Natarajan et al. 2005). The ethanolic extracts of aerial parts of E. hirta

exhibited a broad spectrum of antibacterial activity against E. coli, P. vulgaris, P.

aeruginosa, and S. aureus (Sudhakar et al. 2006).

A compound, isolated from E. paralias, showed a moderate antiviral activity against

HIV-I replication. The activity was based on the inhibition virus induced cytopathicity

in cells (Abdelgaleil et al. 2007). The seven triterpenes, euphol, antiquol B and C,

euphorbol, isohelianol, camelliol C and lemmaphylla-7,21-dien-3-ol isolated from E.

antiquorum were examined for the inhibitory effects on Epstein-Barr-virus early

antigen (EBV-EA) activation induced by TPA. In this assay, the compounds euphol,

antiquol B and C, euphorbol and isohelianol showed 100% inhibition of activation at


36

1000 mol ratio/TPA (Akihisa et al. 2002). The steroids isolated from E. chamaesyce,

also exhibited potent inhibitory effects (100% inhibition of induction at 1000 mol

ratio/TPA, and about 30% inhibition at 100 mol ratio/TPA) in the same assay (Tanaka

e t al. 2000).

2.1.2.2 Antipyretic and analgesic activities

Myrsinane isolated from E. decipiens showed significant analgesic activity when

administered to mice at dose of 5-20 mg/kg. This activity is comparable to that of 100

mg/kg of aspirin or ibuprofen (Ahmad et al. 2006). Resiniferatoxin, an ultra potent

capsaicin analog present in the latex of E. resinifera, interacts at a specific membrane

recognition site, expressed by primary sensory neurons mediating pain perception as

well as neurogenic inflammation. Desensitization to resiniferatoxin is a promising

approach to mitigate neuropathic pain and other pathological conditions in which

sensory neuropeptides released from capsaicin sensitive neurons play a crucial role

(Appendino and Szallasi 1997). Prostratin, obtained in E. fischeriana, showed

significant analgesic and sedative activities. The 92% and 62% inhibitions were

observed in sedative experiments with 20 mg/kg (p.o.) and 1 mg/kg (s.c.) in mice,

respectively (Ma et al. 1997). The ethyl acetate fraction from the residue of an 85%

ethanol extract of the latex of E. royleana showed a dose related peripheral analgesic

effect. The same fraction exhibited a significant antipyretic effect in hyperthermic rats

and rabbits. The oral LD50 was more than 2 g/kg in rats and mice (Bani et al. 1997).

Following an identified use of the plant as analgesic in traditional medicine, the

hexane, chloroform and ethyl acetate extracts of E. heterophylla root were tested for

antinoceptive activity in rats. All extracts showed significant effects (Vamsidhar et al.

2000).

2.1.2.3 Antidiarrheal activity

A significant antidiarrheal effect of the E. paralias extracts against castor oil induced

diarrhea in rats was achieved by 400 mg/kg. It decreased the gastrointestinal

movement as indicated by the significantly decreased distance traveled by the

charcoal meal. The large dose of the extract was slightly more effective than the small

one. The E. paralias methanol extract produced a transient stimulation followed by

inhibition in doses of less than 0.05 mg/kg. Higher concentrations caused rapid

muscle relaxation (Atta and Mouneir 2005). The aqueous leaf extract of E. hirta


37

decreased the gastrointestinal motility in normal rats and decreased the effect of castor

oil induced diarrhea in mice by 300 and 200 mg/kg (Hore et al. 2006).

2.1.2.4 Molluscicidal and antifeedant activities

The molluscicidal and antifeedant activities of diterpenoids from E. paralias reported

(Abdelgaleil et al. 2007, 2002). Kansenonol from E. kansui showed moderate activity

at 500 ppm. The aqueous and serially purified latex extracts of plant E. pulcherima

and E. hirta exhibited potent molluscicidal activity (Singh et al. 2004). Sublethal

doses (40% and 80% of LC50) of aqueous and partially purified latex extract of both

the plants also significantly altered the levels of total protein, total free amino acid,

nucleic acid (DNA and RNA), and the activity of enzyme protease and acid and

alkaline phosphatase in various tissue of the snail Lymnaea acuminate in time and

dose dependent manner.

2.1.2.5 Inhibition of allergic reactions

The water soluble fraction of E. royleana latex, showed dose-dependent anti-

inflammatory and antiarthrirtic effects in different acute and chronic test models in

rats and mice. It reduced the exudates volume and the migration of leukocytes and

showed poor inhibitory effect on the granuloma formation induced by cotton pellets,

while it had a low ulcerogenic score. The oral LD50 was more than 1500 mg/kg in

both rats and mice (Bani et al. 2000). Oral administration of petroleum ether extract

of the aerial parts of E. splendens caused significant inhibition of edema and produced

inhibition of leucocyte migration and exudate volume in the affected tissues. The oral

LD 50 in both rats and mice was approximately 1250 mg/kg (Bani et al. 1997). A 95%

ethanol extract from whole aerial parts of E. hirta showed antihistaminic, anti-

inflammatory and immunosuppressive properties in various animal models (Singh et

al. 2006). In vivo tests, pepluanone, isolated from E. peplus, significantly reduced

carrageenin-induced edema by 40% and 60% (Corea et al. 2005). The lathyrane

diterpenoid obtained from E. nivulia, showed significant PGE2 inhibition using in

vitro assay method employing enzyme immunoassay kits. The IC50 value for the

compound was found to be 0.003 µm compared to that of known PGE2 inhibitor

celecoxib (0.050 µm) (Ravikanth et al. 2002).


38

2.1.2.6 Cytotoxicity

Lathyrane diterpenoids isolated from E. nivulia showed significant cytotoxicity

activity (Ravikanth et al. 2003). 17-Acetoxyjolkinolide B and 13-hexadecanoyloxy-

12-deoxyphorbol, obtained from the dried roots of E. fischeriana, exhibited potent

cytotoxicity activity (Wang et al. 2006).

2.1.2.7 Effects on the cell division

Ingenane-type diterpenoids and euphane triterpeniods, isolated from E. kansui showed

significant effects on the cell diversion of Xenopus laevis cells at the blastular stage

and arrested cleavage significantly (Wang et al. 2003, 2002, Berkwitz et al. 2000).

2.1.2.8 DNA damaging activity

In a mutant yeast bioassay, compound, 3β,5α,15β,17-tetra-O-acetyl-7β-O-benzoyl-

cheiradone, isolated from E. decipiens, showed a positive response to DNA-damaging

activity, camptothecin was used as the standard drug (Ahmad et al. 2003).

2.1.2.9 Modulatority of multidrug resistance

Euphosalicin isolated from E. salicifolia was found to be more active than verapamil

in reserving multidrug resistance in mouse lymphoma cells. Three jatrophane

diterpenoids isolated from E. mongolica, also demonstrated a concentration-

dependent effect in inhibiting the efflux pump activity of the tumour cells in the

range 11.2-112 µm (Hohmann et al. 2003). Two segetane diterpenoids,

euphoportlandols B and A obtained from E. portlandica, as well as three Jatrophane

diterpenoids isolated from E. peplus, found to be inhibitors of P-glycoprotein in the

same test (Hohmann et al. 2002, Madureira et al. 2006, Duarte and Ferriera. 2007).

2.1.2.10 Tumor promoting activity

Diterpene esters of the phorbol and ingenol types are known to be highly active tumor

promoting agents that typically occur in members of the Euphorbiaceae. Latex as

well as total leaf extracts of E. leuconeura exhibited Epstein-Barr-virus (EBV)

inducing activity comparable to TPA (12-O-tetradecanoylphorbol acetate), a well-

known tumor promoter. The activity of individual fractions correlated with their

ingenol ester content (Vogg et al. 1999). Seven ingenol type diterpenoids , obtained

from latex of E. cauducifolia, were evaluated for cocarcinogenic and tumor-

promoting activity on the back skin of mice. After 24 weeks, an average tumor rate of


39

7% and an average tumor yield of 0.07 tumors / mouse were noticed. After 36 weeks,

an average tumor rate of 36 % was observed and the average tumor yield was 0.45

tumors / mouse (Baloch et al. 2005).

2.1.2.11 Proinflammatory activity

Jatrophane diterpenes isolated from E. Peplus were investigated for the irritant

activities. Only one compound from E. segetalis was found to exert a weak pro-

inflammatory activity on mouse ear (the redness of the mouse ear was estimated 4 and

24 h after the application of solutions in acetone). These data indicated that this type

of diterpene does not play a significant role in the skin irritant activity of Euphorbia

species (Hohmann et al. 1999).

2.1.2.12 Inhibitory action on the mammalian mitochondrial respiratory chain

Six diterpenoids isolated from the latex of E. obtusifolia were evaluated for their

inhibition of NADH oxidase activity in submitochondrial particles from beef heart.

Among the six, 12β,13α-diisobutyryloxy-4,20-dideoxyphorbol was the most potent

inhibitor (Betancur-Galvis et al. 2003).

2.1.2.13 Antidipsogenic activity

The effect of the methanol extract obtained from the leaf and stem of E. hirta on the

thirst was examined using rats. Intraperitoneal administration of 10 mg/100 mg body

wt. of the extract significantly decreased the amount of water consumed by rats. This

effect lasted for 2 hours (Williams et al. 1997).

2.1.2.14 Survival effect on fibroblasts PGE2 inhibition activity

Kansuinin E, isolated from the roots of E. kansui, exhibited a specific survival effect

on fibroblasts. In contrast, kansuinins A, D, and F enhanced the survival fibroblasts

(Pan et al. 2004, Ip and Yancopoulos 1996).

2.1.2.15 PEP inhibitory activity

Prolyl endopeptidase (PEP) is the only serine protease that is known to cleave a

peptide substrate in the C-terminal side of a proline residue and plays an important

role in the metabolism of peptide hormones and neuropeptides and is recognized to be

involved in the learning and memory (Anis et al. 2002). The myrsinol-type

diterpenoids isolated from E. decipiens, were active against PEP (Ahmad, et al.

2003).


40

2.1.2.16 Urease inhibtory activity

Studies on the enzyme inhibition have led to the discoveries of drugs. Urease

inhibitors have recently attracted much attention as potential new anti ulcer drugs.

Unfortunately, only a few natural products with this activity have been discovered.

The decipinol ester A was isolated from E. decipiens and was reported as the first

naturally occurring urease inhibitor (Ahmad et al. 2003).

2.1.2.17 Angiotensin converting enzyme inhibiting activity

The methanol extract obtained from the leaves and stems of E. hirta inhibited the

activity of angiotensin converting enzyme (ACE) by 90 and 50% at 500 and 160 µg,

respectively, using enzyme linked immunosorbent assay (Williams et al. 1997).

2.1.2.18 Other activities

3,4-dimethoxycinnamaldehyde, isolated from E. quinquecostata, was significantly

active in the induction of quinine reductase (QR) in hepatoma cells and in the

inhibition of murine epidermal cells (Su et al. 2002). The water and ethanol extracts

(50 and 150 mg/kg) of E. hirta produced time dependent increase in urine output.

Electrolyte excretion was also significantly affected by the plant extracts (Johnson et

al. 1999). The water extract increases the urine excretion of Na+, K+, and HCO3-. In

contrast, the ethanol extract increased the excretion of HCO3-, decreased the loss of

K+, and had little effect on the removal of Na+.

2.2 Literature survey on the phytochemical studies of the genera Vernonia and

Euphorbia

2.2.1 Literature survey on the phytochemical studies of the genus Vernonia

2.2.1.1 Steroids

Misra et al. (1984) isolated first sterol (15) from Vernonia cinerea having double

bond between C-17 and C-20 and belongs to β-sitosterol from the petrolium ether

extract. Bitter steroidal glucosides namely vernoniosides A1 (16), A3 (17), A4 (19) and

it’s aglycone (20) as well as two nonbitter related glucosides vernoniosides B2 (21)

and B3 (22) were isolated from a methanol extract of the leaves of V. amygdalina

(Jisaka, et al. 1992, 1993). V. anthelmintica seeds produce a novel sterol named as 4α-

methylvernosterol (18) and its structure was established by using modern

spectroscopic techniques keeping possible biosynthetic background (Akihisa et al.


41

1992). Five new stigmastane-type steroidal glycosides named as vernoniosides D1

(23), D2 (24), D3 (25), F1 (26) and F2 (27) and a new Androst-8-en-3-O-[D-

glucopyranosyl-(1→3)-D-glucopyranoside (28) were isolated from the root of V.

kotschyana (Sanogo et al. 1998).

H3C CH3

CH3

CH3

CH3

CH3

HO

16

O

O

CH3

CH3

OH

O

O

H3C

CH3

CH3

O

OH

OH

OH

HO

15 17

O

O

CH3

CH3

O

O

O

H3C

CH3

CH3

O

OH

OH

OH

HO

H3C CH3

CH3

CH3

CH3

CH3

HOH

CH3

18

O

CH3

CH3

OH

O

HO

OHOH

OH

O

O

HO

CH3

OCH3

OH

CH3

H3C

H

H

H

21

O

CH3

CH3

OH

O

O

HO

H3C

CH3

CH3

OH

O

OH

OH

OH

HO

19

O

CH3

CH3

OH

O

HO

HO

H3C

CH3

CH3

OH

20

22

O

O

CH3

CH3

O

O

H3C

CH3

CH3

O

OH

OH

OH

HO

OH


42

O

CH3

O

O

OHO

CH3

HO

H3C

CH3H H

H

OHO

O

OH

OH

O

OH

OH

HO

OH

O

CH3

O

O

OHO

CH3

HO

H3C

CH3H H

H

O

HO

OH

OH

OH

O

CH3

O

O

OHO

CH3

HO

H3C

CH3H H

H

O

HO

O

OH

OH

O

OH

OH

OH

23 24

25

O

CH3

CH3

O

OO CH3

OH

H3C

OHOH

O

HO

OH

OH

OH

H H

H

O

CH3

CH3

O

OO CH3

OH

H3C

OHOH

O

HO

OH

OH

O

H H

H

O

OH

OH

OH

26

27

O

CH3

CH3

O

OH

O

OH

HO

O

OH

OH

OH

HO

28

The leaves of V. colorata led to the isolation of six steroids including four steroid

glycosides (29-32) and two trihydroxysteroids (33, 34). The structures of these

compounds were established using sophisticated spectroscopic techniques (Cioffi et

al. 2004).


43

O

CH3

CH3

O

OH

OH

OH

O

OH

OH

OH

HO OO

CH3

CH3

O

OH

OO

OH

OH

OH

HO O

O

OH

OHOH

OH

OH

O

CH3

O

O

OHO

CH3

HO

H3C

CH3H H

H

O

HO

O

OH

OH

O

OH

OH

OHHO

O

CH3

O

O

OHO

CH3

HO

H3C

CH3H H

H

O

AcO

O

OH

OH

O

OH

OH

OHHO

29 30

31 32

HO

CH3

CH3

H3C

OH

CH3

CH3

OH

33

HO

CH3

CH3

H3C

OH

H3C

CH3

CH3

OH

O

34

Seven stigmastane-type steroidal glycosides, vernocuminosides A-G (35-41) have

been isolated from the stem barks of V. cumingiana. The structural elucidation and

stereochemistry determination were achieved by spectroscopic and chemical methods

(Liu et al. 2004). Vernoguinoside (42), a stigmastane derivative (43) and two sucrose

esters, have been isolated from the stem bark of V. guineensis (Tchinda et al. 2003).


44

HOOC

CH3

O

CH3

H3C

CH3

CH3

O

OH

35

HOOC

CH3

O

CH3

H3C

CH3

CH3

O

OH

36

HOOC

CH3

OH

CH3

H3C

CH3

CH3

O

OH

37

HOOC

CH3

OH

CH3

H3C

CH3

CH3

O

OH

38

HOOC

CH3

OH

CH3

H3C

CH3

CH3

O

OH

O

O

OH

HO

O

39

HOOC

CH3

O

CH3

H3C

CH3

CH3

O

OH

O

O

OH

HO

O

40

HOOC

CH3

O

CH3

H3C

CH3

CH3

O

OH

41

O

O

HO

OH

OH

OH

O

HO

OH

OH

OH

O

HO

OH

OH

OH

O

HO

OH

OH

OH

O

HO

OH

OH

OH

O

HO

OH

OH

O

HO

OH

OH

OH

O

O

HO

OH

OH

O

HO

OH

OH

OH

O

O

OHOH

OH

OH

O

OHOH

OH

OH

O

OHOH

OH

OH O

OHOH

OH

OH


45

CH3

CH3

O

42

O

OHO

H

HH

OHCH3

CH3O

H3C

CH3

CH3

O

43

O

OHO

H

HH

OHCH3

CH3O

H3C

O

HO

OH

OH

OH

Two bitter stigmastane derivatives, vernoguinosterol (44) and vernoguinoside (45)

have been isolated from the stem bark of V. guineensis exhibited trypanocidal activity

(Tchinda et al. 2002).

CH3

CH3

HO

O

OHO

H

HH

OHCH3

CH3O

H3C

44

CH3

CH3 O

OHO

H

HH

OHCH3

CH3O

H3C

45O

O

HO

OH

OH

OH

Stigmastane-type steroid glycosides, vernoniosides D (46) and E (47) have been

isolated from the leaves of V. amygdalina, along with vernonioside A3 (Igile et al.

1995).


46

46

CH3

CH3OAc

H3C

CH3

CH3

CH3

O

OH

47

OH

O

CH3

O

O

OHO

CH3

HO

H3C

CH3

O

HO

OH

OH

OH

OH

O

HO

OH

OH

OH

O

2.2.1.2 Sesquiterpene

The investigation of the aerial parts of four Vernonia species (V. galamensis subsp.

galamensis var. petitiana; V. galamensis subsp. galamensis var. ethiopica; V.

galamensis subsp. gibbosa; V. galamensis subsp. afromontana) afforded five

sesquiterpene lactones of the glaucolide type (48-52) (Perdue et al. 1993).

O

O

O

H3C

OO

OH

O

OAc

CH3

CH3

48

O

O

O

H3C

OO

OAc

O

OAc

CH3

CH3

50

O

O

O

H3C

OO

OAc

O

OH

O

CH3

CH3

52

O

O

O

H3C

OO

OAc

CH3O

CH3

OAc

O

O

O CH2OH

H3C

OO

OAc

CH3O

CH3

51

49

8α-(2-methylacryloyloxy)-3-oxo-1-desoxy-1,2-dehydrohirsutinolide-13-O-acetate

(53) and 8α-(5-O-acetoxysenecioyloxy)-3-oxo-1-desoxy-1,2-dehydrohirsutinolide-13-

O-acetate (54), structurally related hirsutinolides were isolated from the

dichloromethane extract of the leaves of V. staehelinoides (Pillay et al. 2007).


47

53

O

OO

OAc

OH3C

OH3C

OCH2

H3C

O

OO

OAc

OH3C

OH3C

OCH3

AcO CH3

54

Two sesquiterpene lactones named as vernolides C (55) and D (56) were isolated

from the dichloromethane fraction of an aqueous extract from V. cinerea (Chea et al.

2006).

55

O O

OAc

O

H3C H

O

H3C

HO

OCl

HO CH3

O O

OAc

O

H3C H

O

H3C

HO

OCH2OH

CH3

56

Phytochemical studies on the leaves of V. amygdalina yielded two sesquiterpene

lactones, namely vernolide (57) and vernodalol (58) (Erasto et al. 2006).

57

O

O

CH2

O

OCH3

CH2

O

OH

H O

O

O

CH2

OOH

O

CH3

CH2

COOCH3OHH

CH2

CH2

58


48

Three sesquiterpene lactones, designated as glaucolides K-M (59-61) were isolated

from Bioassay-guided fractionation of the cytotoxic leaf extract of V. pachyclada.

Glaucolide M showed moderate activity in the human ovarian cancer cell line

(Williams et al. 2005).

O

O

O

OCH3

CH2CH3

CH3O

OH3C

O

OAc

59

O

O

O

OCH3

CH2CH3

60

CH3O

OH3C

O

OHO

O

O

OCH3

CH2CH3

61

OH3C

O

OH3COH

O

CH3

Phytochemical investigations of the aerial part of V. arborea resulted in the isolation

of zaluzanin D (62) (Kumari et al. 2003).

62

O

AcO

H2C

CH2

CH2

O

Glaucolides D (63) and E (64) were isolated from V. liatroides (Campos et al. 2003).

O

O

O

O

CH3CH3

CH3O OAc

AcOO

63

O

O

O

OCH2

CH3CH3

CH3O OAc

AcO

64


49

Two novel sesquiterpene lactones, vernolide A (65) and B (66) were isolated from

bioassay-guided fractionation of an ethanol extract of stems of V. cinerea (Kuo et al.

2003).

65

O O

OH

O

H3C

O

H3C

H3CO

CH3O

CH3

O O

OAc

O

H3C

O

H3C

H3CO

CH3O

CH3

66

Vernolide (67), 11β,13-dihydrovernolide (68) and vernodalin (69) were isolated from

the leaves of V. colorata (Rabe et al. 2002).

68

O

67

O

O

O

OH2COH3C

H2C

OH

O

O

O

O

OH3COH3C

H2C

OHO

O

O

CH2

CH3O

O

OCH2

CH2OH

HO

CH2

69

Five sesquiterpene lactones (70-74) were isolated from the ethyl acetate soluble

fraction of V. fastigiata (Roos et al. 1998).


50

O

O

OOH

H3C

O

OAc

OCOCH2C6H5

70O

O

OOH

H3C

O

OAc

OCH3

CH3

O

71

O

O

OOH

H3C

O

OAc

OAngeloyl

72

O

O

OOAc

H3C

O

OAc

Omethacryloyl

73

O

O

OOH

H3C

O

OAc

O

OO

CH2

CH3

74

Vernodalin (75) was isolated from V. amygdalina and vernolepin (76) from V.

hymenolepis (Magboul et al. 1997).

75

O

O

CH2

O

CH2

O

CH2

H

OCOC(CH2)CH2OH

76

O

O

CH2

O

CH2

O

CH2

H

OH

77

O

i-Bu

H2C

CH2

CH2

O

H

H

A sesquiterpene lactone zaluzanin A isobutyrate (77) was isolated from the non-polar

fraction of the V. leopoldi (Abegaz et al. 1994).

2.2.1.3 Diterpenes

The aerial parts of V. triflosculosa afforded 8α-(4α-hydroxymethacryloyloxy)-10α-

hydroxy-1,13-dimethoxy-hirsutinolide, ent-kaurane diterpenes, 19-[α-L-

arabinopyranosyl-(1→2)-β-D-glucopyranosyl] esters of 16β-hydroxy-ent-kauran-19-

oic acid (78) and 16β,17-hydroxy-ent-kauran-19-oic acid (79). It is the first diterpene

reported from genus Vernonia (Kos et al. 2006).


51

H3C OO O

OO

HOOH

OHOH

OH

OHH

H

CH3

OH

CH3

78

H3C OO O

OO

HOOH

OHOH

OH

OHH

H

CH2OH

OH

CH3

79

2.2.1.4 Coumarins

Cycloisobrachycoumarinone (80) and two new isomeric 5-methylcoumarins, 2'-

epicycloisobrachycoumarinone epoxide (81) and cycloisobrachycoumarinone epoxide

(82) have been isolated from the roots of Vernonia brachycalyx .

O

O

CH3CH3

CH3

CH3

CH3

OO

O

O

CH3CH3

CH3

CH3

CH3

OO

OO

O

CH3CH3

CH3

CH3

CH3

OO

O

80 82 81

2.2.1.5 Flavonoids

A rare flavone, Genkwanin (83), was isolated by column and preparative thin layer

chromatography of a chloroform extract of the leaves of Vernonia fasciculata (Narain,

1976).

O

OOH

H3CO

O OO

OH

HOOH

O

OH

CH3

OH

OH

83


52

A highly oxygenated flavone, namely 8,3-dihydroxy-5,6,7,4'-tetramethoxyflavone

(84), together with hesperidin (85) and p-Hydroxybenzoylvernovan (86) were isolated

from the tropical plant Vernonia saligna.

O

O

OCH3

OH

OH

OO

OH

OH

OH

OO

OH

OH

CH3

OH

85

O

OCH3

OH

O

OCH3

H3CO

H3CO

OH

84

O

OCH3

OH

OO

OH

OH

OH

OO

HO

86

Six phenolic derivatives, including four flavonoids and two benzofuranones (87,88),

were isolated from the aerial parts extract of Vernonia mapirensis (Escobar et al.

2007).

87

O

CH3CH3

H3C

O

OO

OHHO

HO

OH

O

CH3

H3C

O

OO

OHHO

HO

OH

89

The hesperidin (85) was isolated from the methanol extract of the wood of Vernonia

diffusa. The homoesperetin (89) was identified as the aglycone obtained in the

hydrolysis of the new natural flavanone glycoside, homoesperetin-7-O-rutinoside (90)

(Carvalho et al. 1999).


53

O

OOH

O

OMe

OMeOOH

HO

HO

O

O

OH

MeHO

HO

O

OOH

HO

OMe

OMe

9089

2.2.2 Literature survey on the phytochemical studies of the genus Euphorbia

2.2.2.1 Sesquiterpenoids

Shi et al. (1997) reported the isolation of sesquiterpenoids (91,92) from E. wangii.

This is the first investigation on sesquiterpenoids from the genus Euphorbia.

Fattorusso et al. 2002 isolated two novel bisnorsesquiterpene glycosides,

euphorbiosides A (93) as well as its aglycone (94) from E. resinifera.

H3C

H3C

H

H

CH2

OH

OH

CH3H3C

HO

CH3

91 92

OH

CH3

clg-D-O

clg-D-O

CH3

O

OH

CH3

HO

HO

CH3

O

93 94

2.2.2.2 Higher diterpenoids

Polycyclic diterpenoids with a common 6/6/6-tricyclic ring are also major constituents

of Euphorbia. Higher diterpenoids originated from geranylgeranyl diphosphate by

“concertina-like” cyclization, ent-abietanes, ent-atisanes, ent-kauranes, ent-

isopimaranes and ent-pimaranes are introduced as follows.


54

2.2.2.2.1 Ent-Abietanes

Jolkinolide A (95) and B (96) were isolated from E. fischeriana (Liu et al. 1988).

8α,14-dihydro-7-oxojolkinolide E (97) and 8α,14-dihydro-7-oxohelioscopinolide A

(98) were obtained from E. characias (Appendino et al. 2000).

95 96

97 98

O

O

CH3CH3

H3C CH3

H

H O

O

O

CH3CH3

H3C CH3

H

H O

O

O

O

CH3

CH3

H3C CH3

H

H

H

O

O

O

CH3

CH3

H3C CH3

H

H

H

OHO

2.2.2.2.2 Ent-Atisanes

Ent-2-hydroxyatis-1,16(17)-diene-3,14-dione (99), ent-atis-16(17)-ene-3,14-dione

(100) and ent-3α-hydroxyatis-16(17)-ene-2,14-dione (101) were isolated from E.

characias (Appendino et al. 2000). Entatisane-3β,16α,17-triol (102), ent-(13α)-

hydroxyatis-16(17)-ene-3,4-dione (103) and ent-(13α,14α)dihydroxyatis-15-ene-

15,17-dione (104) were obtained from E. fidjana (Lal et al. 1989).


55

CH3

H3C CH3

H

HO

CH2HO

O

H

HCH3H3C

CH3

O

CH2

O

H

HCH3H3C

CH3

O

CH2

HO

O

H

HCH3H3C

CH3

O

HO

OH OH

H

HCH3H3C

CH3

O

CH2

O

CH3

H

HCH3H3C

CH3

OH

CHO

O

OH

99 101

102 103

100

104

2.2.2.2.3 Ent-Kauranes

16β,17-dihydroxy-entkauran-3-one (105) and 17-acetoxy-16β-hydroxy-ent-kauran-3-

one (106) were isolated from E. portulacoides (Morgenstern et al. 1996).

CH3

H3C CH3

H

H

O

OH

OH

CH3

H3C CH3

H

H

O

OH

OAc

105 106

2.2.2.2.4 Ent-Isopimaranes and ent-Pimaranes

3α,12α-dihydroxy-ent-8(14),15-isopimaradien-18-al (107) from E. quinquecostata

and ent-pimara-8(14),15-diene-3α,17-diol (108) from E. fischeriana were obtained

(Su et al. 1989, Wang et al. 2006). Ent-pimara-8(14),15-dien-3α-ol (109), 3β,15,16-

triacetoxy-ent-pimar-8(14)-ene (110) and 3β,15,16-triacetoxy-ent-pimar-8(14)-en-2-

one (111) were isolated from E. characias (Appendino et al. 2000).


56

CH3

H3C CHOH

H

HO

OH

CH3

CH2 CH3

H3C CHOH

H

HO

OH

CH2

OH

CH3

H3C CH3

H

H

HO

OH

CH3

CH2

CH3

H3C CH3

H

H

HO

OH

CH3

AcO

OAc

CH3

H3C CH3

H

H

HO

OH

CH3

AcO

OAc

O

107 109

110 111

108

2.2.2.2.5 Other Diterpenoids

A manoyloxide derivative, 3β-hydroxy-2-oxomanoyloxide (112) was isolated from E.

segetalis (Jakupovic et al. (1998). Langduin C (113), a novel dimeric diterpenoid, and

fischeria A (114), a novel norditerpene lactone were isolated from roots and rhizomes

of E. fischeriana respectively (Zhou et al. 2003, Xu et al. 1998).

OCH3

H3C CH3

H

H

HO

CH3

CH2O

CH3

CH3

H3C CH3

H

O

OOHO

OO

OH

H3CO

OH3C

O

H3C

H3C CH3

CH3

H

H

O

CH2

112

113114

2.2.2.3 Lower Diterpenoids

Considerable attention has been paid to the macrocyclic diterpenoids derived from

cembrane cation because of their high chemical diversity and therapeutically relevant


57

bioactivity. “Euphorbiaceae diterpenoids” include casbanes, jatrophanes, lathyranes,

myrsinanes, tiglianes, ingenanes, segetanes, paralianes, pepluanes, and euphoractines

as shown below.

2.2.2.3.1 Casbanes

Two bicyclic diterpenoids with a casbane skeleton, yuexiandajisu A (115) and B

(116) were isolated from E. ebracteolata (Xu et al. 1998).

OH

COOH

CH3

CH3

CH3

H

HH3CCOOH

CH3

CH3

CH3

H

HH3C

115 116

2.2.2.3.2 Jatrophanes

Euphorbiaceae is a great rich source of jatrophane and the related diterpenoids.

Jatrophanes with various oxygenation stages and stereoisomers are repoted. These

compounds are usually substituted with various acyl groups, such as acetyl,

propanoyl, butanoyl, isobutyryl, benzoyl, tigloyl, nicotinoyl, angeloyl, etc., and are

sometimes called jatrophane polyesters.

Pepluanin A (117), B (118) and C (119) have been isolated from E. peplums (Corea et

al. 2004). Esulatin D (120) and F (121) were isolated from E. esula (Gunther et al.

1999). Amygdaloidins A (122), B (123), C (124) and D (125) were obtained from E.

amygdaloides (Corea et al. 2005).

CH2

HO

H3C

BzOH

AcO

AcO CH3

ONic

CH3

CH3

OAcAcO

CH2

HO

H3C

BzOH

AcO

AcO CH3

ONic

CH3

CH3

OHBuMeO

CH2

HO

H3C

AcOH

BuiO

AcO CH3

OAc

CH3

CH3

OAcBzO

117 118 119


58

CH2

AcO

AcOH

AcO

O CH3

OAc

CH3

CH3

AcO

H3C

AcO CH2

AcO

AcOH

AcO

O CH3

OAc

CH3

CH3

OAcBuiO

H3C

AcO

120 121

Kansuinin E (126) isolated from E. kansui (Wang et al. 2003). Esulatin A (127),

2α,3β,5α,9-tetraacetoxy-11,12-epoxy-7β,8α-diisobutyryloxyjatroph-6(17)-en-14-one

(128) and 2α,3β,5α,6β,9α-pentaaacetoxy-11,12-epoxy-8α-isobutyryloxyjatroph-6(17)-

en-14-one (129) were found in E. salicifolia (Hohmann et al. 2001). Pubescene D

(130) and 3β,9α,15β-triacetoxy-7β-butanoyloxyjatropha-5E,11E-dien-14-one (131)

were isolated from E. pubescens (Valente et al. 2003). Euphoheliosnoid D (132) was

found in E. helioscopia (Zhang and Guo 2006). Jatrophanes with a hemiacetal or a

tetrahydrofuran ring, Kansuinin A (133) from E. kansui while esulatin C (134) from E.

esula were obtained (Hohmann et al. 1997, Wang et al. 2002). 17-

Bishomojatrophanes-1 like terracinolide A (135) and B (136) and isoterracinolide A

(137) were isolated from E. terracina while salicinolide (138) was found in E.

salicifolia (Marco et al. 1996, Hohmann et al. 2001).

CH2

HO

AcOH

AcO

O CH3

ONic

CH3

CH3

AngO

H3C

OH

OAng

CH2

HO

AngOH

HO

O CH3

ONic

CH3

CH3

OAcAngO

H3C

OH

CH2

HO

HydrpOH

HO

O CH3

ONic

CH3

CH3

AngO

H3C

OAc

CH2

HO

AngOH

HO

O CH3

OAc

CH3

CH3

AngO

H3C

OAc

122 123

124 125


59

CH3

HO

AcOH

O CH3

OAc

CH3

CH3

BzO

H

H3C CH3

AcO

AcOH

O CH3

OAc

CH3

CH3

BzO

H3C

H

CH3

AcO

BzOH

O CH3

O

CH3

CH3

HO

H3C

OH

130 131

132

OCH2

H3C O

CH3

CH3

OAc

OBz

OAc

H

H3C

AcO

AcO

HO

OAc

OCH2

H3C O

CH3

CH3

OAc

OBz

OAc

H

H3C

AcO

AcO

HO

OAc

AcO

133134

H3C

AcO

AcO CH3

CH3

OAc

OAcOBui

O

OCH3

OBzO

AcOH

H3C

AcO

AcO CH3

CH3

OAc

OAcOBui

O

OCH3

OAcO

AcOH

H3C

AcO

O CH3

CH3

OAc

OAcOBui

BzO

AcOCH3

OH

AcOH

O

H3C

AcO

AcO CH3

CH3

O

OAcOBui

BzO

HO

OCH3

AcOH

O

O

135 136 137

138

CH2

AcO

AcOH

AcO

O CH3

ONic

CH3

CH3

AcO OBz

H3C

H

O

CH2

AcO

AcOH

AcO

O CH3

OAc

CH3

CH3

BuiO OAc

H3C

AcO

O

CH2

AcO

BzOH

AcO

O CH3

OAc

CH3

CH3

BuiO OBui

H3C

AcO

O

H3C

OBui

CH3

CH3

OAc

CH3O

AcO

HBzO

AcO

AcO CH2

O

AcO

126 127 128

129

2.2.2.3.3 Lathyranes

Lathyranes with a 5/11/3-membered ring are also very common in Euphorbia species.

Several compounds are substituted with methoxy group. Phenylacetyl and

methoxyphenylacetyl groups are rather diagnostic to lathyranes. 3β,12α-diacetoxy-19-

hydroxy-7α,8α-ditigloyloxyingol (139), 3β,12α,19-triacetoxy-7α-hydroxy-8α-

ditigloyloxyingol (140), 12α,19-diacetoxy-3β,7α-hydroxy-8α-ditigloyloxyingol (141)

and 3β,8α,12α-triacetoxy-7α-isovaleryloxyingol (142) were isolated from E.

acrurensis (Marco et al. 1998). 3β,7α,8α,12α-tetraacetoxy-2-epi-ingol (143),

3β,8α,12α-triacetoxy-7α-isobutanoyloxy-2-epi-ingol (144), 3β,8α,12α-triacetoxy-7α-


60

methylbutanoyloxy-2-epi-ingol (145) and 3β,8α,12α-triacetoxy-7α-benzoyloxy-2-epi-

ingol (146) were obtained from E. portulacoides (Morgenstern et al. 1996).

Euphorbia factors L1 (147), L3 (148), L8 (149), L10 (150) and 15β,17-diacetoxy-3β-

benzoyloxyisolathyrol (151) were isolated from E. lathyris (Appendino et al. 1999,

2003, Adolf et al. 1984).

AcO

CH3

O

H3C OAc

OTigl

OTigl

OH

CH3H

H

H3C O

AcO

CH3

O

H3C OAc

OH

OTigl

OAc

CH3H

H

H3C O

HO

CH3

O

H3C OAc

OH

OTigl

OAc

CH3H

H

H3C O

AcO

CH3

O

H3C OAc

OiVal

OAc

OH

CH3H

H

H3C O

139 140 141

142

AcO

CH3

O

H3C OAc

OAc

OAc

CH3

CH3H

H

H3C O

AcO

CH3

O

H3C OAc

OiBU

OAc

CH3

CH3H

H

H3C O

AcO

CH3

O

H3C OAc

OMeBu

OAc

CH3

CH3H

H

H3C O

AcO

CH3

O

H3C OAc

OBz

OAc

CH3

CH3H

H

H3C O

143 144

146145

BzO

AcO

O CH3H

H

CH3

CH3

CH2AcO

H

H

H3C

NicO

AcO

O CH3H

H

CH3

CH3

CH2AcO

H

H

H3C

148149

O

AcO

O CH3H

H

CH3

CH3

AcO

H

H3C

OO

Ph147

C6H11COO

AcO

O CH3H

H

CH3

CH3

H

H3C

OH

BnO

AcO

O CH3H

H

CH3

CH3

H

H3C

OH

150

151

2.2.2.3.4 Myrsinanes, Cyclomyrsinanes, and Premyrcinanes

Myrsinanes and cyclomyrsinanes are derived from lathyranes via premyrsinanes. In

addition to the normal myrsinanes (6,12-cyclojatrophanes) with a 5/7/5-ring carbon

framework, compounds with a hemiacetal ring, a 13,17-epoxy ring, or a 10,13-epoxy

ring are found in the genus. The stereochemistry of all the frameworks and

substituents are the same in myrsinanes. 9-Bishomomyrsinane contains a δ-lactone

ring (Zhang et al. 1998). Premyrsinanes forms a rare acetyl hemiacetal moiety.


61

Premyrsinanes type diterpenoides, eufoboetol (152) from E. boetica while kandovanol

ester A (153) and B (154) from E. decipiens were isolated (Zahid et al. 2001, Ferreira

and Ascenso 1999). Aleppicatine A (155) and B (156), euphoreppine A (157) and B

(158) were isolated from E. aleppica (Oksuz et al. 1996, Shi et al. 1995).

H3C

HO

HO

O

H

OH

H

OH

CH3

CH3H

H

H3C OH

OH

H3C

HO

AcO

O

H

OBz

H

OAc

CH3

CH3H

H

HO CH3

OAc

H3C

HO

AcO

O

H

OBu

H

OAc

CH3

CH3H

H

HO CH3

OAc

152 153

154

13-Deacetylisodecipidone (159), 13-deacetylisodecipinone (160), isodecipidone (161)

and 17-acetoxy-13-deacetyldecipinone (162) are myrinanes type diterpenoides and

were isolated from E. decipiens. Decipinol ester A (163), B (164) and C (165) were

obtained from E. decipiens (Zahid et al. 2001, Ahmad et al. 2002, Ahmad and Jassbi

1998). Euphorprolitherin A (166) and B (167) were isolated from E. prolifera (Zhang

et al. 2004).


62

H3C

AcO

AcO

O

H

BuO

OH

CH3

H

CH2

H3C

OAcHO

H3C

AcO

AcO

O

H

BzO

OH

CH3

H

CH2

H3C

OAcHO

H3C

AcO

AcO

O

H

BuO

OAc

CH3

H

CH2

H3C

OAcHO

H3C

HO

AcO

O

H

BzO

OH

CH3

H

CH2

H3C

OAcAcO

159 160

161 162

H3C

AcO

AcO

HO

H

H

OAc

CH3

CH3H

H

CH3

O

AcOH

TiglO

H3C

TiglO

AcO

HO

H

H

OAc

CH3

CH3H

H

CH3

O

AcOH

TiglO

H3C

AcO

AcO

HO

H

H

H

CH3

CH3H

H

CH3

O

AcOOTigl

AcO

H3C

TiglO

AcO

HO

H

H

H

CH3

CH3H

H

CH3

O

AcOOTigl

AcO

155 156 157

158

H3C

AcO

BuO

HO

H

NicO

OH

CH3

H

CH2

H3C

OAc

O

H3C

AcO

AcO

HO

H

BzO

OH

CH3

H

CH2

H3C

OAc

O H3C

AcO

AcO

HO

H

BuO

OH

CH3

H

CH2

H3C

OAc

O

163

164 165

AcO

O H

AcO

CH3

H

CH3

H3C

OAc

OAcO

H3C

BzO

OAc

O

CH3

AcO

O H

AcO

CH3

H

CH3

H3C

OAc

OAcO

H3C

BzMeO

OAc

CH3

O

166

167 168 169

H

H3C

OH

CH3

OH

CH3

CH3

H2C

O HH

H

H

HO

H3C

OH

CH3

OH

CH3

CH3

H2C

O HH

H

H

2.2.2.3.5 Jatropholanes

Lagaspholones A (168) and B (169) belong to jatropholanes are a new class of

members with a 5,12-cyclojatrophane skeleton were isolated From E. lagascae

(Duarte and Ferreira 2007).

2.2.2.3.6 Daphnanes

Resiniferatoxin (170) and resiniferol (171) forms intramolecular orthoester with

phenylacetic acid are reported in E. poisonii (Schmidt and Evans 1976).


63

H3C

O

H

HO

O

H

O

O

O

Ph

H3C CH2

H3C

O

OH

OMeH3C

O

H

HO

OH

H

O

O

O

Ph

H3CCH2

H3C

170 171 172

173

H3C

O

H

H

H

H3C

OTigl

OBui

CH3

CH3

H

OH

H3C

O

H

H

H

H3C

OBui

OBui

CH3

CH3

H

OH

H3C

O

H

HO

H

H3C OBui

CH3

CH3

H

OAc

OH

H3C

O

H

HO

H

H3COCO(CH2)14CH3

CH3

CH3

H

OH

OH

174 175

176 177

H3C

O

H

H

H

H3CO

CH3

CH3

H

CH3

OH

O

CH3

CH3

H3C

O

H

H

H

H3CO

CH3

CH3

HOH

O

CH3

CH3

OH

2.2.2.3.7 Tiglianes

12-O-Tetradecanoylphorbol 12-acetate (TPA) is a famous tumor promotor. A number

of phorbol derivatives were isolated from Euphorbia species. 13α-isobutyryloxy-4-

deoxy-12β-tigloyloxyphorbol (172) and 12β,13α-diisobutyryloxy-4,20-

dideoxyphorbol (173) were isolated from E. obtusifolia (Appendino et al. 1998,

Marco et al. 1999). 20-acetoxy-13-isobutyryloxy-12-deoxyphorbol (174) and 13-

hexadecanoyloxy-12-deoxyphorbol (175) were obtained from E. fischeriana (Ma et al.

1997, Fattorusso et al. 2002). 13-(2,3-dimethylbutanoyloxy)-4,12,20-trideoxyphorbol

(176) and 20-acetoxy-13-(2,3-dimethylbutanoyloxy)-4,12-dideoxyphorbol (177)

were obtained from E. pithyusa (Appendino et al. 1999).

2.2.2.3.8 Ingenanes

Ingenane diterpenoids have a very unique structural feature, that is, bicyclo [4.4.1]

undecane core adopts a highly strained inside-outside skeleton. A large number of

derivatives have been reported from this genus. 20-O-(2′E,4′E-decadienoyl)ingenol

(178) and 20-O-(2′E,4′Z-decadienoyl)ingenol (179) were found in E. Kansui (Wang et

al. 2003).


64

H3C

HOH

OCH3

CH3

H

H

H

H3C

HO O

O

n-C5H11

H3C

HO H

OCH3

CH3

H

H

H

H3C

HO O

O

n-C5H11

178 179

H3C

AngO H

OCH3

H

H

H

H3C

HO OAc

OAng

H3C

AngO H

OCH3

H

H

H

H3C

HO OH

OAc

180

181

H3C

O HO

OCH3

CH3

H

H

H3C(H2C)10OCO

H3C

HOOAcO

H3C

CH3

H3C

H3C

OHO

OCH3

CH3

H

H

H3C(H2C)10OCO

H3C

HOOHO

H3C

CH3

H3C

182 183

H3C

BzO HO

OCH3

H

H

AcO

H3C

HOCH3

OBz

H3C

AngO HO

OCH3

H

H

AcO

H3C

HOCH3

OBz

184 185

20-acetoxy-3β-O-angeloyl-17-angeloyloxyingenol (180) from E. segetalis while 17-

acetoxy-3β-O-angeloyl-20-deoxyingenol (181) from E. acrurensis has been isolated

(Jakupovic et al. 1998, Marco et al. 1998). 20-O-acetyl-l3β-O-(2,3-dimethyl-

butanoyl)-13α-O-dodecanoylingenol A (182) and 3β-O-(2,3-dimethyl-butanoyl)-13α-

O-dodecanoyl-20-deoxyingenol A (183) were isolated from E. kansui (Wang et al.

2003). 13α-acetoxy-3β-O-benzoyl-17-benzoyloxyingenol (184) and 13α-acetoxy-3β-

O-angeloyl-17-benzoyloxyingenol (185) were obtained from E. segetalis (Jakupovic

et al. 1998).

2.2.2.3.9 Segetanes

The skeleton of these 5/7/6/5-rings could be formed by transannular ring formation

between 8,12 and 13,17-positions of the 12-membered ring of jatrophanes. Segetene

A (186) and B (187) were isolated from E. paralias (Abdelgaleil et al. 2001).


65

H3C

BzO HOO OAc

OAcCH3

OAc

CH3

CH3

O

OH

O

OAc

H3C

BzO HOO OAc

OAcCH3

OAc

CH3

CH3

O

OH

OCH3

AcO

186187

H3C

BzO HAcO OH

OAcCH3

OAc

CH3

CH3

O

OHOAc

H3C

BzO HO OAc

OAcCH3

OAc

CH3

CH3

OAc

OH

O

AcO

OH

OAc

188 189

Euphoportlandol A (188) from E. portlandica while segetanin B (189) from E.

paralias have been isolated (Madureira et al. 2006, Barile and Lanzotti 2007).

2.2.2.4 Triterpenoids

Tetracyclic, pentacyclic triterpenoids, some secotriterpenoids along with several other

kinds of triterpenoids are isolated from many plants of Euphorbia species. Among

lanosterol type compounds, antiquol B (192) has a rare 19(10→9) abeoeuphane

skeleton (Akihisa et al. 2002).

Kansenone (190) from E. kansui while antiquol C (191) and B (192) and euphorbol

(193) from E. antiquorum has been reported (Wang et al. 2003, Akihisa et al. 2002).

Lupeol acetate (194) from E. stygiana and betulin (195) from E. latifolia were

isolated (Lima et al. 2003, Zhang et al. 2006). Lupenone (196) from E. segetalis, D-

friedomadeir-14-en-3-one (197) from E. mellifera, oleanolic acid (198) from E.

latifolia and α-amyrin acetate (199) from E. ebracteolata have been reported (Wang

and Ding 1998, Zhang et al. 2006, Ferreira et al. 1998, Ferreira 1990).


66

H3C CH3

CH3

CH3

CH3

HO

H3C CH3

CH3

HO

H3C CH3

CH3

CH3

CH3

H

H3C CH3

CH3

HO

190 191

H3C CH3

H

CH3

CH3

H3C CH3

CH3

HO

H3C CH3

CH3

CH3

CH3

H

H3C CH3

CH3

HO

CH3

H

CH2

192

193

H3C CH3

CH3

CH3

CH3

HAcO

H

CH3 H

CH2

H3C

CH3

H3C CH3

CH3

CH3

CH3

HHO

H

CH3 H

CH2

H3C

OH

194 195

H3C CH3

CH3

CH3

H

HO

196 197

H

CH3 H

CH2

H3C

CH3

H3C CH3

CH3

CH3

HO

H

CH3 H

H3C

CH3

CH3

H3C CH3

CH3

CH3

HHO

H

CH3 H COOH

CH3H3C

H3C CH3

CH3

HHO

H

CH3 H CH3

CH3

H3C

CH3

198

199

H3C CH3

CH3

HHO

200 201

CH3

H3C

CH2

CH3HO

H3C CH3

CH3

HHO

CH3

H3C

CH3

CH3

OH

2.2.2.4.1 Cycloartanes

Cycloart-25-ene-3β,24-diol (200) and cycloart-23Z-ene-3β,25-diol (201) have been

isolated from E. sessiliflora (Sutthivaiyakit et al. 2000).

2.2.2.5 Steroids

Tanaka et al. (1999) and Ferreira (1990) isolated several ergostane-type steroids

(202,203) from E. chamaesyce. 5α-Stigmastane-3β,6α-diol (204) and 5α-stigmastane-

3β,5,6β-triol (205) were found to be obtained in E. boetica (Ferreira and Ascenso

1999) Rahman et al. (2002) reported the isolation of a new geniculatoside from aerial

parts of E. geniculata. In addition, β-sitosterol and daucosterol are present in many

plants of this species, such as E. boetica, E. segetalis, E. aleppica, E. quinquecostata,


67

and E. latifolia (Mbwambo et al. 1996, Pan et al. 2003, Shi et al. 1995, Zhang et al.

2006, Ferreira et al. 1998).

CH3

CH3

HHO

202 203

CH3

H3C

CH2

CH3

CH3

CH3

O

CH3

CH3

HHO

CH3

H3C

CH2

CH3

CH3

OHCH3

O

HO

CH3

H

HHO

204 205

CH3

H3C

CH2

CH3

CH3

CH3

H

OHHO

CH3

H3C CH3

CH3

CH3

H

H

OH

H

H

OH

2.2.2.6 Phenolics

2-hydroxy-4,6-dimethoxyacetophenone (206), 2,4,6-trimethoxyacetophenone (207),

2-hydroxy-4,6-dimethoxy-3-methylacetophenone (208) and 2,4,6-trimethoxy-3-

methylacetophenone (209) were reported in E. portulacoides (Morgenstern et al.

1996). 2,2′-dihydroxy-4,6-dimethoxy-3-methylacetophenone (210) from E.

quinquecostata and 2,4-dihydroxy-6-methoxyacetophenone (211) from E. fischeriana

have been isolated (Che et al. 1999, Mbwambo et al. 1996).

OH

H3CO OCH3

CH3

O OCH3

H3CO OCH3

CH3

O OH

H3CO OCH3

CH3

O

H3C

OH

H3CO OCH3

CH3

O

H3C

OH

H3CO OCH3

CH2OH

O

H3C

OH

HO OCH3

CH3

O

206 207 208

209 210 211


68

2.2.2.7 Flavonoids

In 2004, a new flavonol glycoside, quercetin 3-O-6′-(3-hydroxyl-3-methylglutaryl)-β-

D-glucopyranoside, and four known flavonoids, kaempferol 3-O-2′′-galloyl-β-D-

glucopyranoside, kaempferol 3-O-rutinoside, quercetin 3-O-β-D-glucopyranoside, and

rutin, were isolated from the aerial parts of E. ebracteolata (Liu et al. 2004).

Nishimura et al. (2005) reported the isolation of one new flavonoid galactoside,

quercetin 3-O-(2″,3″-digalloyl)-β-D-galactopyranoside from E. lunulata, along with

four known ones, quercetin 3-O-(2′′-galloyl)-β-D-galactopyranoside, hyperin, and

quercetin. Recently, Zhang and Guo (2006) reported six known flavonoids such as

licochalcone A, 4,2′,4′-trihydroxychalcone, echinatia, licochalcone B, glabrone, and

5,7,4′-trihydroxyflavanone from E. helioscopia. These compounds were isolated from

the species for the first time. In addition, the common flavonoids such as kaempferol,

kaempferol 3-O-L-rhaside, kaempferol 3-O-β-D-glucopyranoside, quercetin, quercetin

3-O-β-D-glucopyranoside, and astragalin have been isolated from many plants of this

species such as E. latifolia, E. altotibetic, and E. aleppica (Pan et al. 2003, Oksuz et

al. 1996, Shi et al. 1995, Zhang et al. 2006).

2.2.2.8 Miscellaneous Compounds

Lee et al. (2004) isolated a new ellagitannin, jolkinin, from the fresh whole plant of E.

jolkinii which has a unique hexacyclic structure. 3,3′,4′-tri-O-methyl-4-O-[α]-

rhamnopyranosyl-(1′′→6′′)-β-D-glucopyranosyl]ellagic acid has been isolated from E.

quinquecostata (Mbwambo et al. 1996). Su et al. (2002) isolated a new dihydrobenzo

furan neolignan, ()-trans-9-acetyl-4,9′-di-O-methyl-3′-di-O-methyldehydro-

diconiferyl alcohol, from the stem wood of E. quinquecostata along with 3,4-

dimethoxycinnamaldehyde and bicoumarol. Octacosyl cis-ferulate (216) and

octacosyl trans-ferulate (217) were isolated from E. hylonoma. (Ruan et al. 2007)


69

215

O OHO

O

O

OH

O O(CH2)27CH3

H3CO

HO

H3CO

HO

O

O

(CH2)27CH3

O OHO

H3CO

O OH3CO

H3CO

OCH3

O OH3CO

HO

213

216

217

212

214

Coumarins in Euphorbia genus are not very rich. The main compounds are scopoletin

(212), bicoumarol, (213), 6,7,8-trimethoxycoumarin (214) and isoscopoletin, (215)

(Mbwambo et al. 1996, Su et al. 2002). Two alkaloids, uracil and uridine, were

isolated from E. altotibetic (Pan et al. 2003). Che et al. (1999) reported the isolation

of physcion from E. fischeriana. Gallic acid, 3,3′-di-O-methylellagic acid, and 3,4,3′-

tri-O-methylellagic acid 4′-O-β-D-glucopyranoside were isolated from E. fischeriana,

E. sessiliflora, and E. lunulata (Sutthivaiyakit et al. 2000, Marco et al. 1997).

Octacosyl ferulate (216,217), 1-glycerin hexadecanoate, 1-octacosanol, 9-cis-

tricosene, 4-hydroxybenzoic acid, and its methyl ether were found to exist in E.

fischeriana, E. humifusa, E. latifolia, and E. aleppica (Ravikanth et al. 2003, Oksuz et

al. 1996, Berkowitz et al. 2000, Zhang et al. 2006, Ruan, et al. 2007).

CHAPTER # 3 MATERIALS AND METHODS

70

3. MATERIALS AND METHODS

3.1 Plant materials

Both Vernonia cinerascens and Euphorbia granulata were collected from Peruwal

(District Khanewal) in May 2003. Euphorbia serpens was collected from Salt Mine

Rest House Khewara and Shalamar Garden Lahore. The plants were identified by

Prof. Dr. Altaf Ahmad Dasti, Plant Taxonomist, Institute of Pure and Applied

Biology, Bahauddin Zakariya University Multan, Pakistan, where their voucher

specimens (VC09/IPAB/03 for V. cinerascens, EG09/IPAB/03 for E. granulata and

ES09/IPAB/03 for E. serpens) were deposited.

3.2 Extraction

The shade-dried aerial parts and roots of V. cinerascens, aerial parts of E. granulata

and E. prostrata were subjected for extraction successively with dichloromethane and

methanol at room temperature occasionally shaking for 24 hrs. Extracts were

concentrated by Rotavapor-R20 at 35 ºC.

3.3 Chromatographic studies

3.3.1 Analytical

TLC aluuminium sheets 20 × 20 cm, coated with Silica gel 60 F254 were utilized for

the analysis of different components present in dichloromethane and methanol

extracts applied for the separation of different classes of components. Different

solvent systems applied for TLC are given in Table 3.1 and Table 3.2.

3.3.2 Visualization of components on TLC plates

1. Naked eye

2. Under UV 254 nm

3. Under UV 365 nm

4. Spraying with chemical reagents

With regard to detection, TLC plates were observed with naked eye, in UV light 254

nm, in 365 nm and Godine reagent was sprayed on these plates followed by the spray

of 10 percent sulfuric acid. Plates were kept in oven for 5 minutes at 110 °C. The

developed colors were marked.


71

3.3.3 High performance liquid chromatography (HPLC)

The most popular techniques HPLC was used for analytical purposes, methanol:water

and acetonitrile:water were used as eluent (Gradient). The purity of isolated

components was also confirmed by HPLC.

3.4 Isolation

3.4.1 Column chromatography

Large scale isolation of components was carried out by column chromatography using

silica gel 60 as adsorbent. The sizes of the column used were

CR 60/50 (Quickfit-England)




Suitable mobile phase were selected with the help of TLC. Flow rate of the eleunts on

columns was 1 ml/min at room temperature.

3.4.2 Gel chromatography

Different important fractions regarding to isolation were applied for chromatography

on Sephadex. Gel chromatography provided satisfactory means of separation.

Sephadex LH-20 (50 g) was treated with methanol for 24 hours before employing it to

the column. Methanol soluble fractions were subjected for chromatography on

Sephadex LH-20 with flow rate 1 ml/min. Distilled methanol was used as eleunt. The

size of the column used was CR 40/30 (Quickfit-England).


72

Table 3.1. Solvent systems used for the analysis of dichloromethane extracts of Vernonia cinerascens

Solvent System Ratio

Choloroform:Methanol

97.5:2.5

95:5

89:11

n-hexane-Ethyl acetate

80:20

75:25

50:50

30:70

n-hexane:Isopropanol 90:10

80:20

n-hexane:Methanol 80:20

90:10

Ethyl acetate:Chloroform 90:10

80:20

Ethyl acetate:Methanol 90:10

80:20

Dichloromethane:Methanol 80:20

70:30

Dichloromethane:Methanol:Water 80:18:2

Chloroform:Methanol:Water 80:18:2

Ethyl acetate:Methanol:Water 93:5:3

Toluene:Ethyl acetate 80:20


73

Table 3.2. Solvent systems used for the analysis of methanol extracts of Vernonia cinerascens

Solvent System Ratio

Chloroform:Methanol:Water

95:4.5:0.5

90:9:1

85:14:1

80:18:2

70:26:4

60:35:5

55:35:10

Ethyl acetate:Methanol:Water

97:2:1

96:3:1

95:3:2

94:4:2

93:5:2

92:5:3

91:5:4

90:6:4

88:7:5

Ethyl acetate-Methanol 90:10

80:20

Chloroform:Methanol 90:10

80:20

Ethyl acetate:Formic acid:Glacial acetic acid:Water

100:11:11:27

100:10:10:20

3.4.3 Solvents and chemicals

All the solvents used for extraction and isolation like methanol, dichloromethane,

chloroform, n-hexane, ethyl acetate, ethanol, propanol, n-butanol, Vanillin, silica gel

(70-230 mesh) and TLC aluuminium sheets 20 × 20 cm, Silica gel 60 F254 were

imported from Merck KgaA Darmstadt Germany. Sephadex LH-20 25-100μm Fluka

Chemie GmbH (9041-37-6).


74

3.5 Preparation of reagents

The reagents were prepared according to the specification of Pharmaceutical Codex

(11th edition) and British Pharmacopoeia.

3.5.1 Wagner’s reagent (Solution of iodine in potassium iodide)

Composition

Potassium iodide (KI) = 4g

Iodine (I2) = 2g

Procedure

Dissolve 4 g of potassium iodide in minimum quantity of water (10 ml). Add 2 g of

Iodine. Iodine dissolved completely by complex formation. Then volume was made

100 ml with water.

3.5.2 Mayer’s reagent (solution of potassium mercuric iodide)

Composition

Mercuric chloride (HgCl2) = 1.36 g

Potassium iodide (KI) = 5 g

Procedure

Solution (A) of Mercuric chloride was prepared by dissolving 1.36 g of Mercuric

chloride in 60 ml of H2O. Solution (B) was prepared by dissolving 5 g of Potassium

iodide in 20 ml of water. Then added the solution (A) into solution (B), mixed and

made the volume 100 with water.

3.5.3 Hager’s reagent

Picric acid was dissolved in 100 ml of water till the saturation point was achieved the

solution was filtered.

3.5.4 Dragendorff’s reagent (solution of Potassium Bismuth Iodide)

Composition

Tartaric acid = 100 g

Bismuth oxide nitrate = 8.5 g

Potassium iodide = 200 ml 40 % w/v


75

Water = 400 ml

Procedure

25 g of tartaric acid was dissolved in 100 ml of H2O and added 2.1 g of bismuth

oxynitrate. Shacked for 1 hour and added 50 ml of 40 % solution of Potassium iodide

Shacked well allowed to stand for 24 hours and filtered.

3.5.5 Godine reagent (Godine 1954)

Godine reagent was prepared by adding equal volume of two solutions

1- 1% Vaniline in ethanol

2- 3% Perchloric acid in water

3.6 Preparation of solutions

3.6.1 Preparation of dilute HCl

The dilute HCl was prepared according to the requirements of the procedures by

calculating the volume of the acid required according to its strength.

3.6.2 Preparation of dilute ammonia solution

Dilute the 375 ml of strong ammonia solution to 1000 ml with H2O.

3.6.3 Preparation of 70 % alcohol

72.7 ml of alcohol mixed with 27.3 ml of purified water.

3.6.4 Preparation of lead subacetate solution

40 g of lead acetate was dissolved in 90 ml of carbon dioxide free water. Adjust the

pH 7.5 with 10 M Sodium hydroxide solution. Centrifuged and collected supernatant

liquid. It was lead subacetate solution.

3.6.5 10 M NaOH

10 M Sodium hydroxide was prepared by dissolving 40 g of Sodium hydroxide in 100

ml of water.

3.6.6 10 % Ferric chloride solution

10 g of Ferric chloride were dissolved in sufficient amount of purified water and made

the final volume 100 ml.


76

3.6.7 3.5 % Ferric chloride in glacial acetic acid

3.5 % Ferric chloride in glacial acetic acid solution was prepared by dissolving 3.5 g

of ferric chloride in 100 ml of glacial acetic acid.

3.6.8 1 % gelatin solution in 10 % Sodium chloride

1 g gelatin was dissolved in 100 ml of 10 % Sodium chloride solution.

3.6.9 10 % Sulfuric acid

10 % sulfuric acid was prepared by diluting concentrated sulfuric acid available in

ethanol.

3.7 Detection of various classes of secondary metabolites

Phytochemical studies were carried out for the detection of alkaloids, glycosides,

saponins, flavonoids and tannins in different parts of the plants Euphorbia

granulata, Euphorbia serpens and Vernonia cinerascens. The detail of the tests

employed is given.

3.7.1 Detection of alkaloids

3 g of the ground plant material was boiled with 10 ml of acidified water in test tube

for 1 min., cool, and allowed the debris to settle. Filter the liquid in a test tube. 1 ml of

this filtrate was taken and 3 drops of Dragendorff’s reagent were added, there was no

precipitate. The remainder of filtrate was made alkaline by adding dilute ammonia

solution. It was transferred to separating funnel and 5 ml of chloroform solution was

added, two layers were observed. The lower chloroform layer was pipetted out into

another test tube. Chloroform layer was extracted with 10 ml of acetic acid and then

discarded the chloroform. Extracts were divided into three portions; to one portion

added few drops of Dragendorff’s reagent and to second few drops of Mayer’s reagent

were added. Turbidity or precipitate was compared with the third untreated control

portion (Brain and Turner 1975).

3.7.2 Detection of anthraquinones glycosides

1 g of ground plant material was taken and extracted with 10 ml of hot water for five

minutes, allowed it to cool and filtered. Filtrate was extracted with 10 ml of carbon

tetrachloride. Then carbon tetrachloride layer was taken off, washed it with 5 ml

water and then 5 ml dilute ammonia solution was added. No free anthraquinones were

revealed as absence of appearance of pink to cherry red color in the ammonical layer.


77

1 g of second sample of the same plant material was extracted with 10 ml of ferric

chloride solution and 5 ml of hydrochloric acid then it was heated on water bath for

10 minutes and filtered. Filtrate was cooled and treated as above (Brain and Turner

1975).

3.7.3 Detection of cardioactive glycosides

1 g of ground plant material was taken in a test tube and 10 mL of 70% alcohol was

added. It was then boiled for 2 minutes and filtered. Filtrate was diluted twice of its

volume with water and then 1 ml of strong lead subaceatate solution was added. This

treatment leads to the precipitation of chlorophyll and other pigments, which were

then filtered off. Filtrate was extracted with an equal volume of chloroform.

Chloroform layer was pipetted out and evaporated to dryness in a dish over a water

bath. Residue was dissolved in 3 mL of 3.5% ferric chloride in glacial acetic acid and

was transferred to test tube after leaving for 1 min. 1.5 ml of sulphuric acid was then

added, which formed a separate layer at the bottom. Cardio active glycosides were

revealed the appearance of brown color at interface (due to deoxy sugar) on standing,

and appearance of pale green color in the upper layer (due to the steroidal nucleus)

(Brain and Turner1975).

3.7.4 Detection of tannins

Prepared 10% w/v aqueous extract of ground plant material by boiling it with distilled

water for about 10-20 min. Filtered the extract and performed the chemical tests with

clear solution.

3.7.4.1 Ferric chloride test

2 ml of ferric chloride solution was added to 1-2 ml clear solution of extract. A blue

back precipitate indicated the presence of hydrolysable tannin (Trease and Evans

1983).

3.7.4.2 Gelatin test

Test solution (about 0.5-1%) precipitate 1% solution of gelatin containing 10%

sodium chloride (Trease and Evans 1983).


78

3.7.4.3 Catechin test

Dipped the match stick in plant extract, dried and then moist it with concentrated

hydrochloric acid. Warmed near flame, a red or pink wood is produced which showed

the presence of catechin (Trease and Evans 1983).

3.7.5 Detection of flavonoids

2 g of the air dried powdered plant material was boiled with 20 ml of distilled water

for 10 minutes and filtered. The filtrate was acidified with few drops of dilute HCl.

Took 5 ml of aliquot of the filtrate and made it alkaline (pH 10) with sodium

hydroxide (T.S), A yellow colour was developed indicated the possible presence of

flavonoids (El-Olemy et al. 1994).

3.8 Biological activities

3.8.1 Spasmolytic Activity (Gillani et al. 1994)

Locally available rabbits weighing about 1.5 kg, starved for 24 hr were killed by

cervical dislocation by a blow on the back of the head and jejunum was dissected out.

Each segment of rabbit jejunum of about 2 cm length was mounted in 10 ml tissue

bath containing tyrod solution, maintained at 37 oC and continuously bubbled with a

mixture of 95% oxygen and 5% carbon dioxide. A preload of 1 g was applied and

spontaneous contractions were recorded isotonically via a Harvard Transducer

coupled to a Harvard Science Recorder i.e. Harvard Student Oscillograph. The tissue

was allowed to equilibrate for a period of 30 min. before addition of any drug, during

which the tissue was washed with fresh bathing fluid at an interval of every 10

minutes. At equilibrium, each tissue preparation was repeatedly treated with sub

maximal doses of agonists like acetylcholine (0.3 µM) with a 3 min. interval between

doses to stabilize the preparation. The preparation was considered stabilized when

three responses of the same dose were found identical. Spasmolytic actions of test

were observed by administration of test drugs in a cumulative fashion. The drug

induced inhibitory effects of test substances were measured as the percent change in

spontaneous contraction of rabbit jejunum obtained immediately before the addition

of test substances. K+ at high doses was also used as spasmogenic agent to

differentiate the specific and nonspecific nature of spasmolytic action K+ at high doses

(> 30 mM) is known to cause contraction of smooth muscles through opening of

voltage dependent slow calcium channels and the test drug which blocked the effect


79

of high dose of K+ in the study was considered to act through Calcium channel

blockade (Gillani et al. 2000).

3.8.2 Antifungal Assay

A) The in vitro antifungal bioassay of the crude dichloromethane and methanol

extracts was performed by agar tube dilution method (Atta-ur-Rehman et al. 2001).

The crude extracts were evaluated against clinical specimens of Candida albicans,

Aspergillus flavus, Microsporum canis, Fusarium solani and Candida glabrata. A

control experiment with test substance (medium supplemented with appropriate

amount of DMSO) was carried out for verification of the fungal growth. The extracts

(24 mg) dissolved in sterile DMSO (1 ml), served as stock solution. Sabouraud

Dextrose Agar (SDA) (4 ml), was dispensed into screw cap tubes which were

autoclaved at 121 oC for 15 min. and then cooled to 50 oC. The non-solidified SDA

media was poisoned with stock solution (66.6 µl), giving the final concentration of

400 µg of the extract/ml of SDA. Each tube was inoculated with a piece (4 mm

diameter) of inoculum removed from a seven day old culture of fungi. For non-

mycelial growth, an agar surface streak was employed. Inhibition of fungal growth

was observed after 7 days of incubation at 28±1 oC.

B) The antifungal test against Cladosporium cucumerinum was carried out on TLC

plate. After developing with suitable solvent system, the TLC plates were well dried

with an air dryer in order to remove the solvent completely. The developed and dried

TLC plates were sprayed with a conidial suspension of C. cucumerinum in a nutrition

medium and incubated in moist atmosphere for 2-3 days. Inhibition of the fungal

growth was observed as clear zones on the chromatogram that indicates the presence

of antifungal compounds (Chaudhary et al. 2001).

3.8.3 Antibacterial Activity

Antibacterial activity was determined by an agar diffusion method on the already

prepared plates of the inoculated media. The required number of holes was bore using

a sterile cork borer ensuring proper distribution in the periphery and one in the centre.

The solutions i.e. the extract, solvent and reference standard (ampicillin) were poured

into their respective hole with the help of sterilized pipette. The plates were left at

room temperature for 2 hrs to allow diffusion of the sample and incubated at 37 oC for


80

24-48 hrs. The diameter of the zones of inhibition was measured to the nearest mm

(Atta-ur-Rehman et al. 2001).

3.8.4 Brine-Shrimp Toxicity Assay

Eggs of brine shrimp were incubated in a petri dish containing artificial sea water for

24 hrs. Hatched larvae were transferred in another petri dish and incubated for another

24 hrs to allow for molting to 2nd instar. This process allowed obtaining a

homogenous population of 2nd instar larvae. Extracts were solubilized in DMSO with

a maximum concentration of 50 µg/ml. The larvae were counted into groups of 10 and

placed in 1 ml of artificial sea. Survival was evaluated after incubation at 25 oC for 24

hrs by observing under dissection microscope (Atta-ur-Rehman et al. 2001).

3.8.5 Phytotoxicity Assay

Lemna minor (Lemnaceae) is a miniature aquatic Thaloid monocot, consists of a

central oral frond with two attached daughter fronds and a filamentous root. Lemna

assay is a quick measure of phytotoxicity of the material under investigation. An

inorganic medium (E. Medium) of pH 5.5-6.0 was prepared. Vials for testing; 10 vials

per dose (500, 50, 5 ppm, control) were prepared as:

a. 15 mg of extract was weighed and dissolved in 15 ml solvent.

b. 1000, 100, and 10 µl solutions were added to vials for 500, 50, 5 ppm, allowed

solvent to evaporate overnight.

c. 2 ml of E. Medium and then a single plant containing a rosette of three fronds were

added to each vial.

Vials were placed in a glass dish filled with about 2 cm water, and container was

sealed with stopcock grease and glass plate. Dish with vials was placed in growth

chamber for seven days at 26 oC under fluorescent and incandescent light. Number of

fronds per vial were counted and recorded on day 3 and day 7. Data was analyzed as

percent of control with ED50 computer program to determine FI50 values and 65%

confidence interval (Atta-ur-Rehman et al. 2001).

3.5.6 Antioxidant activity

The antioxidant assay was carried out on TLC plate. 100 µg of test sample was

applied on TLC plate and after developing with suitable solvent system, the TLC

plates were well dried with an air dryer in order to remove the solvent completely.


81

TLC plates were then sprayed with a 0.2 % DPPH solution in MeOH. And the plates

were examined 30 min after spraying. The compounds with antioxidant properties

appeared as yellow spots against purple background (Cuendet et al. 1997).

3.8.7 Acetylcholinestrase inhibitory assay

Acetylcholinestrase inhibitory assay was performed on TLC. Acetylcholinestrase

(1000 U) was dissolved in 150 ml of 0.05 M tris-hydrochloric acid buffer at pH 7.8;

bovine serum albumin (150 mg) was added to the solution in order to stabilize the

enzyme during the bioassay. The stock solution was kept at 4 oC. TLC plates were

eluted with an appropriate solvent (acetone or isopropanol) in order to wash them, and

were thoroughly dried just before use. After eluting the sample in a suitable solvent,

the TLC plate was dried with a hair dryer for complete removal of solvent. The plate

was then sprayed with enzyme stock solution and dried again. For incubation of the

enzyme, the plate was laid flat on plastic plugs in a plastic tank containing a little

water; by this means, water did not come directly into contact with the plate but the

atmosphere was kept humid. The cover was placed on the tank and incubation was

done at 37 oC for 20 min. The enzyme had satisfactory stability under these

conditions. For detection of the enzyme, solutions of 1-naphthyl acetate (250 mg) in

ethanol (100 ml) and of Fast Blue B salt (400 mg) in distilled water (160 ml) were

prepared immediately before use (in order to prevent decomposition). After

incubation of the TLC plate, 10 ml of the naphthyl acetate solution and 40 ml of the

Fast Blue B salt solution were mixed and sprayed onto the plate to give a purple

coloration after 1-2 min. (Marston et al. 2002).

3.9 Spectroscopy

Ultraviolet (UV) spectra were recorded on Hitachi U-3200 spectrophotometer.

Infrared (IR) spectra measured on JASCO A-302 Infrared spectrophotometer. Proton

nuclear magnetic resonance (1H-NMR) spectra were recorded in CD3OD using TMS

as internal standard at 400 MHz or 500 MHz on Bruker AM-400 and AM-500 nuclear

magnetic resonance spectrometers with aspect 3000 data systems at a digital

resolution of 32 K. The 13C-NMR spectra were recorded in CD3OD at 100 or 125

MHz on the same instruments. The 2D-NMR (HMQC, HMBC, COSY and NOESY)

spectra were recorded in CD3OD at 400 MHz or 500 MHz on the same instruments.


82

Low-resolution electron impact mass spectra were recorded on a Finnigan MAT 311

with MASSPEC data system. High resolution mass measurements and fast atomic

bombardment (FAB) mass measurements were carried out on Jeol JMS HX 110 mass

spectrometer using glycerol and thioglycerol as the matrix and cesium iodide (CsI) as

internal standard for accurate mass measurements.


83

3.10 Physical and Spectroscopic Data of the isolated Compounds (A-J)

3.10.1 Physical and spectroscopic data of the isolated compound A (Vernonione)

White oil (16 mg)

IR ʋmax (KBr) cm-1: 2962, 1737, 1722, 1651, 1230.

1H-NMR (CDCl3, 500 MHz): δ 6.74 (1H, dd, J = 5.5, 1.0 Hz, H-2), 6.11 (1H, dd, J =

7.0, 1.5 Hz, H-3), 5.79 (1H, d, J = 12.5 Hz, H-5), 5.53 (1H, dd, J = 5.5, 3.5 Hz, H-3),

2.40 (1H, ddd, J = 12.5, 4.0, 3.5 Hz, H-4), 2.06 (3H, s, OAc), 2.00 (1H, m, H-8), 1.99

(3H, d, J = 7.0 Hz, H-4), 1.93 (3H, d, J = 1.5 Hz, H-5), 1.80 (3H, d, J = 1.0 Hz, H-7),

0.99 (3H, d, J = 7.0 Hz, H-10) and 0.96 (3H, d, J = 71.0 Hz, H-9).

13C-NMR (CDCl3, 125 MHz): δ 194.7 (C-6), 170.1 (OAc), 167.1 (C-1), 138.7 (C-3),

137.9 (C-2), 137.3 (C-1), 72.9 (C-5), 68.0 (C-3), 46.2 (C-4), 27.7 (C-8), 21.1 (OAc),

20.4 (C-5), 19.6 (C-10), 19.4 (C-9), 15.7 (C-4) and 15.4 (C-7).

EIMS m/z (rel. int.): 248 (6), 209 (10), 166 (19), 149 (12), 137 (21), 98 (29), 83

(100).

HRFABMS: m/z: 309.1710 [M+H]+ (Calcd. for C17H25O5; 309.1702).


84

3.10.2 Physical and spectroscopic data of the isolated compound B

(Cinerascenone)

Yellow amorphous powder (6 mg)

UV (CH3OH) λmax log nm: 242 (3.7), 250 (4.01), 271 (3.82), 348 (3.91).

IR ʋmax (KBr) cm-1: 3417, 2926, 1655-1502, 831.

1H-NMR (CD3OD, 500 MHz): δ 7.81 (2H, d, J = 8.5 Hz, H-2,6), 7.46 (1H, dd, J =

8.0, 1.5 Hz, H-6), 7.44 (1H, d, J = 1.5 Hz, H-2), 6.91 (1H, d, J = 8.0 Hz, H-5), 6.90

(2H, d, J = 8.5 Hz, H-3,5), 6.54 (1H, s, H-3), 6.41 (1H, d, J = 1.2 Hz, H-8), 6.18

(1H, d, J = 1.2 Hz, H-6) and 3.94 (3H, s, OCH3).

13C-NMR (CD3OD, 125 MHz): δ 184.0 (C-4), 166.7 (C-7), 166.3 (C-7), 166.2 (C-

2), 163.0 (C-5, 4), 159.0 (C-9), 152.5 (C-4), 149.2 (C-3), 129.4 (C-2, 6), 123.0

(C-1), 121.7 (C-6), 117.5 (C-1), 117.0 (C-3, 5), 116.7 (C-5), 110.6 (C-2), 105.2

(C-10), 104.1 (C-3), 100.2 (C-6), 95.1 (C-8) and 56.5 (OCH3).

EIMS m/z (rel. int.): 420 (3), 300 (100), 299 (5), 272 (13), 256 (23), 228 (15), 148

(34), 78 (23).

HREIMS: m/z: 420.0839 [M]+ (calcd. for C23H16O8, 420.0845).


85

3.10.3 Physical and spectroscopic data of the isolated compound C (2-Hydroxy-3-

methoxy-5-(2-propenyl)-phenol)

Yellow oil (26 mg)

UV (CH3OH) λmax log nm: 330 (3.7), 280 (3.9), 236 (4.1).

IR (KBr) max cm-1: 3362, 2968, 1604, 1284 and 1054.

1H-NMR (CDCl3, 500 MHz): δ 6.80 (1H, br s, H-6), 6.77 (1H, br s, H-2), 5.92 (1H,

m, H-8), 5.02 (2H, m, H-9), 3.81 (3H, s, OCH3) and 3.28 (2H, d, J = 7.0, H-7).

13C-NMR (CDCl3, 125 MHz): δ 150.1 (C-3), 141.4 (C-5), 138.9 (C-8), 138.2 (C-4),

132.1 (C-1), 116.5 (C-6), 115.9 (C-9), 110.5 (C-2) and 40.7 (C-7).

EIMS m/z (rel. int.): 180 [M]+ (100), 164 (40), 153 (70), 147 (83), 119 (37), 103 (15),

91 (45), 77 (17) and 64 (25).

HREIMS m/z: 180.0779 [M]+ (calcd. for C10H12O3, 180.0780.


86

3.10.4 Physical and spectroscopic data of the isolated compound D (Vanillic acid)

Crystallized from methanol (3 mg)

MP: 209-211 °C.


IR (KBr) max cm-1: 3445, 2925, 1741, 1599-1515, 1281 and 1032.

1H-NMR (CDCl3, 500 MHz): δ 7.56 (1H, d, J = 1.5, H-2), 7.46 (1H, dd, J = 8.0, 1.5,

H-6), 6.73 (1H, d, J = 8.0, H-6) and 3.87 (3H, s, OCH3).

13C-NMR (CDCl3, 125 MHz): δ 175.0 (C-7), 149.2 (C-4), 148.0 (C-3), 130.0 (C-1),

124.2 (C-6), 115.1 (C-5), 114.0 (C-2) and 56.2 (OCH3).

EIMS m/z (rel. int.): 168 [M]+ (3), 111 (5), 78 (100) and 63 (90).

HREIMS m/z: 168.0415 [M]+ (calcd. for C8H8O4, 168.0422).


87

3.10.5 Physical and spectroscopic data of the isolated compound E (Isoferulic

acid)

Crystallized in methanol (18 mg)

M.P.: 229-231 °C


IR (KBr) max cm-1: 3627, 3427-2500, 1695, 1596 and 1264.

1H-NMR (CDCl3, 400 MHz): δ 7.60 (1H, d, J = 16.0, H-7), 7.04 (1H, d, J = 1.5, H-

2), 6.98 (1H, dd, J = 8.4, 1.5, H-6), 6.71 (1H, d, J = 8.4, H-5), 6.25 (1H, d, J = 16.0,

H-8) and 3.95 (3H, s, OCH3).

13C-NMR (CD3OD, 100 MHz): δ 168.3 (C-9), 149.0 (C-4), 147.5 (C-3), 147.1 (C-7),

127.7 (C-1), 123.1 (C-6), 116.4 (C-5), 115.1 (C-2), 115.1 (C-8) and 56.0 (OCH3).




88

3.10.6 Physical and spectroscopic data of the isolated compound F (Caffeic acid)

Crystallized in methanol (16 mg)

M.P.: 223-225 °C


IR (KBr) max cm-1: 3666, 3422-2395, 1694, 1598, 1264.

1H-NMR (CD3OD, 500 MHz): δ 7.62 (1H, d, J = 15.5, H-7), 7.08 (1H, d, J = 2.0, H-

2), 6.96 (1H, dd, J = 8.0, 2.0, H-6), 6.78 (1H, d, J = 8.0, H-5) and 6.42 (1H, d, J =

15.5, H-8).


128.0 (C-1), 123.3 (C-6), 116.7 (C-5), 115.5 (C-8), 114.5 (C-2).




89

3.10.7 Physical and spectroscopic data of the isolated compound G (Methyl

gallate)

Colorless amorphous powder (4 mg)

MP: 201-203 °C.


IR (KBr) max cm-1: 3597, 2963, 1742, 1697, 1237 and 1016.

1H-NMR (CDCl3, 400 MHz): 7.01 (2H, s, H-2,6) and 3.80 (3H, s, OCH3).

13C-NMR (CDCl3, 100 MHz): δ 169.0 (C-7), 146.5 (C-3,5), 139.9 (C-4), 121.3 (C-1),

110.0 (C-2,6) and 52.2 (OCH3).

EIMS m/z (rel. int.): 184 [M]+ (54), 153 (100), 125 (24), 107 (6) and 79 (12).



90

3.10.8 Physical and spectroscopic data of the isolated compound H (Uridine)

Colorless amorphous powder (11 mg)

UV (CH3OH) λmax log nm: 264 (3.8), 214 (3.9).

IR (KBr) max cm-1: 3451, 2926, 1670, 1595 and 1096.

1H-NMR (CD3OD, 500 MHz): δ 8.00 (1H, d, J = 8.0, H-6), 5.88 (1H, d, J = 5.0, H-

1'), 5.68 (1H, d, J = 8.0, H-5), 4.16 (1H, t, J = 5.0, H-2'), 4.13 (1H, t, J = 5.0, H-3'),

4.00 (1H, m, H-4'), 3.83 (1H, dd, J = 12.0, 2.5, H-5') and 3.72 (1H, dd, J = 12.0, 3.0,

H-5').


90.7 (C-1'), 86.3 (C-4'), 75.7 (C-2'), 71.3 (C-3') and 62.5 (C-5').

EIMS m/z (rel. int.): 226 (4), 133 (10), 112 (21) and 44 (100).

HRFABMS m/z: 245.0770 [M+H]+ (calcd. for C9H13N2O6, 245.0773).


91

3.10.9 Physical and spectroscopic data of the isolated compound I (3'-

Methylquercetin)

Yellow powder (17 mg)


IR (KBr) max cm-1: 3270, 2928, 1730, 1657-1509, 1168 and 1033.

1H-NMR (CD3OD, 500 MHz): δ 8.00 (1H, d, J = 2.0, H-2'), 7.56 (1H, dd, J = 8.5,

2.0, H-6'), 6.89 (1H, d, J = 8.5, H-5'), 6.40 (1H, d, J = 1.2, H-8), 6.17 (1H, d, J = 1.2,

H-6) and 3.94 (3H, s, OCH3).

13C-NMR (CD3OD, 125 MHz): δ 179.1 (C-4), 166.1 (C-7), 163.0 (C-8a), 159.3 (C-5),

151.1 (C-4'), 148.6 (C-3'), 148.6 (C-2), 133.6 (C-3), 123.7 (C-6'), 122.8 (C-1'), 116.0

(C-5'), 114.2 (C-2'), 105.7 (C-8a), 100.0 (C-6), 94.8 (C-8), 56.9 (OCH3).

EIMS m/z (rel. int.): 316 [M]+ (100), 301 (5), 168 (36), 153 (27), 109 (9), 81 (5) and

55 (5).



92

3.10.10 Physical and spectroscopic data of the isolated compound J (Quercetin)

Yellow powder (13 mg)

UV (CH3OH)λmax log nm: 360 (4.1), 257 (4.01), 211 (3.9).

IR (KBr) max cm-1: 3423, 2925, 1658-1598, 1271 and 1058.

1H-NMR (CD3OD, 400 MHz): δ 7.65 (1H, d, J = 2.0, H-2'), 7.63 (1H, dd, J = 8.5,

2.0, H-6'), 6.84 (1H, d, J = 8.5, H-5'), 6.26 (1H, d, J = 1.5, H-8) and 6.09 (1H, d, J =

1.5, H-6).

13C-NMR (CD3OD, 100 MHz): 180.0 (C-4), 166.1 (C-7), 164.0 (C-8a), 158.0 (C-5),

150.0 (C-4'), 146.1 (C-3'), 146.0 (C-2), 133.0 (C-3), 123.2 (C-6'), 123.1 (C-1'), 116.9

(C-2'), 116.2 (C-5'), 104.0 (C-8a), 101.5 (C-6) and 95.9 (C-8).

EIMS m/z (rel. int.): 302 [M]+ (10), 284 (3), 256 (16), 129 (21), 97 (29), 78 (100) and

63 (87).


CHAPTER # 4 RESULTS

93

4. RESULTS

4.1 Extraction

The V. cinerascens and E. granulata were collected from Peruwal (District Khaniwal)

in May 2003 The E. serpens was collected from Salt Mine Rest House Khewara and

Shalamar Garden Lahore. The shade-dried aerial parts and root parts of Vernonia

cinerascens, aerial parts of Euphorbia granulata and Euphorbia serpens were

subjected for extraction successively with dichloromethane and methanol at room

temperature occasionally shaking for 24 hrs. Extracts were concentrated by

Rotavapor-R20 at 35 ºC. The results of the extraction along with the abbreviations

used for different extracts are given in Table 4.1.

Table 4.1. Results of the extraction of plants Vernonia cinerascens, Euphorbia

granulata and Euphorbia serpens

Plant name Part used Solvent Weight of

extract (g)

Abbreviation

for the

extracts

Vernonia

cinerascens

Aerial parts

(1000 g)

Dichloromethane 60 VAD

Methanol 114 VAM

Vernonia

cinerascens

Roots

(1000 g)

Dichloromethane 14 VRD

Methanol 55 VRM

Euphorbia

granulata

Aerial parts

(1000 g)

Dichloromethane 45 EGD

Methanol 68 EGM

Euphorbia

serpens

Aerial parts

(1000 g)

Dichloromethane 37 ESD

Methanol 65 ESM

4.2 Phytochemical Analysis

The dried and powdered aerial parts of Vernonia cinerascens, Euphorbia granulata

and Euphorbia serpens were investigated for presence of alkaloids, anthraquinones,

cardiac glycosides, tannins, flavonoids and saponins. The results of phytochemical

analysis are given in Table 4.2.

CHAPTER # 4 RESULTS

94

Table 4.2. Results of phytochemical screening of V. cinerascens, E. granulate and E. serpens

Name of plants

Alk

aloid

Anth

raqu

inon

e

Card

iac glycosides

Tan

nins

Sap

onin

s

Flavon

oids

V. cinerascens + + +

E. granulata + + +

E. serpens + + +

+ = Present; = Absent

4.3 Biological Screening

Dried and powdered aerial parts and roots of Vernonia cinerascens, aerial parts of

Euphorbia granulata and Euphorbia serpens were extracted successively at room

temperature with dichloromethane and methanol. Dichloromethane and methanol

extracts of aerial parts of Vernonia cinerascens, Euphorbia granulata and Euphorbia

serpens were screened for spasmolytic activity, antifungal bioassay, antibacterial

bioassay, brine-shrimp toxicity, phytotoxicity against Lemna minor, antioxidant

bioautographic assay and acetylcholinestrase inhibitory activity. While

dicholoromethane and methanol extracts of roots of the Vernonia cinerascens were

evaluated for spasmolytic activity, antifungal bioassay, antibacterial bioassay, brine-

shrimp toxicity and phytotoxicity against Lemna minor. The results of these bioassays

are given in Tables 4.3-4.8.

CHAPTER # 4 RESULTS

95

Table 4.3. Results of spasmolytic activities of dichloromethane and methanol extracts of V. cinerascens, E. granulata and E. serpens

Name of

Extract

Spasmolytic activity

Spontaneous (mg/ml) High K+ (mg/ml)

VAD 0.03-1.0 0.03-1.0

VAM 0.1-3.0 0.3-10

VRD 0.01-1.0 0.1-1.0

VRM 0.03-1.0 1.0-5.0

EGD 0.1-1.0 0.03-0.3

EGM 0.1-5.0 0.1-5.0

ESD 0.03-1.0 0.03-1.0

ESM 0.1-3.0 1.0-5.0

Table 4.4a. Results of antifungal activities of dichloromethane and methanol extracts of V. cinerascens

Name of the Fungs

Linear Growth (mm) of Extracts and Control %

InhibitionStandard MIC (µg/ml) VAD VAM VRD VRM Control

Cladosporium cucumerinum

100 100 100 100 100 0 Propiconazole

Candida albicans

100 100 100 100 100 0 Miconazole (110.8)

Candida glabrata

100 100 100 100 100 0 Miconazole (110.8)

Aspergillus flavus

100 100 100 100 100 0 Amphotericin B (20)

Microsporum canis

100 100 20 100 100 80 Miconazole (98.35)

Fusarium solani

100 80* 100 30 100 20*, 70 Miconazole (73.25)

Note: Concentration of extract used, 400 µg/ml of DMSO

CHAPTER # 4 RESULTS

96

Table 4.4b. Results of antifungal activities of dichloromethane and methanol extracts of E. granulata and E. serpens

Name of the Fungs

Linear Growth (mm) of Extracts and Control %

InhibitionStandard

MIC (µg/ml) EGD EGM ESD ESM Control

Cladosporium cucumerinum

100 100 100 100 100 0 Propiconazole

Candida albicans

100 100 100 100 100 0 Miconazole (110.8)

Candida glabrata

100 100 100 100 100 0 Miconazole (110.8)

Aspergillus flavus

50 100 60* 100 100 50, 40* Amphotericin B (20)

Microsporum canis

10 100 60* 100 100 90, 40* Miconazole (98.35)

Fusarium solani

100 100 100 100 100 0 Miconazole (73.25)

Note: Concentration of extract used, 400 µg/ml of DMSO

Table 4.5a. Results of antibacterial activities of dichloromethane and methanol


Name of Bacteria Zone of Inhibition of Extract (mm) Zone of Inhibition

of Standard (mm) VAD VAM VRD VRM

Eschericha coli - - - - 30

Bacillus subtilis - - - - 37

Shigella flexenari - - - - 36

Staphylococcus aureus - - - - 26

P. aeruginosa - - - - 32

Salmonella typhi - - - - 30

Note: Concentration of extract used, 3 mg/ml of DMSO; Concentration of Standard drug Imipenum (10µg/ml)

CHAPTER # 4 RESULTS

97

Table 4.5b. Results of antibacterial activities of dichloromethane and methanol extracts of E. granulata and E. serpens

Name of Bacteria Zone of Inhibition of Sample (mm) Zone of Inhibition

of Standard Drug (mm) EGD EGM ESD ESM

Eschericha coli - - - - 30

Bacillus subtilis - - - - 37

Shigella flexenari 9 - - - 36

Staphylococcus aureus - - - - 26

P. aeruginosa - - - - 32

Salmonella typhi - - - - 30

Note: Concentration of extract used, 3 mg/ml of DMSO; Concentration of Standard drug Imipenum (10µg/ml)

CHAPTER # 4 RESULTS

98

Table 4.6. Results of Brine Shrimp Lethality Bioassay of dichloromethane and methanol extracts of V. cinerascens, E. granulata and E. serpens

Name of Extract

Amount of extract (µg/ml)

Number of Shrimps

Number of Survivor

VAD

1000 30 27

100 30 29

10 30 30

VAM

1000 30 28

100 30 27

10 30 25

VRD

1000 30 20

100 30 25

10 30 26

VRM

1000 30 21

100 30 26

10 30 29

EGD

1000 30 22

100 30 24

10 30 25

EGM

1000 30 22

100 30 29

10 30 30

ESD

1000 30 16

100 30 18

10 30 22

ESM

1000 30 25

100 30 23

10 30 22

Note: Etoposide was used as standard (LD50, 7.4625 µg/ml)

CHAPTER # 4 RESULTS

99

Table 4.7. Results of In Vitro Phytotoxic Bioassay of dichloromethane and methanol extracts of V. cinerascens, E. granulata and E. serpens

Name of extract

Amount of extract (µg/ml)

Numbers of Fronts % Growth regulation

Sample Control

VAD

1000 15

17

11.76

100 16 5.88

10 17 0.00

VAM

1000 13

17

23.50

100 14 17.64

10 15 11.76

VRD

1000 12

17

29.41

100 13 23.52

10 14 17.64

VRM

1000 12

17

29.41

100 15 11.76

10 17 0.00

EGD

1000 13

17

23.52

100 14 17.64

10 15 11.76

EGM

1000 15

17

11.76

100 16 5.88

10 17 0.00

ESD

1000 15

17

11.76

100 16 5.88

10 17 0.00

ESM

1000 15

17

11.76

100 16 5.88

10 17 0.00

Paraquat was used as standard with concentration (0.015 µg/ml)

CHAPTER # 4 RESULTS

100

Table 4.8. Results of antioxidant and acetylcholinestrase inhibitory activities of dichloromethane and methanol extracts of V. cinerascens, E. granulata and E. serpens

Name of

Extract Antioxidant activity

Acetylcholinestrase Inhibitory

activity

VAD – –

VAM + –

EGD – –

EGM – –

ESD – –

ESM + –

+ = Present; = Absent 4.4 Isolation of Compounds A-J:

4.4.1 Isolation of Compound A

The shade dried roots of Vernonia cinerascens were ground and extracted with

dichloromethane and methanol. Dichloromethane extract (10 g) was fractioned by

column chromatography on silica gel using stepwise elution with n-hexane-

ethylacetate (0-100%) and ethylacetate-methanol (0-100%) in increasing order of

polarity. Seven frations (1-7) were obtained. The analysis of fraction 4 (70 mg)

showed the presence of two components. This fraction was subjected to column

chromatography on Sephadex LH-20 using methanol as eluent and then fraction (I,II)

collected. This fraction II (30 mg) was further purified by preparative TLC (toluene-

ethylacetate, 95: 5) which afford a pure compound A (17 mg) .

4.4.2 Isolation of Compounds B-J

The shade dried aerial parts of Vernonia cinerascens were ground and extracted with

dichloromethane and methanol. The methanol extract (20 g) was subjected to column

chromatography on silica gel using stepwise elution with n-hexane-ethyl acetate (0-

100%) and ethyl acetate-methanol (0-100%) in increasing order of polarity. Eight

fractions (1-8) were obtained. The fraction 3 (175 mg) obtained by n-hexane-ethyl

acetate (5.5:4.5) was resolved into two components on TLC. Separation of fraction by

column chromatography on Sephadex LH-20 using methanol as eluent resulted two

fractions (I and II). The fraction II (55 mg) was subjected to preparative TLC (n-

hexane-ethyl acetate, 3.5:6.5) which gave two pure compounds C (26 mg) and D

CHAPTER # 4 RESULTS

101

(3 mg). The fraction 4 (166 mg) obtained from n-hexane-ethyl acetate (4.5:5.5) was

separated by column chromatography on Sephadex LH-20 into two fractions (I and

II). The fraction II (85 mg) was subjected to preparative TLC (n-hexane-ethyl acetate,

3:7) which provided two pure compounds E (18 mg) and F (16 mg). The analysis of

fraction 5 (110 mg) obtained by n-hexane-ethyl acetate (35:75) showed the presence

of two components. A combination of column chromatography on Sephadex LH-20

and preparative TLC (ethyl acetate-methanol, 9.8:0.2) afforded two pure compounds

G (4 mg) and H (11 mg). The fraction 6 (183 mg) obtained by ethyl acetate-methanol

(9.8:0.2) was subjected to column chromatography on Sephadex LH-20 and two

fractions (I and II) were obtained. The fraction II (78 mg) was purified by preparative

TLC (ethyl acetate-methanol-water, 9.5:0.4:0.1) which resulted into the isolation two

pure compounds I (17 mg) and J (13 mg). Fraction 7 (82 mg) obtained from ethyl

acetate-methanol (9.5:0.5) was chromatographed on Sephadex LH-20 using methanol

as eluent afforded two fractions (I and II). Purification of fraction II (35 mg) by

preparative TLC (ethyl acetate-methanol-water, 8.8:1.1:0.1) gave a pure compound B

(6 mg).

CHAPTER # 4 RESULTS

102

IIIIII

A

CH2Cl2 extract of roots (10 g)

1-3 4 5-7

(70 mg)

CC, Sephadex LH-20MeOH

______________________________________

Prep. TLCToluene-EtOAc (95:5)

(30 mg)

(17 mg)

CC, silica gel (40-63 µm)n-Hexane-EtOAc (0-100%)EtOac-MeOH (0-100%)

Figure 4.1: Isolation scheme of compound A from the dichloromethane extract

of the roots of Vernonia cinerascens

______________________________________

CHAPTER # 4 RESULTS

103

I III II I III III II

B

EC FD

G IH J

MeOH extract of aerial parts (20 g)

__________________________________________________________________________

1-2 3 4 5 6 7 8

__________

__________

Sephadex LH-20MeOH

Prep. TLCn-Hexane-EtOAc(35:65)

__________

__________

Prep. TLCn-Hexane-EtOAc(30:70)

Sephadex LH-20MeOH

__________

__________

Prep. TLCEtOAc-MeOH(98:2)

Sephadex LH-20MeOH

__________ __________

__________

Sephadex LH-20MeOH

Sephadex LH-20MeOH

Prep. TLCEtOAc-MeOH-H2O(88:11:1)

Prep. TLCEtOAc-MeOH-H2O(95:5:1)

CC, silica gel (40-63 µm)n-Hexane-EtOAc (0-100%)EtOAc-MeOH (0-100%)

(6 mg)

(17 mg) (13 mg)(4 mg) (11 mg)

(16 mg)(18 mg)(26 mg) (3 mg)

Figure 4.2: Isolation scheme of compounds B-J from the methanol extract of the aerial parts of Vernonia cinerascens

(175 mg)

(55 mg)

(166 mg) (110 mg)

(85 mg) (50 mg)

(183 mg) (82 mg)

(35 mg)(78 mg)

CHAPTER # 4 RESULTS

104

4.5 Structure elucidation of the isolated compounds

4.5.1 Compound A (Vernonione)

CH3

O

OH3C

O

CH3H3C

O

O

CH3

1

2

3

4

5

6

7

89 10

CH3

1'

2'

3'

4'

5'

1''2''

Compound A

Compound A was isolated as oily liquid. In the IR spectrum of compound A

(figure 5.5) the absorptions bands at 2962 cm-1 (CH), 1737, 1230 cm-1 (OAc),

1722, 1651 cm-1 (C=CCO2R) and 1699 cm-1 (C=C-C=O). The molecular

formula was deduced as C17H24O5 through HRFABMS showing molecular ion

peak [M+H]+ at m/z 309.1710 (calcd. for C17H24O5; 309.1702). The HRMS

showed peaks at m/z 248.1415 (C15H20O3) and 209.1180 (C12H17O3) were due

to the loss of acetoxy and angeloyloxy groups from the molecular ion peak.

The detailed mass fragmentation pattern of compound A is shown in figure 4.3.

CHAPTER # 4 RESULTS

105

m/z 308

m/z 209

+

m/z 248

+

AcOH

Figure 4.3: EI-MS Fragmentation pattern of compound A

m/z 83

O

CH3

OH3C

O

CH3H3C

O

O

CH3

CH3

O

CH3

CH3H3C

O

O

CH3

CH3

O

CH3

OH3C

O

CH3H3C

C5H7O2

m/z 166

+O

CH3

OH3C

O

+O

CH3

CH3

C12H17O4

CHAPTER # 4 RESULTS

106

The 1H-NMR spectrum (Table 4.9) of compound A displayed a signal for

acetyl group at 2.06 (3H, s), isopropyl group 0.96 (3H, d, J = 7.0 Hz), 0.99

(3H, d, J = 7.0 Hz) and 2.00 (1H, m), methyl attached to olefinic center 1.80

(3H, d, J = 1.0 Hz), two aliphatic oxygenated protons 5.53 (1H, dd, J = 5.5,

3.5 Hz) and 5.79 (1H, d, J = 12.5 Hz) and an aliphatic proton at 2.40 (1H,

ddd, J = 12.5, 4.0, 3.5 Hz). The signal for angeloyloxy group was appeared at d

1.93 (3H, d, J = 1.5 Hz), 1.99 (3H, d, J = 7.0 Hz) and 6.11 (1H, dd, J = 7.0, 1.5

Hz). By comparing the data with literature it was found that was a 7-

deoxycarvotacetone derivative (Jakupovic et al. 1987, 1990, Zdero et al. 1991,

Ahmad and Mahmoud 1997).

The 13C-NMR spectra (BB and DEPT) of compound A (Table 4.10) disclosed

total seventeen carbon signals for six methyl, six methine and five quaternary

carbons. The downfield signals at 194.7, 137.9 and 137.3 were due to six

membered conjugated ketone. The signals at 167.1, 138.7, 127.4, 20.4 and

15.7 indicated the presence of angeloyloxy group (Appendino et al. 1998).

The substitutions and the linkages were done by 1H-1H COSY and long-range

HMBC correlations (Table 4.9). The position of acetyl and angeloyloxy groups

at C-3 and C-5 was confirmed by HMBC corrections in which H-3 ( 5.53)

showed 3J correlation with ester carbonyl ( 170.1) and H-5 ( 5.79) with C-1'

( 167.1).

The relative stereochemistry of compound A was determined by comparing the

coupling constants with the literature values (Jakupovic et al. 1987, 1990,

Zdero et al. 1991, Ahmad and Mahmoud 1997) and the absolute configuration

was deduced from the positive Cotton effect by application of the helicity rule

for conjugated transoid ketones in CD spectrum. On the basis of above data the

structure of compound A was established as [3-acetoxy-5-angeloyloxy-7-

deoxy-carvotacetone] and it was found to be a new natural product. It was

named on the basis of the genus as vernonione.

CHAPTER # 4 RESULTS

107

Table 4.9. 1H-NMR chemical shifts, 1H→13C, HMQC direct correlations (1J) and

1H→13C HMBC long-range correlations (2J and 3J) in compound A

Proton 1H (J = Hz) C

1J 2J

3J H-2 6.74 dd (5.5, 1.0) 137.9 1, 3 4, 7

H-3 5.53 dd (5.5, 3.5) 68.0 2, 4 1, 5, 1

H-4 2.40 ddd (12.5, 4.0, 3.5) 46.2 3, 5, 8 2, 6, 9, 10

H-5 5.79 d (12.5) 72.9 4, 6 1, 3, 8, 1

H-7 1.80 d (1.0) 15.4 1 2, 6

H-8 2.00 m 27.7 4, 9, 10 3, 5

H-9 0.96 d (7.0) 19.4 8 4, 10

H-10 0.99 d (7.0) 19.6 8 4, 9

H-3 6.11 dd (7.0, 1.5) 138.7 2, 4 1, 5

H-4 1.99 d (7.0) 15.7 3 2

H-5 1.93 d (1.5) 20.4 2 1, 3

-COCH3 2.06 s 21.1 1

CHAPTER # 4 RESULTS

108

Table 4.10. 13C-NMR chemical shift assignments of compound A

Carbon No. 13C-NMR Chemical Shift () Multiplicity (DEPT)

C-1 137.3 (C)

C-2 137.9 (CH)

C-3 68.0 (CH)

C-4 46.2 (CH)

C-5 72.9 (CH)

C-6 194.7 (C)

C-7 15.4 (CH3)

C-8 27.7 (CH)

C-9 19.4 (CH3)

C-10 19.6 (CH3)

C-1 167.1 (C)

C-2 127.4 (C)

C-3 138.7 (CH)

C-4 15.7 (CH3)

C-5 20.4 (CH3)

C=O 170.1 (C)

-COCH3 21.1 (CH3)

CHAPTER # 4 RESULTS

109

Figure 4.4. UV spectrum of compound A

Figure 4.5. IR spectrum of compound A

CHAPTER # 4 RESULTS

110

Figure 4.6. 1H-NMR spectrum of compound A (CDCl3)

Figure 4.7. 13C-NMR spectrum of compound A (CDCl3)

CHAPTER # 4 RESULTS

111

4.5.2 Compound B (Cinerascenone)

Compound B

OOCH3

OH

OOH

O

O

HO

12

3410

5

6

7

81'

2'

3'

4'

5'

6'

7''

1''

2''

3''

4''

5''

6''9

Compound B was isolated as yellow amorphous powder. The IR spectrum

(figure 4.10) showed the absorptions for hydroxyl and aromatic protons (3417,

2926 cm-1), aromatic system and substituted phenyl rings (1655-1502, 831

cm-1). The UV spectrum (figure 4.9) showed absorption bands at 242, 250, 271

and 348 nm which is typical for substituted flavonoids and closely related to

chrysoeriol (Voirin 1983). The high resolution electron impact mass

spectroscopy (HREIMS) deduced the molecular formula C23H16O8 showing

[M]+ peak at m/z 420.0839 (calcd. for C23H16O8, 420.0845) and indicated the

presence of sixteen double bond equivalence. The fragment at m/z 272.0325 in

HREIMS of composition C14H8O6 and 148.0529 (C9H8O2) could arise by the

RDA cleavage of ring C. The detailed mass fragmentation pattern of compound

B is shown in figure 4.8.

CHAPTER # 4 RESULTS

112

O

OOH

O

O

HO OH

OCH3

OH

O

O

HO

O

OOH

O

OH

OCH3

O

OH

O

OH

O

O

OOH

O

OH

O

OOH

O

O

HO

OH

OCH3

H

m/z 420

m/z 299

+

Benzoyl

m/z 267

+

CH3OH

m/z 228

+

m/z 256

+

CH3 + CO

+

++

Figure 4.8: EI-MS Fragmentation pattern of compound B

m/z 272 m/z 148

CHAPTER # 4 RESULTS

113

The 1H-NMR spectrum [Table 4.11] of compound B displayed an ABX splitting

pattern at δ 7.46 (1H, dd, J = 8.0, 1.5 Hz), 7.44 (1H, d, J = 1.5 Hz) and 6.91 (1H, d, J

= 8.0 Hz) indicated the presence of 1,3,4-trisubstituted benzene ring. The two

doublets at δ 6.41 and 6.18 (1H each, J = 1.2 Hz) are typical for flavonoids having

substitution at 5 and 7 position (Park et al. 2007). A singlet at δ 6.54 corresponds to

H-3 in flavones. The 1H-NMR spectrum also showed two doublets each integrating

for two protons at δ 7.81 (d, J = 8.5 Hz) and 6.90 (d, J = 8.5 Hz) indicated the

presence of p-hydroxybenzoyl moeity in the molecule (Riaz et al. 2004). The signal

for methoxy group was appeared at δ 3.94.

The 13C-NMR (BB and DEPT) of compound B (Table 4.12) displayed highly

resolved 21 carbon signals for one methyl, eight methine and twelve quaternary

carbon atoms. The signal for the conjugated carbonyls was appeared at δ 184.0 along

with ester carbonyl δ 166.3. The downfield signals at δ 166.7, 163.0, 159.0, 152.5,

149.2 was due to the presence of oxygenated aromatic quaternary carbons. The

presence of p-hydroxybenzoyl group was also supported by 13C-NMR due to the

signals at δ 166.3, 163.0, 129.4, 117.5 and 117.0 (Riaz et al. 2004). The signal for

methoxy carbon was appeared at δ 56.5. The above data conclude that the compound

B was closely related to chrysoeriol (Park et al. 2007, Nathan et al. 1974) with the

addition of p-hydroxybenzoyl group.

The position of methoxy and p-hydroxybenzoyl group was finally confirmed by

Heteronuclear Multiple Bond Correlations (HMBC) in which CH3O- (δ 3.94) showed 3J correlation with C-3' (δ 149.2). The position of p-hydroxybenzoyl group was

deduced by the RDA fragment in HREIMS at m/z 272.0325 (C14H8O6) and 148.0529

(C9H8O2) indicated the presence of methoxy group in ring B and p-hydroxybenzoyl

group in ring A (Justesen 2001). The downfield shift of C-7 (δ 166.7) indicated the

presence of p-hydroxybenzoyl group at this position and confirmed by the HMBC

correlations (Table 4.11) of H-6 (δ 6.18) and H-8 (δ 6.41) with C-7 (δ 166.7). On the

basis of above data the structure of compound B was established as [5,4'-dihydroxy-7-

(4-hydroxybenzoyl)-3'-methoxyflavone] and found to be a novel natural compound. It

was named on the basis of the species name as cinerascenone.

CHAPTER # 4 RESULTS

114

Table 4.11. 1H-NMR chemical shifts, 1H→13C, HMQC direct correlations (1J) and

1H→13C HMBC long-range correlations (2J and 3J) in compound B

Proton 1H (J = Hz)

C

1J

2J

3J

H-3 6.54 s

104.1 (3) 2, 4

10, 1

H-6 6.18 d (1.2) 100.2 (6) 5, 7 8, 10

H-8 6.41 d (1.2) 95.1 (8) 7, 9 6, 10

H-2 7.44 d (1.5) 110.6 (2) 1, 3 2, 4, 6

H-5 6.91 d (8.0) 116.7 (5) 4, 6 1, 3

H-6 7.46 dd (8.0, 1.5) 121.7 (6) 1, 5 2, 2, 4

H-2,6 7.81 d (8.5) 129.4 (2,6) 1, 3, 5 4, 7

H-3,5 6.90 d (8.5) 117.0 (3,5) 4, 2, 6 1

-OCH3 3.94, s 56.5 3

CHAPTER # 4 RESULTS

115

Table 4.12. 13C-NMR chemical shift assignments of compound B

Carbon No. 13C-NMR Chemical Shift (δ) Multiplicity (DEPT)

C-2 166.2 (C)

C-3 104.1 (CH)

C-4 184.0 (C)

C-5 163.0 (C)

C-6 100.2 (CH)

C-7 166.7 (C)

C-8 95.1 (CH)

C-9 159.0 (C)

C-10 105.2 (C)

C-1 123.0 (C)

C-2 110.6 (CH)

C-3 149.2 (C)

C-4 152.5 (C)

C-5 116.7 (CH)

C-6 121.7 (CH)

C-1 117.5 (C)

C-2,6 129.4 (CH)

C-3,5 117.0 (CH)

C-4 163.0 (C)

-OCH3 56.5 (CH3)

CHAPTER # 4 RESULTS

116

Figure 4.9. UV spectrum of compound B

Figure 4.10. IR spectrum of compound B

CHAPTER # 4 RESULTS

117

Figure 4.11. 1H-NMR spectrum of compound B (CDCl3)

Figure 4.12. 13C NMR spectrum of compound B (CDCl3)

CHAPTER # 4 RESULTS

118

4.5.3 Compound C (2-Hydroxy-3-methoxy-5-(2′-propenyl)-phenol)

Compound C was obtained as yellow oil. The molecular formula C10H12O3 was

deduced by HREIMS showing molecular ion peaks at m/z 180.0779 (calcd. for

C10H12O3, 180.0786). The UV spectrum (figure 4.13) showed the absorption maxima

at 330, 280 and 236 nm indicated the substituted phenyl ring. The IR spectrum (figure

4.14) showed absorption signals at 3362, 2968, 1604, 1284 and 1054 cm-1 for

hydroxyl substituted aromatic system.

The 1H-NMR spectrum of compound C displayed two broad singlets at 6.80 and

6.77 in the aromatic region. It also showed two multiplets at 5.92 and 5.02. The

signal at 3.28 (1H, d, J = 7.2 Hz) assigned to saturated methylene. The signal for

methyl group was appeared at 3.81.

The 13C-NMR spectrum (BB and DEPT) of compound C disclosed total ten carbon

signals including one methyl, two methylene, three methine and four quaternary

carbon atoms. The downfield signals at 150.1, 141.4 and 138.2 were due to the

presence of oxygenated aromatic quaternary carbons. The signals at 138.9 and 115.9

were assigned to terminal non-conjugated double bond. The substitution at various

positions was finally confirmed by long rang (H→C) HMBC and 1H→1H COSY

correlations. Comparison of these data with those reported in literature identified

compound C as 2-hydroxy-3-methoxy-5-(2′-propenyl)-phenol (Bezabih et al. 1997).

OH

OCH31'

2'

3'

Compound C

1

2

35

6

OHCH2

4

CHAPTER # 4 RESULTS

119

4.5.4 Compound D (Vanillic Acid)

Compound D was obtained as crystalline solid. The molecular formula C8H8O4 was

established through HREIMS showing molecular ion peak at m/z 168.0415 (calcd. for

C8H8O4, 168.0422). The UV spectrum (figure 4.17) showed absorption maxima at

283, 251 and 219 nm typical for substituted aromatic acid. The IR spectrum (figure

4.18) exhibited absorption bands at 3445, 2925, 1741, 1599-1515, 1281 and 1032

cm-1 for hydroxyl and substituted aromatic system.

The 1H-NMR spectrum of compound D was showed ABX pattern δ 7.56 (1H, d, J =

1.5 Hz), 7.46 (1H, dd, J = 8.0, 1.5 Hz), 6.73 (1H, d, J = 8.0 Hz) indicated the presece

if 1,3,4-trisustituted benzene ring in the molecule. The signal for methyl group was

appeared at δ 3.87.

The 13C-NMR spectrum (BB and DEPT) of compound D showed highly resolved

eight carbon signals for one methyl, three methine and four quaternary carbon atoms.

The downfield signals at δ 175.0, 149.2 and 148.0 was assigned to conjugated acid

and oxygenated aromatic quaternary carbons. The signal for aromatic methoxy group

was resonated at δ 56.2. The structure was finally confirmed by long range HMBC

corellations. In the light of above evidence as well as by comparison with literature,

the compound D was identified as vanillic acid (Ouattara et al. 2004).

COOH

OH

OCH3

1

2

3

4

5

6

7

Compound D

CHAPTER # 4 RESULTS

120

4.5.5 Compound E (Isoferulic acid)

Compound E was isolated as crystalline solid. The HREIMS of compound E showed

molecular ion peak at m/z 194.0570 corresponding to the molecular formula C10H10O4

(calcd. for C10H10O4, 194.0579). The UV spectrum (figure 4.21) showed absorptions

at 335, 300 and 220 nm typical for substituted cinnamic acid. The IR spectrum (figure

4.22) showed the absorption bands at 3627, 3427-2500, 1695, 1596 and 1264 for

hydroxyl, carboxylic acid and substituted aromatic system.

The 1H-NMR spectrum of compound E displayed ABX splitting pattern in the

aromatic region at δ 7.04 (1H, d, J = 1.5 Hz), 6.98 (1H, dd, J = 8.4, 1.5 Hz), 6.71 (1H,

d, J = 8.4 Hz) along with two doublets at δ7.60 (1H, d, J = 16.0 Hz) and 6.25 (1H, d, J

= 16.0 Hz) indication of a 1,3,4-trisubstituted trans-cinnamic acid. It also showed the

signal for aromatic methoxy group at δ 3.95 (3H, s).

The 13C-NMR (BB and DEPT) spectrum of compound E disclosed the presence of

ten carbon signals for one methyl, five methine and four quaternary carbon atoms.

The downfield signal at δ 168.3 was assigned to conjugated acid carbonyl where as

other signals in the aromatic region at δ 149.0, 147.5, 147.1, 127.7, 123.1, 116.4,

115.1 and 115.1 were due to aromatic methines and aromatic quaternary carbon

atoms. The siganal for aromatic methoxy group was attributed at δ 56.0. The structure

was finally confirmed by the combination HMBC and COSY correlations.

On the basis of above evidences as well as by comparison from the literature

(Hoeneisen et al. 2003), the compound E was identified as isoferulic acid.

OH

O

H3CO

OH

Compound E

1

2

34

5

6

7

8

9

CHAPTER # 4 RESULTS

121

4.5.6 Compound F (Caffeic acid)

Compound F was obtained as colorless crystalline solid. The molecular formula

C10H10O4 was established by HREIMS gave the molecular ion peak at m/z 180.0416

(calcd. for C10H10O4, 180.0422). The UV and IR spectra (Figures 4.25, 4.26) were

very similar to that of compound E showed the absorption bands at 334, 245, 220 nm

and 3666, 3422-2395, 1694, 1598, 1264 cm-1 for substituted cinnamic acid.

The 1H-NMR spectrum of compound F displayed resonances in the aromatic region

as ABX splitting pattern at δ 7.08 (1H, d, J = 2.0 Hz), 6.96 (1H, dd, J = 8.0, 2.0 Hz),

6.78 (1H, d, J = 8.0 Hz) and two doublets at δ 7.62 (1H, d, J = 15.5 Hz), 6.42 (1H, d,

J = 15.5 Hz) was due to 1,3,4-trisubstituted trans-cinnamic acid.

The 13C-NMR (BB and DEPT) spectrum of compound F disclosed the presence of

nine carbon signals for five methine and four quaternary carbon atoms. The downfield

signals at δ 168.9 was assigned to conjugated acid carbonyl whereas other signals at δ

149.0, 146.7, 147.6, 128.0, 123.3, 116.7, 115.5 and 114.5 were due to aromatic

methines and aromatic quaternary carbons. The structure was finally confirmed by the

combination HMBC and COSY correlations.

On the basis of above evidences as well as comparison with literature (Hoeneisen et

al. 2003), the compound F was identified as caffeic acid.

OH

O

HO

OH

1

2

34

5

6

7

8

9

Compound F

CHAPTER # 4 RESULTS

122

4.5.7 Compound G (Methyl gallate)

Compound G was obtained as colorless amorphous powder. The HREIMS showed

the molecular ion peak at m/z 180.0364, corresponding to molecular formula C8H8O5

(calcd. for C8H8O5, 180.0371). The UV spectrum (figure 4.29) showed absorption

bands at 330, 260 and 236 nm closely related to substituted aromatic acids. The IR

spectrum (figure 4.30) showed absorption bands at 3597, 2963, 1742, 1697, 1237 and

1016 cm-1 hydroxyl group, conjugated carbonyl and aromatic system.

The 1H-NMR spectrum of compound G showed only two singlets at 7.01 (2H, s)

and 3.80 (3H, s) typical for methyl gallate.

The 13C-NMR spectra (BB and DEPT) of compound G showed five carbon siganls

which revealed the presence of one methyl, one methine and four quaternary carbon

atoms. The signals at δ 169.0, 146.5, 139.9, 121.3, 110.0 and 52.2 were typical for

methyl gallate. The structure was finally confirmed by long range HMBC

correlations.

The physical and spectral data of compound G was in complete agreement to that

reported in literature for methyl gallate (Lajis and Khan 1994).

Compound G

COOCH3

OH

OHHO

1

2

3

45

6

CHAPTER # 4 RESULTS

123

4.5.8 Compound H (Uridine)

Compound H was obtained as white amorphous solid. The HRFABMS of compound

H gave the molecular ion peak [M+H]+ at m/z 245.0770 corresponding to the

molecular formula C9H13N2O6 (calcd. for C9H13N2O6, 245.0773). The UV spectrum

(figure 4.33) showed the absorption bands at 264 and 214 nm. The IR spectrum

(figure 4.34) showed the absorptions at 3451, 2926, 1670, 1595 and 1096 cm-1 for

hydroxyl and olefinic system.

The 1H-NMR spectrum of compound H displayed a two doublets at δ 8.00 (1H, d, J

= 8.0 Hz) and 5.68 (1H, d, J = 8.0 Hz). The signals for ribose moiety were appeared at

δ 5.88 (1H, d, J = 5.0 Hz), 4.16 (1H, t, J = 5.0 Hz), 4.13 (1H, t, J = 5.0 Hz), 4.00 (1H,

m, Hz), 3.83 (1H, dd, J = 12.0, 2.5 Hz) and 3.72 (1H, dd, J = 12.0, 3.0 Hz).

The 13C-NMR (BB and DEPT) spectrum of compound H disclosed total nine carbon

signals for one methylene, six methine and two quaternary carbon atoms. The

downfield signals at δ 166.0, 152.0 and 142.7 were due to amide carbonyls and

conjugated methane. The signals at δ 90.7, 86.3, 75.7, 71.3 and 62.5 indicated the

presence of ribose sugar attached to nitrogen atom. The structure was further

confirmed by the combination HMBC and COSY correlations.

The spectral data of compound H was in complete agreement to those reported for

uridine (Okuda et al. 1986).

N

NH

O

O

O

OHHO

HO

12

3

4

5

6

5'

2'3'

4' 1'

Coumpound H

CHAPTER # 4 RESULTS

124

4.5.9 Compound I (3'-Methylquercetin)

Compound I was obtained as yellow powder. The HREIMS of compound I deduced

the molecular ion peak at m/z 316.0575 corresponding to the molecular formula

C16H12O7 (calcd. for C16H12O7, 316.0583) indicating the presence of eleven double

bond equivalence. The UV spectrum (figure 4.37) showed the bands at 366, 254 and

209 nm indicated the presence of flavonoid nucleus (Voirin 1983). The IR spectrum

(figure 4.38) showed the absorption bands at 3270, 2928, 1730, 1657-1509, 1168 and

1033 cm-1 for substituted aromatic system.

The 1H-NMR spectrum of compound I displayed two doublets and a double doublet

in aromatic region at δ 8.00 (1H, d, J = 2.0 Hz), 7.56 (1H, dd, J = 8.5, 2.0 Hz), 6.89

(1H, d, J = 8.5 Hz) was due to 1,3,4-trisubstituted benzene ring. It also showed two

doublet at δ 6.40 (1H, d, J = 1.2 Hz), 6.17 (1H, d, J = 1.2 Hz) typical for C-5 and C-7

oxygenated flavonoids. The signal for methoxy group was appeared at δ 3.94 as

singlet.

The 13C-NMR (BB and DEPT) spectrum of compound I disclosed the presence of

sixteen carbon signals for one methyl, five methine and ten quaternary carbon atoms.

The downfield signals at d 179.1, 166.1, 163.0, 159.3, 151.1, 148.6 and 148.6 was

assigned to conjugated carbonyl and aromatic oxygenated quaternary carbons. The

structure was finally confirmed through long range HMBC and COSY correlations.

The spectral data of compound I was in complete agreement to those reported for 3'-

methylquercetin (Mullen et al. 2002).

O

OOH

HO

OH

OCH3

OH

12

34

105

6

7

8

9 1'

2'

3'

4'

5'

6'

Compound I

CHAPTER # 4 RESULTS

125

4.5.10 Compound J (Quercetin)

Compound J was obtained as yellow amorphous solid. The molecular formula

C15H10O7 was deduced from the HREIMS, showing molecular ion peak at m/z

302.0419 (calcd. for 302.0426) having eleven degrees of unsaturation. The UV

spectrum (figure 4.41) displayed absorptions at 360, 257 and 211 nm typical for

flavonoids (Voirin 1983). The IR spectrum (figure 4.42) showed the absorption bands

at 3423, 2925, 1658-1598, 1271 and 1058 cm-1 for hydroxyl, conjugated carbonyl and

aromatic system.

The 1H-NMR spectrum of compound J displayed ABX splitting pattern in aromatic

region at δ 7.65 (1H, d, J = 2.0 Hz), 7.63 (1H, dd, J = 8.5, 2.0 Hz) and 6.84 (1H, d, J

= 8.5 Hz) was due to 1,3,4-trisubstituted benzene ring. It also showed two doublets at

δ 6.26 (1H, d, J = 1.5 Hz), 6.09 (1H, d, J = 1.5 Hz) indicated the presence of C-5 and

C-7 oxygenated flavonoids.

The 13C-NMR (BB and DEPT) spectrum of compound J disclosed the presence of

fifteen carbon signals for five methine and ten quaternary carbon atoms. The

downfield signals at d 180.0, 166.1, 164.0, 158.0, 150.0, 146.1 was 146.0 was

assigned to conjugated carbonyl and aromatic oxygenated quaternary carbons. The

structure was finally confirmed by the combination of Long range HMBC and COSY

correlations.

The spectral data of compound J was in complete agreement to those reported for

quercetin (Ahmed and Ismail 2005).

Compound J

O

OOH

HO

OH

OH

OH

12

34

10

5

6

7

8

9 1'2'

3'

4'

5'

6'

CHAPTER # 5 DISCUSSION

126

5. DISCUSSION

The present studies emphasis on biological and phytochemical studies on three Pakistani

medicinal plants namely Vernonia cinerascens, Euphorbia granulata and Euphorbia

serpens. Dichloromethane and methanol extracts of the aerial parts of the above three

plants along with the roots of Vernonia cinerascens were prepared. All the extracts of

aerial parts were screened for spasmolytic, antifungal, antibacterial, brine-shrimp

lethality, phytotoxic, antioxidant bioassays and acetylcholinestrase inhibitory activity;

while dichloromethane and methanol extracts of root parts of Vernonia cinerascens were

screened for spasmolytic, antifungal, antibacterial, brine-shrimp lethality and phytotoxic

bioassays.

Phytochemical tests on dichloromethane and methanolic extracts of Vernonia

cinerascens, Euphorbia granulata and Euphorbia serpens were performed by using

method described by Parekh and Chanda (2007). Phytochemical analysis releaved the

presence of secondary metabolites like alkaloids, anthraquinones, cardiac glycosides,

tannins, flavonoids and saponins (Table 4.2). Results of these phytochemical tests show

that when test was performed for alkaloids, no brownish red precipitates were produced,

which indicated the absence of alkaloids. When test was performed for saponins, green

blue color was observed which showed the presence of saponins. Steroid were absent

because blue green ring was not formed. When the test was performed for flavonoids

pink tomato red color was observed so flavonoids were present. No green color was

observed when test for cardiac glycoside was performed so absence of cardiac glycoside

was confirmed. Blue black color was observed during test for tannins, so tannins were

present.

All the extracts were found to be active in spasmolytic activity and exhibited inhibition of

spontaneous contractions of rabbit jejunum at different concentrations. To test whether

this effect is mediated through the blockade of Ca++ influx, a high dose of K+ (80 mM)

was used to depolarize the tissue. Addition of dichloromethane and methanol extracts

caused the relaxation of K+-precontracted jejunum, verapamil, a standard calcium

channel blocker was used as a control (Kerins et al. 2001), suggesting a calcium channel

blocking activity of the extracts. Spasmolytic drugs are used as adjunctive therapy in the

treatment of peptic ulcer; functional digestive disorders, including spastic, mucous and


127

ulcerative colitis; and diarrhea, diverticulitis and pancreatitis (Robbers et al. 1996). The

results of spasmolytic activity of dichloromethane and methanol extracts of Vernonia

cinerascens, Euphorbia granulata and Euphorbia serpens showed its potential to be an

antispasmodic agent.

In antifungal bioassay the dichloromethane extract of aerial parts of Euphorbia granulata

exhibited 90% inhibition against Microsprum canis and 50% inhibition against

Aspergillus flavus. Where as the dichloromethane extract of aerial parts of Euphorbia

serpens showed 40% inhibition of both the fungi namely Microsporum canis and

Aspergillus flavus. The dichloromethane extract of the roots of Vernonia cinerascens

showed 80% inhibition against Microsporum canis. Where as methanol extract of the

aerial parts and roots of Vernonia cinerascens exhibited 70% and 20% inhibition against

Fusarium solani respectively. Microsporum canis, a zoophilic dermatophyte most

commonly produces tinea capitis and tinea corporis. Tinea corporis in patients with

advanced HIV infection can extend over large areas of the body (Wright 1991). Azole

antifungals are generally the most effective agents but are among the most expensive.

Fusarium species, are soil saprophytes with a worldwide distribution, are well known as

plant pathogens (Booth 1971). Fusarium has been reported to cause keratomycosis,

mycetoma and onychomycosis. In addition, Fusarium species have increasingly been

reported from colonizing and disseminated infection in patients with severe underlying

diseases (Anaissie 1988, Merz 1988). Fusarium species most often reported from human

infection have been F. solani, F. oxysporum, and F. moniliforme. F. solani reported from

human infections, disseminated disease (Cho 1973, Matsuda and Matsumoto 1986,

Okuda 1987, Wheeler 1981), osteomylitis (Nuovo 1988), Skin infections (Hiemenz et al.

1990) fungemia (Chaulk 1986) and endophthalmitis (Forster and Azchary 1976,

Lieberman et al. 1979). Compounds produced by plants are of interest as a source of

safer or more effective substitutes for synthetically produced antimicrobial agents

(Baladrin et al. 1985). The antifungal activities results showed that the crude

dichloromethane and methanol extracts of roots of Vernonia cinerascens has the potential

to be an antifungal agent against Microsporum canis and Fusarium solani. Further

investigation must be performed for detailed antifungal activities studies including the


128

isolation of bioactive antifungal constituents. The discovery of a potent and safe herbal

remedy will be a great achievement in fungal infection therapies.

Methanol extracts of Vernonia cinerascens and Euphorbia serpens exhibited promising

antioxidant activity. Antioxidants are substances that when present in food or in the body

at low concentrations compared with that of an oxidizable substrate markedly delay or

prevent the oxidation of that substrate (Halliwell et al. 1995). Free radicals may be

involved in a number of diseases and tissue injuries such as those of the lungs, heart, and

cardiovascular system, kidneys, liver, gastrointestinal tract, blood, eye, skin, muscle,

brain, and the process of aging (Aruoma 1994, Burr 1994, Hennig and Toboreek 1993).

Oxidants and radicals which mediate various disorders are mainly reactive species which

are formed from triplet oxygen, water, and unsaturated lipid molecules. Plants provide a

rich source of antioxidants (Caragay 1992). The best known natural antioxidants that

have proven importance in the food industry and human health are tocopherol, vitamin C,

and carotenoids (Packer 1996). Recent results of study of Vernonia cinerascens and

Euphorbia serpens methanol extracts as an antioxidant indicates the potential of the plant

for isolation of antioxidant bioactive compounds.

131

6. CONCLUSION The Ph. D dissertation emphasis on the biological and phytochemical studies of three

Pakistani medicinal plants namely Vernonia cinerascens, Euphorbia granulata and E.

serpens. The roots and aerial parts of Vernonia cinerascens and whole plant materials

of Euphorbia granulata and E. serpens were extracted with dichloromethane and

methanol, respectively. These extracts were subjected to a battery of biological

screening and found that the dichloromethane extracts of V. cinerascens (roots) and E.

granulata (whole plant) exhibited significant antifungal activity against Microsporum

canis; the methanolic extract of V. cinerascens showed significant antifungal activity

against Fusarium solani; the dichloromethane and methanolic extracts of E. serpens

exhibited moderate antifungal activity against Aspergillus flavus; the methanolic

extracts of V. cinerascens and E. serpens showed good antioxidant activity and all the

extracts have dose dependent spasmolytic activity. The dichloromethane extract of V.

cinerascens (roots) afforded a new monoterpene, (3β-acetoxy-5α-angeloyloxy-7-

deoxy-carvotacetone). The methanolic extract of the same plant lead to a new flavone

(5,4'-dihydroxy-7-(4-hydroxybenzoyl)-3'-methoxyflavone) and eight known

compounds, namely, 2-hydroxy-3-methoxy-5-(2′-propenyl)-phenol, vanillic acid,

isoferulic acid, caffeic acid, methyl gallate, uridine, 3'-methylquercetin and quercetin

purified by using classical and modern purification techniques. All the known

compounds showed promising antioxidant and significant urease inhibitory activities

with IC50 values ranging between 51.4-600.9 M for antioxidant and 37.4-56.6 M

for urease inhibition. Quercetin found a standard antioxidant. The remaining

compounds showed significant to moderate antioxidant and urease inhibition and can

be a lead source to boost the physiological systems or can be a better source as

therapeutic agents.

CONCLUSION

131

New Compounds isolated from Vernonia cinerascens

(1) Compound A (Vernonione)

CH3

O

OH3C

O

CH3H3C

O

O

CH3

1

2

3

4

5

6

7

89 10

CH3

1'

2'

3'

4'

5'

1''2''

(2) Compound B (Cinerascenone)

OOCH3

OH

OOH

O

O

HO

12

3410

5

6

7

81'

2'

3'

4'

5'

6'

7''

1''

2''

3''

4''

5''

6''9

CONCLUSION

131

Compounds isolated for the first time from Vernonia cinerascens

COOH

OH

OCH3

COOCH3

OH

OHHO

OH

O

RO

OH

OH

OCH3

O

OOH

HO

OH

OR

OH

2-Hydroxy-3-methoxy-5-(2'-propenyl)-phenol

N

NH

O

O

O

OHHO

HO

1

2

3

4

5

6

7

1

2

3

45

6

7

1

2

34

5

6

7

8

9

1

2

3

4

5

6

78

9

12

34

4a5

6

7

88a 1'

2'

3'

4'

5'

6'

12

3

4

5

6

1'

2'3'

4'5'

Isoferulic acid, R = CH3Caffeic acid, R = H

3'-Methylquercetin, R = CH3Quercetin, R = H

CH2

OH

Vanillic acid

Methyl gallate

Uridine

CHAPTER # 6 REFERENCES

132

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