PHYTOCHEMICAL AND BIOLOGICAL STUDIES OF CONYZA BONARIENSIS (COMPOSITAE), EUPHORBIA PROSTRATA AND EUPHORBIA HELIOSCOPIA (EUPHORBIACEAE)

Thesis submitted For the Fulfillment of the Degree of

DOCTOR OF PHILOSOPHY Pharmacy

By

Muhammad Uzair

FACULTY OF PHARMACY BAHAUDDIN ZAKARIYA UNIVERSITY MULTAN-PAKISTAN 2009

ACKNOWLEDGMENTS

Acknowledgments

I express my deepest gratitude from the core of my heart to Almighty Allah, Who showered upon me all his blessings throughout my efforts for the completion of this laborious and hectic work in the face of following:

Words would barely express my feelings of gratitude for the kind support and valuable guidance I received from my research supervisors, Pof. Dr. Bashir Ahmed Loother and Prof. Dr. Bashir Ahmed Choudhary for their constant care and encouragement was a great source of inspiration during the course of this work.

I am much obliged to Prof. Dr Mohammad Iqbal Choudhary and Prof. Dr. Aatta-ur-Rahman 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 Prof. Dr. Khalid Hussain Junbaz , Chairman department of pharmacy , Bahauddin Zakariya university Multan, Pakistan and Prof. Dr. Abdul-subhan Ijaz for their encouragement and co- operation throughout my research work.

I am grateful to all my staff members and laboratory staff for their cooperation during all this period.

I am morally obliged to mention the name of my friends and colleagues, Irshad Ahmad, Dr. Saeed Ahmad Rao, Dr. Mohammad Ashraf , Dr. Mazhar Hussain, Imran Manzoor, Muhammad Arif and Majid Javed.

Hearty appreciations are due to my father, mother, wife, kids, brother, sisters and all other well-wisher who never failed in helping me whenever their assistance was needed.

Muhammad Uzair

IX

Dedicated to my

Parents

And

Family

SUMMARY

Summary

The present research work describes the biological and phytochemical studies of the medicinal plants Conyza bonariensis (Compositae), Euphorbia prostrata and Euphorbia helioscopia (Euphorbiaceae) found locally.

Dichloromethane and methanol extracts of the different parts of Conyza bonariensis, Euphorbia prostrata and Euphorbia helioscopia were prepared.

Biological screening of these extract was carried out and it was observed that dichloromethane extract of the aerial parts of Euphorbia helioscopia exhibits significant antifungal activity against Fusarium solani. Fusarium solani is a pathogen responsible for fungal infection in plant and human.

Dichloromethane extract of the root parts and aerial parts of Conyza bonariensis also showed significant antifungal activity against Microsporum canis and Aspergillus flavus respectively. Dichloromethane extract of the aerial parts of Euphorbia helioscopia when phytochemicaly investigated it gave two compounds (1 & 2) namely 4-hydroxy 3-methoxy cinnamic acid and

7-hydrooxy coumarin. After chemical analysis of Euphorbia prostrata methanolic fraction was considered for further isolation. It offered two compounds (3 & 4) 2-hydroxy cinnamic acid and 3,5,7-trihydroxy-2-(4- hydroxyphenyl)-4H-chromen-4-one. Methanolic extract of the roots of Conyza bonariensis offered compound (5) 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H- chromen-4-one. The structures of the isolated compounds were confirmed by different spectroscopic method.

X LIST OF CONTENTS

List of Contents

NO. Contents Page

Acknowledgments IX Summary X Chapter 1: Introduction 1.1. Introduction of Euophorbiaceae 7 1.2. Genus Euphorbia 7 1.3. Euphorbia helioscopia 7 1.4. Euphorbia prostrata 11 1.5. Asteraceae 14 1.6. Genus Conyza 14 1.7. Conyza bonariensis 14 Chapter 2: Literature Review 2.1. Medicinal and biological importance of genus Euphorbia 18 2.1.1. Analgesic, antipyretic and anti-inflammatory activities 18 2.1.2. Antibacterial activity 18 2.1.3. Antifungal activity 18 2.1.4. Antiamoebic and spasmolytic activities 18 2.1.5. Antimalarial activity 19 2.1.6. Antioxidant activity 19 2.1.7. Diuretic effect 19 2.1.8 Anti-asthmatic activity 20 2.1.9. Cytotoxicity 20 2.1.10. Antiviral activity 21 2.1.11. Hepatoprotective activity 21 2.1.12. Antitumor activity 21 2.1.13. Anti-hyperglycemic activity 21 2.1.14. Central nervous system depressant activity 22 2.1.15. Effects on the cell division 22 2.1.16. Tumor promoting activity 22 2.2. Medicinal and biological importance of genus Conyza 23 2.2.1. Analgesic, antipyretic and anti-inflammatory activities 23

I

LIST OF CONTENTS 2.2.2. Antidiarrhoeal activity 23 2.2.3. Antiviral activity 23 2.2.4. Smooth muscle relaxing activity 24 2.2.5. Antibacterial activity 24 2.2.6. Antimalarial activity 24 2.2.7. Antirheumatic activity 24 2.2.8. Diuretic activity 24 2.2.9. Anticoagulant activity 24 2.3. Phytochemical studies of Euphorbia species 25 2.3.1. Lipids and its derivatives 25 2.3.2. Alkaloids 26 2.3.3. Flavonoids 27 2.3.4. Coumarins 31 2.3.5. Terpenes 33 2.3.6. Sesquiterpenoids 33 2.3.7. Diterpene 34 2.3.8. Triterpenes 39 2.3.9. Steroids 40 2.3.10. Sugars 42 2.4. Phytochemical studies of Conyza species 42 2.4.1. Alkaliods 42 2.4.2. Monoterpenoid 43 2.4.3. Sesquiterpnoid 43 2.4.4. Diterpnoid 44 2.4.5. Triterpnoid 45 2.4.6. Flavonoids 47 2.4.7. Saponins 48 2.4.8. Glycosides 51 2.4.9. Coumarin 51 2.5. Medicinal Uses of Euphorbia helioscopia 52 Reported phytochemical studies of Euphorbia 2.6. 53 helioscopia

2.7. Medicinal Uses of Euphorbia prostrata 62

II

LIST OF CONTENTS 2.8. Reported Phytochemical Studies of Euphorbia prostrata 62 2.9. Medicinal uses of Conyza bonariensis 71 2.10. Phytochemical studies of Conyza bonariensis 71 Chapter 3: Biosynthesis 3. Biosynthetic aspects of primary metabolic products 74 3.4. Biosynthesis of coumarin 78 3.5. Biosynthesis of 4-hydroxy-3-methoxy cinnamic acid 79 3.6.. Biosynthesis of flavonoid 80 3.8. Flavonoid classes 80 Chapter 4: Material Methods 4.1. Plant materials 82 4.2. Extraction 82 4.3. Chromatographic studies 82 4.3.1 Analytical 82 4.3.2. High performance liquid chromatography ( HPLC ) 83 4.4. Isolation 83 4.4.1. Column chromatography 83 4.4.2. Gel chromatography 83 4.4.3. Solvents and chemicals 86 4.5. Preparation of reagent 86 4.6. Preparation of solutions 87 4.7. Detection of various classes of secondary metabolites 89 4.7.1. Detection of alkaloids 89 4.7.2. Detection of anthraquinones glycosides 89 4.7.3. Detection of cardio active glycosides 90 4.7.4. Detection of tannins 90 4.7.5. Detection of flavonoids 91 4.8. Biological methods 91 4.8.1. Antifungal assay 91 4.8.2. Antibacterial assay 92 4.8.3. Antioxidant assay 92 4.8.4. Phytotoxicity bioassay 92 4.8.5. Brine shrimp lethality bioassay 93 4.9. Spectroscopic methods 94

III

LIST OF CONTENTS 4.9.1. Physical constants 95 4.10. Phytochemical isolation 96 4.10.1. Isolation of compounds 1 and 2 96 4.10.2. Isolation of compounds 3 and 4 99 4.10.3. Isolation of compound 5 102 Chapter 5: Results 5.1. Extraction 104 5.2. Biological screening 104 5.2.1. Antibacterial activity 104 5.2.2. Antifungal bioassay 104 5.2.3. Brine shrimp lethality bioassay And phytotoxic bioassay 105 5.2.4. Antioxidant activity 105 5.3. Phytochemical analysis 105 5.4. Phytochemical isolation 113 5.4.1. Isolation of compounds 1 and 2 113 5.4.2. Isolation of compounds 3 and 4 115 5.4.3. Isolation of compound 5 117 5.5. Structure determination 119 5.5.1. Structure determination of compound 1 119 5.5.2. Structure determination of compound 2 124 5.5.3. Structure determination of compound 3 129 5.5.4. Structure determination of compound 4 134 5.5.5. Structure determination of compound 5 139 Chapter 6: Discussion 144 Chapter 7: References 148

IV

LIST OF FIGURES

List of Figures

Figure Title Page No. 1 Euphorbia helioscopia Gallery 9 1.1 Seeds of E.heliosopia 10 1.2 Seedling of E.heliosopia 10 1.3 Plant of E.heliosopia 10 1.4 Roots of E.heliosopia 10 1.5 Shoot of E.heliosopia 10 1.6 Leaf of E.heliosopia 10 1.7 Flowers of E.heliosopia 10 1.8 Flower of of E.heliosopia 10 1.9 Bunch of E.heliosopia 10 2 Euphorbia prostrata Gallery 12 2.1 Seeds of E. prostrata 13 2.2 Plant of E. prostrata 13 2.3 Seedling of E. prostrata 13 2.4 Shoot of E. prostrata 13 2.5 Root of E.prostrata 13 2.6 Shoot with latex of E. prostrata 13 2.7 Cluster of shoots E. prostrata 13 2.8 Shoot with flowers E. prostrata 13 2.9 Flowers E. prostrata 13 3 Conyza bonariensis Gallery 16 3.1 Stem of C.bonariensis 17 3.2 Seeds of C.bonariensis 17 3.3 Leaves of C.bonariensis 17 3.4 Shoot with Flowers of C.bonariensis 17 3.5 Seedling of C.bonariensis 17 3.6 FlowerOf C.bonariensis 17 3.7 Roots of C.bonariensis 17 3.8 Immature flower C.bonariensis 17 3.9 shoot C.bonariensis 17

VI LIST OF FIGURES

4 Interrelations of biosynthetic pathways leading to 75 secondary metabolites in plants 5 Acylopolymalonate pathway 76 6 Shikimate Pathway 77 7 Biosynthesis of Coumarin 78 8 Biosynthesis of 4-Hydroxy-3-methoxycinnamic acid 79 9 Biosynthesis of apigenin and kaempferol. 81

10 Isolation of compound 1 and 2 from dichloromethane 114 extract of E. helioscopia 11 Isolation of compound 3 and 4 from methanol extract of E. 116 prostrata 12 Isolation of compound 5 from methanol extract of C. 118 bonariensis 13 UV spectrum of compound 1 121 14 IR spectrum of compound 1 121 15a H NMR spectrum of compound 1 122

15b Elaborated H NMR spectrum of compound 1 122 16 13C spectrums of compound 1 123 17 Mass spectrum of compound 1 123 18 UV spectrum of compound 2 126 19 IR spectrum of compound 2 126 20a 1H NMR spectrum of compound 2 127 20b Elaborated H NMR spectrum of compound 2 127 21 13C spectrums of compound 2 128 22 Mass spectrum of compound 2 128 23 Figure 23: UV spectrum of compound 3 131 24 IR spectrum of compound 3 131 25a 1H NMR spectrum of compound 3 132 25b Elaborated H NMR spectrum of compound 3 132 26 13C spectrums of compound 3 133 27 Mass spectrum of compound 3 133 28 UV spectrum of Compound 4 136 29 IR spectrum of compound 4 136 30a 1H NMR spectrum of compound 4 137 30b Elaborated H NMR spectrum of compound 4 137

VII LIST OF FIGURES

31 13C spectrum of compound 4 138 32 Mass spectrum of compound 4 138 33 UV spectrum of compound 5 141 34 IR spectrum of the compound 5 141 35a 1H NMR spectrum of the com1pound 5 142 35b Elaborated H NMR spectrum of compound 5 142

36 13C spectrum of compound 5 143 37 Mass spectrum of compound 5 143

VIII 1 LIST OF PUBLICATIONS

LIST OF PUBLICATIONS

1. Uzair, M., Loothar, B. A. and Choudhary, B. A. (2009). Biological screening of Euophorbia helioscopia L. Pak. J. Pharm. Sci.,Vol.22.No.2, 184-186. 2. Bashir, A. Ch., Khalid, H. J., Uzair, M. and Ijaz, S. A. (2001). Biological studies of Conyza and Euphorbia species. Journal of Research (Science). 12, 85-88.

3. Loothar, B. A., Uzair, M., Ansari, M. T. and Jahangir, Q. (1998). Pharmacokinetic interaction between cimetidine and rifampicin in human volunteers. Pakistan Journal of (Science). 50, 30-33.

4. Loothar, B. A., Uzair, M., Ansari, M. T. and Rana, M. A. (1996). Influence of cimetidine on the pharmacokinetic parameters of isoniazid in human volunteers. Journal of Research ( Science). 8, 21.

5. Arif, M., Loothar, B. A., Nawaz, R., Uzair, M. and Pervez, H. (1993). The influence rifampicin on the pharmacokinetic parameters of chlorpropamide. Journal of Research ( Science). Bahauudin Zakariya University, Multan Pak. 5, 17-20.

6. Arif, M., Loothar, B. A., Nawaz, R., Uzair, M. and Pervez, H. (1993). Study of the interaction between isoniazid and chlorpropamide in human volunteers. Science International (Lahore). 5, 173-174.

LIST OF TABLES

List of Tables

Table Tables Page NO.

Solvent systems used for the analysis of dichloromethane 1 84 extracts of E. helioscopia, E. prostrata and C. bonariensis

Solvent systems used for the analysis of methanol extracts of 2 85 E. helioscopia, E. prostrata and C. bonariensis

Results of the extraction of plants Euphorbia helioscopia 3 ,Euphorbia prostrata and Conyza bonariensis with different 106 solvents

Results of antibacterial activity of the different extracts of 4 107 E.helioscopia, E. prostrata and C. bonariensis

Results of antifungal activity of different extracts of 5 108 E.helioscopia, E. prostrata andC. bonariensis

6 Results of brine shrimp lethality bioassay of the different 109 extracts of E.helioscopia, E. prostrata andC. bonariensis

Result of phytotoxicity bioassay of the different extracts of 7 110 E.helioscopia, E. prostrata and C. bonariensis

Result of antioxidant bioassay of the different extracts of 8 111 E.helioscopia, E. prostrata and C. bonariensis

Result of phytochemical analysis of E. helioscopia ,E. prostrata 9 112 and C. bonariensis

V CHAPTER # 1 INTRODUCTION

1. GENERAL INTRODUCTION Throughout history natural products from the plants have played a major role in the life of human beings regarding for food source and for medicinal products (Leland et al., 2006). Early documents about the use of medicinal plants are sparse. History of mankind describes a strong relationship between man, plants and the drugs derived from plants. It is a fact that majority of the world population still depend upon plant for the treatment of different kinds of ailments. The Greeks are considered to have a great knowledge about the uses of herbs. Babylon about 2000 B.C. gives instruction for the preparation and administration of medicinal herbs by the early first century A.D. Greek herbalist listed 500 plants with their medicinal properties. Romans also have a great deal with medicinal herbs. About 200 herbs were introduced to Britain by the invading Romans (Peter et al., 1994). Herbal remedies have got a popular position among the patient with medical problems like arthritis, diabetes, cancer, depression, eczema, insomnia, and cardiac disorders. The role of plants in health care system is still under estimated. Unani system of Pakistan, Ayurvedic system of India and Chinese herbal system are the pillars of modern medicine system (Trops et al., 2001). Most important medicinal plants including Belladonna (Atropa belladonna), Digitlis (Digitalis purpurea), Henbane (Hyoscyamus niger), Datura (Datura stramonium) have also been used in ancient Greek and American shamanic rituals. (Schultes et al.,1992). Medicinal plants are widely known for their curative potential in certain diseases like Acorus calamus as tranquilizer, Aegle marmelos and plantago ovate in chronic diarrhea, Butea frondosa as an anthelmintic and Curcuma longa as anti-inflamatory (Bhattacharya et al., 2001). Rosemary herb was considered to stimulate the mind in particular the memory (Strange et al., 1977). Since 4000 B.C. Greeks and Romans used opium poppy to relieve pain and induce sleep (Mann et al., 1992). Coca leaves are still used by South American Indians to sustain energy and physical activities (Karch et al., 1997). Nowadays medicinal chemists all around the world are emphasizing the valueable herbal remedies for enjoying good health. According to World Health Organization (WHO) that 80 % of the world‘s inhabitants still rely on traditional medicine for their health related problems, remaining 20% of the population belongs to developed countries. 1

CHAPTER # 1 INTRODUCTION

Large number of synthetic drugs has natural origin from animals, plants and microorganisms. Reserpine, Scopolamine and Atropine are important drugs derived from different plants families. Aspirin for the first time was extracted from willow tree bark (Kiefer et al., 1997). It is assumed that cancer is easier to prevent than to treat, consumption of certain fruits and vegetables can reduce the risk of cancer (Aggarwal et al., 2004). Researchers are expecting new molecules from herbs that can provide us with tools for fighting this dreaded disease. Allamanda cathratica (allamandin), Elephatopus elatus (elephantpoin), Helenium autmnale (helenalin) Vernonia hymenlepis, Heliotropium indicum (Indicine-N-oxide) Daphne mezereum (mezerien) and Stereospermum suaveolans (laphacol) are medicinal plants that have shown significant tumor inhibiting effect. Diabetes mellitus is another area where a lot of research is going on. Ajuga reptens (the active principle is said to potentiate effects of insulin), Galagea officinalis (galagine), Bougainvillea spectabilis (pinitol), Momordica charantia (chirantin), Gymnema sylvestre (gymnemic acid) are some medicinal plants that have shown effectiveness in non-insulin dependent diabetes. Arthritis a very common disease its no satisfactory treatment is present in modern medicine. Commiphora mukul (guggulsterones), Boswellia serrata (boswellic acid), Withania somnifera (withanolides), Ruscus acueleatus (ruscogenin), Harpagophytum procumbens (harpagoside) are prominent plants with anti- arthritic activity. Medicinal plants may be used directly or in the form of extracts or they may also be used in the production of drugs ultimately used for the cure of ailments. Plants produce biochemical compounds as a result of their metabolic activities which can be divided into two categories, (a). Primary metabolites; which include amino acids, sugars, fats and (b). Secondary metabolite; which are much more specialized substances with a wide variety of functions. Over many years, plant pharmacologists and other scientists have accumulated a wealth of knowledge on the chemical constituents of plants and their pharmacological actions. At present, use of medicinal plants in the primary health care is prevailing in most of the countries. The goal of modern phytochemical research is to develop preparation derived from medicinal plants to meet present day standards of quality, safety and efficacy. Achievement of this goal will be possible by proper isolation and structural 2

CHAPTER # 1 INTRODUCTION elucidation of major constituents of medicinal plants, elucidation of the structures of the isolated components and complete profile of pharmacological effects including the molecular and biological mechanisms followed by toxicological studies and controlled clinical studies according to modern clinical practice. The efficacy of some plant products is beyond doubt, the most recent examples being Silybum marianum (silymarin), Artemisia annua (artemesinin) and Taxus baccata (taxol). WHO monographs contain information on selected medicinal plants that contain chemical compounds meeting the modern standards of health care. The use of modern isolation techniques and pharmacological testing procedures means that new plant drugs usually find their way into medicines as purified substances rather than in the form of galenical preparations. Therefore the herbal scenario in the 21st century is marked by stiff competition amongst the pharmaceutical industries. Such competition is promoted by the fact that herbal drugs are user-friendly due to their natural origin and lesser side effects. Traditional herbalism is a popular system along with modern health care system. There is a need to learn more about the efficacy, adverse effects and the quality of phytomedicines remedies. More Clinical trial evidences about the efficacy and safety of herbal remedies. along with their relevant pharmacological activities are required to be demonstrated (Fencial et al.,1993). Knowledge of the medicinal plants has been mostly inherited. Preservation and spreading this knowledge along with conservation, cultivation and assessment of all medicinal plants has become important for mankind existence. The best-studied dietary plants with pharmacomimetic phytochemicals include Glycine max L., (Fabaceae), Vitis vinifera L., (Vitaceae), Vccinium L., (Ericaceae), Lycopersicon esculentum, Solanum lycopersicum L. and Capsicum annuum L. (Solanaceae),. Curcuma longa L, Curcuma domestica and Zingiber officinale (Zingiberaceae), Fragaria L. and Rubus L. (Rosaceae) . Ocimum basilicum L. and Rosmarinus officinalis L., (Lamiaceae), Brassica L. (Brassicaceae). Silybum marianum. and Cynara cardunculus L., (Asteraceae), Foeniculum vulgare, (Apiaceae), Syzigium aromaticum, Allium sativum L., (Liliaceae) and Thea sinensis L ., (Theaceae). (Leland et al., 2006). About 500,000.natural products produced by the plants have been estimated and 3

CHAPTER # 1 INTRODUCTION

16,000 from these have been identified (Mendelson et al.,1995). Medicinal herbs and food plants are assumed to contain valuable phytochemicals having medicinal properties. Pharmacologically safe phytochemicals that have been identified from plants includes genistein, resveratrol, dially sulfide, S-ally cysteine, allicin, lycopene, capsaicin, curcumin, 6-gingerol, ellagic acid, ursolic acid, betulinic acid, flavopiridol, , anethol, catechins and eugenol (Aggarwal et al., 2004).

OH

N

OH NH2 HS N

OH HO O

Genistein Resveratrol, Cysteine

4

CHAPTER # 1 INTRODUCTION

S H S

O

Allicin Lycopene

OO OO

HO OH O OOH N H OO HO HO OH O R

Capsaicin Curcumin (Tautameric structures)

O OH O O OH HO OH CH3 O HO HO

OCH3 O

6-Gingerol Ellagic Acid

5

CHAPTER # 1 INTRODUCTION

OH OH

O O

HO HO

Ursolic acid Betulinic acid

OH O O OH OH OH OH

O HO O HO O

O OH OH OH

Silymarin Catechin

O

OH

Eugenol

6

CHAPTER # 1 INTRODUCTION

1.1. INTRODUCTION TO EUPHORBIACEAE

Euphorbiaceae is the largest family of Angiosperm having 300 genera and 5000 species. In Pakistan 24 genera of the Euphorbiaceae are found of which 11 genera are not native (Ali et al., 1986). Dioecious or monoecious often poisonous, prostrate, erect or scan dent annual, biennial or perennial herbs, shrubs or trees succulent or not spiny or unnamed, some times with phylloclade, ith or without a milky latex or colored sap ( Ali et al ., 1986). The genera include Euphorbia (2000 species), Malloyus (2 species), Ricinus(1 species), Caroton (750 species), Hevea (12 species), Jatropha (175 species), Manihot (170 species), Pranthera ( 10 species),Secrinega (25 species), Aleurites (2 species) and Hippomance (5 species) (Trease et al., 1983).

1.2. Genus Euphorbia.

One of the sixth largest genera of the flowering plants having about 2000 species. Genus Euphorbia is cosmopolitan, restricted to tropical, subtropical and warm temperate region monocious herbs, shrbs or trees often succulent, with milky layex and with a simple indumentums when present. Leaves often of 3 types lower, median and upper or ray–leaves whorled or opposite free or connate. All or most leaves usually sessile rarely shortly petiolate, stipulate or not , simple, entire or toothed, penni- or palm nerved (Ali et al., 1986).

1.3. Euphorbia helioscopia (Sun Spurge)

An erect sparingly piloes to glabrescent bright green fleshy annual herb up to 50 cm tall but more commonly 10-30 cm tall. All parts of the plant emit a milky sap when cut. Stems of Euphorbia helioscopia are distinctly red-tinged and emit a milky sap when cut. Stems radiate outward from a central point. Stems may be sparsely hairy, leaves alternate, shortly petiolate , the petioles 1-2 mm long; leaf-blades obovate-spathulate 1-3 (-4.5) x 0.5-1.5(-2) cm, obtuse, rounded or retuse at apex, tapered to the base, serrulate towards the apex. All leaves generally attach directly to the stem and do not occur on petioles. Inconspicuous, light green to pale yellow in color. Inflorescences

7

CHAPTER # 1 INTRODUCTION

(cyathias) occur at the ends of stems and are 4-lobed. Each lobe has a round yellowish gland. Fruit roundly trilobite with deep grooves and subglobose cocci, 2.5-3x32.5-3.5 mm, smooth, glabrous, pale green. Styles are spreading and swollen (Ali et al., 1986).

Botanical Classification of Euphorbia helioscopia L

Kingdom Plant

Sub kingdom Vascular plant

Divison Flowering plant

Supper Divison Seed plant

Class Dicotyledons

Family Euphorbiaceae (spurge family)

Genus Euphorbia

Species Euphorbia helioscopia L

8

CHAPTER # 1 INTRODUCTION

Figure 1: Euphorbia helioscopia Gallery

9

CHAPTER # 1 INTRODUCTION

Fig.1.1. Seeds of E.heliosopia Fig.1.2..Seedling of E.heliosopia Fig.1.3. Plant of E.heliosopia

Fig.1.4. Roots of E.heliosopia Fig.1.5. Shoot of E.heliosopia Fig.1.6. Leaf of E.heliosopia

Fig.1.7. Flowers of E.heliosopia Fig.1.8.Flower of E.heliosopia Fig.1.9. Bunch of E.heliosopia

10

CHAPTER # 1 INTRODUCTION

1.4. Euphorbia prostrata

A prostrate annual herb with the stems flattened and purberulous or pubescent above, terete and glabrous beneath, extending to 30 cm, thought usually not exceeding 20 cm in length. Leaf-blades obovate, surborbicular, 3- 8 x 1-5 mm, obtuse or rounded at the apex, obliquely rounded at the base, minutely serrulate in the upper half, otherwise ± entire palminerved reticulate, green. Stipuls triangular , laciniate ,the upper paired, narrow, 0.5 mm long the lower fused broader 1 mm long (Ali et al., 1986). Flowers are small, pink, and grow from leaf axils. Flower parts are difficult to discern without magnification. Prostrate spurge is a dicot. It is also a prolific seed producer, so often hundreds of seed will germinate at the same time. Spurge develops a deep taproot, though it's been my experience that this weed is fairly easy to handweed.

Botanical Classification of Euphorbia prostrata

Kingdom Plants

Sub kingdom Vascular plants

Division Flowering plants

Supper division Seed plants

Class Dicotyledons

Family Euphorbiaceae

Genus Euphorbia

Species Euphorbia prostrata

11

CHAPTER # 1 INTRODUCTION

Figure 2: Euphorbia prostrata Gallery

12

CHAPTER # 1 INTRODUCTION

Fig.2.1.Seeds of E.prostrata Fig.2.2.Plant of E.prostrata Fig.2.3. Seedling of E.prostrata

Fig.2.4.Shoot of E.prostrata Fig.2.5.Root of E.prostrata Fig.2.6.Shoot with latix

Fig.2.7. Cluster of shoots Fig.2.8.Shoot with flowers Fig.2.9.Flowers

13

CHAPTER # 1 INTRODUCTION

1.5. INTRODUCTION TO ASTERACEAE

The family Asteraceae or Compositae (known as the aster, daisy, or sunflower family) is the largest family of flowering plants, in terms of number of species. The family name Aster, is a Greek word referring to the star shaped flower head of its members (Walter et al., 1996) while Compositae an older but still valid name. Asteraceae is the largest family of angiosperms comprising about 1535 genera and 23000 species distributed in three families and seventeen tribes. It is also the largest family in Pakistan, represented by 650 species distributed in fifteen tribes. The largest genera are Senecio (1,500 species), Vernonia (1,000 species), Centaurea (600 species), Conyza (50 species) Erigeron (200 species) (Ali et al., 1986).

1.6. Genus Conyza

Conyza (horseweed, butterweed or fleabane) is a genus of about 50 species of flowering plants in the family Asteraceae, native to tropical and warm temperate regions throughout the world, and also north into cool temperate regions in North America and eastern Asia. The genus is closely related to Erigeron (also known as fleabanes).The species are annul or perennial herbaceous plants, rarely shrubs, growing to 1-2 m tall. The stems are erect, branched, with alternate leaves. The flowers are produced in inflorescences, with several inflorescences loosely clustered on each stem.It is met with on the tropical Himalaya from Nepal to Shikhim, altitude to 4000 feet extending to Assam, Khasia Hills, Chittagong Burma and the Straits (Watt et al., 1962).

1.7. Conyza bonariensis

The species C. bonariensis is found in many part of Punjab (Pakistan ). It is also found in the surrounding of Multan. C. bonariensis is distributed in ecotonal areas along the edges of roads and maize lands.

14

CHAPTER # 1 INTRODUCTION

Plant Description

Erect perennial, stem rough branching extensively at the base of the plant with tapered leaves covered in stiff hairs, 20-75 cm in height. Erect stems with stiff hairs, branching extensively at the base, decreasing upwards and stems can be 20-75 cm in height. Narrow lanceolate leaves are grey to green in colour, measuring 2-6 cm in length, coarsely toothed and covered in fine hairs. Upper leaves are smaller and linear.Flowers are numerous on poorly arranged pyramidal panicles (much branched inflorescence). The capitulum (flower head) is greater than 2 mm in diameter and looks in fact like a flower bud. Flowers occur at the ends of the branches. Surrounding each flower are involucral (bell-shaped leaf-like) bracts 3-5 mm in length, the inside of each bract is white sometimes tinged purple or red. The cypsela (fruit) is a linear shaped seed approximately 1.5 mm long, straw colored, covered in hairs with 16-20 noticeably longer at the top that are white or pink. Flowering is in spring-autumn. (Jessop et al., 1981).

Botanical Classification Conyza bonariensis

Kingdom Plant

Sub kingdom Vascular plants.

Super division Seed plants.

Division Flowering plants.

Class Dicotyledons.

Subclass Asteridae

Order Asterales

Family Asteraceae

Genus Conyza Less,-horseweed

Species Conyza bonariensis (L) Cronq. (Asthma weed)

15

CHAPTER # 1 INTRODUCTION

Figure 3: Conyza bonariensis Gallery

16

CHAPTER # 1 INTRODUCTION

Fig.3.1.Stem of C.bonariensis Fig.3.2.Seeds of C.bonariensis Fig.3.3.Leaves of C.bonariensis

Fig.3.4.Shoot with Flowers Fig.3.5.Seedling of C.bonariensis Fig.3.6.Flower Of C.bonariensis

Fig.3.7.Roots of C.bonariensis Fig.3.8.Immature Flowers. Fig.3.9.Shoot of C.bonariensis

17

CHAPTER # 2 LITERATURE REVIEW

2.1. Medicinal and Biological importance of genus Euphorbia

2.1.1. Analgesic, antipyretic and anti-inflammatory activities

Different tribes of Central India have been used the paste of rhizomes of E. acaulis as cure of inflammatory disorders (Satti et al., 1988). The hydro- soluble fraction of E. royleana administered by gavage at doses of 50-200 mg/kg, showed dose-dependent anti-inflammatory and anti-arthritic effects in different acute and chronic test models in rats and mice (Bani et.al., 2000). In an other study lyophilised aqueous extract of E. hirta L. has been evaluated for analgesic, antipyretic and anti-inflammatory properties in mice and rats. This study suggested that the plant extract exerts central analgesic properties (Lanhers et al., 1991).

2.1.2. Antibacterial activity Roots of E. ebracteolata having two phenolic compounds 2,4-dihydroxy-3- methyl acetophenone and 2-dihydroxy-6-methoxy-3-methyl acetophenone-4- β-glucoside inhibited the growth of tubercle bacilli (Zhang et al.,1993).

2.1.3. Antifungal activity Extracts obtained from fresh, healthy plant E. tirucalli (stems) was screened for its antifungal activity against Aspergillus flavus. The extract exhibited significant activity against Aspergillus flavus (Perumal et al., 2004). A less polar fraction of the methanolic extract of E. peplis L exhibited antifungal activity (Francesca et al., 2003).

2.1.4. Antiamoebic and spasmolytic activities E. hirta whole plant traditionally used as antidiarrhoeal agent. The plant was investigated for its putative antiamoebic and spasmolytic activities in vitro. Results indicated that both biological activities are concentrated in the polyphenolic fraction, and not in the saponin or alkaloid containing fractions (Tona et al., 2000).

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2.1.5. Antimalarial activity Extracts from E. hirta, exhibited significant chemosuppression of parasitaemia in infected mice (Tona et al., 1999).

2.1.6. Antioxidant activity Methenol extracts of E. supina was identified as potent antioxidant in all the assay systems. It exhibited antioxidant activity (> 80%) in DPPH radical assay, (MinKyun et al., 2003). Ethanol extracts of E. acanthothamnos and E. macroclada showed higher antioxidant activity against DPPH assay (Asli et al., 2007). E. kansui has antioxidative and antifatigue properties and can be given as prophylactic or therapeutic supplements for increasing antioxidant enzyme activities and preventing lipid peroxidation during strenuous exercise (Farong et al., 2006). Aqueous extract of the aerial parts of E. antiquorum Linn. was evaluated for its hepatoprotective and antioxidant activity to validate its use in traditional therapeutic indications. This extract exerted significant antioxidant activity as evidenced by its reducing power for hydroxyl and superoxide anion radical scavenging activities (Jyothi et al., 2008).

2.1.7. Diuretic effect E. hirta is locally used in Africa and Australia to treat numerous diseases, including hypertension and edema. The diuretic effect of the E. hirta leaf extracts were assessed in rats using acetazolamide and furosemide as standard diuretic drugs. The study suggests that the active components in the water extract of E. hirta leaf had diuretic spectrum similar to that of acetazolamide. These results validate the traditional use of E. hirta as a diuretic agent (Johnson et al., 1999).

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2.1.8. Anti-asthamatic activity

The ethanolic extract of E. stenoclada is traditionally used as a herbal remedy against asthma and acute bronchitis (Chaabi et al., 2007). A 95% ethanol extract from whole aerial parts of E. hirta showed antihistaminic, antiinflammatory and immunosuppressive properties in various animal models. The findings demonstrated that E. hirta possessed significant activity to prevent early and late phase allergic reactions (Singh et al., 1984). The phytomedicine E. hirta used to treat asthma and has effect on the ultra structure of fibrin as well as platelets, both play an important role in the coagulation process. Hydrocortisone was used as positive control. Ultra structure of the fibrin networks and platelets of control mice were compared to mice that were asthmatic. E. hirta does not impact on the fragility of the fibrin and that it prevents the minor fibers to form the dense netlike layer over the major fibers, as was seen in untreated asthmatic mice. This ultra structural morphology might give us better insight into asthma and the possible new treatment regimes (Pretorius et al., 2007).

2.1.9. Cytotoxicity

Diterpenoids from E. nivulia showed significant cytotoxic activity against Colo 205 MT2 and CEM cell lines (Ravikanth et al., 2002). Two compounds 17- Acetoxyjolkinolide B and 13-hexadecanoyloxy-12-deoxyphorbol obtained from the dried roots of E. fischeriana, exhibited potent cytotoxic activity to Ramos B cells (Wang et al., 2006). Diterpenoids isolated from the activity guided fractionation of the latex of E. poisonii, showed strong cytotoxic selectivity for the human kidney carcinoma (A-498) cell line with potencies exceeding that of adriamycin (Fatope et al., 1996). Different compounds from E. kansui had been assayed for their cytotoxicity activity.The relations between structures and bioactivities have also been noted (Zheng et al., 1998).

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2.1.10. Antiviral activity Extract of E. australis has activity against HCMV (Semple et al., 1998). In an other study the ethyl acetate extract of E. thymifolia and 3OG46HG affected virus infectivity in a dose-dependent manner. The ethyl acetate extract significantly reduced virus infectivity at a concentration of 4.0 µg/ml (Chien et al., 2005).

2.1.11. Hepatoprotective activity The extract from E. nematocypha showed antihepatotoxic activity in primary cultured rat hepatocytes (Shimura et al., 1990) Aqueous extract of the aerial parts of E. antiquorum Linn. was evaluated for its hepatoprotective activity. Hepatoprotection (at 125 mg/kg and 250 mg/kg dose) was observed by decreasing the activity of serum enzymes, bilirubin, cholesterol, triglycerides and lipid peroxidation. The hepatoprotective activities of the extract are being comparable to standards Silymarin and Sodium metabisulphite respectively. The results indicate that E. antiquorum is a potential source of natural hepatoprotectives. The present study justifies the claim of the native practitioner that the decoction of the plant is useful in treating jaundice (Jyothi et al., 2008).

2.1.12. Antitumor activity The extract from the roots of E. kansui, which has been widely used in Chinese folk medicine for the treatment of cancer, demonstrated antileukemic activity against the P-388 lymphocytic leukemia in mice (Wu et, al., 1991). Similarly extracts of E. esula L. showed antileukemic activity against the P- 388 lymphocytic leukemia in mice (Kupchan et al., 1976). The seeds extracts of E. lathyris L. showed antitumor activity against Sarcoma 180 ascites in mice ( Atta-ur-Rahman et al., 1988). E. fischeriana is a Chinese herbal medicine which has been reported to possess chemotherapeutic effects (Liu et al., 2002).

2.1.13. Anti-hyperglycemic activity E. prostrata. significantly decreases the hyperglycemic peak or the area under the glucose tolerance curve (Alarcon-Aguilara et al., 1998). 21

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2.1.14. Central nervous system depressant activity Crude methanolic extracts from both root and cell cultures of E. calyptrata were investigated and found to be active on the Central Nervous System. An active fraction isolated from the methanolic extract of suspension cultures, possesses significant depressant activity on the Central Nervous System (Speroni et al., 1991).

2.1.15. Effects on the cell division Diterpenoids like Ingenane and euphane triterpenoids all isolated from E. kansui, showed significant effects on the cell diversion of Xenopus laeVis cells at the blastular stage (Wang et al., 2003).

2.1.16. 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 (Vogg et al., 1999). Different compounds obtained from the latex of E. cauducifolia, were evaluated for cocarcinogenic and tumor- promoting activity on the back skin of mice. After 36 weeks it was found an average tumor rate of 36% with the average tumor yield 0.45 tumors/mouse (Baloch et al., 2005).

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2.2. Medicinal and biological importance of genus Conyza

2.2.1. Analgesic, antipyretic and anti-inflammatory activities

C. pyrifolia and C. sumatrensis are used for back pains, diarrhea, dysentery, pimples, postpartum pains, stomach and toothache (Njoroge et al., 2006). C. Canadensis the whole plant posses antirheumatic properties and is used in the treatment of inflamed tonsils, ulceration and inflammation of the throat. (Grieve et al., 1984). Roots of C. schimperiana are used for toothache, tonsillitis and influenza (Esther et al., 2003). C. sumatrensis has significant central and peripheral analgesic properties as well as antiinflammatory activities (Asongalemh et al., 2004). Aerial parts of C. blinni. are used for the treatment of bronchitis and other inflammatory diseases. Now it is confirmed that the polar extracts of C. blinni posses antibacterial, anti-inflammatory, anti- tussive, anti-ulcer and expectorant effects. C. dioscaridis significantly inhibited the nociception to acetic acid-induced writhes with a protection of 85.5–61.3% (Atta et al., 2004).

2.2.2. Antidiarrheal activity Oral administration of methanol extract from C. dioscoridis in a 200 mg kg−1 dose exhibits a significant antidiarrhoeal effect against castor oil-induced diarrhea (Atta et al., 2004). C. canadensis is used in the treatment of gastro- intestinal problems such as diarrhea and dysentery (Chevallier et al., 1996). The spasmolytic activity exhibited by the extract of C. filaginoides and its active principles support the traditional use of C. filaginoides as an antispasmodic agent (Mata et al., 1997).

2.2.3. Antiviral activity C. aegyptiaca was found active against all three test viruses Herpes simplex, sindbis and poliovirus (Anani et al., 2000).

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2.2.4. Smooth muscle relaxing activity Chloroform-methanol (1:1) extracts of C. filaginoides (Asteraceae) posses smooth muscle relaxing activity (Mata et al., 1997).

2.2.5. Antibacterial activity Aqueous, methanolic and ethyl acetate extracts used in traditional Zulu medicine for the treatment of infectious ailments were screened for antibacterial activity. The highest antibacterial activity was found in methanolic extracts of C. viridis (Jonathan et al., 2000).

2.2.6. Antimalarial activity Leaves of C. schimperiana are thought to be active in malaria (Esther et al., 2003).

2.2.7. Antirheumatic activity C. linifolia is a green herbacous plant used as an antirheumatic (Tackholm et al., 1974 ).

2.2.8. Diuretic activity C. linifolia is reported to posses diuretic activity (Tackholm et al., 1974 ).

2.2.9. Anticoagulant activity Methanolic extract of C. linifolia exhibited anticoagulant effects in a dose of 400 mg /kg (Amani et al., 2004 ).

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2.3. Phytochemical Studies of Euphorbia The chemistry of the Euphorbia species is much interesting and diverse among the flowering plants families (Evans et al., 1986). The major secondary metabolites present in Euphorbia species can be classified as lipids and its derivatives, terpenes, aromatics, amines, alkaloids and amino acids found to be present in the family.

2.3.1. Lipids and its derivatives The seeds of E. hiberna contain 33% of an oil and fatty acids like oleic acids, linoleic acid, palmitic acid, stearic acid, and myristic acid (Salgues et, al., 1956). In another study it was found the E. lathyris and E. chrcias contain 30% and 27 % of fatty acids respectivally (Conti et al., 1984).

O OH

O OH

Myristic acid Stearic acid

O OH HO O

Palmitic acid Linoleic acid

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CHAPTER # 2 LITERATURE REVIEW

O OH

Oleic acid

2.3.2. Alkaloids Alkaloids are the nitrogenous compounds founds in plants. They are basic in nature and form salts with plant acids. Glyco alkaloids are formed when they combine with sugars. Simple and highly complex alkaloids are found in the family Euphorbiaceae. From E. hitra for the first time an alkaloid xanthorhamine was isolated (Ueda et al.,1949). Later on, an alkaloid, (+)-9- aza-1-methyl-bicyclo-(3,3,1)-nonan-3-one, was isolated from E. atoto and its absolute configuration was established. (Beecham et al., 1968). Two alkaloids, uracil and uridine were isolated from E. altotibetic (Pan et al., 2003).

O

O NH

HO N O NH O

N O H OH OH

Uracil Uridine

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2.3.3. Flavonoids The flavonoids are polyphenolic compounds possessing 15 carbon atoms with two benzene rings joined by a linear three carbon chain. Flavonoids constitute one of the most characteristic classes of compounds in higher plants. Many flavonoids are easily recognised as flower pigments in most angiosperm families (flowering plants). However, their occurence is not restricted to flowers but include all parts of the plant. Numerous flavonoids have been reported in the members of family Euphorbiaceae. Leaves and stems of E. thymifolia contain 5, 7, 4-trihydroxy-flavone-7-glycoside (Nagase et al., 1941). Steppogenin stepposide and isomyricitrin were isolated from E. stepposa (Sotrikova at al., 1968). Kaempferol is reported in E. helioscopia, E. retusa, E. vergata, and E. paralias. Quercetinan other valuable flavonoid are present in E. hirta, E. lucida, E. minuta, E. granulata and E. tinctoria. Quercetin-5,3-di- galactoside ,Quercetin-3-glacto-2”-gallate, and Quercetin-3- galactoside has been reported in E. helioscopia. Rutin has been reported in E. granulate, E. larica, E. tinctoriaand and E. vergata. 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-Dglucopyranoside, kaempferol 3-O- rutinosid quercetin 3-O-β-D-glucopyranoside, and rutin, were isolated from the aerial parts of E. ebracteolata (Liu et al., 2004). 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 (Nishimura et al., 2005). 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 (Zhang et al., 2006).

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OH OH OH O OH HO O OH HO O OH OH O OH

Quercetrin Kaempferol

OH OH

HO O

O OH O OH O

OH OH OH Quercetin-3- galactoside

OH HO

HO OH

O OHHO OH O O O OH O O

HO OH

Rutin

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CHAPTER # 2 LITERATURE REVIEW

OH OH

HO O

OH O HO OH O O HO O O O HO HO O HO OH OH

Quercetin 3-O-(2″,3″-digalloyl)-β-D- galactopyranoside

OH OH

OH

O OH O OH

O

OH HO O

OH

OH

Isomyricitrin

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CHAPTER # 2 LITERATURE REVIEW

OH

OH

HO O

O

OH O O HO O O HO

HO

HO OH

OH

Quercetin 3-O-(2′′-galloyl)-β-D-galactopyranoside

HO

OH OH HO OH

O O O O O

O

HO OH OH

Hyperin

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CHAPTER # 2 LITERATURE REVIEW

O OH

O

O

OH

HO O

OH OH Licochalcone B Licochalcone A

HO O

O

O

HO Glabrones 2.3.4. Coumarins The main Coumarins compounds found in the genus Euphorbia are scopoletin, 7,7-dihydroxy-6,8-bicoumarin (bicoumol) 6,7,8- trimethoxycoumarin and 6-hydroxy-7-methoxycoumarin (isoscopoletin,) (Qing- Wen et al., 2008 ) . Benzocoumarin have also been reported in E. royleana (Rizk et al., 1987). Aesculetin is the constituent of E. lanata, E. lathyris, E. lunulata and E. terraciana. E. Formosa, E. discolor, E. granulata, E. eberica, E. tirucalli and E. wallichii is reported to have Ellagic acid.(Singla et al., 1989)

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H3CO

H3CO

H3CO O O

HO O OCH3 O

6,7,8-trimethoxycoumarin Scopoletin

HO O O

OH

HO

O

O H3CO O O

Bicoumol Isoscopoletin

HO O-CO

HO OH

CO-O OH

Ellagic Acid.

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2.3.5. Terpenes

Terpenes are a group of molecules whose structure is based on a various but definite number of isoprene units. Terpenes are probably the largest group of compounds, at least 25,000 have been reported and many yet to be described. They are common in plants and fungi and rarely accumulate in bacteria. Terpenes are also synthesized in some animals. Terpenes can be clacified a Monoterpenes, Sesquiterpenes, Diterpenes, Sesterpenes, Triterpenes, Carotenoids and Rubber having depending upon the number of isoprene units 2,3,4,5,6,8 and > 100 respectively. Many terpenes are hydrocarbons, but oxygen-containing compounds such as alcohols, aldehydes or ketones are also found. These derivatives are frequently name as terpenoids.

2.3.6. Sesquiterpenoids

The first sesquiterpenoids from the genus Euphorbia was euphanginol along with three known sesquiterpenoids cyclocaryophylla-4-en-8-ol(2) , 4β,5α- epoxy-4,5-dihydrocaryophyllen-14-ol (3), clovandiol(4) from E. wangii. (Shi et al., 1997) From a polar fractions of E. resinifera two novel bisnorsesquiterpene glycosides, euphorbiosides A and B. (Qing-Wen et al., 2008 ). Two known norsesquiterpenoids, 4,5-dihydroblumenol A and aglycone of icariside B2, from E. helioscopia (Zhang et al., 2006).

H H H

OH

H

OH

Euphanginol Cyclocaryophylla-4-en-8-ol

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H OH

O

H HO OH 4β,5α-epoxy-4,5- Clovandiol dihydrocaryophyllen-14-ol

OH OH

D-glc-O RO H H O O D-glc-O RO

Euphorbiosides A Euphorbiosides B

2.3.7. Diterpene

A large variety of diterpenes occur in the Euphorbiaceae (Singla et al., 1989). 12-deoxyphorbol is to be reported in E. balsamifera, E. paganorum, E. poisonii, E. triangularis and E. unispina. Sixteen new diterpene polyesters were isolated and identified from Hungarian Euphorbiaceae species. Two of them were unknown diterpene core. The structures of three jatrophane type diterpene heptaester were elucidated which are diterpenoids with the highest degree of esterification. (Evanics et al., 2001). A new diterpenoid, named euphpekinensin, along with three known diterpenoids, was isolated from the roots of E. pekinensis for the first time and the structures were elucidated by spectral analysis (Kong et al., 2002). The structures of two new macrocyclic jatrophane diterpenoid esters from the whole herb of E. esul were established

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as 7beta11,14-epoxy-3beta,5alpha,8alpha,9alpha,15beta-hexaacetoxy-12- oxo-13alphaH- jatropha-6(17)-ene and 1alpha,3beta-diacetoxy-5alpha,7beta- dibenzoyloxy-9,14-dioxo-11beta,12alpha-epoxy -2alpha,8alpha,15beta- trihydroxy-13betaH-jatropha-6(17)-ene by a combination of 1D- and 2D-NMR techniques as well as UV, IR and mass spectral data (Liu et al., 2002). The latex of E. nivulia afforded two ingol diterpenes 3,12-diacetyl-8-benzoylingol and 3,12-diacetyl-7-benzoyl-8-nicotinylingol along with three known ingol diterpenes (Ravikanth et al., 2002). Two segatane diterpenoids containing a bicyclic ring system were isolated, together with 11 known diterpenoids, four segetanes, five jatrophanes and two paralianes, from the aerial parts of E. paralias (Abdelgaleil et al., 2001). Five new and one known jatrophane diterpenoid esters were isolated from the ethanol extract of the whole herb of E. turczaninowii. Their structures were established by extensive spectroscopic methods (Liu et al., 2001). From the ethanolic extract of the roots of E. ebracteolata a new isopimarane diterpene, namely yuexiandajisu C has been isolated. The structure of yuexiandajisu C was elucidated by spectral analysis (Xu et al., 2000). From a pro-inflammatory active extract of E. peplus, two new diterpene polyesters based on the pepluane and jatrophane skeletons were isolated, together with four known ingenane and jatrophane diterpenes (Hohmann et al., 2000). E. characias from Sardinia afforded 13 oxygenated diterpenoids of the atisane, abietane, pimarane, and kaurane type (Appendino et al., 2000). The aerial parts of E. pithyusa collected in Sardinia afforded eleven novel diterpenoids belonging to the lathyrane , premyrsinane , and tigliane types. Structures were elucidated by spectroscopic and chemical methods (Appendino et al., 1999). Two novel pentacyclic diterpenoid esters of the cyclomyrsinane type and one tetracyclic diterpenoid ester of the myrsinane type were isolated from the aerial parts of the plant E. teheranica (Ahmad et al., 1999). From the extract of E. peplus, a new diterpene polyester based on the jatrophane skeleton was isolated (Hohmann et al., 1999). A new lathyrane diterpene has been isolated and characterized from a dichloromethane extract of the roots of E. lathyris (Hohmann et al., 1999). Pepluane diterpene polyester was isolated from a dichloromethane extract of the whole, undried plant of E. peplus (Hohmann et al., 1999). From E. seguieriana, seven new diterpene polyesters five of them are having a new 35

CHAPTER # 2 LITERATURE REVIEW

parent alcohol. Named as 17-hydroxymyrsinol, were structurally related to myrsinol. The other two compounds were new derivatives of the known parent alcohols cyclomyrsinol and lathyrane. The structure elucidations of the new compounds by highfield spectroscopic methods, including 1D and 2D NMR techniques (Oksuz et al., 1998). E. semiperfoliata gave an abietanolide, 13 jatrophane polyesters two 4-deoxyphorbol diesters , and a pair of epimeric diterpenes with a novel carbon skeleton, which was named euphoperfoliane (Appendino et al., 1998). Manoyloxide derivative, 3β-hydroxy-2- oxomanoyloxide was isolated from E. segetalis (Jakupovic et al., 1998). Langduin C, a novel dimeric diterpenoid,was isolated from the roots of E. fischeriana. Fischeria A, a novel norditerpene lactone was isolated from the rhizomes of the same specie (Qing-Wen et al., 2008). A diterpenoid named Jolkinolide A and Jolkinolide B were isolated from E. ebracteolata and E. fischerina respectivally (Yao et al., 2005) (Liu et al., 2002).

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CHAPTER # 2 LITERATURE REVIEW

O O

H O O HO H

Fischeria A 3β-hydroxy-2-oxomanoyloxide

CHO OH O

H OH

H Euphpekine Euphpekinensin

H

H H

O O

OH O O H

HO H O

O O

H H H Langduin C

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CHAPTER # 2 LITERATURE REVIEW

OH H3C

O O CH3 OH H3C CH3 HO H O H O H3C HO H OH OH

O HO OH OH

12-deoxyphorbol Cyclomyrsinol

O

O O O OH

O

O HO HO HO

Jolkinolide Myrsinol

kaurane

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CHAPTER # 2 LITERATURE REVIEW

2.3.8. Triterpenes

The plants of the family Euphorbiaceae are rich in terpenes. Triucllol and euphorbol are often found in the latex of the member of the genus Euphorbia (Singla et al., 1989). The structures of three triterpene alcohols isolated from the latex of E. antiquorum were established to be eupha-7,9(11),24-trien- 3beta-ol(2;antiquolC),19(10-->9)ab8alpha,9beta, 10alpha-eupha-5,24-dien- 3beta-ol(3; antiquol B), and 24-methyltirucalla-8,24(24(1))-dien-3beta-ol (4; euphorbol) on the basis of spectroscopic methods (Akihisa et al., 2002). Investigation of the acetone extract of the whole plant of E. tuckeyana afforded a new cycloartane-type triterpene named as cyclotucanol. Its structure was established as cycloartane-24-methylene-3beta,25-diol. The known cycloartane triterpenes cycloeucalenol, 3beta-hydroxycycloart-25-en- 24-one, cycloart-25-ene-3beta,24-diol, 25,26,27-trisnor-3beta- hydroxycycloartan-24-al, and cycloart-23-ene-3beta,25-diol were also isolated and identified from the same species. (Ferreira et al., 2001). The latex of E. nivulia afforded two known triterpenes cycloart-25-en-3beta-ol and cyclonivulinol (Ravikanth et al., 2002). Seven triterpenoids have been isolated from the roots of E. nematocypha (Cao et, al., 1992). Antiquol C an other triterpnoid was reported in. E. antiquorum (Akihisa et al., 2002). A triterpene named cyclotucanol is found in E. tuckeyana (Ferreira et al., 2001).

H H3C CH3 CH3 CH3 H CH3

CH3 CH3

H CH3

HO H H H3C H3C CH3 CH3 Euphol Atisane

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CHAPTER # 2 LITERATURE REVIEW

HO HO

Euphorbol Cycloartenol

HO

Cycloeucalenol

HO

Antiquol C

2.3.9. Steroids Several ergostane-type steroids (1-6) from E.chamaesyce were isolated. (Tanaka et al.,1999). A new geniculatoside F from aerial parts of E. geniculata Linn (Rahman et al., 2002).

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CHAPTER # 2 LITERATURE REVIEW

R2

HO R H 1

R1 R2 1) 3β-hydroxy-4α,14 α -dimethyl-5 α -ergosta-8,24(28)-dien-11-one H2 =O 2) 3β, α R-dihydroxy-4 α,14 α dimethyl-5 α -ergosta-8,24(28)-dien-7-one = O α-OH 3) 3β,7 α -dihydroxy-4 α,14 α -dimethyl-5 α -ergosta-8,24(28)-dien-11-one -OH =O 4) 3β hydroxy-4 α,14 α -dimethyl-5 α -ergosta-8,24(28)-dien-7-one =O H2 5) 3β -hydroxy-4 α,14 α -dimethyl-5 α -ergosta-8,24(28)-dien-7,11-one =O =O 6) obtusifoliol H2 H2

H

H H

a-D-gal-O H

Gniculatoside F

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2.3.10. Sugars

From E. pulcherrima glucose and arabinose have been isolated Sucrose and glucose are reported from the roots of E. ebracteolata and leaves of E. paralias Seeds of E. caudicifolia contain free sugars (Singla et al., 1989).

2.4. Phytochemical studies of Conyza species

After going a Comprehensive literature survey on genus Conyza of family Compositae it is found that a variety of secondary metabolites including alkaloids, terpenes, aromatics, amines and amino acids are found to be present in the family Compositae.

2.4.1. Alkaliods

Methanol extract of the seeds of C. cyanus yielded four indole alkaloids which were characterised as moschamine, cis-moschamine, N-(4- hydroxycinnamoyl)-5-methoxytryptamine and N-(4-hydroxycinnamoyl)-5- methoxytryptamine (Satyajit et al., 2001).

H H N O N O OH OH H HO H HO N N O O

Moschamine cis-Moschamine

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2.4.2. Monoterpenoid

Monoterpenes like α-thujene , β-pinene , myrcene , limonene , cis and trans ocimene , cosmene and isomer of cosmene were reported in C. canadensis (Hrutfiord et al., 1998).

CH2

α-Thujene β-Pinene Myrcene

CH2 CH3

CH2

H3C H3C CH3 CH2

Myrcene Limonene Cosmene

2.4.3. Sesquiterpnoid

From C. canadensis sesquiterpenes named trans-caryophyllen e, α-cis- bergamotene, β-trans-bergamotene, cis- β-farnesene, trans- β-farnesene, β- himachalene, β-cubebene, β-bisabolene, δ-cadinene and α-curcumene (Hrutfiord et al., 1998). A sesquiterpnoid named β-caryophyllene 4,5- α-oxide was isolated from C. filagenoides (Mata et al., 1997).

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α-cis-Bergamotene β-Himachalene

β-Bisabolene δ-Cadinene

2.4.4. Diterpnoid

A diterpnoid named strictic acid was reported from C. sitricta (Buckingham et al., 1994). From the same plant labdane diterpene and farnesylic acid were isolated (Ahmed et al., 1990). A New trans-Clerodane Diterpene lactone named Variant was reported from C. blinii (XU et al.,1999). Labdane diterpenoid glycosides named Blinoside A and Blinoside B are reported from C. blinii (Su et al.,2002).

OH O O

OOH

Strictic Acid Farnesylic Acid

44

CHAPTER # 2 LITERATURE REVIEW

O O

O O O

Variant

OH OH HO OH OH O O OH O OH O OH OH

R

Blinoside A Blinoside B

2.4.5. Triterpnoid

In a phytochemical study three triterpenoids α-spinasterol β-amyrin and n- triaconatane were isolated from C. filagenoides (Dominguez et al., 1972). Two trterpenoids erythrodiol and 3β-tridecanoyloxy-28-hydroxyolean-12-ene were reported from C. filagenoides (Mata et al., 1997).

45

CHAPTER # 2 LITERATURE REVIEW

HO

α-Spinasterol

H3C CH3

CH3 CH3 H CH3

H CH3 HO H C 3 H CH3

β-Amyrin

H3C CH3

CH3 CH3 H COOH

H CH3 HO H3C H CH3

Erythrodiol

46

CHAPTER # 2 LITERATURE REVIEW

2.4.6. Flavonoids

A known flavones named flindulatin, was isolated from C. stricta (Buckingham et al., 1994) A new flavone named conyzatin along with 5,7- dihydroxy-3,8,4-trimethoxyflavoné were isolated from the same sepies. In an activity guided fraction, isolation of three flavonoids quercetin 3-glycoside, rutin and pinostrobin from the extract of C. filaginoides was confirmed (Mata et al., 1997). A flavones flindulatin was isolated from C. stricta (Buckingham et al., 1994). Phytochemical screening of C. linifolia revealed the presence flavonoids. Bioassay-guided fractionation of C. filaginoides led to the isolation of three new flavonol caffeoyl glycosides namely, kaempferol 3-O-(6' '-O-E- caffeoyl)-beta-D-galactopyranoside, isorhamnetin 3-O-(6''-O-E-caffeoyl)-beta- D-galactopyranoside , and quercetin 3-O-(6' '-O-E-caffeoyl)-beta-D- glucopyranoside (Calzada et al., 2001). The presence of flavonoids was confirmed in C. ulmifolia (Oluwole et al., 2007). Conyzatin was isolated from C. stricta (Buckingham et al., 1994). 5-Hydroxy-3,4',7,8-tetramethoxyflavone (Flindulatin) was reported in C. stricta (Calvert et al.,1979).

O O O O

O O O O O O O O

O OH HO OH

Flindulatin Conyzatin

47

CHAPTER # 2 LITERATURE REVIEW

O

O

Pinostrobin

OH OH OH OH HO O

HO O

OH O O OH O OH OH O O O O OH OH OH OH OH OH OH OH

Quercetin-3-glycoside, Rutin

2.4.7. Saponins

Three new bisdesmosidic saponins named conyzasaponins A, B, and C and one new monodesmosidic saponin, conyzasaponin G were isolated from the aerial parts of C. blinii. 28-O- β-D-apiofuranosyl-(1 3)- β -D-xylopyranosyl- (1 4)-α -L-rhamnopyranosyl-(1 2)- α -L-arabinopyranosyl ester (Conyzasaponin A), 28-O- β -D-apiofurano- syl-(1 3)- β -D-xylopyranosyl- (1 4)-[ α -L-arabinopyranosyl-(1 3)]-α -L-rhamnopyranosyl-(1 2)- α -L- arabinopyranosyl ester (conyzasaponin B), and 28-O- α -L-rhamnopyranosyl- (1 3)- β -D-xylopyranosyl-(1 4)-[ β -D-apiofuranosyl-(1 3)]- -L- rhamnopyranosyl-(1 2)- α -L-arabinopyranosyl ester (conyzasaponin C) of the prosapogenin, respectively (Yan-fang et , al., 2000).

48

CHAPTER # 2 LITERATURE REVIEW

HO O HO HO HO O O O O

HO O O O OH OH OH OH HO OH O

HO O O

HO OH O O

HO HO OH

Conyzasaponin A

HO OH O HO OH O O HO O O O HO O OH O HO OH OH O OH O HO O O O

HO HO OH OH O O

HO HO OH

Conyzasaponin B

49

CHAPTER # 2 LITERATURE REVIEW

HO OH O HO O OHHO O O O HO O O O OH

OH HO OH O OH

O

O O HO O

HO HO OH

HO O O

HO OH

OH

Conyzasaponin C

HO OH HO HO HO O O O

HO O O OH OH OH

Conyzasaponin G

50

CHAPTER # 2 LITERATURE REVIEW

2.4.8. Glycosides

Three new pyrone glucosidic derivatives, together with the known pyromeconic acid glucoside, three acytelenes and two eudesmanes, were obtained from the aerial parts of C. albida (Ahmed et al., 2002 ). Presence of glycosides was confirmed in C. ulmifolia (Oluwole et al., 2007). A new phenolic glycoside, 4-propionyl-2,6-dimethoxyphenyl β-D-glucopyranoside was isolated from the aerial parts of C. blinii (Yan-Fang et al., 2001).

Eudesmane

2.4.9. Coumarin The phytochemical investigation of C. linifolia offer 6 coumarins identified as scopoletin , isoscopoletin , esculetin , phenylacetoxycoumarins, lacarol and methyl lacaryl (Amani et al., 2004 ). From C. obscura two naturally occurring coumarins virgatol and virgatinol were isolated (Dominick et al., 2004 ).

Me O HO

HO O O MeO O O Scopoletin Isoscopoletin

51

CHAPTER # 2 LITERATURE REVIEW

HO

HO O O

Esculetin

O OH O O OH

O HO

OH

O HO O

Lacarol Virgatol

2.5. Medicinal Uses of Euphorbia helioscopia E. helioscopia is reported to be used as anthelmintic, antiperiodic, cancer, febrifuge and antiperiodic. The leaves and stems are febrifuge and vermifuge (Panda et al.,1985). The root of E. helioscopia possess anthelmintic properties. The said plant is used as cathartic .The milky sap of the plant is applied externally to skin eruptions. The seeds, mixed with roasted pepper, have been used in the treatment of cholera. The oil from the seeds of E. helioscopia has purgative properties (Chopra et al., 1986). Young leaves are used as a tea substitute (Kunkel et al., 1984). E. helioscopia is a common herbaceous weed found in the different parts of the world and Europe. It has been responsible for poisoning of livestock resulting a severe inflammation particularly of mucous membranes and the eyes (Richard et al., 2004). Plant- induced toxic dermatitis is of increasing importance in dermatology ( Wilken et al., 2005). Antitumor activity of the aquatic extract the root of E. helioscopia in

52

CHAPTER # 2 LITERATURE REVIEW

vitro were studied. The results indicated that E. helioscopia had obvious antitumor activity and it could be considered as a potent antitumor herb (Cai et al., 1999).

2.6. Reported Phytochemical Studies of Euphorbia helioscopia Different types of secondary metabolites such as diterpnoids triterpnoids, flavonoids, tannins, steroids, and lipids have been isolated from E. helioscopia . Palmitic acid is predominated among other fatty acids Euphorbia helioscopia Β Sitosterol is to be reported in E. helioscopia (Wen et al., 2006). Licochalcone A, and Licochalcone B, 2', 4, 4'-trihydroxychalcone(4), echinatia, glabrone, 4' , 5, 7 trihydroxy flavanone from the E. helioscopia (Wen et al., 2006). Quercetin, Quercetin-5,3-di-glactoside (Singla et al.,1989), Quercetin-3-glactoside, Quercetin3-glacto-2”-gallate (Pohl et al., 1975). Kaempferol , and Quercetin-3-glucoside are reported in the same plant. ( Singla et al., 1989) Diterpene named euphornin, euphornin B, C, euphoscopins A,B,C, D, E ,F,J epieuphorscopins A and B, euphohelioscpoin A, jalkinolide E, helioscopinolide A, B and 2α-hydroxy helioscopinolide B, two nor-sesquiterpenoids, were obtained from E. helioscopia Euphoheliosnoid D , a new jatrophone-type diterpenoid, was isolated together with 22 known metabolites from the Chinese medicinal herb E. helioscopia L (2006). Three new jatrophone-type diterpenoids, euphoheliosnoids A-C, together with two known related compounds, were isolated from the Chinese medicinal plant E. helioscopia L (Wen et al.,2006). A new cytotoxic macrocyclic diterpenoid, euphornin L and euphoscopin F were isolated from E. helioscopia exhibited significant cytotoxicity against HL-

60 cell lines with IC50 values of 2.7 and 9.0 μM, respectively. Epieuphoscopin B , euphoscopin B and euphoscopin C were also reported for the first time ( Hong-Wen et al., 2008). A new series of diterpenes, the jatrophanes euphoscopin M , euphoscopin N and euphornin L , and the lathyrane euphohelioscopin C were isolated from plants of E. helioscopia L., together with four other known analogues, euphoscopin C , epieuphoscopin B and euphohelioscopin A (Barile et al., 2008). Four new jatrophane-type diterpenoids , 7-β,9α,14 β-Triacetoxy- 3β- Benzoyloxy-12 β,15 β-Epoxyjatroph-5-E-en-11 β-OL, 14β-acetoxy-3 β - 53

CHAPTER # 2 LITERATURE REVIEW

benzoyloxyjatropha-5E, 11 E-diene-7 β,9R,15 β -triol , 7β,9R,14β-triacetoxy-3 β -benzoyloxyjatropha-5 E,11 E-diene-15 β,17-diol and 14α,15β -diacetoxy-3 β,7 β -dibenzoyloxy-17-hydroxy-(2 β H,13 β H) jatropha-5 E,11 E-dien-9-one, together with 16 known related compounds, were isolated from the Chinese medicinal plant E. helioscopia (Lu et al., 2008).

CH3

H3C CH3 H3C CH3 H3C H

H R HO

Β Sitosterol

CH2 CH3 CH3

HO OH

O O CH3 Licochalcone A

54

CHAPTER # 2 LITERATURE REVIEW

OH O OH

OH HO O

OH

Quercetin

OH OH

HO O

O OH O OH O

OH

OH OH

Quercetin-3-glactoside

O HO OH

H O BzO HO

Euphoheliosnoid D

55

CHAPTER # 2 LITERATURE REVIEW

AcO OH O

BzO H OAc AcO

7-β,9α,14 β-Triacetoxy- 3β-Benzoyloxy-12 β,15 β-Epoxyjatroph-5-E-en-11 β-OL

AcO HO

BzO H OH HO 14β-acetoxy-3 β -benzoyloxyjatropha-5E,11 E-diene-7 β,9R,15 β -triol

AcO AcO

H AcO OAc

AcO OAc

7β,9R,14β-triacetoxy-3 β -benzoyloxyjatropha-5 E,11 E-diene-15 β,17-diol - AcO HO HO

H OH BzO AcO 14α,15β -diacetoxy-3 β,7 β -dibenzoyloxy-17-hydroxy-(2 β H,13 β H) jatropha-5 E,11 E-dien-9-one

56

CHAPTER # 2 LITERATURE REVIEW

O

O H

H

H H H H H

Jolkinolide E O

O H

H

H HO H H H

Helioscopinolide A

O

O H

H

H H H HO H

Helioscopinolide B

57

CHAPTER # 2 LITERATURE REVIEW

O

O O

O O OO

O

O OH

O

3-Benzoyl, 7,9,11-tri-Ac(Euphohelin A)

O

O O O O O O

O

O O O

O

3,14-Dibenzoyl, 7,9,11-tri-Ac (Euphohelin C)

58

CHAPTER # 2 LITERATURE REVIEW

O

O O O O OO

O

O OH

11-Ketone, 3-benzoyl, 7,9-di-Ac (Euphohelin E )

O

O O O O OO

O

O O

11,14-Diketone, 3-benzoyl, 7,9-di-Ac (Euphohelin D)

HO

HO

O OH

O HO OH

5,6;12,15-Diepoxy-3,7,9,11,14-jatrophanepentol

59

CHAPTER # 2 LITERATURE REVIEW

O O O O O O

OH

O

O

Euphornin

O O

O O O

O O

O

Euphoscopin F

60

CHAPTER # 2 LITERATURE REVIEW

O O

O O O O

O O

O

Euphoscopin B

O O

O O O O

O

O O

Euphoscopin C

61

CHAPTER # 2 LITERATURE REVIEW

2.7. Medicinal Uses of Euphorbia prostrata

E. prostrata (Euphorbiaceae) is traditionally used in Cameroon for the treatment of many diseases, including diarrhea. The effect of the aqueous ethanol extract of the plant on gastrointestinal propulsion, in vitro bacterial growth and in vivo bacillary dysentery were studied. The results suggested that E. prostrata possesses bactericidal and antidiarrheic properties and could be a therapeutic alternative for diarrheas of bacterial etiology (Rene et al., 2007). Decoction of the leaves of E. prostrata is given to kill intestinal worms in claves (Ishtiaq et al., 2006). The whole plant E. prostrata is taken, crushed and eaten with bread as a useful remedy for kidney stone (Hamayun et al., 2003). E. prostrata significantly decrease the hyperglycemic peak and/or the area under the glucose tolerance curve. The results suggest the validity of their clinical use in diabetes mellitus control, after their toxicological investigation (Alarcon et al., 1998).

2.8. Reported Phytochemical Studies Of Euphorbia prostrata

Ellagitannin, Euphorbin G and Euphorbin H were isolated from E. prostrata . In another study Prostratin A, Prostratin B and Ellagitannin constitutes were reported from the leaves of same plants. (Atta-ur- Rahman et al., 1988). Ten compounds have been isolated from E. prostrata and identified as gallic acid, corilagin, 1,2,3-tri-O-galloyl-D-glucose, geraniin, tellimagradin I and II, rugosin A, rugosin E, rugosin D and rugosin G on the basis of physicochemical and spectroscopic methods (Chen et al., 1992).

62

CHAPTER # 2 LITERATURE REVIEW

OH HO O

O HO O HO O O O HO O O O O OH O OH HO HO OH HO OH

HO OHHO OH

HO O OH

O O HO OH O OH O O OH O O O O OH O O

OOHHO OH HO O OH

Euphorbin G

63

CHAPTER # 2 LITERATURE REVIEW

OH HO O

O HO O HO O O O O HO O OO OH O OH OH HO OH HO OH

HO OH HO OH

HO O OH

HO OH O OO O O HO O O OH OH

HO OH Euphorbin H

64

CHAPTER # 2 LITERATURE REVIEW

OH OH

OH HO OHHO OH

O

HO O

OH O O O O HO O

HO O HO O O O O

HO O OH

O O HO OH

OH O OH

Prostratin A

65

CHAPTER # 2 LITERATURE REVIEW

OH HO OH HO OH OH

OH

HO O O OO

HO OH OH HO HO O OH O O HO OHHO O O

O HO OH OH O O HO OH HO O O OH

O OH O O HO OH O O OH HO O O O HO O O O O O HO HO O O HO O OH O O

O HO HO OH OH OH

HO OH

Prostratin B

66

CHAPTER # 2 LITERATURE REVIEW

HO OH HO HO OH HO HO HO O O HO O O O O O O O HO O HO O HO HO O O OH O OH OH

Prostratin C

HO OH HO

O O O HO HO

O O O O

HO OH HO OH HO OH

Corilagin

67

CHAPTER # 2 LITERATURE REVIEW

OH HO

O OH

OH HO O OH

O O

O O OH

O O O OH

O O

O O

HO OH HO OH

HO OH

Geraniin

68

CHAPTER # 2 LITERATURE REVIEW

HO OH HO HO OH HO

O OH OH HO OH HO O O OH O O OH

HO O O O O O O O HO O HO

HO OH

Rugosin A

69

CHAPTER # 2 LITERATURE REVIEW

OH HO OH OH

HO O HO OH

HO O O OH O HO OH OH O OH

HO OH O HO O OH O OH O O O HO HO O O O O O HO OH O

HO O O O OH

HO O OH

OH

Rugosin E

70

CHAPTER # 2 LITERATURE REVIEW

2.9. Medicinal uses of Conyza bonariensis

Aerial parts of the C. bonariensis were investigated for their pharmacological studies and it was observed that it increases the force of contraction with transient fall off blood pressure and did not demonstrate any effect on central nervous system, it was also observed that it antagonizes Acetylcholine (Tariq et al., 1987). The methanol extract of C. bonariensis (Asteraceae), inhibited xanthine oxidase which was comparable to that of allopurinol a positive control used in the study (Kong et al., 2001). Plant extracts of C. bonariensis have marked inhibitory effects against BChE with weak action on AChE. Both AChE and BChE are involved in the pathophysiology of AD. The inhibitory activity of the plant extract against these enzymes, which indicates its potential in the treatment of AD (Rafeeq et al., 2006). The herb C. bonariensis (L) Cronquist (Local name : Paleet ) is used as homeostatic, stimulant, astringent and diuretic. It is used in dysentery, diarrhea and haemorrhage (Saeed et al., 2007).

2.10. Phytochemical studies of Conyza bonariensis

The essential oils from the roots, leaves, stems and inflorescences of C. bonariensis L. were investigated. A total of 17 constituents were identified by GC-MS, including monoterpenes, acetylenes, sesquiterpenes and diterpenes. The major constituents were matricaria methyl ester (Luiz et al., 2004). Conyza bonariensis afforded known compounds including amyrin, β-sitosterol daucosterol, syringic acid (4-Hydroxy-3,5-dimethoxybenzoic acid), 3-hydroxy- 5-methoxybenzoic acid, eugenol 4-O-glucopyranoside, luteolin, apigenin and takakin 8-O-glucuronide (Kong et al., 2001).

71

CHAPTER # 2 LITERATURE REVIEW

H3C CH3

CH3 CH3 CH3

CH3 HO HO H3C CH3

Amyrin β-Amyrin

CH3

H3C CH3 H3C

CH3

H3C H

H R

HO

β Sitosterol O O

HO

OH O O HO O OH

4-Hydroxy-3,5-dimethoxybenzoic acid 3-hydroxy-5-methoxybenzoic acid

72

CHAPTER # 2 LITERATURE REVIEW

OH OH HO O HO O OH

OH O OH O

Luteolin Apigenin

OH HO OH OH OH O O OH OH HN

O

N-(9E-Hexadecenoyl), 1-O--D-glucopyranoside(Bonaroside)

C

C O O

5-(1,2,4-Hexatrienylidene)-2(5H)-furanone

73

CHAPTER # 3 BIOSYNTHESIS

3. Biosynthetic aspects of primary metabolic products with corresponding secondary metabolites

3.1. Interrelations of biosynthetic pathways leading to secondary

metabolites in plants.

3.2. Acylopolymalonate pathway.

3.3. Shikimate pathway.

3.4. Biosynthesis of coumarin.

3.5. Biosynthesis of 4-hydroxy 3-methoxy cinnamic acid.

3.6. Biosynthesis of flavonoids.

3.7. Biosynthesis of Apigenin and kaempferol.

74 CHAPTER # 3 BIOSYNTHESIS

Figure 4: Interrelations of biosynthetic pathways leading to secondary metabolites in plants (Tyler et al., 1996)

75 CHAPTER # 3 BIOSYNTHESIS

Figure 5: Acylopolymalonate pathway

Glycolysis pyruvic acid Acetic acid CO2+H2O Carbohydrate

O O

COOH R n-1

Polyketides

R R O OH HO OH COOH O R O O HO OH OH

76 CHAPTER # 3 BIOSYNTHESIS

Figure 6: Shikimate Pathway

COOH OH H COOH HO O P O H2C O P OCH 2 P O OH H2C OH OH 3-Deox-D-arabino-heptulo sonate7-phosphate ( DAHP)

P1

COOH COOH HO COOH NADPH

Shikimate dehydrogenase OH HO O OH H2O O OH OH OH HO 3-Dehydroshikimate ATP

Pi COOH COOH COOH

CH2 CH2 O C P O OH PEP P O O C Pi COOH COOH OH OH OH

NH2 H CCOOH O O H2C CH2 C COOH HOOC CH2 C COOH

CO2 H2O OH

77 CHAPTER # 3 BIOSYNTHESIS

3.4. Biosynthesis of Coumarin Coumarins are derived from 1,2-benzopyrones. These molecules are found in higher plants where they originate from the general phenylpropanoids pathway (Harborne et al., 1999) The coumarin structure is derived from cinnamic acid via ortho-hydroxylation (a), trans-cis isomerisation of the side chain double bond (b) and (c), and lactonisation (d). The trans form is stable and could not cyclise, therefore, there should be isomerisation of some sort and the enzyme isomerase is implicated.

Figure 7: Biosynthesis of Coumarin

COOH COOH COOH

OH O - glucose Cinamic acid o-cumaric acid o -coumaric acid-ß-D-Gluciside

COOH O-glucose O O Coumarin o -coumaric acid-ß-D-Gluciside

78 CHAPTER # 3 BIOSYNTHESIS

3.5. Biosynthesis of 4-Hydroxy-3-methoxy cinnamic acid

Phenylpropanoids are a class of plant-derived organic compounds that are biosynthesized from the amino acid phenylalanine. Phenylalanine is first converted to cinnamic acid by the action of the enzyme phenylalanine ammonia-lyase (PAL). A series of enzymatic hydroxylations and methylations leads to 4-Hydroxy-3-methoxy cinnamic acid (Leland et al., 1990).

Figure 8: Biosynthesis of 4-Hydroxy-3-methoxycinnamic acid

COOH COOH COOH COOH NH2

OH OCH3 OH OH OH OH Phenylalanine Cumaric acid Caffeic acid Fe rulic acid

79 CHAPTER # 3 BIOSYNTHESIS

3.6. Biosynthesis of Flavoniod Flavonoids hold two benzene rings separated by propane unit. They are generally biosynthesized via a combination of the shikimic acid pathway (Fig. 6) and acylpolymalonate pathway (Fig. 7). The starting material is phenylalanine. The final step is ring closure, catalyzed by chalcone isomerase to yield the flavanonenaringenin. Thus in flavonoids, ring A is formed from acetate, whereas ring B originates from shikimc acid and the three carbon atoms connecting rings A and B come from phosphoenol pyruvate.

3, 2, 4, , B 1 , O 6, 5 8 1 7 2 A 6 3 5 4 The glycosidation is probably one of the last reactions in the biosynthesis of the various plant pigments. 3.8. Flavonoids Classes

O O O

OH O O O Flavonols Flavones Flavanones

O O

OH O O O Anthocyanins Isoflavones Chalcones

80 CHAPTER # 3 BIOSYNTHESIS

Figure 9 : Biosynthesis of apigenin and kaempferol.

+ H N H3 OH

-O2C + -O C -O2C NADPH NADP 2 L-phenylalanine NH trans-cinnamate + 3 +H +O2 +H2O CoA-SH+ATP

AMP+PPi OH

CoA-S 4-coumaroyl-CoA O O 3CoA-CO-CH2-CO2

3CoA-SH+3CO2

O S-CoA OH O

O O CoA-SH OH

HO OH

OH O naringenin chalcone

OH

OH HO O

HO O OH O succinate+ 2-oxoglutarate CO2+H 2O +O2 naringenin OH O (a flavanone ) ap igen in (a flavone ) 2-oxo glutarate + O2

succinate + CO2

OH OH HO O HO O 2oxoglutarate + O 2 OH OH succinate OH O +CO2 +H2O OH O armadendrin dihydrokaempferol Kaempferol (a dihydroflavonol)

81 CHAPTER # 4 MATERIAL METHODS

4. MATERIAL METHODS

4.1. Plant materials All three plants Conyza bonariensis, Euphorbia helioscopia L and Euphorbia prostrata were collected from the surrounding of Bahauddin Zakariya University Multan Pakistan in September 2002 and air dried under the shade. The plants were identified by Professor Dr Altaf Husain Dasti , plant taxonomist Institute of Biological Sciences B Z university Multan . A voucher specimens are deposited in the herbarium of the department.

4.2. Extraction Aerial parts and roots parts of Conyza bonariensis , aerial parts Euphorbia helioscopia and Euphorbia prostrata were subjected for extraction with dichloromethane and methanol successively at room temperature occasionally shaking for 24 hrs. Extracts were concentrated by Rotavapor- R20 at 35ºC.

4.3. Chromatographic studies

4.3.1. Analytical

TLC aluuminium sheets 20 x 20 cm, coated with Silica gel 60 F254. of Merck KgaA Darmstadt Germany 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 1 and table 2.

Visualization of components on TLC plates 1- Naked eye. 2- Under UV 254nm. 3- Under 365nm . 4- Chemical reagent. With regard to detection that TLC plates were observed with naked eye, in UV light 254 nm , in 365 nm and Godine reagent was sprayed on these

82

CHAPTER # 4 MATERIAL METHODS 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.

4.3.2. High performance liquid chromatography ( HPLC ) The most popular techniques HPLC was used for analytical purposes methanol : water and acetonitril : water were used as eluent ( Gradient ). The purity of isolated components was also confirmed by HPLC.

4.4. Isolation

4.4.1. 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 ) CR 40/50 ( Quickfit-England ) CR 40/30 ( Quickfit-England ) CR 20/30 ( 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.

4.4.2. Gel chromatography Different important fractions regarding to isolation were applied for chromatography on Sephadex. Sephadex chromatography provided satisfactory means of separation. Sephadex LH-20 100μm. 50 grams of Sephadex LH-20 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 / minute. Distilled methanol was used as eleunt. The size of the column used was CR 40/30 ( Quickfit-England ).

83

CHAPTER # 4 MATERIAL METHODS

Table 1: Solvent systems used for the analysis of dichloromethane extracts of E. helioscopia, E. prostrata and C. bonariensis

Solvent System Ratio 97.5 : 2.5 Choloroform : Methanol 95 : 5

89 : 11 80 : 20 Ethyl acetate : n-hexane 75 : 25 50:50 30 : 70 90 : 10 n-hexane : Isopropanol 80 : 20 80 : 20 n-hexane : Methanol 90 : 10 90 : 10 Ethyl acetate : Chloroform 80 : 20 90 : 10 Ethyl acetate : Methanol 80 : 20 80 : 20 Dichloromethane : Methanol 70 : 30

Dichloromethane : Methanol : Water 80 : 20 :2

Chloroform : Methanol : Water 80 :20 : 2

Ethyl acetate : Methanol : Water 95 : 5 : 3

Toluene : Ethyl acetate 80 : 20

84

CHAPTER # 4 MATERIAL METHODS

Table 2: Solvent systems used for the analysis of methanol extracts of E. helioscopia, E. prostrata and C. bonariensis

Solvent System Ratio 95 :10 : 0.5 90 :12 :1 85 :15 : 1 Chloform : Methanol : Water 80 : 20 :2 70 : 30 : 4 60 : 40 : 5 65 : 35 : 5 64 : 40 : 10

100 : 17 : 13 100 : 14 : 7 100 : 13.5 : 10 100 : 10 : 5 Ethyl acetate : Methanol : Water 98 : 4 : 2

95 : 10 : 5 95 : 5 : 3 85 : 15 : 10 70 : 20 : 10

80 : 20 n-hexane : Ethyl acetate 90 : 10 80 : 20 Chloroform : Methanol 90 : 10

Ethyl acetate :Formic acid :Glacial acetic acid : Water 100 : 11 : 11 : 27 100 : 10 : 10 : 20

85

CHAPTER # 4 MATERIAL METHODS

4.4.3. Solvents and chemicals All the solvents used for extraction and isolation like methanol, dichloromethane, chloroform, n-hexane, ethyl ecetate, ethanol, proponol, n- butanol Vanillin, silica gel (70-230 mesh) and TLC aluuminium sheets 20 x 20 cm, Silica gel 60 F254. , were imported from Merck KgaA Darmstadt Germany. Sephadex LH-20 25-100μm Fluka Chemie GmbH (9041-37-6).

4.5. Preparation of reagent The reagents were prepared according to the specification of Pharmaceutical Codex (11th edition) and British Pharmacopoiea.

4.5.1. Wagner’s reagent. (solution of iodine in potassium iodide ) Composition Potassium iodide (KI) = 4g

Iodine (I2) = 2g

Procedure Dissolve 4.0g of potassium iodide in minimum quantity of water (10ml). Add

2.0 g of Iodine into it. Iodine completely dissolved by complex formation. Then volume was made 100 ml of water.

4.5.2. Mayer’s reagent (solution of potassium mercuric iodide) Composition

Mercuric chloride (HgCl2) = 1.36g Potassium iodide(KI) = 5g

Procedure

Solution (A) of Mercuric chloride was prepared by dissolving 1.36g 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) of Mercuric chloride into solution (B) of Potassium iodide Mixed and made the volume 100 by addition of water. 86

CHAPTER # 4 MATERIAL METHODS

4.5.3. Hager’s reagent Picric acid was dissolved in 100ml of water till the saturation point was achieved the solution was filtered.

4.5.4. Dragendorff’s reagent (solution of potassium Bismith iodide ) Composition Tartaric acid = 100 g Bismith oxide nitrate = 8.5 g Potassium iodide = 200 ml 40 % w/v Water = 400 ml

Procedure

25 g of tartaric acid was dissolved in 100 ml of H2O and added 2.1 g of bismuth oxynitrate. Shaked for 1 hour and added 50 ml of 40 % solution of Potassium iodide Shaked well allowed to stand for 24 hours and filtered.

4.5.5. Godine reagent Godine reagent was prepared by adding equal volume of two solutions 1- 1% Vaniline in ethanol 2- 3% Perchloric acid

4.6. Preparation of solutions

4.6.1. 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.

4.6.2. Preparation of dilute ammonia solution

Dilute the 375 ml of strong ammonia solution to 1000 ml with H2O.

4.6.3. Preparation of 70 % alcohol 72.7 ml of alcohol mixed with 27.3 ml of purified water

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CHAPTER # 4 MATERIAL METHODS

4.6.4. Preparation of lead subacetate solution 40 g of lead acetate was dissolved in 90 ml of carbon dioxide free water. Adjusted the pH 7.5 with 10 M Sodium hydroxide solution. Centrifuged it and collected supernant liquid. It was lead subacetate solution.

4.6.5. 10 M NaOH 10 M Sodium hydroxide was prepared by dissolving 40 g of Sodium hydroxide in 100 ml of water.

4.6.6. 10 % Ferric chloride solution 10 g of Ferric chloride were dissolved in 100 ml of purified water.

4.6.7. 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.

4.6.8. 1 % gelatin solution in10 % Sodium chloride 1 g gelatin was dissolved in 100 ml of 10 % Sodium chloride solution .

4.6.9. 10 % Sulfuric acid 10 % sulfuric acid was prepared by diluting concentrated sulfuric acid available in ethanol.

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4.7. 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 helioscopia, Euphorbia prostrata and Conyza bonariensis. The detail of the tests employed is given.

4.7.1. Detection of alkaloids

3 g of the ground plant material was boiled with 10ml of acidified water in test tube for 1 minute, cool, and allowed the debris to settle. Filtered the supernatant liquid into another 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 addition of dilute ammonia solution. It was transferred to separating funnel and 5 ml of chloroform solution was added to the solution, 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 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 et al., 1975).

4.7.2. 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. 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 et al., 1975).

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CHAPTER # 4 MATERIAL METHODS

1g of ground plant material was taken in a test tube and 10mL 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 3mL of 3.5% ferric chloride in glacial acetic acid and was transferred to test tube after leaving for 1 minute. 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 et al., 1975).

4.7.4. Detection of tannins

Prepare 10% w/v aqueous extract of grinded drug by boiling it with distilled water for about 10-20 minutes. Filtered the extract and performed the chemical tests with clear solution.

4.7.4.1. 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 et al., 1983).

4.7.4.2. Gelatin test

Test solution (about 0.5-1%) precipitate 1% solution of gelatin containing 10% sodium chloride (Trease et al., 1983).

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CHAPTER # 4 MATERIAL METHODS

4.7.4.3. Catechin test

Dip the match stick in plant extract, dry and then moist it with concentrated hydrochloric acid. Warm near flame, a red or pink wood is produced which shows the presence of catechin (Trease et al., 1983).

4.7.5. Detection of flavonoids

2 Grams of the air dried powdered drug was boiled with 20 ml of distilled water for 10 minutes and filtered. The filtrate is 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 that indicate the possible presence of flavonoids (El-Olemy et al., 1994)

4.8. Biological methods

4.8.1. Antifungal assay

Test fungi such as Trichphyton longifusus, Candia albicans, Aspergillus flavus, Microsproum canis, Fusarium solani and Candia glabrata were employed for preliminary screening. Extracts were dissolved in sterile DMSO to serve as stock solution. Sabouraud dextrose agar was prepared by mixing

Sabouraud 4 % glucose agar and agar in distilled water. Known amount of media was dispensed into screw capped test tubes. Test tubes containing media were autoclaved 121 C° for 15 minutes. Tubes were allowed to cool to

50 C° and the desired concentration of extract was added into non-solidified media. The tubes were allowed to solidify at room temperature. Each tube was inoculated with a 4 mm diameter piece of inoculums removed from a seven-day-old culture of fungi. All culture-containing tubes were inoculated at optimum temperature of 28-30 C° for growth for 7-10 days. Culture was examined at least twice a weekly during the incubation. The concentration of

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CHAPTER # 4 MATERIAL METHODS the sample was 400ug /ml of DMSO and incubation period was 7 days at 28

C° ( Atta-ur-Rehman et al., 2001 ).

4.8.2. Antibacterial assay (Agar Well Diffusion Method)

The Petri plates were prepared with an inoculated media. Three wells of 6 mm diameter on one plate were cut with a borer and sealed with a drop of inoculated sterile media. All the solutions i.e. the extract, solvent and the reference standard were poured into their respective well by sterilized pipette.

The concentration of extract was 3 mg / ml of DMSO. The Petri dishes were incubated at 37C° for 24-48 hrs. Zones of inhibition were measured with vernier caliper and Imipenum 10 ug / disc was used as standard drug ( Atta- ur-Rehman et al., 2001 ).

4.8.3. Antioxidant assay

Reduction of 2,2-Diphenyl-1-picrylhydrazyl (=2,2-Diphenyl-1-(2,4,6 trinitrophenyl) hydrazyl; DPPH) Radical. TLC autographic assay after developing and drying, TLC plates were sprayed with a 0.2 % DPPH solution in methanol. The plates were examined after 30 min after spraying. Active compounds appear as yellow spots against a purple background. (Cendet et al.,1997).

4.8.4. Phytotoxicity bioassay

Prepared inorganic medium of 5.5 – 6.0 pH attained with KOH pellets.10 vials per dose 500, 50, 5, and control were prepared.15 mg of the extract was dissolved in 15 ml of the solvent.1000, 100, and10 µl of solution to vials for

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CHAPTER # 4 MATERIAL METHODS testing allow the solvent to evaporate overnight. 2 ml of medium was added in each vial containing a single plant a rosette of three fronds. Placed the vials in a glass dish filled with 2 cm of water, sealed the container with stopcock grease and glass plate. Place the dish along with vials in growth chamber for seven days at 25 C° under fluorescent and incandescent light. Count the number of fronds per vials on day 3 and 7.Analyzed the data as percent of control with ED 50 computer program ( Atta-ur-Rehman et al., 2001). Plant used in this assay was Lemna minor The standard drug used was paraquat at the concentration of 0.015 μg/ml .

4.8.5. Brine shrimp lethality bioassay

Artificial sea water was prepared by dissolving 3.8 g sea salt per liter of water and filtered. Sea water was placed in a small unequally divided tank and shrimp eggs were added to the larger compartment of the tank which was darkened by covering it with aluminum foil. The eliminated compartment attracted the shrimp larvae (nauplii) through perforations in the dam. The shrimp was allowed for two days at room temperature (22-29 C°) prepared three vials for testing for each fraction, tested initially at 1000, 100, and 10 µ / mg; prepared three replicates of each concentration making total of 9 vials weighed 20 mg of sample and 2 ml of organic solvent ( 20 mg / 2 ml ); from this 500, 50, or 5 µl were transferred to vials corresponding to 1000, 100, or

10 µg/ml, respectively. Solvent was evaporated under nitrogen and then placed under high vacuum for about 30 min; the volatile organic solvent was allowed to evaporate overnight. Alternatively, polar insoluble materials may be dissolved in DMSO and up to 50 µl was added per 5 ml of sea water before

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DMSO affected the results. After two days ( the brine shrimp larvae have matured), and added 5ml of sea water to each vial, and added 10 shrimps per vial with the help of Pasteur pipette ( 30 shrimps per dilution). The vials were maintained under illumination. After 24 hours Chad clasped, the number of surviving shrimps were counted and recorded with the aid of 3x magnifying glass. The data was analyzed with the Finney computer program ( Probit analysis ) to determine LC50 values and 95 % confidence intervals. ( Atta-ur-

Rehman et al., 2001).

4.9. Spectroscopic methods

Ultraviolet (UV) spectra were recorded in chloroform on a Shimadzu UV 240

(Shimadzu Corporation, Kyoto, Japan) and Perkin-Elmer spectrophotometers.

Infrared (IR) JASCO A-302 (Japan Spectroscopic Co. Limited) spectrophotometers. Proton magnetic resonance (1H-NMR) spectra were recorded either in CDCl3, 400 MHz on Bruker AM-300, AM-400 and AMX-500 nuclear magnetic resonance spectrometers, respectively. The 13C-NMR spectra were recorded in the solvents CDCL3 and CD3OD at 100 MHz, on the same instruments. Mass spectra were recorded on Finnigan MAT 312 double- focusing mass spectrophotometer both connected to PC 386 computer system or Peak matching, field desorption (FD) measurements performed on the MAT 312 mass spectrophotometer. High-resolution electron impact mass

(HREI MS) spectra were recorded on JEOL JMS HX 110 mass spectrophotometer.

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CHAPTER # 4 MATERIAL METHODS

4.9.1. Physical Constants

Fraction collector used was Spectra / Chrom CF1, Oven of Memmert UVIS of

DESAGA, weighing balance of SHAIMADZU, Vortexmixer of Stuart and

Melting points were determined in glass capillary tubes using Gallenkamp melting point apparatus.

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CHAPTER # 4 MATERIAL METHODS

4.10. Phytochemical isolation

4.10.1. Isolation of compounds 1 and 2 10 gram dichloromethane extract of E. helioscopia dissolved in minimum quantity of methanol was loaded on silica gel. CR 60/50 ( Quickfit-England ) column was packed with 400 grams of silica gel dissolved in the mobile phase. Flow rate of the mobile phase chloroform : methanol (95 : 5) was kept 1 ml/min . Each subfraction of 25 ml was collected and analyzed by TLC. On the basis of TLC results total 11 fractions were prepared by combining same fractions. Fraction EHADJ on the basis of chemical analysis was selected for further isolation and was chromatographed using silica gel as stationary phase and n-hexane : methanol (95 : 5) as mobiile phase with flow rate 1 ml/min. Each fraction of 10 ml was collected with the help of fraction collector. All the fractions were analyzed carefully by TLC. It gave 8 fractions finally fraction EHADJg was applied to Sephadex LH-20 using distilled methanol as eleuent at flow rate 1ml/min. This gave a compound 1. Similarly fraction

EHADK on the basis of TLC results was selected for further investigation and was chromatographed using silica gel as stationary phase and ethyl acetate : methanol (95 : 5) as mobile phase. Each fraction of 10 ml was collected with the help of fraction collector. Further 9 fractions were prepared. Fraction

EHADKh was subjected to Sephadex LH-20 and it yield compound 2. 1 Differents spectroscopic techniques like UV, IR (FTR), H NMR [CDCl3, ( = 13 7.24,) 300 MHz, C NMR [CDCl3, ( = 77.00,) 100 MHz, BB], MS (70 eV) were used to establish the structure of isolated compounds. The spectroscopic data of compound 1 and 2 is as under.

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Compound 1

Chemical Formula: C10H10O4

Molecular Weight: 194.18

Melting point: 168-172 °C

Physical State: Off white crystalline powder

UV: λmax = 390.0 nm, 378.4 mn, 321.2 mn, 26.0 nm, 234.0 nm, 225.6 nm, 218.6 nm, 199.0 nm, 198.4 nm and 193.4 nm.

IR (FTR): ~ = 3437 cm¯1 (br), 3016 (w), 2968 (w) 2921 (w), 2842 (w), 2662 (w), 2594 (w), 2516 (w) 1825 (w), 1690 (m)1665 (m), 1620 (m), 1515 (s), 1464 (w), 1431 (m), 1379 (w), 1324 (w), 1272 (s), 1205 (s), 1176 (m), 1113 (w), 1034 (w), 944 (w), 852 (w), 804 (w) and 576 (m).

1 3 H NMR [CDCl3, ( = 7.24,) 300 MHz]:  = 3.80 (s, 3 H, OCH3), 6.18 (d, JHH = 3 4 15.8 Hz, 1 H, 2-H), 6.76 (dd, JHH = 8.5 Hz, JHH = 4.2 Hz, 1 H, 5’-H), 6.93 (overlapped with 6’-H , 1 H, 2’-H), 6.95 (overlapped with 2’-H, 1 H, 6’-H), 7.53 (d, 3 JHH = 15.8 Hz, 1 H, 3-H) ppm. 13 C NMR [CDCl3, ( = 77.00,) 100 MHz, BB]:  = 55.6 (OCH3), 109.7 (C-2’), 114.3 (C-2), 121.3 (C-1’), 122.8 (C-5’), 126.4(C-6’), 145.5 (C-3), 147.2 (C-4’), 148.3 (C- 3’), 169.6 (C-1).

+ + + MS (70 eV): m⁄z (%) = 194 (100) [M ], 179 (28) [M – CH3], 133 (38) [M – CH3-

COOH], 105 (24) [C7H5O], 77 (45).

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CHAPTER # 4 MATERIAL METHODS

Compound 2

Chemical Formula: C9H6O3

Molecular Weight: 162.14

Melting point: 230-232°C (Sublimation)

Physical state: Off-white crystalline powder

UV: λmax = 389.4 nm, 375.8 nm, 324.0 nm, 260.6 nm, 216.0 nm, 210.8 nm, 201.2 mn and 195.4 nm.

IR (FTR): ~ = 3703 cm¯1 (w), 3558 (w), 3168 (br), 2923 (w), 1709 (m), 1682 (s), 1605 (s), 1566 (s), 1508 (m), 1455 (w),1385 (w), 1320 (m), 1233 (s), 1133 (m), 986 (w), 835 (m), 685 (w), 538 (w), 483 (w), 449 (w) and 423 (w).

1 3 H NMR [CDCl3, ( = 7.24,) 300 MHz]:  = 6.08 (d, JHH = 9.4 Hz, 1 H, 3-H), 6.64 4 4 3 (d, JHH = 1.3 Hz, 1 H, 8-H), 6.68 (d, JHH = 2.2 Hz, 1 H, 6-H), 7.2 (d, JHH = 6.5 Hz, 3 1 H, 5-H), 7.57 (d, JHH = 9.4 Hz, 1 H, 4-H).

13 C NMR [CDCl3, ( = 77.00,) 100 MHz, BB]:  = 102.7 (C-8), 111.4 (C-6). 113.5 (C-3), 129 (C-5), 144.3 (C-4).

+ + + MS (70 eV): m⁄z (%) = 162 (70) [M ], 134 (100) [M – CO], 105 (34) [M – C2HO],

78 (38) [C5H2O].

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4.10.2. Isolation of compounds 3 and 4 E. prostrata (aerial parts) methanol extract was carried out for isolation of different components and compounds 3 (22 mg) of light yellow color and compound 4 off white crystalline powder (27 mg) were isolated. 15 grams of methanol extract of Euphorbia prostrata was dissolved in minimum quantity of methanol. The solution was filtered and loaded on 16 grams of silica gel. The column of size CR 60/50 (Quickfit-England) was packed with 450 grams of silica gel dissolved in the mobile phase. The column was allowed to be stable with a flow rate 1 ml /min. When the silica was settled properly then after 1 hour with continous flow of mobile phase, the dried sample was applied at the top of the column.The column was eluted continously with mobile phase ethyl acetate ; methanol :water (95:5:3) for 36 hours. Each subfraction of 25 ml was collected and analyzed by TLC. On the basis of TLC results total 9 fractions were prepared. Fraction EPAMC on the basis of chemical analysis was again chromatographed using silica gel as stationary phase and n-hexane : ethyl acetate (90 : 10) as mobiile phase. Each fraction of 10 ml was collected with the help of fraction collector. It gave further 5 fractions. All the fractions were analyzed by TLC, and on the basis of TLC results fraction EPAMC2 was applied to Sephadex LH 20 using distilled methanol as eleuent. This fraction give a compound 3. TLC analysis of fraction EPAMD was carried out and on the basis of the results this fraction ws again chomatographed using silica gel as stationary and chloroform : methanol : water (85 : 15 : 1) as mobile phase with flow rate of 1 ml/min. It gave four fractions which were analyzed and the fraction EPAMD3 was subjected to Sephadex LH-20 . It gave compound 4. 1 13 The UV, IR (FTR), H NMR [CDCl3, ( = 7.24,) 300 MHz, C NMR [CDCl3, ( = 77.00,) 100 MHz, BB], MS (70 eV) of compound 3 and 4 are given below.

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Compound 3

Chemical Formula: C15H10O6

Molecular Weight: 286.24

Melting Point: 276-278 °C

Physical State: Light yellow crystalline solid

UV: λmax = 365.4 nm, 329.6 nm, 324nm, 282 nm, 265.8 nm, 238.8 nm, 204.6 nm, 202.2 nm, 198.8 nm, 195.8 nm, 192.8 nm, 190.2 nm and 189.4 nm.

IR (FTR): ~ = 3697 cm¯1 (w), 3380 (br), 2957 (br), 1661 (m), 1612 (s), 1568 (w), 1507 (m), 1443 (w) 1380 (m), 1306 (m),1252 (m), 176 (s), 1088 (w), 1008 (w), 974 (w), 796 (w), 723 (w), 673 (w) and 585 (w).

1 4 H NMR [CDCl3, ( = 7.24,) 300 MHz]:  = 6.12 (d, JHH = 1.3 Hz, 1 H, 6-H), 6.27 4 3 3 (d, JHH = 1.3 Hz, 1 H, 8-H), 6.81 (d, JHH = 6.5 Hz, 2 H, 3’-H), 7.94 (d, JHH = 6.5 Hz, 2 H, 2’-H).

13 C NMR [CDCl3, ( = 77.00,) 100 MHz, BB]:  = 93.7 (C-8). 98.3 (C-6). 103.1 (C- 10), 115.2 (C-3’,5’), 122.1(C-1’),129.3 (C-2’,6’), 135.2 (C-3), 146.3 (C-2), 156.7 (C- 4’), 158.7 (C-9), 160.4 (C-5), 163.7 (C-7), 175.3 (C-4).

+ + + MS (70 eV): m⁄z (%) = 286 (100) [M ], 258 (13) [M – CO], 229 (13) [M – C2HO2], + + 153 (9) [M – C8H5O2], 121 (31) [M – C8H5O4].

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CHAPTER # 4 MATERIAL METHODS

Compound 4

Chemical Formula: C9H8O3

Molecular Weight: 164.16

Melting Point: 211.2 C

Physical State: Off white crystalline powder

UV: λmax = 389.8 nm,377.4 nm, 324 nm, 300.6 nm, 273.6 nm, 244.8 nm, 211 nm, 210 nm, 201.8 nm, 196.4 nm, 194.6 nm and 187.8 nm.

IR (FTR): ~ = 3720 cm¯1 (w), 3360 (br), 3073 (w) 2724 (w), 2531 (w), 1668 (m), 1617 (s), 1600 (m) 1459 (w), 1332 (m), 1218 (s), 1185 (w), 1092 (w), 993 (w), 913 (w), 751 (w), 591 (w) and 492 (w).

1 3 H NMR [CDCl3, ( = 7.24,) 300 MHz]:  = 6.43 (d, JHH = 12.0 Hz, 1 H, 3-H), 6.69 3 3 (dd, JHH = 6.9, 7.8 Hz, 1 H, 5’-H), 6.95 (dd, JHH = 7.7, 7.9 Hz, 1 H, 4’-H), 7.08 (dd, 3 4 3 4 JHH = 6.7, JHH =1.4 Hz, 1 H, 3’-H), 7.32 (dd, JHH = 6.7, JHH = 1.2 Hz, 1 H, 6’-H), 3 7.88 (d, JHH = 12.0 Hz, 1 H, 4-H).

13 C NMR [CDCl3, ( = 77.00,) 100 MHz, BB]:  = 115.8 (C-2), 117.6 (C-3’), 119.5 (C-5’), 121.3 (C-1’), 128.7 (C-6’), 131.1 (C-4’), 141.2 (C-3), 156.3 (C-2’) 170.0 (C- 1).

+ + + MS (70 eV): m⁄z (%) = 164 (29) [M ], 146 (55) [M – H2O], 118 (100) [M – CO2H2],

90 (45) [C7H6], 77 (20).

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4.10.3. Isolation of compound 5 Methanol extract of C. bonariensis (roots) was carried out for isolation and different components. A compound 5 (29mg) light yellow colour was isolated. 11 grams of methanol extracts of C. bonariensis was dissolved in sufficient quantity of methanol. The solution was filtered and loaded on silica gel. Glass column CR 40/50 ( Quickfit-England) was packed with 350 grams of silica gel dissolved in the mobile phase ethyl acetate : methanol : water (80 : 20 : 2). Dried sample was loaded on the top of the column when it became stable and the silica was settled properly. The column was eluted at a flow rate 1 ml/min at room temperature about 30 C° for 30 hours. Each fraction of 25 ml was collected and analyzed carefully. On the basis of TLC results subfractions were combined to give finally 12 fractions. All these fractions were concentrated under reduced pressure at 30 C°. Fraction CBRMC was subjected again to column chromatography using silica gel as stationary phase and chloroform : methanol (95 : 5) as mobile phase. Each fraction of 10 ml was collected with the help of fraction collector. All the fractions were analyzed by TLC. It gave further 5 fractions. On the basis of analysis fraction

CBRMC4 was subjected to Sephadex LH-20 using distilled methanol as an eluent at a flow rate 1 ml/min. it gave a compound 5. 1 13 The UV, IR (FTR), H NMR [CDCl3, ( = 7.24,) 300 MHz, C NMR [CDCl3, ( = 77.00,) 100 MHz, BB], MS (70 eV) data of the isolated compoud 5 is givrn below.

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Compound 5

Chemical Formula: C15H10O5

Molecular Weight: 270.24

Melting Point: 345-350 °C

Physical State: Yellow crystalline solid

UV: λmax = 335 nm, 280.8 nm, 268nm, 246.4nm, 210.6nm, 197.6nm, 196.6nm, and195.4nm.

IR (FTR): ~ = 3805 cm¯1 (w), 3699 (w), 3287 (br) 3094 (w), 3021 (w), 2924 (w), 2495 (w), 2423 (w) 1654 (s), 1609 (s)1556 (s), 1499 (s), 1446 (m), 1398 (w), 1355 (s), 1297 (w), 1270 (m), 1244 (s), 1223 (m), 1180 (s), 1116 (w), 1030 (w), 907 (w), 829 (s).

1 4 H NMR [CDCl3, ( = 7.24,) 300 MHz]:  = 6.12 (d, JHH = 1.5 Hz, 1 H, 6-H), 6.29 4 3 (d, JHH = 1.5 Hz, 1 H, 8-H), 6.38 (s, 1 H, 3-H), 6.80 (d, JHH = 6.5 Hz, 2 H, 3’-H), 3 7.65 (d, JHH = 6.5 Hz, 2 H, 2’-H).

13 C NMR [CDCl3, ( = 77.00,) 100 MHz, BB]:  = 94.1 (C-8). 99.0 (C-6), 115.7 (C- 3’,5’), 128.0 (C-2’,6’), 164.5 (C-7).

+ + + MS (70 eV): m⁄z (%) = 270 (100) [M ], 242 (11) [M – CO], 153 (30) [M – C8H5O], + + 121 (23) [M – C8H5O3], 118 (9) [M – C7H4O4], 78 (60).

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CHAPTER # 5 RESULTS

5.1. Extraction Conyza bonariensis, Euphorbia helioscopia and Euphorbia prostrata were collected from the surrounding of Bahauddin Zakariya University Multan Pakistan. The air dried aerial parts and roots Conyza bonariensis, aerial parts of Euphorbia helioscopia and Euphorbi prostrata were grounded and extracted with dichloromethane and methanol successively at room temperature occasionally shaking for 24 hrs. Extracts were concentrated by Rotavapor-R20 at 35 C º. The results of the extraction along with the abbreviations used for different extracts are given in table 3.

5.2. Biological screening Both dichloromethane and methanol extract of aerial parts and roots of Conyza bonariensis , the aerial parts of Euphorbia helioscopia and Euphorbia prostrata were screened for antibacterial activity , antifungal activity, brine shrimp (artemia salina) Lethality bioassay , phytotoxic bioassay and anti- oxidant activity. The results are as under.

5.2.1. Antibacterial activity Antibacterial bioassay was performed against Escherichia coli, Bacillus subtilis, Shigella flexenari, Staphylococcus aureus, Pseudomonas aeruginosa and Salomnella typhi . Methanol and dichloromethane extractsof Conyza bonariensis roots showed non significant activity against Bacillus subtilis and Salomnella typhi. The results are given in the table 4.

5.2.2. Antifungal bioassay E. helioscopia (aerial parts) dichloromethane extract exhibited significant activity against Fusarium solani with 90 % inhibition. Similarly dichloromethane extract of C. bonariensis aerial parts and roots demonstrated 70 % and 60% inhibition against Aspergillus flavus and Microsporum canis respectively. While all other extracts under investigation remain inactive as shown in table 5.

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5.2.3. Brine shrimp lethality bioassay and phytotoxic bioassay Brine shrimp lethality bioassay and phytotoxic bioassay were perfomed. All the extracts did not exhibit cytotoxicity and showed non significant phytotoxicity. Results are reflected in tables 6 and 7.

5.2.4. Antioxidant activity Dichloromethane and methanol extracts of aerial parts and roots of Conyza bonariensis, aerial parts of Euphorbia helioscopia and Euphorbia prostrata were employed for this test. The methanol extract of all the medicinal plants investigated exhibited antioxidant activity where as dichloromethane extracts did not respond to DPPH as observed in the table 8.

5.3. Phytochemical analysis Phytochemical studies were carried out for the detection of alkaloids, glycosides, saponins, flavonoids and tannins in different parts of the plants Euphorbia helioscopia, Euphorbia prostrata and Conyza bonariensis. The detail of the tests employed is given in material methods. The results of phytochemical analysis are given in table 9.

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EPAD EPAD EHAD EHAD CBAD CBRD EPAM EPAM EHAM EHAM CBAM CBRM For The Extracts Extracts Abbreviations Euphorbia prostrata Euphorbiaprostrata , 9.6 9.6 19.8 32.1 19.1 35.9 33.2 15.4 38.3 Weight of Weight of Extract (gms) (gms) Extract Solvent Solvent Methanol Methanol Methanol Methanol Euphorbia helioscopia Dichloromethane Dichloromethane Dichloromethane Dichloromethane Dichloromethane Roots (1000gm) (1000gm) (1000gm) (1000gm) Part Used Used Part Aerial parts parts Aerial parts Aerial parts Aerial with different solvents solvents different with

Plant Name Plant Name Conyza bonariensis Conyza Conyza bonariensis Euphorbia helioscopia Euphorbia prostrata Euphorbia and Table 3: Results of the Extraction of plants Extraction the of 3: Results Table

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30 37 36 26 32 30 of Std. Zone of Zone inhibition inhibition Drug (mm) - - - - 9 10 10 CBRM CBRM - - - - 10 10 CBRD

------CBAM E. helioscopia, E. prostrata and and E. prostrata helioscopia, E. ------CBAD ------EPAM EPAM ------EPAD EPAD Zone of inhibitionExtracts of DifferentZone of - - - - 9 11 11 vity of thedifferent extracts of vity of EHAM EHAM - - - - 10 13 EHAD EHAD

Name ofBacteria Escherichia coli coli Escherichia Bacillus subtilis flexenari Shigella aureus Staphylococcus aeruginosa Pseudomonas typhi Salmonella Table 4: Results of antibacterial acti antibacterial of Results 4: Table C. bonariensis

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20 70 MIC 98.35 98.35 73.25 110.8 110.8 110.8 µg/ml Std. Drug Drug Std. Miconazole Miconazole Miconazole Miconazole Miconazole Amphotericin

0 0 0 % 70 70 60 90 Inhibition

100 100 100 100 100 100 Ctr. 100 100 100 100 100 100 CBRD 40 100 100 100 100 100 CBRD CBRD E.helioscopia, and E. prostrata E.helioscopia, 100 100 100 100 100 100 100 CBAM

30 100 100 100 100 100 CBAD 100 100 100 100 100 100 EPAM 100 100 100 100 100 100 EPAD Linear Growth (mm) of Extracts and Control Control (mm) of Extracts and Growth Linear 100 100 100 100 100 100 EHAM 10 100 100 100 100 100 EHAD

Fungus Name of the Trichphyton Trichphyton longifusus Aspergillus Aspergillus flavus Microsporum Microsporum canis Table 5: activitydifferent extractsTable of Results of antifungal of bonariensis C. Candida albicans Fusarium solani Candida glabrata

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Table 6: Results of brine shrimp lethality bioassay of the different extracts of E. helioscopia, E. prostrata and C. bonariensis Number of Number of LD50 Dose Extract (µg/ml) Shrimps Survivor (µg/ml) 1000 30 23 - EHAD 100 30 27 - 10 30 28 - 1000 30 18 - EHAM 100 30 21 - 10 30 24 - 1000 30 22 - EPAD 100 30 18 - 10 30 19 - 1000 30 21 - EPAM 100 30 19 - 10 30 21 - 1000 30 19 - CBAD 100 30 19 - 10 30 23 - 1000 30 23 - CBAM 100 30 24 - 10 30 24 - 1000 30 21 - CBRD 100 30 23 - 10 30 26 - 1000 30 22 - CBRM 100 30 24 - 10 30 23 -

*The standard drug used in the assay was Etoposide with LD 50 (7.4625μg/ml).

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Table 7: Result of Phytotoxicity bioassay of the different extracts of E. helioscopia, E. prostrata and C. bonariensis

Name of Dose Number of fronts Control % Growth regulation Extract (µg/ml) 1000 14 17 17.64 EHAD 100 15 17 11.76 10 17 17 0.00 1000 11 17 35.29 EHAM 100 12 17 29.41 10 13 17 23.52 1000 16 20 20.0 EPAD 100 18 20 10.0 10 20 20 0.00 1000 18 20 10 EPAM 100 18 20 10 10 18 20 10 1000 15 17 11.76 CBAD 100 16 17 5.88 10 17 17 0.00 1000 13 17 23.52 CBAM 100 15 17 11.76 10 16 17 5.88 1000 13 17 23.52 CBRD 100 15 17 11.76 10 16 17 5.88 1000 15 17 11.76 CBRM 100 16 17 5.88 10 17 17 0.00

Plant used in this assay was Lemna minor & the concentration of the standard drug (paraquat) was of0.015μ.

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+ CBRM CBRM - CBRD + CBAM CBAM E. helioscopia, E. prostrata E. prostrata E. helioscopia, - CBAD CBAD

+ EPAM

- EPAD EPAD + EHAM - EHAD Name of plant Name of plant Antioxidant activity activity Antioxidant Table 8:Table of Antioxidant bioassayResult of the extracts different of and C. bonariensis

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+ + + + Flavonoids

+ + + + Saponins + + + + Tannins - - - - Cardiac glycosides E. prostrata and C. bonariensis E. prostrataand C. bonariensis ,

- - - - Anthraquinones E. helioscopia - - - + Alkaloids Part Part used Aerial Aerial Aerial Aerial Roots Roots

Name of plant plant of Name Euphorbia helioscopia Euphorbia prostrata Euphorbia bonariensis Conyza Table 9: Result of Phytochemical analysis of analysis Phytochemical of Table 9: Result

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5.4. Phytochemical isolation

5.4.1. Isolation of compounds 1 and 2 Dichloromethane E. helioscopia (aerial parts) was carried out for isolation of different components. Two compounds 1 (23 mg) and 2 (18 mg) were isolated from E. helioscopia (Figure 10). 10 grams of the dichloromethane extract was dissolved in minimum quantity of methanol. The solution was filtered and loaded to 11grams of silica gel. The column of size CR 60/50 ( Quickfit-England ) was packed with 400 grams of silica gel dissolved in the mobile phase. The column was allowed to be stable with a flow rate 1 ml /min. When the silica was settled properly then after 1 hour with continous flow of mobile phase, the sample was applied at the top of the column.The column was eluted continously with mobile phase chloroform : methanol (95 : 5) for 40 hours. Each subfraction of 25 ml was collected and analyzed by TLC. On the basis of TLC results total 11 fractions were prepared. Fraction EHADJ on the basis of chemical analysis was again chromatographed using silica gel as stationary phase and n-hexane : methanol (95 : 5) as mobiile phase. Each fraction of 10 ml was collected with the help of fraction collector. All these fractions were analyzed by TLC. It gave 8 fractions

finally. Fraction EHADJg was applied to Sephadex LH-20 using distilled methanol

as eleuent. This fraction give a compound 1. Similarly fraction EHADK on the basis of TLC results was chromatographed using silica gel as stationary phase and ethyl acetate : methanol (95 : 5) as mobile phase. Each fraction of 10 ml was collected with the help of fraction collector. All the fractions were analyzed

carefully. It gave finally 9 fractions. Fraction EHADKh was subjected to Sephadex LH-20 using distilled methanol as mobile phase. Each fraction of 10 ml was collected with the help of fraction collector. All the fractions were analyzed by TLC. and it yielded compound 2.

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Figure 10: Isolation of compound 1 and 2 from dichloromethane extract of E. helioscopia Dichloromethane extract of Euphorbia helioscopia 10 gm silica gel chloroform : methanol 95 : 5

A C E G I 0.0288gm 0.0238gm 0.0621gm 0.0516gm 0.1371gm B 0.0227gm D H 0.2079 gm F 0.092gm 0.0295gm

J 0.430gm K 0.341gm

n-hexane : methanol silica gel 95 : 5

Ja Jb Jc Jd Je Jf Jh 0.0331gm 0.0256gm 0.0307gm 0.0699gm 0.0367gm 0.0271gm 0.0536gm Jg 0.0840gm

Sephadex LH 20 silica gel

Ethyl acetate : methanol 1 95 : 5

Ka Kb Kc Kd Ke Kf Kg Kh Ki 0.0131gm 0.0329gm 0.0385gm 0.0273gm 0.0201gm 0.0486gm 0.0244gm 0.0344gm 0.0267gm

Sephadex LH 20

2

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5.4.2. Isolation of compounds 3 and 4

E. prostrata (aerial parts) methanol extract was carried out for isolation of different compounds and two compounds 3 (22 mg) of light yellow color and 4 (27mg) was isolated according to (Figure 11). 15 grams of methanol extract of Euphorbia prostrata was dissolved in minimum quantity of methanol. The solution was filtered and loaded on 16 grams of silica gel. The column of size CR 60/50 (Quickfit-England) was packed with 450 grams of silica gel dissolved in the mobile phase. The column was allowed to be stable with a flow rate 1 ml /min. When the silica was settled properly then after 1 hour with continous flow of mobile phase, the dried sample was applied at the top of the column.The column was eluted continously with mobile phase ethyl acetate : methanol :water (95:5:3) for 36 hours. Each subfraction of 25 ml was collected and analyzed by TLC. On

the basis of TLC results total 9 fractions were prepared. Fraction EPAMC on the basis of chemical analysis was again chromatographed using silica gel as stationary phase and n-hexane : ethyl acetate (90 : 10) as mobiile phase. Each fraction of 10 ml was collected with the help of fraction collector. All the fractions were analyzed by TLC. As a result 5 fractions were obtained. On the basis of

TLC results fraction EPAMC2 was applied to Sephadex LH-20 using distilled methanol as eluent. This fraction gave a compound 3. TLC analysis of fraction EPAMD was carried out and on the basis of results this fraction ws again chomatographed using silica gel as stationary phase and chloroform : methanol : water (85 : 15 : 1) as mobile phase with flow rate of 1 ml/min. Each fraction of 10 ml was collected with the help of fraction collector. All the fractions were

analyzed by TLC. It gave four fractions. The fraction EPAMD3 was subjected to Sephadex LH-20 . It gave compound 4.

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Figure 11: Isolation of compound 3 and 4 from methanol extract of E. prostrata

Methanol extract of Euphorbia prostrata 15 gm

Silica gel ethyl acetate:methanol: water 95 : 5 : 3

EPAMA EPAMD EPAMH 0.1409 gm EPAMF 0.8451 gm 0.8362 gm 0.3457 gm EPAMB EPAME 0.5134 gm 0.7698 gm EPAMG EPAMI 0.9770 gm 2.461 gm EPAMC 2.2962 gm n-hexane : ethyl acetate silica gel 90 : 10

EPAMC1 EPAMC2 EPAMC3 EPAMC4 EPAMC5 silica gel 0.1917 gm 0.4720 gm 0.2436 gm 0.3207 gm 0.6531 gm chlorof orm : methanol : water 85 : 15 : 1

Sephadex LH-20 EPAMD 1 EPAMD2 EPAMD EPAMD 0.1143gm 3 4 0.1732 gm 0.2314gm 0.2411 gm

Sephadex LH-20 EPAMC2b1 EPAMC2b2 0.0187 gm 0.0163 gm 3

4 EPAMD3b 0.1276gm

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5.4.3. Isolation of compound 5 Methanol extract of C. bonariensis (roots) was selected for isolation of different components. A compound 5 (29 mg) light yellow colour was isolated from the methanol extract of C bonariensis . 11 grams of methanol extracts of C. bonariensis was dissolved in sufficient quantity of methanol. The solution was filtered and loaded on silica gel. Glass column CR 40/50 ( Quickfit-England) was packed with 350 grams of silica gel dissolved in the mobile phase Ethyl acetate : methanol : water (80 : 20 : 2). Dried sample was loaded on the top of the column. The column was eluted at a flow rate 1 ml/min at room temperature about 30 C°. Each fraction of 25ml was collected and analyzed carefully. On the basis of TLC results subfraction were combined to give finally 12 fractions. All these fractions were concentrated under reduced pressure at 30 C°. Fraction CBRMC was subjected further to column chromatography using silica gel as stationary phase and chloroform: methanol (95 : 5) as mobile phase. Each fraction of 10 ml was collected with the help of fraction collector. All the fractions were analyzed by TLC. It gave further 5 fractions. All the fraction were analyzed carefully. On the

basis of analysis fraction CBRMC4 was subjected to Sephadex LH-20 using distilled methanol as an eluent at a flow rate 1 ml/min. It gave compound 5.

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Figure 12: Isolation of compound 5 from methanol extract of C. bonariensis

Methanol extract of Conyza bonariensis (Roots)

11 gm

silica gel Ethyl acetate : Methanol : Water 80 : 20 : 2

CBRMA CBRMI CBRMG CBRMK 0.226 gm CBRMC CBRME 0.9743 gm 0.5722 gm 0.6235gm 0.8711 gm 0.694 gm

CBRMD CBRMB CBRMF CBRMH CBRML 0.7329 gm 0.4039 gm CBRMJ 0.6423 gm 0.3291 gm 0.4372 gm 1.067 gm

silica gel chloroform: methanol 95 : 5

CBRMC

CBRMC1 CBRMC3 CBRMC5 0.0326 gm 0.0357 gm 0.0469 gm

CBRMC6 CBRMC2 CBRMC4 0.0327 gm 0.0129 gm 0.08631 gm

Sephadex LH-20

CBRMC4a 5 0.0217 gm

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5.5. Structure determination 5.5.1. Structure determination of Compound 1

Structure of the isolated compound 1 was established with the help of UV, IR, 1H NMR and 13C NMR spectroscopy including EIMS spectrometry. Two characteristics band observed at 218nm and 321nm in UV spectra suggests for cinnamic acid and its derivatives (Fig .13). The IR spectra was recorded from 400-4000 cm.¯1 The IR absorption of the interest in the spectra of compound 1 are a band at 3437(broad) cm¯1 is for O-H stretching. Another significant absorption is 1690 (sharp) cm¯1 for C=O stretching. The absorption band observed at 1515 (medium) cm¯1 is assigned to olefinic C=C stretching (Fig.14). The 1H NMR spectrum shows one singlet at  = 3.8 ppm which corresponds to

(OCH3). One doublet at  = 6.18 ppm for one olefinic proton (3-H) and other doublet at  = 7.53 ppm for the other olefinic proton (2-H) Both protons showed coupling 15.8 Hz which is trans coupling. While other three aromatic protons showed absorption with ABC spin system which is a close resemblance with the structure of Cinnamiacid (Fig 15a) and (Fig 15b). In the 13C NMR spectrum the

carbon atom (OCH3) resonates at  = 55.6 ppm and absorptions found at  = 114.3 for (C-2) and  = 145.5 for (C-3). Signals observed for aromatic carbon are in close accord with the structure of the Cinnamicacid. The downfield shift at  = 169.6 assigned to (C=O) (Fig.16) .The LREIMS mass spectrum exhibits the molecular ion peak [M+] at m⁄z = 194 (100), which is a base peak, along with previous information confirms that actual molecule is cinnamic acid derivative . A + fragment signal [M – CH3] was observed at m⁄z = 179 (28) obtained by the loss

of CH3 (Fig.17). All other signals observed in the mass spectrum are in complete accord to the fragmentation pattern of the constitution of 4-hydroxy3-methoxy cinnamic acid. On the basis of these measurements, the assigned constitution of 4-hydroxy3-methoxy cinnamicacid is confirmed.

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Compound 1

H 1 2 COOH 6' 3 5' 1' 4' 2' H 3' HO

OCH3

Chemical Formula: C10H10O4

Molecular Weight: 194.18

Melting point: 168-172 °C

Physical State : Off white crystalline powder

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Figure 13: UV spectrum of compound 1

Figure 14: IR spectrum of compound 1

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Figure 15a: 1H NMR spectrum of compound 1

Figure 15b: Elaborated 1H NMR spectrum of compound 1

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Figure 16: 13C spectrums of compound 1

Figure 17: Mass spectrum of compound 1

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5.5.2. Structure determination of Compound 2

The structure of the isolated compound 2 was established by UV, IR, 1H NMR and 13C NMR spectroscopy including EIMS spectrometry. The UV spectra for the compound 2 a strong band at 216nm and other strong band at 324 nm ascribable to benzene and pyrone ring respectively. (Fig. 18). The IR absorption band at 3437(broad) cm¯1 is for O-H stretching. The absorption band for C=O (pyrone carbonyl) of α, β-unsaturated lactone in the IR spectrum observed at 1682 (sharp) cm¯1. The absorption band observed at 1605 (medium) cm¯1 is assigned to olifinic C=C stretching (Fig.19). The 1H NMR spectrum shows one doublet at  = 6.08 ppm for one olefinic proton (3-H) and other doublet at  = 7.57 ppm for (4-H). Both protons show coupling of 9.4 Hz which is cis coupling. While aromatic proton (8-H) shows one doublet at  4 = 6.64 ppm which experiences meta coupling ( JHH = 1.3 Hz) with (6-H). The signal observed at  = 6.68 ppm splited twice is assigned to (6-H) giving 3 4 information of orhto coupling ( JHH = 6.5 Hz) with (5-H) and meta coupling ( JHH = 3 2.2 Hz) with (8-H). The proton (5-H) absorbs as one doublet ( JHH = 6.5 Hz) at  = 7.20 ppm (Fig.20a and Fig.20b). In the aromatic region of absorption, ABC spin system is observed which is in resemblance with the structure of compound 2 that may coumarin in nature. In the 13C NMR spectrum the absorption signals observed at  = 113.5 ppm and  = 144.4 ppm are assigned to (C-3) and (C-4) respectively (Fig.21). The chemical shift values of these carbons are in compliance with the literature values indicating α, β-unsaturated system. Signals observed for aromatic carbons are in close accord with the structure of coumarin. It is concluded that 1H and 13C NMR spectra are in close agreement to the assigned substitution pattern of the aromatic ring. The LREIMS mass spectrum exhibits the molecular ion peak [M+] at m⁄z = 162 (70), which along with previous information confirms that actual molecule is hydroxy coumarin . A fragment signal [M+ – CO] was observed at m⁄z = 134 (100) obtained by the loss of CO (Fig.22). All other signals observed in the mass

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spectrum are in complete accord to the fragmentation pattern of the constitution of 7-hydroxycoumarin. On the basis of these measurements, the assigned constitution of 7-hydroxy coumarin (compound 2) is confirmed. 7-hydroxy coumarin is for the first time reported in the species Euphorbia helioscopia but already in the genus Euphorbia in the species Euphorbia pekinensis (Kong et al., 1996)

compound 2

5 4 6 10 3 7 9 2 8 1 HO O O

Chemical Formula: C9H6O3

Molecular Weight: 162.14

Melting point: 230-232°C (Sublimation)

Physical state: Off-white crystalline powder

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Figure 18: UV spectrum of compound 2

Figure 19: IR spectrum of compound 2

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Figure 20a: 1H NMR spectrum of compound 2

Figure 20b: Elaborated 1H NMR spectrum of compound 2

127

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Figure 21: 13C spectrums of compound 2

Figure 22: Mass spectrum of compound 2

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5.5.3. Structure determination of Compound 3

Structure of the isolated compound 3 was established by UV,IR, 1H NMR and 13C NMR spectroscopy including EIMS spectrometry. Two strong bands observed in UV spectra one at 265.8nm and the other at 365.4nm (Fig.23) are due to the absorption of benzoyl and cinnamoyl system respectively proposed flavonol moiety. The IR absorption band at 3380 (broad) cm¯1 is for O-H stretching. The absorption band for C=O of α, β-unsaturated lactone in the IR spectrum observed at 1612 (sharp) cm¯1. The absorption band observed at 1568 (medium) cm¯1 is assigned to olifinic C=C stretching (Fig.24). The 1H NMR spectrum exhibits one doublet at  = 6.12 ppm for aromatic proton (6-H) and other doublet at  = 6.27 ppm for other aromatic proton of ring A (8-H). Aromatic protons of ring B show typical values in the spectrum and exhibit AA’BB’ spin system in close accordance with the structure of flavonol moiety (Fig.25a and Fig.25b). In the 13C NMR spectrum the carbon atom C-3 and C-2 resonate at  = 135.2 and 146.3 ppm respectively. The absorptions found at  = 93.7 and 98.3 ppm are assigned to (C-8). The chemical shift values at  = 115.2 and 129.3 ppm are matched with (C-3’,5’) and (C-2’,6’) respectively. The signals for quaternary carbons of ring are observed at  = 160.4 ppm for (C-5) and 163.7 ppm for (C-7). Similarly in ring B the quaternary carbons (C-1’) absorbs at  = 122.1 ppm and (C-4’) shows  = 156.7 ppm. The typical signal observed in the spectrum for the absorption of (C=O) is shown at  = 175.3 ppm. (Fig.26) The LREIMS mass spectrum exhibits the molecular ion peak [M+] at m⁄z = 286 (100), which along with previous information confirms that actual molecule is 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one. A fragment signal [M+ – CO] was observed at m⁄z = 258 (13) obtained by the loss of CO.(Fig.27) All other signals observed in the mass spectrum are in complete accord to the fragmentation pattern of the constitution of 3,5,7-trihydroxy-2-(4-hydroxyphenyl)- 4H-chromen-4-one. On the basis of these measurements, the assigned

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constitution of 3,5,7-trihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (Kaempherol) is confirmed.

OH Compound 3 3' 4' 2' 5' 1' 6' HO O 8 7 1 9 2 6 10 3 5 4 OH OH O

Chemical Formula: C15H10O6

Molecular Weight: 286.24

Melting Point: 276-278 °C

Physical State: Light yellow crystalline solid

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Figure 23: UV spectrum of compound 3

Figure 24: IR spectrum of compound 3

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Figure 25a: 1H NMR spectrum of compound 3

Figure 25b: Elaborated 1H NMR spectrum of compound 3

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Figure 26: 13C spectrums of compound 3

Figure 27: Mass spectrum of compound 3

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5.5.4. Structure determination of Compound 4 The structure of the isolated compound 4 was established with the help of UV, IR, 1H NMR and 13C NMR spectroscopy including EIMS spectrometry. Two strong bands at 210.0 nm and 324 nm are indication for cinnamic acid and its derivatives (Fig 28). The IR absorption band at 3360 (broad) cm¯1 is for O-H stretching. The absorption band for C=O of α, β-unsaturated carboxylic acid group in the IR spectrum is observed at 1600 (sharp) cm¯1. The absorption of aromatic conjugated C=C bond is observed at 1617(medium) cm¯1 (Fig.29). The 1H NMR spectrum shows one doublet at  = 6.64 ppm for one olefinic proton (2-H) and other doublet at  = 7.88 ppm for (3-H). Both protons showed coupling 16.0 Hz which is trans coupling. While other four aromatic protons showed absorption with ABCD spin system which is in close resemblance with the structure of cinnamicacid (Fig.30a and Fig.30b). In the 13C NMR spectrum the absorptions found at  = 117.6 for (C-2) and  = 141.2 for (C-3). The down field shift of (C-3) is due deshielding of the benzylic position. Signals observed for aromatic carbon are in close accord with the structure of the compound. The highly downfield shift at  = 169.6 assigned to (C=O) indicates for the cinnamic acid (Fig.31). The LREIMS mass spectrum exhibits the molecular ion peak [M+] at m⁄z = 164 (29), which is a base peak indicating the stability of the molecule, along with previous information confirms that actual molecule is cinnamicacid derivative. A + fragment signal [M – CO2H2] was observed at m⁄z = 146 obtained by the loss of

CO2H2.(Fig.32) All other signals observed in the mass spectrum are in complete accord to the fragmentation pattern of the constitution of 2-hydroxy cinnamicacid On the basis of these measurements, the assigned constitution of 2-hydroxy cinnamicacid is confirmed.

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Compound 4

H 1 2 COOH 6' 3 5' 1' H 4' 2' 3' OH

Chemical Formula: C9H8O3

Molecular Weight: 164.16

Melting Point: 211.2 C

Physical State: Off white crystalline powder

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Figure 28: UV spectrum of Compound 4

Figure 29: IR spectrum of compound 4

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Figure 30a: 1H NMR spectrum of compound 4

Figure 30b: Elaborated 1H NMR spectrum of compound 4

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Figure 31: 13C spectrum of compound 4

Figure 32: Mass spectrum of compound 4

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5.5.5. Structure determination of Compound 5

The Structure of he isolated compound 5 was established with the help of UV, IR, 1H NMR and 13C NMR spectroscopy including EIMS spectrometry. The UV spectra provides information that strong band at 268 nm and other at 335 nm are for flavone moiety (Fig. 33). The IR absorption band at 3728 cm¯1 is for O-H stretching. Another significant absorption is 1654 (sharp) cm¯1 for C=O stretching indicating lactone ring. The absorption band observed at 1556 (medium) is assigned to olefinic C=C stretching (Fig.34). The 1H NMR spectrum exhibits one doublet at  = 6.12 ppm for aromatic proton (6-H) and other doublet at  = 6.29 ppm for other aromatic proton of ring A (8-H). The significant absorption band in the spectrum of isolated compound are for olefinic proton (3-H) a singlet is observed as the case at  = 6.38 ppm. Aromatic protons of ring B show typical values in the spectrum and exhibit AA’BB’ spin system close accordance with the structure flavone moiety (Fig.35a and Fig.35b). In the 13C NMR spectrum the carbon atom C-3 resonates at  = 94.1 ppm. The absorptions found at  = 99.0 and 103 ppm is assigned to the carbon atoms (C-4) and (C-8) of ring A. (Fig.36) Other signals observed in the spectrum are in fully accord to the structure of flavonone. The LREIMS mass spectrum exhibits the molecular ion peak [M+] at m⁄z = 270 (100), A fragment signal [M+ – CO] was observed at m⁄z = 242 (100) obtained by the loss of CO (Fig.37) All other signals observed in the mass spectrum are in complete accord to the fragmentation pattern of the constitution of 5,7-dihydroxy- 2-(4-hydroxyphenyl)-4H-chromen-4-one. On the basis of these measurements, the assigned constitution of 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4- one (apiginin) is confirmed that is for the first time reported in roots Conyza bonariensis. 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (apiginin) was reported in the aerial part of the same plant (Kong et al., 2001).

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Compound 5 OH 3' 4' 2' 5' 1' 6' OH O 8 7 1 9 2 6 10 3 5 4

OH O

Chemical Formula: C15H10O5

Molecular Weight: 270.24

Melting Point: 345-350 °C

Physical State: Yellow crystalline solid

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Figure 33: UV spectrum of compound 5

Figure 34: IR spectrum of the compound 5

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Figure 35a: 1H NMR spectrum of the compound 5

Figure 35b: Elaborated 1H NMR spectrum of compound 5

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Figure 36: 13C spectrum of compound 5

Figure 37: Mass spectrum of compound 5

143

RESULTS

144

CHAPTER # 6 DISCUSSION

6. Discussion: The presents work emphasis on biological and pytochemical studies of medicinal plants Euphorbia helioscopia, Euphorbia prostrata and Conyza bonariensis. Dichloromethane and methanol extracts of the said plants were prepared. All the extracts were subjected for antimicrobial activity, antifungal activity, antioxidant activity, brine shrimp lethality bioassay and phytotoxic bioassay Dichloromethane extract of E. hhelioscopia exhibited significant antifungal activity against Fusarium solani with 90 % Inhibition while miconazole used as standard drug in this assay inhibited 98% (Uzair et al., 2009). Fusarium solani is one of the most frequently isolated fungi from soil and plant debris. It is a host-specific pathogen for number of agriculturally important plants, including cucurbits, pea, sweet potato, and and responsible for hyalohyphomycosis in humans (Anaissie et al., 1997). It is also associated with serious invasive mycoses in immunocompromised and immunosuppressed patients (Summerbell et al., 2002). The optimal treatment regimen for patients with disseminated infections has not yet been established, but rapid diagnosis and recovery of the neutrophil count seem to be essential for survival (Pujol et al., 1997). The results suggests that after significant clinical trails the dichloromethane extract of Euphorbia helioscopia may be used a new antifungal agent against Fusarium solani in any suitable dosage form.

The two compound namely 4-Hydroxy-3-methoxycinnamic acid and 7- hydroxy-2H-chromen-2-one (Umbelliferon) isolated from dichloromhthane extract of Euphorbia helioscopia possess different biological activities. Compound 1 is an antioxidant in the sense that it is reactive toward free radicals such as reactive oxygen species. Reactive oxygen species and free radicals are implicated in DNA damage, cancer, accelerated cell aging. Animal studies and in vitro studies suggest that 4-Hydroxy-3- methoxycinnamic acid may have direct antitumor activity against breast cancer (Graf et al.,1992) 4-Hydroxy-3-methoxycinnamic acid may has effect on cancer cells, leading to their destruction (Fry et al.,1986). It is found to be effective in preventing cancer induced by exposure to the carcinogenic compounds benzopyrene and 4-nitroquinoline 1-oxide (Fry et al.,1995). It may

144 CHAPTER # 6 DISCUSSION reduce oxidative stress by formation of thiamine dimers in skin, when added to a topical preparation of ascorbic acid and vitamin E (Bolwell et al., 1993). 4- Hydroxy-3-methoxycinnamic acid may be useful as a precursor in the manufacturing of vanillin. (Ralph et al., 1994) An other isolated compound from the same species is 7-hydroxy coumarin (Umbelliferone) a natural antioxidant of benzopyrone in nature. 7-hydroxy coumarin has a protective effect on membrane fatty acid composition of liver and kidney due to its antioxidant and antihyperlipidemic effects, by improving histopathological changes, hepatic and nephritic markers, and reduces the risk of diabetic complications. (Ramesh et al.,2007). Umbelliferon absorbs short wave UV radiations is employed for protection against Sun (Gunnar et al., 1999). 7-hydroxycoumarin is a strong xanthine oxidase inhibitors.(Chang et al., 1995) Some Mannich bases of 7-hydroxycoumarin and their simple derivatives were reported to be effective against viruses containing single- stranded, positive-sense RNA genomes (ssRNA+) 7-hydroxycoumarin and its derivatives are found to be moderately active in antiviral tests (Mazzei et al.,2008)

Our investigation regarding to biological screening of the plant Euphorbia prostrata we found the methanolic extract has potential for free radical- scavenging properties. An antioxidant component 3,5,7-trihydroxy-2-(4- hydroxyphenyl)-4H-chromen-4-one (Kaempferol) is isolated from the methanol extract of the E. prostrata. Kaempferol is known to suppress growth of a number of human malignancies (Sharma et al., 2007). It also inhibits the ability of cigarette smoke condensate to induce growth of immortalized lung epithelial (BEAS-2B) cells in soft agar (Puppala et al., 2007). As micromolar concentrations of kampferol in the blood protects against brain damage induced by transient focal cerebral ischemia, a new potential therapeutic role of kaempferol to acute treatment of ischemic stroke is suggested (López- Sánchez et al., 2007). Kaempferol offers protection against Ischemia/Reperfusion-Induced cardiac dysfunctions through the regulation of endoplasmic reticulum stress (Kim et al., 2008) 3,5,7-trihydroxy-2-(4- hydroxyphenyl)-4H-chromen-4-one had an inhibitory activity against IL-2 gene expression. It is suggested that it could be a new immunosuppressant (Wang

145 CHAPTER # 6 DISCUSSION et al., 2008). Isolated compound 3 is known to be a natural compound found in edible plants, has promising anti-cancer activity (Yoshida et al., 2008). Aspergillus flavus is the second most important Aspergillus among the species responsible for human infections. Aspergillus flavus causes a broad spectrum of disease in humans, ranging from hypersensitivity reactions to invasive infections associated with angioinvasion (Denning et al., 1998, Morgan et al., 2005). The importance of this fungus increases in regions with a dry and hot climate. Aspergillus flavus isolates produce aflatoxin B1, the most toxic and potent hepatocarcinogenic natural compound ever characterized. Common clinical syndromes associated with Aspergillus. flavus include chronic granulomatous sinusitis, keratitis, cutaneous aspergillosis, wound infections and osteomyelitis following trauma and inoculation (Hedayati et al., 2007). Dichloromethane extract of Conyza bonaiensis aerial and roots parts exhibited sinnificant antifungal activity against Aspergillus flavus and microsporum canis 70% and 60% respectivally. Microsporum canis is a pathogenic fungus that causes a superficial cutaneous infection called dermatophytosis. Dermatophytosis is a general term used to define the infection in hair, skin or nails due to any dermatophyte species. Microsporum canis can cause pseudomycetoma in an immunosuppressed patient (Jena et al., 2007). The results suggests that crud extracts of the aerialparts and root of C. bonariensis having significant activity may employed as antifungal agent in any suitable dosage form after satisfactory clinical trails.

From the methanolic root extract of C. bonariensis an antioxidant compound 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one (Apigenin) is isolated. Apigenin is a naturally occurring, nonmutagenic flavonon, that inhibits UV induced skin tumorigenesis in mice when topically applied (McVean et al 2000). Apigenin may be important in the prevention of carcinogenesis and inflammation (Liang et al., 1999). It is reported that apigenin is a potent regulators which may play roles in prevention of prostate cancer cells (Kobayashi et al 2002) In-vitro superoxide anion radical and peroxyl radical scavenging properties of apigenin indicate that its antioxidant properties contribute to the anti-inflammatory activity (Fuchs et al., 1993). It is to be reported that 5,7-dihydroxy-2-(4-hydroxyphenyl)-4H-chromen-4-one inhibited

146 CHAPTER # 6 DISCUSSION hypoxia-inducible factor-1 and vascular endothelial growth factor expression in human ovarian cancer cells (Jing et al 2007). Apigenin inhibits human pancreatic cancer cell growth in vitro through G2/M phase cell cycle arrest (Michael et al.,2006). Apigenin holds promise as a new therapeutic agent against antiestrogen-resistant breast cancer, it acts as both an estrogen and an antiestrogen in a dose-dependent manner (Xinghua et al 2008).

.

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