Echinops Heterophyllus Family

Republic of Iraq Ministry of Higher Education and Scientific Research University of Baghdad College of Pharmacy

PHYTOCHEMICAL INVESTIGATION AND TESTING THE EFFECT OF IRAQI ECHINOPS HETEROPHYLLUS FAMILY COMPOSITAE ON WOUND HEALING A Thesis Submitted to the Department of Pharmacognosy and Committee of the Graduate Studies of the College of Pharmacy - University of Baghdad in A Partial Fulfillment of the Requirements for the Degree of Doctor of Philosophy in Pharmacy (Pharmacognosy)

By Enas Jawad Kadhim

(M.Sc. Pharmacognosy, 2001) Supervisor: Prof. Dr. Alaa A. Abdulrasool

Co-supervisor: Assist Prof. Dr. Zainab J. Awad

2013 1434

بسى هللا انشحًٍ انشحٍى

﴿ٌَشفع ٱلل ه ٱن زٌ ٍَ َءا َي ُ هٕ ا ي ُ هك ى َٔ ٱن ز ٌ ٍَ أٔح هٕ ا ٱن ع ه َى َد َس َ خ ج َٔ ٱلل ه ب ًَ ا ح َع ًَ ه هٌٕ َخ ب ٍ ش ﴾

طذق هللا انعظٍى

سورة المجادلة : االٌة ۱۱ Certificate

We certify that this thesis entitled (Phytochemical investigation and testing the effect of Iraqi Echinops heterophyllus Family Compositae on wound healing) was prepared under our supervision at the Department of Pharmacognosy, College of Pharmacy- University of Baghdad in a partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacy (Pharmacognosy)

Signature: Supervisor: Prof. Dr. Alaa A. Abdulrasool Date: Department:

Signature: Co-supervisor: Ass. Prof. Dr . Zainab J. Awad Date: Department

In view of the available recommendation, I forward this thesis for debate by the Examining Committee:

Signature: Name: Chairman of the Committee Graduate Studies in the College of Pharmacy Date:

Certificate

We, the Examining Committee after reading this thesis entitled (Phytochemical investigation and testing the effect of Iraqi Echinops heterophyllus Family Compositae on wound healing) and examining the student (Inas Jawad Kadhim ) in its content, found it adequate as a partial fulfillment of the requirements for the degree of Doctor of Philosophy in Pharmacy (Pharmacognosy).

Signature: Signature: Signature: Name: Name: Name: (Member) (Chairman) (Member) Date: Date: Date:

Signature: Signature: Name: Name: (Member) (Member) Date: Date:

Approved for the University Committee for Graduated Studies.

Signature:

Name: Dr. Ahmed A. Hussein

Dean of College of Pharmacy-University of Baghdad

Date:

Dedication

My Parents, Husband, Sisters,Brothers,my sweet daughters "Sarah & Rand" and all my Friends with respect

Enas

ACKNOWLEDGEMENTS

Prays and thanks be to Allah and prayer and peace upon Prophet Mohammed and his Descendants.

I would like to express my greatest thank to my supervisor Prof. Dr. Alaa A. Abdulrasool ( Chairman of Baghdad University) for his scientific guidance, kindness, and prompt help whenever needed.

My great thanks go to my co-supervisor Ass. Prof. Dr . Zainab J. Awad, for her encouragement, endless support during the study

My appreciation to Ass. Prof. Dr. Ahmed A. Hussein (Dean of College of Pharmacy- University of Baghdad) for his continuous support and facilities to the postgraduate students.

I would like to express my gratitude and appreciation to Lecturer Maha Noori Hamad (Head of Pharmacognosy Department, College of Pharmacy, University of Baghdad) for her cooperation, continuous guidance, patience, and help in providing me the research materials and all possible facilities.

Also, I wish to express my deepest grateful to Ass. Prof Dr. Abed AL-Jabbar Khalaf, (College of Science- Al-Mustansiriya University) and the doctors: Ass. Prof Mohammed Hasan, Lecturer Ahlam Qasear Lecturer Ammar Abed-Al-Razaq, , (College of Pharmacy-Baghdad University), Dr. Munther Faisal (Dean of College of Pharmacy Al-Mustansiriya University) for their help in explaining the NMR analysis.

I am also thankful to Asst. Lecturer Zena Qaragholi for her assistance throughout the work.

I would like to express my sincere appreciation to Prof. Ali Al-Shammaa for his advice, supervision throughout this work

I can never thank enough the staff members in the Department of Pharmacognosy College of Pharmacy-Baghdad University for their continuous praying, love, and encouragement. Special thanks are extended to my friends, Dr. Nada-Al-Shawi , Dr. Dair Arif and Dr. Nawal Ayash for their valuable advice , support and helping during my study.

I wish to express my gratefulness to Mrs. Zaineb, Mr. Ali and Mr. Salah Mahdy Baker, in College of Science in Al- Mustansiriya University for their great help in running FTIR, CHN and GC-MS spectrometer.

Special thanks to lecturer Salema Sultan (College of Pharmacy-Baghdad University) for her help in computer application.

My greatfulness to lecturer Suaad H. Moslem and Miss.Muna K. Shaker (College of Pharmacy-Baghdad University) for their help in the library.

I'm greatly indebted to my family( specially my father , mother and my husband Dr. Yasser Al-Shammaa ) for their patience, encouragement and care.

Thanks are also due to all those whom I forgot and the wholeness is only for Allah.

Enas

List of Contents

No Subject Page

Acknowledgements II

List of contents IV

List of figures VIII

List of tables XIII

List of abbreviations XV

Abstract XVI

CHAPTER ONE : INTRODUCTION

Introduction 1

1.1 Asteraceae or Compositae (Aster Family) 2

1.2 The genus Echinops Linn 4

1.3 Echinops heterophyllus P.H.Davis 6

1.3.1 Classification 6

1.3.2 Description of the plant 7

1.3.3 Distribution of the plant 7

1.3.4 The Folkloric uses 9

11 Pharmacological activities of different species of 1.4 Echinops extracts

Antibacterial activity 11 1.4.1

1.4.2 Antifungal activity: 12

1.4.3 Antileishmanial activity 14

1.4.4 Antioxidant activity 15

1.4.5 Anticancer action 15

1.4.6 Protective effects of Echinops on testosterone- 16 induced prostatic hyperplasia

1.4.7 Hepato-protective activity of Echinops 17

1.4.8 Anti-ulcerogenic activity 17

1.4.9 Anti-inflammatory action 18

1.4.10 Diuretic action of Echinops 18

1.4.11 Analgesic activity of Echinops 19

1.4.12 Effects on C.N.S 19

1.5 Phytochemical constituents of Echinops 20

1.5.1 Alkaloids of Echinops species 20

1.5.1.1 Echinopsine 23

1.5.1.2 Echinopsidine 24

1.5.1.3 Echinorine 24

1.5.1.4 Echinozolinone 25

1.5.2 Flavonoids 26

1.5.2.1 Reported pharmacological activities of flavonoids 28

1.5.2.1.1 Flavonoids as antioxidants 28

1.5.2.1.2 Flavonoids in the treatment of gastric ulcer 28

1.5.2.1.3 Effect of flavonoids on inflammation 29

1.5.2.1.4 Effect of flavonoids on cancer-related pathways 30

1.5.2.1.5 Antimicrobial activity of flavonoids 30

1.5.2.1.6 Flavonoids in treatment of cardiovascular diseases 32

1.5.2.1.7 Flavonoids in treatment of diabetes mellitus 32

1.5.2.1.8 Role of flavonoids in treatment of hepato-toxicity 33

1.5.2.1.9 Effect of flavonoids on depression 33

1.5.3 Terpenoids 34

1.5.3.1 Beta-sitosterol 35

1.5.3.2 Stigmasterol 36

Aim of this study 38

CHAPTER TWO : EXPERIMENTAL WORK

2.1 Reagents and Materials 39

2.2 Instruments 40

2.3 Plant material 41

2.4 Experimental work 41

2.4.1 Preliminary qualitative phytochemical analysis 42

2.4.2 Extraction and fractionation of different active 44 constituents

2.4.3 Isolation and purification of different active 48 constituents

2.4.3.1 Preparative HPLC 48

2.4.3.2 Preparation of preparative TLC plates 50

2.4.3.3 Isolation of flavonoids glycosides by (CC) 51

2.4.4 Identification and characterization of the isolated 52 compounds

2.4.4.1 Thin layer chromatography (TLC) 52

2.4.4.2 Melting point(M.P.) 52

2.4.4.3 Ultra violet (UV) spectrum analysis 52

2.4.4.4 Fourier transforms infrared(FT-IR) spectra 52

2.4.4.5 Elemental microanalysis (CHN) 53

2.4.4.6 One proton and thirteen carbon nuclear magnetic 53 resonance spectroscopy1H and 13C (NMR) analysis

2.4.4.7 Qualitative and quantitative estimation of isolated 53 compounds by HPLC

2.4.5 Investigation of some pharmacological activity of 54 the different isolated fractions

CHAPTER THREE : RESULTS and DISCUSSION

3.1 Preliminary qualitative phytochemical analysis 57

3.2 Extraction and fractionation of different active 58 constituents

3.3 Preliminary identification of different Echinops parts 60 by TLC

3.4 Isolation and purification of different active 78 constituents

3.4.1 Isolation and purification of alkaloids 78

3.4.1a Isolation and purification of alkaloids by preparative 78 HPLC

3.4.1b Isolation and purification of alkaloids by preparative 80 TLC

3.4.1.2 Characterization and identification of the isolated 82 alkaloids (E1, E2 and E3)

3.4.1.2.1 M. P. 82

3.4.1.2.2 U.V. spectra 82

3.4.1.2.3 3.4.1.2.3- FT.IR spectra 84

3.4.1.2.4 CHN 88

3.4.1.2.5 1H &13C NMR analysis 88

3.4.2.1 Isolation and purification of flavonoids glycoside by 93 column chromatography (CC)

3.4.2.2 Characterization and identification of the isolated 95 flavonoids glycoside (EJ1 and EJ2)

3.4.2.2.1 M. P. 95

3.4.2.2.2 U.V. spectra 95

3.4.2.2.3 FT.IR spectra 96

3.4.2.2.4 CHN 100

3.4.2.2.5 1H &13C NMR analysis 100

3.4.3.1 Isolation and purification of flavonoids as (aglycon) 109 by preparative TLC

3.4.3.2 Characterization and identification of the isolated 110 myricetin, quercetin and kaempferol

3.4.3.2.1 TLC 110

3.4.3.2.2 M. P. 110

3.4.3.2.3 U.V. spectra 112

3.4.3.2.4 FT- IR 113

3.4.3.2.5 HPLC analysis 118 3.5 A relative assess on wound healing activity of crude 124 LIST Echinops extract and some of its bioactive fractions OF 3.5.1 Visual remarks 124 FIGU 3.5.2 Histology 129 RES

Conclusions & Recommendation 137

References 139 NO. Figure Page

1.1 Iraqi Echinops heterophyllus 8

1.2 Basic structure of quinoline nucleus 22

1.3 Chemical structure of echinopsine 23

1.4 Chemical structure of Echinopsidine 24

1.5 Chemical structure of echinorine 25

1.6 Chemical structure of Echinozolinone 25

1.7 Chemical structure of (a) and (b) 26

1.8 Basic structure of flavonoids 26

1.9 Chemical structure of different types of flavonoids 27

1.10 Numbering of atoms in flavonoids aglycone at which 27 substitution may occur

1.11 Formation of peroxy radical 28

1.12 Chemical structures of some flavonoids 30

1.13 Chemical structure of β-sitosterol . 35

1.14 Chemical structure of stigmasterol 36

2.1 General scheme for separation of different plant constituents 46

2.2 Preparative HPLC apparatus used in the separation of 49 alkaloids 2.3 (1x2 cm²) Wound incision at the back region 54

2.4 The application of the crude plant extract 74

2.5 The application of the bioactive fraction alkaloids 56

2.6 The application of the bioactive fraction flavonoids 56

3.1 TLC of fraction one (F-1) for different Echinops parts 61

( roots, seeds, aerial parts) using silica gel GF254nm as

adsorbent and S1a as a mobile phase.

3.2 TLC of fraction one (F-1) for different Echinops parts 62

( roots, seeds, aerial parts) using silica gel GF254nm as

adsorbent and S2a as a mobile phase.

3.3 TLC of fraction one (F-1) for different Echinops parts 62

( roots, seeds, aerial parts) using silica gel GF254nm as

adsorbent and S3a as a mobile phase.

3.4 TLC of fraction two (F-2) for different Echinops parts 65

( seeds, aerial parts, roots) using silica gel GF254nm as

adsorbent and S1f as a mobile phase.

3.5 TLC of fraction two (F-2) for different Echinops parts 66

( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S2f as a mobile phase. 3.6 TLC of fraction two (F-2) for different Echinops parts 67

( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S3f as a mobile phase. 3.7 TLC of fraction three (F-3) for different Echinops parts 70

( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S4f as a mobile phase.

3.8a TLC of fraction three (F-3) for different Echinops parts ( 71 seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S5f as a mobile phase. Detection by UV-light at 254nm 3.8b TLC of fraction three (F-3) for different Echinops parts ( 72 seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S5f as a mobile phase. Detection by UV-light at 366nm 3.9 TLC of fraction three (F-3) for different Echinops parts ( 73

seeds, aerial parts, roots) using silica gel GF254nm as

adsorbent and S6f as a mobile phase.

3.10 TLC of fraction four (F-4) for different Echinops parts (aerial 75

parts, roots) using silica gel GF254nm as adsorbent and S1s as a mobile phase.

3.11 TLC of fraction four (F-4) for different Echinops parts (aerial 76

parts, roots) using silica gel GF254nm as adsorbent and S2s as a mobile phase..

3.12 TLC of fraction four (F-4) for different Echinops parts (aerial 77

parts, roots) using silica gel GF254nm as adsorbent and S3s as a mobile phase..

3.13 Preparative HPLC analysis of fraction-1 obtained from seeds 79 plant

3.14 Co-TLC of three alkaloids (E1, E2, E3) isolated by 80 preparative HPLC .

3.15 Chromatogram of preparative TLC for fraction one (F-1) , 81

using silica gel GF254 as adsorbent and S1a as a mobile phase

3.16 Co-TLC of three bands (E1, E2, E3) isolated by preparative 81 TLC from fraction-1 (F-1) of seeds part using silica gel

GF254nm as adsorbent and S1a as a mobile phase.

3.17 UV spectrum of the isolated alkaloids ( E1, E2, E3) 83

3.18 FT-IR spectrum of the isolated alkaloid (E1) 85

3.19 FT-IR spectrum of the isolated alkaloid (E2) 86

3.20 FT-IR spectrum of the isolated alkaloid (E3) 87

3.21 13C-NMR analysis of the isolated E2 compound 89

3.22 1H-NMR analysis of the isolated E2 compound 90

3.23 13C-NMR analysis of the isolated E3 compound 92

3.24 1H-NMR analysis of the isolated E3 compound 92

3.25 TLC of fraction- B and C obtained from CC using silica gel 95

GF254nm as adsorbent and S2f as a mobile phase

3.26 UV spectrum of the two flavonoids glycoside EJ1 and EJ2 96

3.27 FT-IR spectrum of the isolated EJ1 98

3.28 FT-IR spectrum of the isolated EJ2 99

3.29a 13C-NMR analysis of the isolated EJ1 compound 102

3.29b Expansion of 13C-NMR analysis of the isolated EJ1 103 compound

3.30a 1H-NMR analysis of the isolated EJ1 compound 104

3.30b Expansion of 1H-NMR analysis of the isolated EJ1 compound 105

3.31 13 C-NMR analysis of the isolated EJ2 compound 107

3.32 1H-NMR analysis of the isolated EJ2 compound 108

3.33 Chromatogram of preparative TLC for fraction-3 , using 109

silica gel GF254 as adsorbent and S4f as a mobile phase

3.34 TLC chromatogram of qualitative analysis of isolated 111

myricetin, using silica gel GF254 as adsorbent and S4f as a

mobile phase

3.35 TLC chromatogram of qualitative analysis of isolated 111

quercetin, using silica gel GF254 as adsorbent and S4f as a mobile phase.

3.36 TLC chromatogram of qualitative analysis of isolated 112

kaempferol, using silica gel GF254 as adsorbent and S4f as a mobile phase.

3.37 UV spectrum of the isolated flavonoids (myricetin, 113 quercetin, kaempferol)

3.38 FT-IR spectrum of the isolated myricetin 115

3.39 FT-IR spectrum of the isolated quercetin 116

3.40 FT-IR spectrum of the isolated kaempferol 117

3.41 HPLC of aerial parts 119

3.42 HPLC of roots parts 120

3.43 HPLC of seeds parts 120

3.44 HPLC of myricetin standard 121

3.45 HPLC of isolated myricetin 121

3.46 HPLC of quercetin standard 122

3.47 HPLC of isolated quercetin 122

3.48 HPLC of kaempferol standard 123

3.49 HPLC of isolated kaempferol 123

3.50 Visual remarks of group-1 day-1. 124

3.51 Visual remarks of group-1 day-6. 124

3.52 Visual remarks of group-1 day-12. 125

3.53 Visual remarks of group-2 day-1. 125

3.54 Visual remarks of group-2 day-6. 125

3.55 Visual remarks of group-2 day-12. 126

3.56 Visual remarks of group-3 day-1. 127

3.57 Visual remarks of group-3 day-6. 127

3.58 Visual remarks of group-3 day-12. 128

3.59 Visual remarks of group-4 day-1. 128

3.60 Visual remarks of group-4 day-6. 128

3.61 Visual remarks of group-4 day-12. 129

3.62 Histology of group one day one 130

3.63 Histology of group one day six 130

3.64 Histology of group one day twelve 131

3.65 Histology of group two day one 131

3.66 Histology of group two day six 132

3.67 Histology of group two day twelve 132

3.68 Histology of group three day one 133

3.69 Histology of group three day six 133

3.70 Histology of group three day twelve 134

3.71 Histology of group four day one 134

3.72 Histology of group four day six 135

3.73 Histology of group four day twelve 146

LIST OF TABLES

NO. Table Page

1.1 Antibacterial, antimicrobial and antiviral activities of 31 flavonoids

1.2 Flavonoids isolated from different species of Echinops . 34

1.3 Terpenoids isolated from different species of Echinops 37

2.1 Reagents and materials used in the study 39

2.2 Instruments used in the study 40

3.1 Phytochemical screening of different parts of 57

Echinops heterophyllus

3.2 Percentage of different fractions obtained from different plant 59 parts (seeds, aerial parts, roots)

3.3 Rf values of alkaloids obtained from different plant parts in 60 different developing solvent systems in TLC

3.4 Rf values of flavonoids (as glycoside) obtained from different 64 plant parts in different developing solvent systems in TLC

3.5 Rf values of flavonoids (quercetin, myricetin and kaempferol 69 ) obtained from different plant parts and their standard in different developing solvent systems in TLC. 3.6 Rf values of steroids (stigmasterol and β-sitosterol ) obtained 75 from different plant parts and their standards in different developing solvent systems in TLC 3.7 Characteristic FT-IR absorption bands( in cm-1) of the 84 isolated alkaloids 3.8 Elemental microanalysis of the unknown isolated alkaloids 88

3.9 Major fractions obtained from column chromatography 94

3.10 Characteristic FT-IR absorption band (cm-1) of the isolated 96 EJ1 &EJ2

3.11 Elemental microanalysis of the unknown isolated flavonoids 100 glycoside

3.12 Characteristic FT-IR absorption band (cm-1) of the isolated 114 flavonoids

3.13 Percentage of flavonoids in the different plant 118 parts.

LIST OF ABBREVIATIONS

Abbreviation Meaning

13C NMR 13 Carbon nuclear magnetic resonance cm Centimeters

C Degree Centigradeه

CC Column chromatography

FT-IR Fourier transforms infrared spectra

GC-MS Gas chromatography –mass spectroscopy

HPLC High Performance Liquid Chromatography

1H NMR 1Proton nuclear magnetic resonance m/z Mass-to-charge- ratio

M.P. Melting point ml Milliliters

MIC Minimum inhibitory concentration

26 min Minutes nm Nanometer

Rf value Retention factor (mobility relative to solvent front) silica gel Silica gel with gypsum & fluorescence material

GF254nm

TLC Thin layer chromatography

U.V Ultra violet spectra

Abstract

Echinops heterophyllus ( Arabic name: Chouk el djamal, local name: Shakroka), of the family Compositae, is an indigenous wild plant, widely distributed in the North of Iraq, used by public people to treat wounds injury , burns and against snake bite. Literature survey have revealed that no previous phytochemical investigation work had been done on this species, so it was deemed desirable to carry out a research on this plant , and if possible to be a good source for economical value. This study concerned with extraction, fractionation, isolation, purification and identification of some biologically important compounds that belong to different chemical classes (alkaloids, flavonoids, sterols) from different plant parts (seeds, aerial parts, roots).

Preliminary qualitative phytochemical screening of various secondary metabolites by a specific chemical tests was carried out on the ethanolic extract of the different plant parts, and the results indicated that all plant parts contained alkaloids, flavonoids, and terpenoids in different percentage in addition to the presence of sterols compounds in the aerial parts and roots only. General procedure for extracting different plant parts and fractionating into different classes was done using 80% ethanol in soxhlet apparatus. Different fractions were obtained :

Fraction-1 which contained alkaloids.

Fraction-2 which contained flavonoids in the glycosidic linkage.

Fraction-3 which contained flavonoids as a free aglycon.

Fraction-4 which contained steroidal compounds.

Thin layer chromatography of fraction-1 using three different mobile phases demonstrated the occurrence of three alkaloids in the seeds named (E1, E2, E3), and two alkaloids in the roots (E1, E2) with very traces amount of E1 in the aerial parts. These three alkaloids were isolated in a pure form from seeds using two chromatographic methods preparative thin layer chromatography (PTLC), and preparative performance pressure liquid chromatography (PHPLC) which was used to isolate in a very pure form these components. Chemical structure of E2 and E3 was confirmed using different physio-chemical spectral analysis: melting point (M.P.), ultra violet (UV) spectrum analysis, fourier transforms infrared spectra(FT-IR), elemental microanalysis (CHNO),1H nuclear magnetic resonance spectroscopy(1H-NMR) analysis and (13C-NMR) and identified as 1-methyl-2,3- dihydro-4(1H)-quinolinone (E2) and 3-methyl-4-amino-quinoline (E3). Unsuccessful attempt was tried to identify the exact structure of first alkaloid (E1) although all the previous spectral analysis was done. Mass spectroscopy and two- dimensional NMR analysis are required for structure elucidation of E1 compound, therefore, it is left for further study.

Thin layer chromatography of fraction-2 using three different mobile phases revealed the presence of two flavonoids glycoside named (EJ1 and EJ2) in the aerial and root parts with one flavonoids glycoside (EJ1) in the seeds. These two compounds were isolated in a pure form from aerial parts using column chromatography packed with polyamide-6 adsorbent substance and identified as Kaempferol-3-O-rhamnoside(EJ1) and Rutin (EJ2) depending on data obtained from M.P, UV, FT-IR, CHN, 1H-NMR and 13C-NMR.

Three flavonoids (myricetin, kaempferol , and quercetin,) were detected in TLC of fraction-3 obtained from aerial parts and roots and the two former

29 flavonoids were detected in the same fraction of seeds part using three different solvent systems. The isolated myricetin, quercetin and kaempferol obtained from preparative TLC for aerial parts were identified by HPLC method by comparison of retention times obtained at slandered chromatographic conditions of analyzed samples and authentic standards. Thin layer chromatography (TLC) of fraction-4 detected the presence of steroids in the aerial and roots part but didn’t give a clear idea about identity of these components so it is left for further study.

A relative asses was conducted to study the effect of the crude extract of local Iraqi Echinops heterophyllus plant and the some of its bioactive fractions (F-1and 3) on wound healing process and the possible anti-scar property in vivo. Twenty four adult male rabbits were used. Aged between six to 12 months. The effect on wound healing and the anti-scar activity was evaluated visually and through histopatholigical changes. Treatment was applied three times daily in a concentration of 50% using a cotton swab. The results showed that Echinops extract served to accelerate the wound healing process and specifically increased epithelialization in the treated compared to the untreated group. Alkaloidal fraction was more effective than the flavonoids fraction in treating wounds. Both groups treated with either the crude extract or alkaloids showed no scar formation, so it can be concluded that this study is a good step to show that Echinops crude extract is effective in stimulating the enclosure of wounds and has an anti-scar effect; also these results may shed a light on the scientific basis for traditional uses of the genus Echinops in the treatment of wounds .

1.INTRODUCTION

Man ever since his first appearance on earth, has used plant throughout his historical development as a source of medicines. Medicinal plants have formed the basis of the folkloric medicine which was the main source for new medicines discoveries(1). By the middle of the nineteenth century at least 80% of all medicines were derived from plants. Then, after the scientific revolution which leads to development of the pharmaceutical industry, the synthetic drugs dominated(2), but even; herbal drugs are prescribed widely because of their effectiveness, fewer side effects and are relatively low in cost(3).

Natural products of folk medicine have been used for centuries in every culture throughout the world. Scientists and medical professionals have shown increased interest in this field as they recognized the true health benefits of these remedies. While searching for food, the ancient found that some foods had specific properties of relieving or eliminating certain diseases and maintaining good health. It was the beginning of herbal medicine. For thousands of years, cultures around the world have used herbs and plants to treat illness and maintain health. Many drugs prescribed today in modern medicinal system are derived from plants (4).

Herbs and plants are valuable not only for their active ingredients but also for their minerals, vitamins, volatile oils, glycosides, alkaloids, acids, alcohols, esters etc., Complementary and alternative medicine (CAM) can be defined as any treatment used in conjugation (complementary) or in place of (alternative) standard medical treatment. In alternative medicine, medicinal plant preparations have found widespread use particularly in the case of diseases not amenable to treatment by modern method (5).

Iraq was known as the valley of the two rivers “Mesopotamia”, occupies an excellent geographic position where it encompasses mountainous areas in the north, the temperature of which drops below zero; a desert areas around the middle and south part of the country of a very high temperatures and pelagic humidity impregnated areas. All those factors gave Iraq a peculiar geographic position led to the creation of different environments that helped considerably the diversification of its flora.

Prof. H.L.Chakravarty mentioned in his book “ Plant Wealth of Iraq” that there are more than three thousands species of plants in Iraq. He mentioned also that about 1500 species are of economical value. Those economic plants were classified as : plants needed for basic food; others of medicine and drug industry needs, which are called as “medicinal plants”. In addition, there are a large number of plants that are considered as raw materials for numerous transformative industries (6).

Quite a large number of medicinal and poisonous plants occur in Iraq, which are mostly used for home remedies. Investigation and study of the active constituents of these plants might bring a good revenue for the drug industries; analysis of some of wild drugs gave very satisfactory results(6). Of these wildly grown and widely distributed plant species, Iraqi Echinops heterophyllus P.H.Davis Family: Compositae which was chosen for this study.

1.1-Asteraceae or Compositae (Aster Family)

The family Asteraceae or, alternatively, family Compositae, known as aster, daisy, or sun flower family, is a taxon of dicotyledonous flowering plants. The family name is derived from the genus Aster and referred to the star-shaped flower head of its members, typified well by the daisy. The Asteraceae is the second

largest family in the division Magnoliophyta, with some 1,100 genera and over 20,000 recognized species. Only the Orchidaceae is larger than Asteraceae, with about 25,000 described species. Plants belonging to the Asteraceae share all the following characteristics:  Inflorescence: a capitulum or flower head.  Syngenesious anthers, i.e. with the stamens fused together at their edges by the anthers, forming a tube.  Ovary with basal arrangement of the ovules (7,8). The Asteraceae are cosmopolitan in distribution, but partial to open or semi-open habitats rather than deep woods. In most parts of the temperate zone, including Iraqi region, they are by far the largest family. Many genera and species are cultivated for ornament. The family is one of the easiest groups to recognize, but many of the genera are poorly defined or confluent. The flower heads vary from small to large, and are often brilliantly colored. The number of flowers in a head is seldom less than 5, and ranges upward into the hundreds or even more than a thousand, as in the common cultivated sunflower. A few species have only a single flower in each head. Echinops and some other genera have one-flowered, individually involucrate heads aggregated into a secondary head with a secondary involucre. Compound heads with more than one flower in each individual head also occur in some genera, such as Elephantopus (9).

The composite nature of the inflorescence of these plants led early taxonomist to call this family the Compositae. This family has a remarkable ecological and economical importance, and is present from the polar regions to the tropics, colonizing all available habitats. Asteraceae may represent as much as 10% of autochthon flora in many regions of the world. Most members of Asteraceae are

herbaceous, but a significant number are also shrubs, vines and trees. The family has a worldwide distribution, and is most common in the arid and semi-arid regions of subtropical and lower temperate latitudes (10,11).

1.2- The genus Echinops Linn.

Echinops a genus includes many plants which are individually referred to as globe thistle, is made up of more than 120 species of perennials, annuals, and biennials(12,13). The genus belongs to the daisy family Asteraceae, and its species are found in Eastern and Southern Europe, Tropical and North Africa and Asia(14). These plants are hardy and are often considered to be highly ornate. This genus receives its common name from its globe-like flower that grow in shades of purple and white. The leaves of these plants are spiky from the edges and woolly and greenish grey in color, while the fruits borne are cylindric achene. The blossoms of these plants are round flower heads that grow in groups. These flower heads are on top of the ribbed stems of the plant, making the total height of the plant nearly 5 feet (1.5 m). The plants attract swarms of bees and butterflies and are usually planted behind the borders in gardens. These plants are often utilized as cut flowers, as they can last for weeks when placed in vases indoors. They are also used as dried floral arrangements, and for its ornamental uses (15).

Many globe thistle plants are very popular. One such popular species is Echinops sphaerocephalus, known by the common name Great globe thistle or Pale globe-thistle, the genus name derives from the Greek words "ekhinos"

meaning "hedgehog" and "opisis" meaning "aspect", with reference to the appearance of the inflorescence, while the species name sphaerocephalus derives from the words "sphaera" meaning "round" and "kephalos" meaning head, also known as arctic glow, which is much taller than other species in the genus, usually reaching up to 7 feet (2.1 m) in height. This species is native to Eurasia but it lives on other continents where it was introduced, including North America where it is a widespread weed. It is very common in the mountains of southern France and southern and central Europe (16).

Another popular species is Echinops ritro L., or the taplow blue, which has bright blue flowers and is often used as a border plant because it is 3 feet (1 m) high. It is native to Europe and western Asia(17). Other prominent species that are referred to as Indian globe thistle Echinops echinatus (Roxb.) native to India, Afghanistan, Pakistan and Myanmar (18).

Echinops galalensis and Echinops hussoni are found practically throughout Saudi Arabia(19). Echinops spinosus Turra. very common in the Algerian Sahara and Egypt (20). In Turkey, the genus comprises 19 species, 2 subspecies and 3 varieties among them : Echinops ritrodes Bunge, E. gaillardotii Boiss., E. adenocaulos Boiss., E. chardinii Boiss. & Buhse, and E. tenuisectus Rech. (21-24). In Iran , 14 species of Echinops were identified in 75 habitats in different parts of Fars province in growth season during 2001-2003 among them: E. endotrichus, E. dichrous, E. tenuisectus and E. persepolitanus (25). In Iraq Ali Al-Rawi was mentioned 11 species of Echinops in his book (26):

E. armatus Boiss. distribution in Erbil. E. bicolor Nab. distribution in Rawanduz.

E. blancheanus Boiss. distribution in Southern desert district, Kirkuk district, Persian foothill district, Central alluvial district and Rawanduz district. E. cephalotes DC distribution in Mirjani. E. descendens Hand,-Mzt distribution in Jazirah (HZ). E. heterophyllus P.H. Davis distribution in Rawanduz. E. horridus Desf. distribution in Amadia district. E. inermis Boiss distribution in Sulaimaniya district. E. rectangularis Rech. F. distribution in Baghdad University Herbarium. E. tournefortii Ledeb distribution in Rawanduz. E. viscosus DC. distribution in Sulaimaniya district, Amadia district and Persian foothill district. Nature Iraq recorded five new species in Kurdistan Iraq for the first time : E. cyanocephalus (in Barzan), E. beteromorpbus (in Chamirazan 30km west of Sulaimani and in Barzan north of Erbil), E. haussknechtii, E. adenocaulos and E. phaeocephalus (27). 1.3- Echinops heterophyllus P.H.Davis 1.3.1- Classification(28) Kingdom: Plantae

Division: Spermatophyta

Subdivision: Angiospermae

Class: Dicotyledones

Order: Asterales

Family: Compositae/ Asteraceae

Genus: Echinops

Species: heterophyllus

Botanical Name: Echinops heterophyllus P.H.Davis

English name: Globe thistle

French : Chardon à fleurs globuleuses

Arabic name: Teskra, chouk el hmir, chouk el djamal, sorr

In Hanara village and surrounding area in Wadi Bastora and Shaklawa in Erbil governorate, the plant is called (Shakroka). The term (Shakroka) is come from the circle-like part of the plant, before getting harder in the late spring, is eaten and the taste is sweet, therefore, it is called (Shakroka):

Shakr means sugar , Shakroka------sweet like sugar.(oral communication).

1.3.2- Description of the plant

Echinops heterophyllus (figure1.1)is a perennial, 40-100 cm high. Stems are simple or branching from the base, sparsely cobwebby-canescent. Leaves are lanceolate or oblong-lanceolate, the lower ones are 10-15 cm long, 4-6cm wide, with triangular-lanceolate, prickly lobes, greenish, shiny, subglabrous above, densely whitish-tomentose below; stem–leaves are gradually smaller, subpinnatisect, prickly and the uppermost ones are narrow liner – lanceolate, diminute. Heads are 5-7 cm in diameter and penicil is about 1/3 as long as the involucres the bristles scabrous. Involucral bracts 12-14, the outer bracts as long as the penicil, narrow spathulate – deltoid, the intermediate ones subulate- attenuate, up to 2.5-3.5 cm long, produced into a long slender prickly horn, twice to twice and a half times as long as the outer ones, the innermost ones are about equal length,

acute, fimbriate, connate to the middle. Pales of pappus barbellate, connate at base into a contiguous corona (29).

1.3.3- Distribution of the plant

E. heterophyllus is endemic in Iraq: Erbil, Kirkuk and Rawanduz (29). Until 2012 this species was endemic in Iraq only, but a certain article indicates the presence of heterophyllus species in Turkey too, specific in the meeting boundary of Iraqi- Turkey- Iranian frontiers (30).

Figure (1.1) - Iraqi Echinops heterophyllus

1.3.4- The Folkloric uses:

The plant increases the appetite and stimulates liver; used in India in diseases of the brain, pains in the joints, inflammations. Roots and root bark of the plant are used in various indigenous systems of medicine for treating different ailments. The root is used as abortifacient and aphrodisiac (31), infusion of the root is given in seminal debility, impotence, hysteria, and its decoction is given in dyspepsia, scrofula, syphilis and fevers (32). Also the whole plant is used against skin itching by boiling 2kg of the whole plant with 12-15 liters of water and bath with that water twice a day for 3-4 days (33). In Egypt, the plant is taken to cure diseases related to the circulatory system (a haemostat, a vasodilator for hypertension, varices, varicocele). The tender part of the flowers is eaten like an artichoke. The plant used to be taken for tinder. It is much appreciated pasture for dromedaries and goats. The stems, leaves and roots are also considered abortive, diuretic and depurative and are taken for liver disease, dysmenorrhoea, metrorrhagia and prostatic problems (34,35). In Morocco, it is mainly used to ease childbirth. A decoction of the roots in either water or olive oil is given to help the woman evacuate the placenta. It is also given before the birth to stimulate contractions. In Marrakech and Salé, a decoction of the roots is used for stomach pain, indigestion and lack of appetite as well as diabetes. In Casablanca, the entire plant, in a powder or decoction, is used as a diuretic or depurative and to cure liver diseases. Everywhere in Morocco, the plant is used as an abortifacient. The aerial part of the plant is edible and sold in small bundles in traditional markets (36). In Saudi Arabia, whole herb of Shook Algamal E. spinosissimus Turr is used in Splenic diseases and sore throat (37).

In Ethiopia, root powder of Kebercho (E. kebericho Mesfin ) is sprinkled on burning charcoal and smoke is inhaled for evil eye (38).

Reports and ethnobotanical surveys also evidence long traditional use of this plant for preparation of medicines against migraine, mental illness, heart pain, lung tuberculosis TB, leprosy, kidney disease, malaria, billharzia, syphilis and amoebic dysentery (39).

Extracts and essential oils of the roots of E. kebericho were also assessed for their antimicrobial, antihelmintic and molluscicidal activities (40). The history of Echinops displays that it has been used since 6000 BC in Iran. It’s used for anti-tussive, laxative effect and anti-fever (41,42). The use of plants against the effect of snake bite has long been recognized. In Brazil , public people used root of E.amplexicaulis (as paste) against snake bite (43), also this is the main traditional uses of E. heterophyllus in Iraq, but public people in Iraq used seeds and aerial parts of the plant only. (oral communication)

In Pakistan E. graffithianus Boiss., it is considered as spiny weed. Stem and leaves are diuretic and aphrodiasic(44). In India all parts of Astrakhar (E. echinatus Roxb.) is appetizing, carminative, diuretic and used in liver diseases and sexual impotency. Powder of root bark is used along with honey for asthma and cough. Juice of flowers is poured in eyes(45). Fresh root decoction of E. echinatus is taken twice a day till cured to relieve scanty urination , strangury and urinary discharges (46-47). In China, Japan and Korea, the root of E. setifer lljin. is anthelmintic, it has a weak antitumor action, it is used as an emmenagogue (48,49).

Although many literatures had been published about Echinops ,but it was no one about heterophyllus species, so the following literature survey revealed pharmacological activities of different species of Echinops plant:

1.4-Pharmacological activities of different species of Echinops extracts:

1.4.1- Antibacterial activity :

The antibacterial activity of the plant extract of E. spinosissimus were evaluated in vitro against both gram-positive and Gram negative bacteria known to cause infectious diseases in humans. Methanolic extract of the plant showed antibacterial activity at 6mg/ml against Staphylococus aureus , Bacillus cereus, Escherichia coli and Klebsiella oxytoca, and antibacterial activity against Klebsiella pneumonia at 23mg/ml(50).

Phytochemicals are routinely classified as antimicrobial on the basis of susceptibility tests that produce minimum inhibitory concentration (MIC) in the range of 100 to 1000 μg/ml(51). Activity is considered to be significant if MIC values are below 100 μg/ml for crude extract and moderate when 100 < MIC < 625μg/ml. Therefore, the recorded activity of the methanolic extract for Cameroonian species of E. giganteu on Enterobacte aerogenes and Klebsiella pneumonia can be considered significant (52).

Essential oils extract of the roots of Ethiopian Echinops (E. kebericho) were screened for antibacterial activity against Staphylococcus aureus, Bacillus cereus, Streptococcus faecalis, Pseudomonas aeruginosa, Salmonella paratyphi, Citrobacter spp., Shigella dysenteriae, Klebsiella pneumonia and Escherichia coli. 42

The antibacterial investigation showed that essential oils extract have antibacterial activity against the tested strains at test concentrations ranging from 0.2 to 25 μl/ml. The observed activities were significantly higher against gram-positive bacteria (with mean MICs 0.25 to 1.6μl/ml) than gram-negative bacteria (with average MICs > 7.63μl/ml)(53).

Also 80% methanolic extracts of the leaf of E. ellenbeckii and the leaf and stem of E. longisetus inhibited the growth of Staphylococcus aureus in a dose

dependent manner.

Antimicrobial activities of the ethyl acetate, acetone, methanol and ethanol extracts of E. viscosus and E. microcephalus were studied by disc diffusion method and revealed various levels of antimicrobial activity. The methanol, ethyl acetate, and acetone extracts of E. microcephalus showed more antibacterial activity against Staphylococcus aureus than standard antibiotics . The methanol extracts of E. viscosus showed antibacterial activity against Escherichia coli equal to standard antibiotics: vancomycin 30 μg/disc (V30), erythromycin 15 μg/disc (E15). The ethyl acetate extracts of E. viscosus showed antibacterial activity against Bacillus megaterium equal to standard antibiotic (V30), so E. viscosus and E. microcephalus contain antimicrobial components against different microorganisms(54).

Previous study aimed to evaluate the in vitro antimycobacterial activities of methanol crude extracts prepared from root of E. giganteus for their ability to inhibit the growth of or kill Mycobacterium tuberculosis strains H37Rv (ATCC

27294) and H37Ra (ATCC 25177). Results indicated that the extract of E. giganteus exhibited the most significant activity with a MIC value of 32 μg/ml and (55) 16 μg/ml, respectively against H37Ra and H37Rv .

Recent study indicated that 80%methanol extracts from the aerial part of Egyptian E. spinosissimus have antibacterial activity against, Escherichia coli, Pseudomonas aerogenes, Klebsiella pneumonia and Providencia stuartii with (MIC) of 1024μg/ ml(56).

1.4.2- Antifungal activity:

Root extracts of E. ritro were evaluated for their antifungal activity using direct-bioautography assays with three Colletotrichum species that cause strawberry anthracnose: Colletotrichum acutatum, C. fragariae, C. gloeosporioides, Botrytis cinerea, Fusarium oxysporum, Phomopsis viticola, and P. obscurans. Among the bioactive extracts, the dichloromethane extract of the radix of E. ritro was the most potent. Bioassay-guided fractionation of this extract led to the isolation of eight thiophenes which have very potent antifungal activity . A class of phytochemical called thiophenes were isolated and further studied for their biological activity. Three thiophenes were shown to be active at 30 uM against Colletotrichum acutatum, C. fragariae, C. gloeosporioides, Fusarium oxysporum, Phomopsis viticola, and P. obscurans(57).

Hydro-alcohol extracts of the root, flower head, leaf and stem of E. ellenbeckii and E. longisetus , were investigated for their antifungal activity. The flower extract of E. ellenbeckii showed strong inhibitory activity against Candida albicans(58).

Another study showed that ethanol extracts of E. viscosus presented the best -1 antifungal activity against Kobresia fragilis (12 mm 50 μL inhibition zone). However methanol extracts of E.viscosus displayed antifungal activity against -1 Miniopterus pusilus with 10 mm 50 μL inhibition zone while ethyl acetate and

acetone extracts of the same plant showed antifungal activity against only M. pusilus with 12 mm 50 μL-1 and 13 mm 50 μL-1 inhibition zone(54), respectively.

1.4.3- Anti-protozoal activity

Potential toxicity, costs, and drug-resistant pathogens necessitate the development of new antileishmanial agents. Medicinal and aromatic plants constitute a major source of natural organic compounds. In this study, essential oils of E. kebericho were investigated by gas chromatography (GC) and gas chromatography-mass spectrometry (GC-MS) analysis. Isolated oils were screened for antileishmanial activity against two Leishmania strains (L. aethiopica and L. donovani), and toxicity on the human monocytic leukemia (THP-1) cell line and red blood cells in vitro. GC-MS analysis revealed 43 compounds (92.85%) for E. kebericho oil. The oils contained sesquiterpene lactones (41.83%) as major constituents, the oils showed activity against promastigote (MIC 0.0097-0.1565 μL/ml) and axenic amastigote forms (0.24-0.42 μL/ml) of both Leishmania species. Weak hemolytic effect was observed for the oils, showing a slightly decreased selectivity index (SI 0.8-19.2) against the THP-1 cell line. E. kebericho exerted strong antileishmanial activity that was even higher than that of amphotericin B with significant cytotoxicity. This study, therefore, demonstrated the potential use of plant oils as source of novel agents for the treatment of leishmaniasis(59).

It was indicated that aerial parts of E. ritro L. and E.spinosissimus from the Greek island of Crete could be extracted, and the extracts obtained have been investigated for in-vitro anti-protozoal activity. The activity against chloroquine- sensitive (D6) and resistant (W2) strains of Plasmodium falciparum and Leishmania donovani promastigotes was determined as well as the cytotoxicity on a mammalian kidney fibroblast (Vero) cell line was tested. Dichloromethane of aerial part extract of E. ritro and E. spinosissimus had moderate activity against L. donovani with no significant anti-malarial activity or cytotoxicity(60).

1.4.4-Antioxidant activity

The antioxidant activities of methanolic extracts of the E. kotschyi were determined via 2,2- diphenyl-1-picrylhydrazyl (DPPH) radical scavenging assay, and also screened for cytotoxic activity against three human cancerous cell lines (MOLT-4, K562 and MCF7) using the MTT assay (3-[4,5-dimethylthiazol-2-yl]- 2,5 diphenyl tetrazolium bromide) assay. The methanolic extract of E. kotschyi exhibited potent cytotoxic activity against MOLT-4 and K562 cell lines among all extracts tested in this study (61).

Previous study revealed that phenolic content of E. giganteus which is used as spices in Cameroon were analyzed by using ferric iron reducing activity (FIRA), hydroxyl radical scavenging activity (HRSA) and free radical scavenging activity (FRSA) . The result showed that the plant has moderate levels of FIRA, HRSA and FRSA(62).

A natural alkynol group-substituted thiophene, 2-(penta-1,3-diynyl)-5-(3,4- dihydroxybut-1-ynyl)-thiophene (PDDYT), was isolated from the roots of E. grijsii. It possessed potent NAD(P)H: quinone oxidoreductase1 (NQO1) inducing

activity and could activate Keap1-Nrf2 pathway effectively in murine hepatoma Hepa 1c1c7 cells(63).

Other research evaluated free radical scavenging activity of E. echinatus and E. spinosissimus extracts by using different in vitro models like scavenging of 2, 2 diphenyl-1-picrylhydrazyl (DPPH) radical, nitric oxide radical and superoxide anion(64,65).

1.4.5- Anticancer action

The history of plant as a source of anti-cancer agents started in earnest in the 1950s with the discovery and development of the vinca alkaloids (vinblastine and vincristine)(66) . Some studies revealed anti-cancer activity of E. grijisii(67,68) and E. latifolius(69). Other study investigated some important medicinal plants used against cancer and their plant parts among them E. setifer since whole plant contain echinopsine alkaloid which has anti-tumor activity(70).

1.4.6-Protective effects of Echinops on testosterone-induced benign prostatic hyperplasia:

Many reports on E. echinatus suggest an anti-androgenic action for the plant, and it can be used as clinically effective medicine for the treatment of benign prostatic hyperplasia (BPH) where anti-androgenic agents are useful. So this study suggest that, the use of E. echinatus as Brahmadandi is not justifiable in light of its anti-androgenic action, E. echinatus proved to be a promising agent for the treatment of BPH (71).

The effect of terpenoidal fraction prepared from the petroleum ether extract of the roots of E. echinatus on male reproductive parameters has been investigated , and the study was carried out at two different dose levels of 30 and 60 mg/kg body

weight using wistar albino rats. Treatment with terpenoidal fraction showed a decrease in the relative weight of the reproductive organs without affecting the final body weight of the animals, and a significant decrease (P < 0.01) in serum testosterone levels and cauda epididymal sperm concentration compared with animals in the control group(72).

The effect of this species of Echinops on prostate production, play very important role in the development of new contraceptive modalities for male, since one of the most challenging pursuits in this program, is searching for newer, more potent, additionally safe and less expensive method that require infrequent and self administration and should have long lasting but complete reversible anti-fertility effect. Recently efforts are being made to explore the hidden wealth of medicinal plants for male contraceptive use, many studies showed that 50% ethanol root extract of E. echinatus in rat reduces sperm density in cauda epididymis and caused sperm anti-motility(73). 1.4.7- Hepatoprotective activity of Echinops

The Iraqi Echinops ethanolic extract (E. tenuisectus) was evaluated for its hepato-protective effect in rats by inducing hepato-toxicity with CCl4. Single oral dose of 250mg/kg of seeds extract was given to rats for 7 days. Serum activities of transaminases, aspartate aminotransferase and alanine aminotransferase (ALT and

AST) were used as the biochemical marker of hepato-toxicity. Histopatholigical changes in rats liver section were also examined. The results of the study indicated that, the pretreatment of rats with Echinops extract before the hepatotoxins agent (CCl4) offered a hepato-protective action(74).

The protective effect of ethanolic extract of E. grijisii and E. latifolius on CCl4- induced hepato-toxicity has been studied. The results suggested that both E.

grijisii and E. latifolius could correct the hepatocyte necrosis and functional disorder induced by the CCl4 treatment(75). Another research evaluated hepatoprotective activity of roots of E. echinatus which contained high percentage of flavonoids compounds(76).

1.4.8- Antiulcerogenic activity:

Echinops persicus extract exhibited both prophylactic and curative effects in rat gastric ulcer models. Latex of watery extract of plant was prepared by grounding the whole plant to a very fine powder and made suspension with water to be given in a dose of 500mg/kg body weight. This dose was administered intraperitoneally and orally through gastric intubation. Oral administration of Echinops extract at dose 500 mg/ kg and intraperitoneally administration with same dose significantly inhibited the development of gastric lesions in all experiments. The extract also caused significant decreased of the pyloric-ligation induced basal gastric mucosal injury(77).

1.4.9- Anti-inflammatory action

Anti-inflammatory studies were conducted on an ethanol extract of E. echinatus whole plant. The extract effectively inhibited the acute inflammation induced in rats by carrageenan, formaldehyde and adjuvant and the chronic arthritis induced by formaldehyde and adjuvant. The extract was more effective parentrally than orally. The toxicity studies showed reasonable safety warranting further studies(78). On the other hand, the ethanolic extract of E. spinosus has efficient action on muscular fibers; anti-inflammatory activity; The ethanolic extract of E. spinosus (100 mg/kg, intraperitoneal ) exhibited a very good anti-inflammatory activity

against carrageenan-induced paw edema in mice and rats, and it selectively (79) inhibited prostaglandin E2 (PGE2) -induced inflammation .

A new anti-inflammatory active flavanone glycoside 5,7 -8,4 - dimethoxyflavanone-5-O-α -L-rhamnopyranosyl- 7-O-β -D-arabinopyranosyl-(1 4)-O-β -D-glucopyranoside along with another anti-inflammtory active known compound dihydroquercetin-4'-methyl ether have been isolated from the leaves of E. echinatus(80).

1.4.10-Diuretic action of Echinops

The dried roots and aerial parts of E. echinatus were subjected to methanolic extraction. The prepared extracts were then subjected to preliminary phytochemical analysis. It was found that roots and aerial parts possess alkaloids, carbohydrates, flavonoids, tannins and phenolic compounds. The diuretic potential of methanolic extracts of the aerial parts and roots was assessed in albino rats using in-vivo Lipschitz test model. The volumes of urine, urinary concentration of sodium and potassium ions were the parameters of the study. Frusemide was used as standard. The results indicated that methanolic extracts at 250 mg/kg and 500 mg/kg body weight show a significant increase in the urine volume and electrolyte excretion when compared to control. Both extracts showed a significant diuretic activity so, this may be concluded that the constituents present in methanolic extracts may be responsible for diuretic activity(81).

1.4.11- Analgesic activity of Echinops

The analgesic potential of methanolic extracts of the aerial parts and roots of E. echinatus was assessed in albino rats using hot plate, tail immersion and tail flick models. The reaction time was the parameter of the study. Pentazocine was used as

standard. The results indicate that methanolic extracts at 250 mg/kg and 500 mg/kg body weight shows a significant increase in reaction time when compared to control. Both the extracts show significant analgesic activity. From this study it may be concluded that the constituents present in methanolic extracts may be responsible for analgesic activity(82).

1.4.12- Effects on Central Nervous System

Korolen is a bio-information product with a wide-spectrum regenerative effect that is manufactured using the latest achievements in the fields of phytotherapy, psychotronics, crystal therapy and bio-resonance. This product contained many herbal plants, among them Echinops sphaerocephalus which regulates the function of the parasympathetic autonomous nervous system. It improves memory, hearing and vision. It restores the function of fine nerves and of neural centers in the brain. It is used in paralysis, neuralgia, inflammation and damage of the spinal cord. The drug is administered orally at a dilution of 1:250 for 10-20 drops to receive 2 times a day and subcutaneous injection in 0.4%

solution of 1ml per day. The course of treatment lasted for 20-30 days. Good results of treatment were noted in patients with paralysis of the facial nerve, especially in the early stages of paralysis.(83)

1.5- Phytochemical constituents of Echinops

Echinops plant was reported to possess variety of compounds belonging to various classes like: alkaloids, flavonoids, terpenoids, lipids, steroids and polyacetylenes (84).

1.5.1-.Alkaloids of Echinops species

Alkaloids are a group of naturally occurring chemical compounds that contain mostly basic nitrogen atoms(85). This group also includes some related compounds with neutral and even weakly acidic properties(86). Also some synthetic compounds of similar structure are attributed to alkaloids(87). In addition to carbon, hydrogen and nitrogen, alkaloids may also contain oxygen, sulfur and more rarely other elements such as chlorine, bromine, and phosphorus(88) .

Alkaloids are produced by a large variety of organisms, including bacteria, fungi, plants, and animals, and are part of the group of natural products (also called secondary metabolites)(89). Many alkaloids can be purified from crude extracts by acid-base extraction. Many alkaloids are toxic to other organisms. They often have pharmacological effects. The boundary between alkaloids and other nitrogen- containing natural compounds is not clear-cut(90). Compounds like amino acid peptides, proteins, nucleotides, nucleic acid, amines, and antibiotics are usually not called alkaloids. Natural compounds containing nitrogen in the exocyclic position (mescaline, serotonin, dopamine, etc.) are usually attributed to amines rather than alkaloids(89). Some authors, however, consider alkaloids a special case of amines(91).

Compared with most other classes of natural compounds, alkaloids show great variety in their botanical and biochemical origin , in chemical structure and in pharmacological action . Consequently, many different system of classification are possible(92). More recent classifications are based on similarity of the carbon skeleton (e.g., indole-, isoquinoline-, and pyridine-like) or biogenetic precursor (ornithine, lysine, tyrosine, tryptophan, etc.). Chemical classification, it is probably the most widely accepted and common mode of classification of alkaloids for which the main criterion is the presence of the basic heterocyclic nucleus (i.e., the chemical entity)(92,93):-

1. Non-Heterocyclic Alkaloids or Atypical Alkaloids:

These are also sometimes called proto-alkaloids or biological amines. These are less commonly found in nature. These molecules have a nitrogen atom which is not a part of any ring system. Examples of these include ephedrine, colchicine.

2. Heterocyclic Alkaloids or Typical Alkaloids:

Structurally these have the nitrogen as a part of a cyclic ring system. These are more commonly found in nature. Heterocyclic alkaloids are further subdivided into different groups based on the ring structure containing the nitrogen:

 Pyrrole and Pyrrolidine alkaloids.  Pyridine and Piperidine alkaloids.  Tropane alkaloids.  Quinoline alkaloids.  Isoquinoline alkaloids.  Quinolizidine alkaloids.  Imidazole alkaloids.  Indole alkaloids.  Purine alkaloids.  Steroidal alkaloids.

All the alkaloids isolated from different species of Echinops related to the quinoline type so; the biosynthetic origin of quinoline alkaloids is the aromatic amine anthranilic (2-aminobenzoic) acid involved in the metabolism of the amino acid tryptophan. The skeleton of quinoline alkaloids constitutes a bicyclic system with a fused benzene and pyridine ring (figure 1.2). The attachment of a furan ring to the pyridine nucleus generates furoquinolines (e.g. furacridone), an important subgroup of quinoline alkaloids. The plant family Rutaceae represents the major source of quinoline alkaloids. Included in this group are quinine, the anti-malaria

medication, and quinidine, which calms the heart in tachycardiasis and arrhythmia. Chinchonine is an astringent. The main classes of quinoline alkaloids are: simple quinolines, 2(1H)-quinolinones, 4(1H)quinolinones, furoquinolines, and pyranoquinolines(94).

Some of these naturally occurring quinolines have profound medicinal properties while others have served as lead structures and provided inspiration for the design of synthetic quinolines as useful drugs(95).

Figure (1.2) (94) -Basic structure of quinoline nucleus

1.5.1.1- Echinopsine

Echinopsine was quinoline alkaloid isolated in 1900 by M. Greshoff from seeds of the blue globe thistle, E. ritro and its presence was also demonstrated in 14 other species of Echinops(96) like E. latifolius(97), E. setifer Iljin(98). Echinopsine is a weak base, solution of its salts, which exhibits no fluorescence gives

precipitates with the usual alkaloid reagent . it is very bitter ; its physiological action is similar to, but not identical with, that of a mixture of brucine and strychnine(96). Echinopsine (figure1.3) with properties similar to strychnine, but it is less toxic. Drug to use it in small doses, echinopsine has a stimulating effect, increases the reflex excitability of the spinal cord, tones the muscles of the system. Moreover, this alkaloid is used for the normalization of pressure: at a reception in small doses, it increases blood pressure in the large – lowers. Echinopsine is used in official medicine and has been licensed for use since 1957 in the form of nitrate. It is used in the treatment of paralysis, paresis, impotence, incontinence of urine, but in large doses can cause convulsions(96).

Figure (1.3) (96) - Chemical structure of echinopsine 1-methyl-4(1H)-quinolone; 1,4-dihydro-1-methyl-4-oxoquinoline; N-methyl-4-

quinolone (C10H9NO).

1.5.1.2-Echinopsidine

(Adepren) is an antidepressant used in Bulgaria for the treatment of depression. It increases serotonin, nor-epinephrine, and dopamine levels in the

brain and is believed to act as a monoamine oxidase inhibitor (MAOI)(99,100). Echinopsidine (figure1.4) is found naturally in E. echinatus along with the related alkaloids echinopsine(101).

Figure (1.4) (101) - Chemical structure of echinopsidine

1-methyl-2,3-dihydroquinolin-4-imine (C10H12N2)

1.5.1.3- Echinorine

The alkaloid echinorine isolated from fruits of E. ritro L. is shown to be a 1- methyl-4-methoxyquinolinium-compound (figure1.5) . The synthesis and some of the chemical and physical properties of this compound are described. Echinorine is decomposed in alkaline solution to echinopsine [1-methyl-quinolone-(4)] and methanol(102) . Echinorine is the only alkaloid detectable by chromatography in fruits of E. ritro and E. sphaerocephalus, the other alkaloids found previously are artifacts formed during storage or isolation of this compound, but recent studies reported the isolation of echinopsine from different species of Echinops without any mention to the detection of echinorine. In Egypt, echinopsine was proved to be a natural alkaloid after its isolation from E. spinosus without any alkaline treatment(103).

Figure (1.5) (102)- Chemical structure of echinorine 1-methyl-4-methoxyquinolinium (C11H12NO)

1.5.1.4- Echinozolinone

In addition to echinopsine and echinopsidine, a new alkaloid, echinozolinone, has been identified in E. echinatus as: 3(2-hydroxyethyl)-4(3H)-quinazolinone from its spectral data(104) (figure1.6). Quinazolinones are also a class of drugs which function as hypnotic-sedatives that contain a 4-quiazolinone core. Their use has also been proposed in the treatment of cancer (105).

CH 2 CH2 OH N

Figure (1.6) (104) -Chemical structure of echinozolinone 3(2-hydroxyethyl)-4(3H)-quinazolinone (C10H10N2O2)

Recently two new glycosidic quinoline alkaloids, 1-methyl-4-methoxy-8-(beta- D-glucopyranosyloxy)-2(1H)-quinolinone (a) and 4-methoxy-8-(beta-D-

glucopyranosyloxy)-2(1H)-quinolinone (b) (figure1.7) , have been isolated from the 1-butanol extract of the aerial parts of E. gmelinii . Structural elucidation of the two new glycol-alkaloids was based on their one proton(1H), thirteen carbon(13C) nuclear magnetic resonance spectroscopy( NMR) spectra and high-resolution fast atom bombardment- mass spectrometry (FAB-MS) data. These two compounds are rare examples of quinoline alkaloidal glycosides from natural sources(106).

a: R = H b: R = Methyl

Figure (1.7)( 106) -Chemical structure of (a) and (b) (a)[(−)-4-methoxy-8-(b-D-glucopyranosyloxy)quinolin-2(1H)-one ] and (b)[(−)-1-methyl-4-methoxy-8-(b-D-glucopyranosyloxy) quinolin-2(1H)-one]

1.5.2-Flavonoids Flavonoids are low molecular weight, bioactive polyphenols, which play a vital role in photosynthesizing cells. Flavonoids are secondary metabolites characterized by C6-C3-C6 carbon-skeleton(107). The basic structural feature of flavonoids is 2- phenyl-benzo-γ-pyrane nucleus consisting of two benzene rings (A and B) linked through a heterocyclic pyran ring (C) as shown in (figure1.8)(108).

Figure (1.8) (108)- Basic structure of flavonoids

Flavonoids differ in their arrangement of hydroxyl, methoxy and glycosidic side groups and in the conjunction between A and B rings, a variation in C ring provides division of subclasses. According to their molecular structure, they are divided into eight classes (figure 1.9)(109):

(109) Figure (1.9) - Chemical structure of different types of flavonoids

In plants, flavonoids are often present as O-glycosides or C-glycosides. The O- glycosides possess sugar substituent bound to –OH of aglycone, usually at position 3 or 7, whereas, C-glycosides possess sugar groups bound to carbon of aglycone usually 6-C or 8-C(108). The flavonoids are a class of natural product that gains interest due to its great variety and the number of its members. The flavonoids are often hydroxylated in positions 3, 5, 7, 3′, 4′, and 5′ as shown in (figure1.10) which are frequently methylated, acetylated, or sulphated(109).

Figure (1.10) (109)- Numbering of atoms in flavonoid aglycone at which substitution may occur 1.5.2.1-Reported pharmacological activities of flavonoids:

1.5.2.1.1-Flavonoids as antioxidants

Flavonoids are powerful antioxidants against free radicals and are described as free-radical scavengers(110). This activity is attributed to their hydrogen-donating ability. Indeed, the phenolic groups of flavonoids serve as a source of a readily available "H" atoms such that the subsequent radicals produced can be delocalized over the flavonoids structure(111).

Free radical scavenging capacity is primarily attributed to high reactivity of hydroxyl substituent that participate in the reaction as shown in the following equation(107) : F-OH + R. F-O. + RH Flavonoids inhibit lipid peroxidation in vitro at an early stage by acting as scavengers of superoxide anion and hydroxyl radicals. They terminate chain radical reaction by donating hydrogen atom to a peroxy radical as in (figure1.11), thus, forming flavonoids radical, which, further reacts with free radicals thus terminating propagating chain(112).

Figure (1.11)(112)- Formation of peroxy radical

1.5.2.1.2-Flavonoids in the treatment of gastric ulcer

Flavonoids protect the gastrointestinal mucosa from lesions produced by various experimental ulcer models and against different necrotic agents. Several mechanisms of action may be involved in this protective effect. Quercetin has an anti-secretory mechanism of action, this flavonol has antihistaminic properties, thus, decreases histamine levels, as well as preventing the release of histamine from gastric mast cells and inhibiting the gastric H+/K+ proton pump, diminishing acid gastric secretion(113). However, the most important mechanism of action responsible for the anti-ulcer activity of flavonoids is their antioxidant properties, seen in myricetin, rutin and quercetin, which involves free radical scavenging, transition metal ions chelation, inhibition of oxidizing enzymes, increase of proteic and nonproteic antioxidants and reduction of lipid peroxidation. These effects are correlated with presence in the structures of an ortho-dihydroxy in the ring B (catechol), and additionally a 2,3 double bond in conjugation with a 4-oxo function, as well as the presence hydroxyl groups in positions 3, 5 and 7. Besides the gastro-protective activity, quercetin and myricetin accelerate the healing of gastric ulcers, these polyphenolic compounds have anti-H. pylori activity and may be utilized as an alternative or additive agent to the current therapy. Therefore flavonoids could have an ideal more effective and less toxic therapeutic potential for the treatment of gastrointestinal diseases, particularly for peptic ulcers(114).

1.5.2.1.3-Effect of flavonoids on inflammation Flavonoids have been found to be prominent inhibitors of cyclooxygenase (COX) and lipoxygenase (LOX), as well as prevent synthesis of prostaglandin

(PGs) that suppress T-cells, also inhibit the activity of protein kinas C (PKC) at ATP-binding site, also promote IFN synthesis(115). Flavonoids (e.g., quercetin, kaempferol, myricetin)(figure1.12) also inhibit cytosolic and tyrosine kinase and also inhibit neutrophil degranulation(116). Many studies reported that quercetin and hesperedin given at a daily dose of 80 mg/kg inhibit both acute and chronic phase of inflammation while rutin was found to be effective only in chronic case(117).

1.5.2.1.4-Effect of flavonoids on cancer-related pathways:

Flavonoids are potent bioactive molecules that possess anti-carcinogenic effects since they can interfere with the initiation, development and progression of cancer by the modulation of cellular proliferation, differentiation, apoptosis, angiogenesis and metastasis(118) .

Myricetin Quercetin Kaempferol

Figure(1.12) (119)- Chemical structures of some flavonoids

1.5.2.1.5-Antimicrobial activity of flavonoids : Large number of flavonoids showed antibacterial, antimicrobial and antiviral activities at different concentrations, the following table illustrated antimicrobial activity of some flavonoids. (Table1.1)(120,121) .

Table (1.1)-Antibacterial, Antimicrobial and Antiviral Activities of Flavonoids(120,121)

No. Activity Organism Flavonoids 1. Antibacterial Staphylococcus aureus Quercetin, Baicalin, activity Hesperitin, Rutin. Staphylococcus albus Fisetin. Streptococcus pyogenes Apigenin. Streptococcus viridians Apigenin . Streptococcus jaccalis Chrysin. Streptococcus baris Chrysin. Streptococcus pneumonia Chrysin. Pseudomonas aeruginosa Rutin,naringin,baicalin, hydroxyethylrutosine. Escherichia coli Quercetin. Baccilus subtilis Quercetin. Bacillus anthracis Rutin. Proteus vulgaris Datisetin. Clostrium perfingens Hydroxyethylrutoside. 2. Antiviral Rabies virus Quercetin, quercetrin, rutin. activity Herpes virus Quercetin. Para influenza virus Quercetin, rutin. Herpes simplex virus Galangin, quercetin,

Respiratory synctial virus kaempferol, apigenin. Auzesky virus Quercetin,naringin. Polio virus Quercetin. Mengo virus Quercetin, apigenin. Pseudorabies virus Quercetin.

3. Antifungal Candida albicans Chloroflavonin. activity Candida tropicalis Quercetin. Fusarium solani Chrysoeriol. Botrytis cinerea Chrysoeriol. Verticillum dahlia Chrysoeriol. Azotabacter vinelandii Quercetin, rutin, epicatechin. 63

Alternacia tennisima Apigenin, echinacin. Cladosporium herbarum Phaseolinisoflavan.

1.5.2.1.6- Flavonoids in treatment of cardiovascular diseases :

Studies ensure that long-term administration of flavonoids can decrease, or tend to decrease the incidence of cardiovascular diseases(122), and their consequences by different mechanisms(123-126) :  Decrease in low density lipoprotein (LDL) oxidation by LOX inhibition and attenuation of oxidative stress, inhibition of leucocyte-leucocyte adhesion,

myeloperoxidase, decreased expression of inducible nitric oxide synthase (iNOS) and COX-2 .  Inhibition of platelet aggregation.  Decrease in oxidative stress (direct reactive oxygen species ROS scavenging) inhibition of metalloproteinase.  Vasodilatory properties, inhibition of nicotinamide adenine dinucleotide

phosphate-oxidase (NADPH), recovery of nitric oxide (NO) due to inhibition of superoxide production. Many studies reported that quercetin protects LDL against oxidative modifications effect, and it is the most protective acutely in situations of oxidative stress(126, 127).

1.5.2.1.7- Flavonoids in treatment of diabetes mellitus:

All flavonoids cannot cure diabetes mellitus because most types of this disease are basically genetic and no single drug can correct an inborn error. However, flavonoids can ameliorate some of the consequences of diabetes mellitus(128). Flavonoids have been identified to be good inhibitors of aldose reductase(129). It has been reported by several researchers that some flavonol possess anti-diabetic

activity, since it brings about regeneration of pancreatic islets and increases insulin release in streptozotocin-induced diabetes, and also, it has been reported to stimulate Ca2+ uptake from isolated islet cells thus suggesting it to be effective even in type-2 D.M.(130).

1.5.2.1.8- Role of flavonoids in treatment of hepatotoxicity:

Flavonoids bind to subunit of DNA-dependent RNA polymerase I, thus activating the enzyme. As a result, protein synthesis gets increased leading to regeneration and production of hepatocytes(131). Silymarin, apigenin, quercetin and kaempferol, are reported to be potent therapeutic agents against microcrystin LR-induced hepatotoxicity(131). Rutin and myricetin are reported to show regeneration and hepatoprotective effects in experimental cirrhosis(132).

1.5.2.1.9- Effect of flavonoids on depression: Depression is caused by functional deficiency of monoamine transmitters at certain sites in brain(133) . Flavonoids have found to be ligand for GABA-A receptors in the central nervous system and it led to hypothesis that they act as benzodiazepine-like molecules(134).

Many studies reviewed that dietary flavonoids possess multiple neuro- protective actions in central nervous pathophysiological conditions including depression(134).

Some of the flavonoids isolated from different species of Echinops plant are listed in the following table (table 1.2):

Table (1.2) - Flavonoids Isolated from Different Species of Echinops

Flavonoids Sources References Kaempferol, kaempferol 4'-methylether, kaempferol 7-methylether, Echinops echinatus 135 kaempferol 3-O- alpha- L- rhamnoside, myrecetin-3-O-alpha-L-rhamnoside Dihydroquercetin-4'-methyl ether, 5,7 -8,4 -dimethoxyflavanone-5-O- -L- Echinops echinatus 80 rhamnopyranosyl- 7-O- -D-arabinopyranosyl- (1 4)-O- -D-glucopyranoside Silymarine Echinops tenuisectus 136 Quercetin Echinops tenuisectus 137 Apigenin (4',5,7-trihydroxyflavone, luteolin Echinops niveus 138 Kaempferol Echinops galalensis and 139 Echinops hussoni Apigenin, hispidulin, 5,4dihydroxy flavone and Echinops spinosissimus 140 apigenin 7-O- glucoside Apigenin Echinops latifolius 141 Kaempferol, myricetin Echinops spinosus 79

Neoflavonoid nivetin Echinops niveus 142

Apigenin, apigenin-7-O-glucoside, echinacin, 143 and echinaticin Echinops echinatus

1.5.3-Terpenoids:

Terpenoids are defined as secondary metabolites with molecular structures containing carbon backbones made up of isoprene (2-methylbuta- 1, 3-diene) units,

CH2=C(CH3)-CH=CH2. Isoprene contains five carbon atoms and thus, terpenoids

are all based on the isoprene molecules and their carbon skeletons are built up from the union of two or more of these C5 units. They are then classified according to whether they contain two (C10), three (C15), four (C20),six (C30) or eight (C40) such units. They range from the essential oil components, the volatile mono-and sesquiterpenes (C10 and C15), through the less volatile diterpenes (C20) to the (144) involatile triterpenoids and sterols (C30) and carotenoid pigments (C40) . The terpenoids group show significant pharmacological activities, such as antiviral, anti-bacterial, anti-malarial, anti-inflammatory, inhibition of cholesterol synthesis and anti-cancer activities(145). The most important terpenoids isolated from different species of Echinops: 1.5.3.1-Beta-sitosterol

Is one of the most prevalent vegetable-derived phytosterols in the diet. It is structurally related to cholesterol (figure1.13)(119), but since it is slowly absorbed from the intestinal tract, it may interfere with the absorption of cholesterol. β- sitosterol also appears to modulate the immune function, inflammation, and the pain levels by controlling the production of inflammatory cytokines(146,147). This last effect may help to control allergies and reduce prostate enlargement(148).

The compound can affect the structure of cell membranes and alters the signaling pathways that regulate tumor growth and apoptosis(149). Moreover, β- sitosterol has shown a decrease in proliferative changes and tumor yields when added to diets of mice and rats treated with colon carcinogens(150).

β-Sitosterol compound isolated from E. nanus, E. transiliensis(151) , and from roots of E. grijisii (152), also β-sitosterol and β-sitosterol glucoside have been identified in the whole plant of E. niveus(138). β-sitosterol-3-O-β-D-glucopyranoside isolated from E. ritro(153) .

Figure (1.13) (119)- Chemical structure of β-sitosterol 1.5.3.2-Stigmasterol

Stigmasterol is an unsaturated plant sterol occurring in the plant fats or oils of soybean, calabar bean, and rape seed, and in a number of medicinal herbs(154).

Stigmasterol (figure1.14) is used as a precursor in the manufacture of semisynthetic progesterone a valuable human hormone that plays an important physiological role in the regulatory and tissue rebuilding mechanisms related to estrogen effects, as well as acting as an intermediate in the biosynthesis of androgens, estrogens, and corticoids(155), it is also used as the precursor of vitamin (156) D3 .

It was demonstrated that stigmasterol inhibits several pro-inflammatory and matrix degradation mediators typically involved in osteoarthritis-induced cartilage degradation (154), it also possesses potent antioxidant, hypoglycemic and thyroid inhibiting properties(157). This compound was isolated from E. nanus, E. transiliensis(151) and E. grijisii (152) .

Figure (1.14) (119) -Chemical structure of stigmasterol

The other terpenoids isolated from different species of Echinops plant are listed in the following table: (table 1.3).

Table (1.3) -Terpenoids Isolated from Different Species of Echinops

No. Terpenoids Plant species Reference

1. Taraxasterol E. niveus 138

2. Taraxasterol acetate E. ritro 153

3. pseudo taraxasteryl acetate together with B- E. spinosissimus 158 amyrin acetate

4. methyl chavicol, 1,8-cineole , p-cymene E.graecus& 159 E.ritro 5. dehydrocostus lactone, β-phellandrene, E. kebericho 53 germac-rene B, α-selinene, α and β-pinene and caryophyllene oxide

6. τ-cadinol . β-cubebene , β-patchoulene E. kebericho 160 longifolene and cyperene

7. Sesquiterpenes:beta-selinene , beta-maaliene , E. ellenbeckii 161 caryophyllene oxide & cyperene

8. Sesquiterpenoids: Echinopines A and B E. spinosus 162

9. tricyclic sesquiterpenes: α and β- E. giganteus 163 caryophyllene, α and β-bisabolene,α and β- santalene, guaiene

10. sesquiterpene alcohols (e.g. (-)-nopsan-4-ol E. grijsii 164 and (+)-prenopsan-8-ol, silphiperfol-6-ene) 11 sesquiterpene lactones (e.g.α and β- E. grijsii 165 caryophyllene epoxide

Aims of this study

Echinops heterophyllus (Family: Compositae) is an indigenous plant, widely distributed in Iraq. Literature survey have revealed that no previous phytochemical investigation work had been done on this species, so it was deemed desirable to carry out a phytochemical investigation on this plant. This study is emphasized on the isolation and identification of different active components from Iraqi E. heterophyllus family , this is done through extraction of the different plant parts (seeds, roots and aerial parts) using soxhlet apparatus, fractionation, isolation, purification and confirmation of different components utilizing different physio-chemical and spectral analysis. A relative asses was conducted to study the effect of the crude extract of Echinops plant and some of its bioactive fractions in wound healing and as an anti-scar agent in vivo.

2. EXPERIMENTAL WORK

2.1- Reagents and Materials:

The reagents and materials that were used in this study with their suppliers are listed in (table 2.1).

Table (2.1)- Reagents and Materials Used in the Study

Chemical Supplier Acetic anhydride BDH, Ltd. Poole , England Acetone Scharlab S.L. Spain Ammonia 25% BDH, Ltd. Poole, England Beta-sitosterol standard Chengdu Biopurify Phytochemicals Benzene GCC. UK Chloroform Scharlab S.L. Spain Diethylamine Sigma-Aldrich, USA Ethanol 99.9% Scharlab S.L. Spain Ethyl acetate Scharlab S.L. Spain Formic acid BDH, Ltd. Poole, England Glacial acetic acid BDH, Ltd. Poole , England Hexane BDH, Ltd. Poole, England Hydrochloric acid 37 % GCC. UK Iodine Sigma-Aldrich, USA Kaempferol standard ˃ 98.0% Fluka. Austria Mercuric chloride HDPE Nalgene lab-quality bottle Methanol HPLC grade 99.9% GCC.UK.

Methanol 99.8% Scharlab S.L. Spain Myricetin standard ˃ 98.0% Sigma-Aldrich, USA N-butanol BDH, Ltd. Poole, England Petroleum ether ( 60-80 ِC ) BDH, Ltd. Poole, England Polyamide 6 for column Sigma-Aldrich Chemie GmbH, chromatography Germany Potassium iodide Sigma-Aldrich, USA Quercetin standard Chengdu Biopurify Phytochemicals Rutin standard Chengdu Biopurify Phytochemicals xylazine and Ketamine Bayer/ Germany haematoxylin and eosin Pureview®/ UK Stigmasterol standard Chengdu Biopurify Phytochemicals Sulfuric acid BDH, Ltd. Poole , England Toluene BDH, Ltd. Poole, England

2.2- Instruments : -

The instruments used in this study are listed in (table 2.2).

Table (2.2)- Instruments Used in the Study

Instruments Manufacturer Chiller: Ultratemp 2000, Julabbo F30 Buchi/ Germany Elemental microanalysis (CHN) : EuroEA Elemental IRMS/ Italy analyzer Electrical sensitive balance Sartorius/ Germany Fourier transforms infrared spectra (FT-IR) spectra Shimadzu /Japan were scanned on Shimadzu FT-IR-8400S Infrared

Spectrometer Gas chromatography –mass spectroscopy GC-MS-QP Shimadzu /Japan Ultra Shimadzu. Instrument model :AOC-2 Oi High Performance Liquid Chromatography (HPLC) Waters / Germany Jobling Laboratory Division thin layer Germany chromatography TLC Coater.

Melting point: melting point was determined by Stuart / UK electro–thermal melting point 1H and 13C NMR was carried out in Al-Albayt Italy University, Al-Mafraq, Jordan (Euro-vectorEA 3000A) Oven: Memmert 854 Buchi /Germany Preparative HPLC (JASCO FC-2088-30) Jasco/ Japan Rotatory evaporator: Buchi rotatory evaporator Buchi/ Germany attached to vacuum pump.

Ultraviolet light (DESAGA HEIDELBERG) of 254 DESAGA/Germany nm and 366 nm wave lengths. Ultra violet (UV) spectra were recorded in methanol Japan using computerized spectrophotometer Shimadzu (UV-1700)

2.3-Plant material 73

The whole plant of Echinops heterophyllus of the Family (Compositae) was collected from Nazali, 71Km north of Erbil . The plant was authenticated by Dr. Abdul-hussien Alkhait specialist in plant taxonomy in Science College/ Erbil University .

The plant seeds were collected during the month of November (2011), while aerial parts (leaves/stem) and roots were collected during the months of May and June (flowering time) and were cleaned, dried at room temperature in the shade then pulverized by mechanical mills and weighed.

2.4- Experimental work

The experimental work is divided into :

2.4.1- Preliminary phytochemical screening of various secondary metabolites like alkaloids, flavonoids, steroids, tannins, saponins, anthraquinioin, terpenoids and cardiac glycosides) in the different parts of Echinops plant.

2.4.2- Extraction and fractionation of different active constituents.

2.4.3- Isolation and purification of different active constituents.

2.4.4- Identification and characterization of the isolated compounds.

2.4.5- Investigation of the some pharmacological activity of the different isolated fractions.

2.4.1- Preliminary qualitative phytochemical analysis:

Chemical tests were carried out using the ethanolic extracts from plants and or the powdered specimens, using standard procedures to identify the active constituents.(93,166,167)

Test for alkaloids

Alcoholic extract (10 ml) was stirred with 5 ml of 1% HCL on a steam bath. Mayer’s (1.35gm mercuric chloride in 60ml water + 5gm potassium iodide in 10ml water )and Wagner’s reagents (1.27g of iodine and 2g of potassium iodide in 100ml of water) were added, white and reddish brown color precipitate respectively, were taken as evidence for the presence of alkaloids.

Test for flavonoids

(i)Lead acetate test: Lead acetate 10% (1 ml) solution was added to 5ml of alcoholic extract, The formation of a yellowish- white precipitate was taken as a positive test for flavonoids.

(ii)NaOH test: The extract (5 ml) was treated with aqueous NaOH and HCl, and looking for the formation of a yellow orange color.

Tests for steroids

(i) Liebermann-Burchard test: Extract (3ml) was treated with chloroform, acetic anhydride and drops of sulphuric acid was added. The formation of dark pink or red color indicates the presence of steroids.

(ii)H2SO4 test: The development of a greenish color was considered as indication for the presence of steroids, when the organic extract (2 ml) was treated with sulphuric and acetic acids.

Test for tannins

Plant material (10mg) in 10ml distilled water was filtered, then the filtrate (3ml) + 3ml of FeCl3 solution (5%w/v) were mixed. The formation of a dark green or blue black precipitate was considered an indication for the presence of tannins.

Tests for anthraquinones

Borntrager’s test: 3ml of alcoholic extract was shaken with 3 ml of benzene, filtered and 5 ml of 10% ammonia solution was added to the filtrate. The mixture was shaken and the development of a pink, red or violet color in the ammonical (lower) phase indicates the presence of free anthraquinones.

Test for terpenoids

Alcoholic extract (2ml) was dissolved in chloroform (2ml) and evaporated to dryness. concentrated sulphuric acid (2ml) was then added and heated for about 2 min. A grayish color was considered an indication for the presence of terpenoids.

Test for cardiac glycoside

Keller-kiliani test: Alcoholic extract (2ml) +1ml glacial acetic acid+

FeCl3+con.H2SO4. Formation of green-blue color indicates the presence of cardiac glycoside.

2.4.2-Extraction and fractionation of different active constituents

Shade-dried coarsely powdered seeds, aerial parts and roots (120, 500, 200gm) separately were defatted with hexane for 24 hours then allowed to dry at room

temperature. The defatted plant materials was extracted with 80% ethanol (1.250, 3, 2 L) in soxhlet apparatus until complete exhaustion .

The alcoholic extract was evaporated under reduced pressure at a temperature C to give a dark greenish-yellow residue designated as a crudeه not exceeding 40 fraction .

Crude fraction was acidified with hydrochloric acid (5%) to pH 2 and partitioned (three times) with equal volume of ethyl acetate to get two layers (aqueous acidic and ethyl acetate layer). The aqueous acidic layer was then separated and basified with equal volume of sodium hydroxide 5% to pH 10 and extracted with chloroform in the separatory funnel (three times) to get two layers, the chloroform layer which was separated and evaporated under reduced pressure at a temperature not C to give blackish residue designated as fraction 1(F-1) and aqueousه exceeding 40 basic layer which is designated as fraction 2 (F-2).

The ethyl acetate layer of the original alcoholic extract (crude fraction) was evaporated to dryness under reduced pressure and basified with 300ml of sodium hydroxide5% to pH 10 and extracted with chloroform in the separatory funnel to get two layers, the aqueous basic layer and chloroform layer.

The aqueous basic layer was separated, evaporated to dryness and acidified with 5% hydrochloric acid to pH 2 then extracted with ethyl acetate to get fraction designated as fraction 3 (F-3) .

Chloroform layer was also separated and evaporated to dryness under reduced pressure then partitioned with methanol 80% and petroleum ether to get two

layers petroleum ether fraction and methanol 80% fraction which designated as fraction 4 (F-4)(166). (figure 2.1). each fraction was tested using specific chemical test

Powdered plant material

Defatting step by maceration with hexane

Hexane extract Defatted plant material

(Fat)

80% Ethanol

in the soxhlet

Exhausted plant material Alcoholic extract (crude extract) (crude extract)

5% HCl / ethyl acetate

Ethyl acetate layer Aqueous layer

(neutral and acids components) (alkaloids and water soluble components)

5%NaOH / CHCl3

CHCl3 layer (neutral) Aqueous layer (acids)

90% methanol/

petroleum ether 5%HCl /ethyl acetate

CHCl3 layer

(F-1)

( Alkaloids)

ethyl acetate layer

- (Flavonoids) F-3 Aqueous OH layer (F-2)

(Water soluble components)

Petroleum ether (waxes, fats)

(MeOH) F-4 (Terpenes & steroids)

Figure (2.1) (166)- General scheme for separation of different plant constituents

The components of all of the above designated fractions were examined by TLC using the following systems:-

 Readymade plates of silica gel GF254nm (20x20cm) of 0.25mm thickness C forه MERCK) . The plates were activated at 110) 30 minutes before use(168).  Developing solvent systems: different solvent systems were used for the detection of different components obtained from different plant parts. Solvent system was prepared and placed in a glass tank (22.5 cm x 22 cm x 7 cm) covered with a glass lid. The atmosphere of the glass tank should be saturated with the solvent vapors before running samples, so part of the inside of tank was lined with filter paper (Whatman No.2 )to aid in this saturation process and allow to stand for 45 minutes before use. Solvent systems were used for fraction-1 : (168)  S1a= Benzene : methanol: (80 : 20) (168)  S2a = Chloroform: acetone: diethylamine (50 : 40 :10) (169)  S3a = Toluene: ethylacetate: diethylamine (70 : 20 : 10) For fractions 2 and 3 : (169)  S1f = Ethylacetate: formic acid : glacial acetic acid : water (100: 11: 11:27 ) (169)  S2f = n-Butanol : glacial acetic acid : water (40: 10: 50 ) (168)  S3f = Acetic acid: water (15:85) (169)  S4f = Chloroform: aceton : formic acid (75: 16.5 :8.5) (170)  S5f = Toluene: chloroform : aceton (40: 25: 35) (169)  S6f = Ethylacetate : methanol: formic acid (50: 50 :1) For fraction 4: 80

(167)  S1s = Chloroform : methanol (100 : 10 ) (167)  S2s = Hexane : ethyl acetate (50: 50) (171)  S3s = Chloroform : ethyl acetate (80: 20) A small amount of each fraction (1 mg dissolved in 1 ml solvent) was applied with or without standard samples (1mg/ml) to TLC plates manually, using capillary tubes, in form of spots and allowed to dry, then developed by ascending technique. The solvent migration limit is being 14-16 cm from the base line. After development of the solvent system, the plates were examined either by UV light at 254 and 366 nm (for flavonoids) or by chemical detection (for alkaloids & steroids) . The spots were marked with a pencil, the value for each compound as evident from the florescent spots under UV or colored spots was calculated as the Rf value (retention factor) for that compound:

Rf value = Distance traveled by the compound Distance traveled by the solvent system

2.4.3-Isolation and purification of different active constituents

Different chromatographic analysis was carried out to isolate different active constituents as follows :

Isolation of alkaloids by :  Preparative HPLC  Preparative TLC. Isolation of flavonoids glycoside by column chromatography (CC). Isolation of flavonoids (as aglycon) by preparative TLC.

2.4.3.1-Preparative High Pressure Liquid Chromatography

The term preparative HPLC is usually associated with large columns and high flow rates. The objective of an analytical HPLC run is the qualitative and quantitative determination of a compound, while preparative HPLC run , it is the isolation and purification of a valuable product .With increasing demand for production of highly pure valuable compounds in varying amounts in the chemical and pharmaceutical industry , the field of operation for preparative HPLC is increased (172), so preparative HPLC (figure 2.2) was used in this study (for the first time in Iraq) to isolate in a very pure form three alkaloids from Echinops plant.

The traditional task of natural product chemistry is the isolation of active compounds from active crude natural product extracts, as the crude extract is a very complex mixture, the purification process usually consists of several consecutive purification until the active compound is available in pure form for structure elucidation. Requirements for the isolation and purification system of large number and good quantity of natural compounds were required, which was depending mainly on preparative HPLC(173).

Figure (2.2)- Preparative HPLC apparatus used in the separation of alkaloids

2.4.3.2- Preparation of preparative thin layer chromatography plates:

On a 20cm x 20cm glass plates a slurry of 75 gm of silica gel GF 254 suspended in 150 ml of distilled water was applied in 1mm thickness manually by using Jobling laboratory division plate coater. The freshly coated plates were left until the transparency of the layer disappears. After 10 minutes, the plates stacked in a dry rack and heated in vertical position for 1 hour at 110oC with occasional opening of the oven door from time to time in order to allow moisture escape. The completely dried and activated plates were kept in a dry and moisture free container containing adsorbent silica gel . 83

Fractions -1 and 3 were applied as a concentrated solution in a row of spots using capillary tube four times on each plate (the spots should dry before the next application), then the plates placed inside glass tank which contained the proper solvent system.

The detection was done using dragendorffʼs reagent (for alkaloids), and UV light at a wave length of 254 nm (for flavonoids). The bands corresponding to each compound were scraped out and collected in a beaker, mixed with chloroform or methanol, stirred and left a side for one hour, then filtered. For more purification, each isolated compound was dissolved in a hot ethyl acetate or methanol and a small amount of decolorizing charcoal was added so that the solution turns black. Then the hot solution was poured through filter paper into another flask. Then the solvent was evaporated to give solid product .

After evaporation of the solvent, the obtained residues were subjected to co- chromatography using different mobile phases for identification.

2.4.3.3-Isolation of flavonoids glycosides by column chromatography

Fraction-2 was subjected to (CC) using glass column (50cm in height x 2cm in diameter) packed with polyamide 6 slurry in ethanol. The top of the column had a perforated filter paper disc; approximately 0.5cm of sea sand layer, followed by another perforated filter paper disc.

Three gram of the sample (F-2) was dissolved in 10 ml of methanol and applied to the column. The column was eluted by simple elution technique using 45% ethanol as a mobile phase (experimental work) . The column was developed by adding 1.5L of eluent with collecting 15 ml fractions, then monitored by TLC. A total number of 100 fractions were obtained. Those consecutive fractions, which have the 84

same number of spots with the same Rf values, were combined and concentrated to dryness to get major fraction.

2.4.4-Identification and characterization of the isolated compounds:

2.4.4.1- Thin layer chromatography (TLC):-

Analytical TLC was performed for each separated compound by using the same system mentioned in page 47.

2.4.4.2- Melting point(M.P.):-

The melting points of the isolated compounds were done and compared with that of the available standards.

2.4.4.3- Ultra violet (UV) spectrum analysis:-

The UV spectra of the isolated compounds are taken in double beam Shimadzu spectrophotometer (UV-1700) in between range 200 nm to 700 nm. Methanol was taken as reference solvent. The pure isolated compounds was dissolved in pure methanol then subjected to UV spectro-photometric measurements .

2.4.4.4- Fourier transforms infrared(FT-IR) spectra:-

The FT-IR spectra for each separated compound was recorded in KBr disc. The structural assignments have been correlated for characteristic

bands as mentioned in results.

2.4.4.5- Elemental microanalysis (CHN) :-

The CHN analysis for separated compounds was done.

2.4.4.6- 1H and 13C nuclear magnetic resonance spectroscopy (NMR) analysis :-

The proton NMR spectra was taken by dissolving the sample in dimethyl sulphoxide (DMSO) – d6 and run on NMR Spectrometer. All chemical shifts reported are in reference to tetra methyl silane (TMS) at 0 part per million (ppm).

1H-and 13C-NMR are the most efficient method for identification and elucidation of structure of various types of components. The NMR measurement was carried out on Euro-vectorEA 3000A NMR spectrometer apparatus (300MHz for 1H-NMR and 75.4 MHz for13C-NMR). Chemical shifts are given on a δ (ppm) scale with TMS as internal standard

2.4.4.7- Qualitative and quantitative estimation of isolated compounds by HPLC :

Qualitative and quantitative estimations of isolated components were done by using HPLC in which identifications were made by comprise of retention times obtained at identical chromatographic conditions of analyzed samples and authentic standards .

The following equation was used to calculate the percentage of the compound in the plant: -

(AUC of plant sample / AUC of the standard)

× Conc. St× DF×100 86

Percentage of compound in the plant =

Weight of the dried plant used in the extraction Where:-

AUC = Area under the curve. DF = Dilution factor.

Conc. St. = Concentration of the standard used in HPLC.

2.4.5- Investigation of some pharmacological activity of the different isolated fractions:

A relative assess on wound healing activity of crude Echinops heterophyllus extract and some of its bioactive fractions (F-1and 3) was done as follow: In vivo experiment:

 Plant material Crude plant extract, bioactive fractions (alkaloids and flavonoids)

 Experiment Animals

Twenty four adult male rabbits were used. Aged between six months to one year, obtained from the local market and placed in sterilized cages subjected to constant environmental conditions.

 Induction of wounds:

Surgical preparations were made at the upper back region after clipping, shaving and washing the area with tap water and drying. Then, standard longitudinal incisions (1x2 cm²) were implemented using a surgical scalpel(177,178). (Figure 2.3)

Figure (2.3)- (1x2 cm²) Wound incision at the back region.

 Blood collecting: Blood samples were obtained from each animal (heart) at day one, four, six and twelve. Measuring the blood glucose, protein, albumin, AST and ALT, to ensure that animals are in a healthy state and the wound healing process was not affected by other factors. Two milliliters of blood was obtained through percutaneous cardiocentesis in anesthetized rabbits approaching the heart from the lateral left side and the midline under the sternum side aiming the needle toward the heart. Using 19 to 25G needle with 3 to 5 ml syringe; and blood sample collection tubes with an anticoagulant agent(179-181).

 Experimental Design: Adult male rabbits were divided into four equal groups (6 animal each). The effect of crude Echinops extract and its bioactive fractions (alkaloids and flavonoids ) were evaluated visually and through histopatholigical changes. Treatment was applied as (50% concentration)(182-184) three times daily using a cotton swab.

 Group 1: six rabbits were surgically wounded and considered as untreated control group  Group 2: six rabbits were wounded and treated with 50% crude Echinops extract (figure 2.4).  Group 3: six wounded rabbits were treated with 50% alkaloid fraction obtained from Echinops extract (figure 2.5).  Group 4: six wounded rabbits were treated with 50% flavonoids fraction obtained from Echinops extract (figure 2.6).

 Histological evaluation At day 12, the experiment was terminated and the wound area was removed from the surviving animals for histological examination. The tissue was processed in the routine way for histological evaluation. Five micrometer thick sections were stained with haematoxylin and eosin.

Specimens (skin) were taken from day one, fourth , sixth and twelfth. Animals were anesthetized using (xylazine and ketamine) in a dose of 5mg/Kg and 15 mg/Kg respectively(185). Later, the specimens were kept in buffered formalin (10%) solution and examined.

Figure (2.4)- The application of the crude plant extract

Figure (2.5)- The application of the bioactive fraction alkaloids

Figure (2.6)- The application of the bioactive fraction flavonoids

3. RESULTS AND DISCUSSION

3.1 - Preliminary qualitative phytochemical analysis:

The results of phytochemical screening are given in (table-3.1)

Table (3.1)- Phytochemical Screening of Different Parts of

Echinops heterophyllus

Plant part Alkaloids Flavonoids Steroids Tannins Saponins Anthraquinoin Terpenoids Cardiac glycoside Seeds + + - - - - + - Aerial Traces part + + - - - + - Roots + + + - - - + - +, - represent presence and absence of phytoconstituents respectively.

The results of preliminary phytochemical screening of plant extracts showed the presence of alkaloids, flavonoids, steroids, and terpenoids in different parts of Iraqi species in different percentage, and the absence of, tannins, saponins, anthraquinoin and cardiac glycosides in all plant parts. These results can be compared with phytochemical screening of other Echinops species for example,: the aerial part of Iraqi heterophyllus species was contained traces amount of alkaloids, unlike Egyptian species E. spinosissimus, its aerial parts was contained about 11.3% alkaloids(56), also quinoline alkaloids and flavonoids found in the aerial part of Indian E. echinatus with the presence of tannins in the root parts only(82). In the Saudian species E. hussoni, only aerial parts have alkaloids, anthraquinoin, terpenoids, coumarine without any percentage of flavonoids

compounds(186). Many researchers reported that the concentration of secondary metabolites are varying from plant to plant belong to the same genus and even in the different parts of the same plant(187) , this is due to many factors like environmental heterogeneity, since the effect of environmental heterogeneity is highly scale-dependent. It may create high niche diversity and hence allow species to coexist at a large spatial scale(188), also the high complexity and heterogeneity of soil, like( soil structure, texture and depth, moisture retention characteristics, aeration) create a big variation in the chemical constituents even in the same country (189), good example seen in two Iraqi species of Echinops plant : E. tenuisectus and E. heterophyllus, phytochemical analysis of E. tenuisectus revealed the presence of high percentage of silymarine in the seeds (0.878%) and aerial parts (0.095)(136) with the absence of this compound in the heterophyllus species.

3.2-Extraction and fractionation of different active constituents

The precise mode of extraction naturally depends on the type of substance that is being isolated. In general, the standard defatting method is continuous extraction of the plant materials in a soxhlet extractor, using petroleum ether (boiling point 40-60°C) or hexane as solvent, or use maceration in the hexane for overnight in percolator.

Next day remove the solvent by filtration, and extract defatting plant materials with alcohol (80% ethanol) to get crude extract .

Since different plant parts contain different chemical classes of active constituents, alkaloids (basic compounds), flavonoids (acidic compounds) and

steroids (neutral compounds) so the fractionation based on the conversion of basic compound to its salt by aqueous mineral acids, and when the salt of an alkaloid is treated with hydroxide ion, nitrogen gives up a hydrogen ion and the free amine is liberated which is taken or extracted by specific organic solvent like (chloroform) to get free alkaloids (F-1) leaving quaternary alkaloids and water soluble compounds in the aqueous layer (F-2).

Testing F-2 with mayer’s and wagner’s reagents gave negative results indicating that there is no quaternary alkaloids in fraction-2 , on the other hand, testing this fraction with lead acetate, NaOH test and reducing sugar test (Fehling test) after acid hydrolysis showed positive results indicating that fraction-2 contains flavonoids in the glycosidic linkage, not as a free aglycon.

The same principle was applied to the acidic compounds to get flavonoids as a free aglycon in fraction-3 leaving neutral components in the organic layer which was then extracted with 80% methanol to get fraction-4.

The following table(3.2) shows the percentage of active constituents (secondary metabolites) obtained from each part of Iraqi Echinops

Table(3.2)- Percentage of Different Fractions Obtained from Different Plant Parts (Seeds, Aerial parts, Roots)

Plant part Fractions Weight % of active constituents Seeds Crude extract 27 gm F-1 8.8 gm 7.4%

F-2 0.8 gm 0.7% F-3 5 gm 4.2% F-4 1.3 gm 1.1% Aerial parts Crude extract 86 gm F-1 2.5 gm 0.5% F-2 9.5 gm 1.9% F-3 30 gm 6 % F-4 17 gm 3.4%

Roots Crude extract 30 gm F-1 4 gm 2% F-2 6 gm 3% F-3 7.5 gm 3.8 % F-4 5 gm 2.5%

3.3- Preliminary identification of different Echinops parts by TLC

Thin layer chromatography of different fractions ( F- 1, 2, 3, 4, ) obtained from different parts of the Echinops, confirms the following:

(a) The presence of three different alkaloids in fraction-1 (named E1, E2 and E3) which is obtained from seeds part and two alkaloids in the same fraction obtained from roots part (E1 and E2) with very traces one compound (E1) in the alkaloidal fraction of aerial plant parts, these different alkaloids appeared as a single spot on

TLC plates, using three different developing solvent systems ( S1a, S2a,S3a,) and detected by dragendorffʼs spraying reagent as shown in figures from (3.1 to 3.3) without using standard.

The Rf values of these compounds in the different solvent systems were calculated, table (3.3)

Table (3.3)- Rf Values of Alkaloids Obtained From Different Plant Parts in Different Developing Solvent Systems in TLC.

Compound Plant part S1a S2a S3a E1 Seed 0.16 0.22 0.25 E2 Seed 0.58 0.68 0.66 E3 Seed 0.75 0.8 0.79 E1 Root 0.17 0.25 0.26 E2 Root 0.6 0.7 0.67 E1 Aerial part 0.15 0.21 0.25

R S A

Figure (3.1)- TLC chromatogram of fraction one (F-1) for different Echinops parts

( roots, seeds, aerial parts) using silica gel GF254nm as adsorbent and S1a as a mobile phase. Detection by dragendorffʼs spraying reagent

R : Roots S : Seeds A : Aerial parts

S R A

Figure (3.2)- TLC chromatogram of fraction one (F-1) for different Echinops parts( seeds, roots, aerial parts) using silica gel GF254nm as adsorbent and S2a as a mobile phase. Detection by dragendorffʼs spraying reagent

S : Seeds R : Roots A : Aerial parts

A R S

Figure (3.3)- TLC chromatogram of fraction one (F-1) for different Echinops parts

( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S3a as a mobile phase. Detection by dragendorffʼs spraying reagent S : Seeds R : Roots A : Aerial parts

Chromatographic separation techniques are multi-stage separation methods in which the components of a sample are distributed between 2 phases, one of which is stationary, while the other is mobile(190). The separation may be based on adsorption, mass distribution (partition), ion exchange, etc., or may be based on differences in the physico- chemical properties of the molecules such as size, mass, volume(191).

All the techniques in chromatography depend upon the same basic principle i.e. variation in the rate in which different components of a specific sample migrates through a stationary phase under the influence of a mobile phase, so there is many factors affecting rates of migration , one of them; polarity of the mobile and

stationary phase(192). Polar mobile phase will cause desorption of the polar compound.

Silica gel and alumina are highly polar materials that adsorb molecules (specially polar one) strongly. Activity is determined by the overall polarity and the number of adsorption site. In silica gel, the adsorption sites are the oxygen atom and silanol groups (-Si - OH) which readily form H – bonds with polar molecules(193).

The previous TLC figures of fraction one (F-1) of different plant parts in three different mobile phase revealed that the three spots related to three different compounds. In the mobile phase S1a (Benzene : methanol) (8 : 2), E1 have the smallest Rf value (i.e. more adsorbed on the silica gel, so it is the more polar compound among the three compounds). E3 was moved more than E2 (i.e. E3 was contained functional groups make it less polar than E2, since the mobile phase was contained high percentage of benzene). The same idea was applied on the other mobile phases.

(b) The presence of two flavonoids (named EJ1 and EJ2) in the glycosidic linkage soluble in the aqueous fraction (F-2) obtained from aerial and root parts with one compound (EJ1) in the same fraction obtained from seed parts . These different compounds appeared as a single spot on TLC plates, using three different

developing solvent systems ( S1f , S2f,S3f ) and detected by UV in two different wave length 254, 366nm as indicated in figures from (3.4 to 3.6) .

The Rf values of these compounds in the different solvent systems were calculated, table (3.4)

Table (3.4)- Rf Values of Flavonoids (as Glycoside) Obtained From Different Plant Parts in Different Developing Solvent Systems in TLC.

Compound Plant part S1f S2f S3f EJ1 Aerial part 0.4 0.48 0.31 EJ2 Aerial part 0.47 0.52 0.38 EJ1 Roots 0.42 0.42 0.33 EJ2 Roots 0.42 0.57 0.39 EJ1 Seeds 0.44 0.43 0.37

100

101

Figure (3.4)- TLC chromatogram of fraction two (F-2) for different Echinops parts( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S1f as a mobile phase. Detection by UV-light at 254 and 366nm. S : Seeds R : Roots A : Aerial parts

102

Figure (3.5)- TLC chromatogram of fraction two (F-2) for different Echinops parts

( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S2f as a mobile phase. Detection by UV-light at 254 and 366nm.

S : Seeds R : Roots A : Aerial parts

103

Figure (3.6)- TLC of fraction two (F-2) for different Echinops parts

( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S3f as a mobile phase. Detection by UV-light at 254 and 366nm.

S : Seeds R : Roots A : Aerial parts

Fraction two (F-2) contains flavonoids in the glycosidic linkage (polar components) which were adsorbed strongly by silica gel, so polarity of mobile phase should be increased here to ensure the separation of different components since chromatographic separation is based on a balanced state among the components to be separated, an adsorbent agent in the stationary phase and a solvent flowing through it (mobile phase)(194).

104

A high adsorption capacity between the components of interest and the stationary phase means that there is a high retention of these components and that there is a considerable delay in the running from the base line by mobile phase. The separation of a mixture into its individual components is only possible if the individual components in a combination of stationary and mobile phases have different adsorption/desorption properties(195).

Retention factor "Rf" values of EJ1 and EJ2 compounds obtained by using S1f as a mobile phase (Ethyl acetate: formic acid : glacial acetic acid : water ) (100: 11: 11:27 ) were very closure to each other( i.e. there is small differences in the distribution of EJ1 and EJ2 molecules which are polar compounds between two phases, stationary and mobile phase. In the second mobile phase S2f ( n-butanol : glacial acetic acid : water ) (40: 10: 50 ), polarity of solvent systems increased ,so the more polar compound among EJ1 and EJ2 will run larger than the other "if the force of mobile phase overcome the retardation force of stationary phase" , but if the retardation stationary force will overcome, Rf values of more polar compound will be smaller than the other.

The third mobile phase (acetic acid : water) (15:85) gave bad separation, there is overlapping and tailing in the spots, this is may be due to the high percentage of water in this mobile phase.

(c) The presence of three flavonoids: quercetin, myricetin and kaempferol (as free aglycon) in fraction-3 obtained from aerial and root parts, and two flavonoids: myricetin and kaempferol in the same fraction obtained from seed parts. These compounds appeared as a single spot in three different developing system (S4f,

105

S5f, S6f) as shown in figures from (3.7 to 3.9 ). The spots of quercetin, myricetin and kaempferol having the same color and Rf values as that of standards on the TLC plates after detection by UV light in two different wave length 254, 366nm. Table (3.5).

Table (3.5)- Rf Values of Flavonoids (Quercetin, Myricetin and Kaempferol ) Obtained From Different Plant Parts and their Standard in Different Developing Solvent Systems in TLC.

Compound S4f S6f S5f Quercetin standard 0.46 0.82 0.62

Quercetin isolated from aerial parts 0.46 0.81 0.6

Quercetin isolated from roots 0.44 0.8 0.6

Myricetin standard 0.25 0.78 0.43

106

Myricetin isolated from aerial parts 0.23 0.77 0.41

Myricetin isolated from roots 0.24 0.77 0.41

Myricetin isolated from seeds 0.23 0.78 0.42

Kaempferol standard 0.71 0.87 0.74

Kaempferol isolated from aerial part 0.7 0.86 0.72

Kaempferol isolated from roots 0.69 0.86 0.71

Kaempferol isolated from seeds 0.68 0.85 0.73

107

Figure (3.7)- TLC of fraction three (F-3) for different Echinops parts

( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S4f as a mobile phase. Detection by UV-light at 254and 366nm.

M : Myricetin standard

K : Kaempferol standard

Q : Quercetin standard

S : Seeds A : Aerial parts R : Roots

108

109

Figure (3.8 a)- TLC of fraction three (F-3) for different Echinops parts ( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S5f as a mobile phase. Detection by UV-light at 254nm.

M : Myricetin standard

K : Kaempferol standard

Q : Quercetin standard

S : Seeds A : Aerial parts R : Roots

110

Figure (3.8 b)- TLC of fraction three (F-3) for different Echinops parts ( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S5f as a mobile phase. Detection by UV-light at 366nm.

M : Myricetin standard

111

K : Kaempferol standard

Q : Quercetin standard

S : Seeds A : Aerial parts R : Roots

112

Figure (3.9 )- TLC of fraction three (F-3) for different Echinops parts ( seeds, aerial parts, roots) using silica gel GF254nm as adsorbent and S6f as a mobile phase. Detection by UV-light at 254 and 366nm.

M : Myricetin standard

K : Kaempferol standard

Q : Quercetin standard

S : Seeds A : Aerial parts R : Roots

Among the three flavonoids found in the Iraqi Echinops plant, myricetin (3,3',4',5,5',7-hexahydroxyflavone) was more polar than quercetin (3,3',4',5,7- pentahydroxyflavone) and kaempferol (3,4',5,7-tetrahydroxyflavone). In S4f mobile phase (chloroform: acetone : formic acid) (75: 16.5 :8.5) and S5f (Toluene: chloroform : acetone) (40: 25: 35) Rf value of kaempferol was the largest one while the smallest Rf value was for myricetin since the polarity index of these both solvent systems was less than polarity of silica gel so a good separation was obtained .

Polarity of S6f solvent system (ethyl acetate : methanol: formic acid) (50: 50 :1),was increased by the existence of methanol, therefore bad separation was take place because the difference in the distribution of sample molecules between stationary phase and mobile phase was decreased.

(d) The presence of a number of steroidal compounds in the neutral fraction (F-4) obtained from aerial and root parts with the absence of these components in the

113

seeds, and presence of terpenoids components in the same fraction of all plant parts.

Stigmasterol and beta-sitosterol standards have very closer Rf value, so one (or two) of these steroidal compounds was identified as either stigmasterol or beta- sitosterol since they appeared as a single spot match with the spots of both standards in three different developing system (S1s, S2s, S3s) as seen in figures from (3.10 to 3.12 ) .Table (3.6)

Table (3.6)- Rf Values of Steroids (Stigmasterol and β-Sitosterol ) Obtained from Different Plant Parts and their Standards in Different Developing Solvent Systems in TLC.

Compound S1s S2s S3s Stigmasterol standard 0.75 0.8 0.83 β-Sitosterol standard 0.73 0.85 0.88 Steroid isolated from aerial 0.75 Upper spot 0.93 Upper spot 0.89 part Lower spot 0.8 Lower spot 0.88 Steroid isolated from root 0.75 Upper spot 0.93 one spot 0.86 Lower spot 0.79

114

Figure (3.10)-TLC of fraction four (F-4) for different Echinops parts (aerial parts, roots) using silica gel GF254nm as adsorbent and S1s as a mobile phase.

Visualization by Liebermann-Bur chard spray reagent, followed by heating for ه 10 mints at 105 C

S : Stigmasterol standard B : Beta-sitosterol standard

A : Aerial parts R : Roots

115

Figure (3.11)-TLC of fraction four (F-4) for different Echinops parts (aerial parts, roots) using silica gel GF254nm as adsorbent and S2s as a mobile phase. Visualization by Liebermann-Bur chard spray reagent followed by heating for 10 ه mints at 105 C

S : Stigmasterol standard B : Beta-sitosterol standard

A : Aerial parts R : Roots

116

Figure (3.12)-TLC of fraction four (F-4) for different Echinops parts (aerial parts, roots) using silica gel GF254nm as adsorbent and S3s as a mobile phase.

117

Visualization by Liebermann-Bur chard spray reagent, followed by heating for ه 10 mints at 105 C

S : Stigmasterol standard B : Beta-sitosterol standard

A : Aerial parts R : Roots

The TLC doesn’t give a clear idea about identity of steroidal compounds in this fraction. The only difference between stigmasterol and β‐sitosterol is the presence of C22=C23 double bond in the first one and C22 C23 single bond in the later one, hence; because of the lack of practical difference in their Rf values despite the use of several solvent systems , the GC-MS analysis was used to identified these components in both aerial and root parts.

3.4-Isolation and purification of different active constituents

3.4.1-Isolation and purification of alkaloids:

Two chromatographic analysis were carried out to isolate in a pure form three alkaloids (named E1, E2, E3) found in the plant which are: preparative HPLC and preparative TLC, since seeds contain the largest number and highest quantity of the alkaloids so alkaloids fraction obtained from seeds part was used to separate and isolate these compounds in a pure form.

3.4.1a- Isolation and purification of alkaloids by preparative HPLC

118

One gram (1 gm) of F-1 obtained from plant seeds was dissolved in a minimum quantity of chloroform and injected in to preparative HPLC using :- acetonitrile : water (65:35) as a mobile phase (experimental work)

Column: mediterranea C18 , 5 µm 15 X 2.12 cm.

Flow rate: 5 ml / min.

Injection volume: 1 ml.

Detection: UV. Detector at λ 254 nm.(experimental work)

Chromatogram gave three peaks which represent three different compounds one of them (E2) is a major peak. Each compound was collected by fractions collector after monitoring it according to the time (time from the beginning of each peak appearance until disappearance of peak). (Figure 3.13).

119

Figure (3.13 )- Preparative HPLC analysis of fraction-1 obtained from seeds plant observing three peaks represent three different compounds, one of them (E2) is a major one.

The three samples obtained from preparative HPLC were weighted and subjected to co-TLC as shown in figure (3.14).

Weight of E1 = 0.07 gm

Weight of E2 = 0.56 gm

Weight of E3 = 0.16 gm

120

Figure-(3.14) Co-TLC of three alkaloids (E1, E2, E3) isolated by preparative HPLC from fraction-1 (F-1) of seeds part using silica gel GF254nm as adsorbent and S1a as a mobile phase. Detection by dragendorffʼs spraying reagent.

3.4.1b Isolation and purification of alkaloids by preparative TLC

One gram (1 gm) of F-1 obtained from plant seeds (highest quantity) was dissolved in a minimum quantity of chloroform and applied on a number of preparative TLC plates using S1a solvent system. The solvent was allowed to rise to a height of 15cm from the base line. One major and two minor bands were observed after spraying a side of plates with dragendorffʼs reagent as shown in figure (3.15), three bands had been scrapped off, eluted with chloroform, then filtered. The filtrate evaporated to dryness, in vacuo to give white crystals, upon re-crystallization out of boiling ethyl acetate, a fluffy white crystals of E1, E2 and E3 were obtained. The three samples obtained from preparative TLC were weighted and subjected to co-TLC as shown in figures (3.16)

121

Weight of E1 = 0.037 gm

Weight of E2 = 0.247 gm

Weight of E3 = 0. 063 gm

Figure(3.15 )- Chromatogram of preparative TLC for fraction one (F-1) , using silica gel GF254 as adsorbent and S1a as a mobile phase. Detection by spraying a side of plates with dragendorffʼs spraying reagent.

122

Figure (3.16 ): Co-TLC of three bands (E1, E2, E3) isolated by preparative TLC from fraction-1 (F-1) of seeds part using silica gel GF254nm as adsorbent and S1a as a mobile phase. Detection by dragendorffʼs spraying reagent.

From the above results, the quantity of compounds obtained in a (pure form) by preparative HPLC is higher than that obtained by preparative TLC. Classical preparative TLC suffers from several drawbacks, the main disadvantage being the removal of purified substance from the plate and its subsequent extraction from the sorbent, other drawbacks include the length of time required for the separation and degree of purity for the separated compounds (196), compare with preparative HPLC, which is consider know, the most powerful and versatile (197) method for purification tasks in the pharmaceutical industry . Despite the fact that among the tools used in the large scale purification of pharmaceuticals, preparative HPLC is one of the more expensive and solvent-consuming approaches, it yields the highest-purity drug substance. The interest in preparative HPLC will continue to grow because of the increasing uncertainty in the market expectations for product purity. Its nearly linear scalability makes preparative HPLC one of the more viable approaches to compound purification(198).

3.4.1.2- Characterization and identification of the isolated alkaloids

(E1, E2 and E3):

3.4.1.2.1- M. P. :

The isolated compound which is named E1 had a sharp melting point of 145- .C هC and E3 of 123-125هC, while E2 had a melting point 160-162 ه146

123

3.4.1.2.2- U.V. spectra: The unsaturated heterocyclic compounds (hetero- aromatic compounds) like quinoline compounds usually show absorption in the near ultraviolet region 218, 265, 313nm in the cyclohexane(199). So the isolated compounds show strong new triplet-triplet absorption bands in the ultraviolet region and were assigned to transitions from the lowest triplet state to a triplet state which is doubly excited with respect to the closed shell ground state.

124

Figure (3.17 )- UV spectrum of the isolated alkaloids ( E1, E2, E3)

3.4.1.2.3- FT.IR spectra :

The identification of the unknown alkaloids ( E1, E2 and E3) was further confirmed by using FT-IR spectroscopy figures (3.18 to 3.20).

The characteristic IR absorption bands of the isolated alkaloids are listed in table(3.7).

Table (3.7)- Characteristic FT-IR Absorption Bands( in cm-1) of the Isolated Alkaloids(199)

Group Functional frequency Assignment group wave number ( in cm-1) (For E1: N-H 3302 N-H stretching(2ِ amine, one &very weak band C-H 3090 C-H aromatic

125

C-H 2976, 2943 Asymmetric and symmetric stretching of CH3

C=O 1681 C=O of quinolone N-H 1645 N-H bending

C=C 1593,1510,1491 C=C stretching of aromatic

For E2: =C-H 2875, 2773, C-H stretching C=O 2713 C=O stretching C-N 1660 C-N stretching C-H 1329 C-H of aromatic group out of plane 943, 862, 765 (For E3: N-H 3190, 3144 N-H stretching (two band for 1ِ amine C-H 2910, 2852 Asymmetric and symmetric stretching of CH3 N-H 1640 N-H bending C-N 1333, 1336 C-N stretching bands of tertiary amine C=C 1489, 1431 C=C aromatic stretching C-H 914, 815, 750 C-H of aromatic group out of plane

126

127

Figure (3.18 )- FT-IR spectrum of the isolated alkaloid (E1)

128

129

Figure (3.19 )- FT-IR spectrum of the isolated alkaloid (E2)

130

131

Figure (3.20 )- FT-IR spectrum of the isolated alkaloid (E3)

3.4.1.2.4- CHN :

Elemental microanalysis was performed for unknown isolated compounds alkaloids to confirm their chemical structure. The result of this analysis (table 3.8) illustreated that the unknown compounds consist of carbon , hydrogen, oxygen and nitrogen in different percentage.

Table( 3.8)- Elemental Microanalysis of the Unknown Isolated Alkaloids

Name C% calculator H% calculator O% calculator N% calculator (found) (found) (found) (found) E1 64.41% ---- 11.65 ---- 15 --- 8.99% ----- E2 74.07 77.41 6.208 7.09 10.25 10.3 9.463 9 E3 74.66 75.9 5.58 6.32 0 0 19.4 18

3.4.1.2.5- 1H &13C NMR analysis :

The E2 compounds presented 13C NMR spectra (DMSO, 75 MHz): with chemical shifts typical of quinoline rings(199) in the ranges of δC 21.12 (C-3), 24.77 (C-11), 170.12(C-4),126.987 (C-5), 121.825 (C-6), 127.640 (C-7), 114.951 (C-8), 138.26 (C-9), 123.47 (C-10), 30.4 (C-2).

Figure (3.21).

132

1H NMR (DMSO-d6-, 300 MHz) revealed that E2 compound undergo tautomerism which lead to the appearance of chemical shifts of the hydroxyl group at 10.02 at (C-4), 2.4 (3H, as a singlet of the methyl protons ), 2.6 (2H, d, H-2), 5.09 (1H, H-3), 6.84-7.15 (4H, m, H-5 ,H-6 , H-7 , H-8). Figure (3.22).

133

Figure (3.21 )- 13C-NMR analysis of the isolated E2 compound

134

135

H H

Figure (3.22 )- 1H-NMR analysis of the isolated E2 compound

136

Depending on the above results, the expected chemical structure for the isolated E2 compound is may be :

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

CH3 11

1-Methyl-2,3-dihydro-4(1H)-quinolinone, it is a new compound isolated (for the first time) from Iraqi Echinops heterophyllus plant, it seen to be the hydrogenated form of echinopsine (1-Methyl-4(1H)-quinolinone), an alkaloid isolated from 14 species of Echinops plant.

The E3 compounds presented 13C NMR spectra (DMSO, 75 MHz): with chemical shifts in the ranges of δC 152.194 (C-2), 146.076 (C-4), 134.140 (C-9), 130.383 (C-8),128.667 (C-7), 128.272 (C-6),127.684 (C-5), 127.254 (C-10), 126.372 (C-3), 18.042 carbon of methyl group (C-11). Figure (3.23).

1H NMR of E3 (DMSO-d6-, 300 MHz) gave the following results: δH 8.680 (1H, s, H-2), 7.965 (1H,d,H-8), 7.750 (1H,d, H-5), 7.607 (1H,d, H-6), 7.478 (1H,d,H-7),

137

4.070 (2H of amino group at carbon number-4), 2.314 (3H- singlet of methyl group at carbon number-3). Figure (3.24).

Figure (3. 23)- 13C-NMR analysis of the isolated E3 compound

138

H H H H H H H

H H

Figure (3.24 )- 1H-NMR analysis of the isolated E3 compound.

Depending on all previous chemical analysis, the expected chemical structure for the isolated E3 compound could be :

139

3-Methyl-4-amino-quinoline, it is a new compound isolated (for the first time) from Echinops plant, this is the first report of the occurrence of this alkaloid in this genus among all phytochemical investigation of different Echinops species .

All chemical analysis (M.P., FT.IR, CHN, 1H-NMR, 13C-NMR ) was done for the third alkaloid (E1) without reaching to the exact structure, since1H-NMR analysis indicated that there is a sugar molecules in the structure so it may be a type of glycoside alkaloid. Mass spectroscopy and two-dimensional NMR analysis are required for structure elucidation of E1 compound, there for its left for further study.

3.4.2.1.Isolation and purification of flavonoids glycoside by column chromatography (CC) :

One hundred fractions obtained from column chromatography of fraction-2 of aerial part (highest quantity) were monitored by TLC. The consecutive fractions that have the same number of spots and the same Rf values were combined to get 3 major fractions, which were concentrated to dryness , re-crystallized out of hot methanol and weighed, as listed in table(3.9).

In the first 17 fractions there was no indication for spots presence. Fractions (18- 25) gave one spot in the TLC and were collected to give the first fraction called fraction-A. Fractions (26-29) gave no spot, while fractions (30-55) gave one spot which were collected to give second fraction designated as fraction-B. Fractions (56-58) gave no spot, while fractions (59-79) gave one spot which were collected to give third fraction called fraction-C. fractions (80-100) gave no spot. Fractions B

140

and C gave positive result with lead-acetate test (flavonoids glycoside), while fraction-A gave negative test, so it is left for further study.

Selected chromatograms for the separated fractions B and C are illustrated in figure (3.25).

Table(3.9)- Major Fractions Obtained from Column Chromatography

Major fractions No.of collections No. of spots Weight (gm) 20ml each F-A 18-25 1 0.08 F-B 30-55 1 0.89 F-C 59-79 1 0.51

For further purification of the isolated compounds obtained from CC fractions which are named (EJ1 and EJ2), each isolated compound was dissolved in a hot methanol and a small amount of decolorizing charcoal was added so that the solution turns black, then the hot solution was poured through filter paper in to another flask, the solvent was evaporated to give solid product(200) , a pale yellow powder of EJ1(0.48gm) and dark yellow powder of EJ2(0.84gm)

141

Figure (3.25 )-TLC of fraction- B and C obtained from CC using silica gel GF254nm as adsorbent and S2f as a mobile phase. Detection by UV-light at 254nm.

3.4.2.2- Characterization and identification of the isolated flavonoids glycoside (EJ1 and EJ2)

3.4.2.2.1-M. P. :

The isolated compound EJ1 had a melting point at 160-161°C, while EJ2 showed a melting point at 195-197°C which is identical with that reported for rutin(119)

142

3.4.2.2.2- U. V. spectra:

Flavonoids contain conjugated aromatic systems and thus show intense absorption band in the UV and visible region of the spectrum. The first compound EJ1 gave maximal absorbance peaks at λmax 265 and 342 nm, which were characteristic of a flavonoid with a flavone skeleton while two major absorption bands at 359 and 370nm were appeared for EJ2 which indicate the presence of flavonol structure. Figure (3.26)

Figure (3. 26)- UV spectrum of the two flavonoids glycoside EJ1 and EJ2.

3.4.2.2.3- FT.IR spectra :

The characteristic IR absorption bands revealed by EJ1 and EJ2 are listed in table 3.10, figures (3.27 &28) , since absorption bands at 1675 and 3197 nm of the IR spectrum indicated where the molecule harbors conjugated carbonyl and hydroxyl groups, respectively.

143

Table(3.10)- Characteristic FT-IR Absorption Band (cm-1) of the Isolated EJ1 &EJ2(199)

Functional Group frequency Assignment group wave number (cm-1) EJ1 broad band (3600- O-H stretching of phenol O-H 3083) central at 3334 C-H 3052, 3142 C-H stretching of aromatic ring C=O 1660 C=O stretching of keton conjugated system C=C 1614-1569 C=C stretching of aromatic ring

O-H 1363 O-H bending of phenol

C-O-C 1130, 1124 C-O-C stretching

O-H 1296 O-H bending of alcohol

C-H 975, 885, 848 C-H of aromatic group out of plane

EJ2: broad band (3600- O-H stretching of phenol O-H 3070) central at 3334 C-H 3033, 3100 C-H stretching of aromatic

C=O 1652 C=O stretching of keton conjugated system C=C 1600-1592 C=C stretching of aromatic

144

O-H 1363 O-H bending of phenol

C-O-C 1132, 1124 C-O-C stretching

O-H 1296 O-H bending of alcohol

C-H 943, 879, 808 C-H of aromatic group out of plane

145

146

Figure (3.27 ) FT-IR spectrum of the isolated EJ1

147

148

Figure (3.28 ) FT-IR spectrum of the isolated EJ2

3.4.2.2.4- CHN analysis :

Elemental microanalysis was performed for unknown isolated compounds (EJ1, EJ2), and the data of this analysis indicated that both of them consist of carbon , hydrogen and oxygen in different percentage, (table 3.11)

Table( 3.11)- Elemental Microanalysis of the Unknown Isolated Flavonoids Glycoside

Compound C% (calculator) H% (calculator) O% (calculator) EJ1 56.21% 58.3% 4.46% 4.6% 39.25% 37.03% EJ2 53.11 % 53.1% 4.95% 4.91% 41.93% 41.9%

3.4.2.2.5-1H &13C NMR analysis :

The 1H NMR spectrum of the compound (EJ1) gave two aromatic hydrogen signals with ‘meta coupling’ at δ 6.20 (1H, s) and 6.42 (1H, s) which was predicted by the hydrogens at C-6 and C-8 of the A ring of the flavone skeleton. Accordingly, this compound was suggested to have a hydroxyl group at C-5 and C-7. Furthermore, its 1H NMR spectrum revealed two signals with ‘ortho coupling’ at δ 6.8 (2H, d) and 8.0 (2H, d), the signals of which were approximated from the hydrogens at C-2′, C-3′, C-5′ and C-6′ of the B ring. The absence of a specific signal for an olefinic hydrogen at C-3 and the presence of an anomeric hydrogen signal

149

at δ 5.24 (1H, d) suggested that the compound was a flavonol glycoside. The appearance of an anomeric carbon signal at δ 93.5 in the 13C NMR spectrum indicated the presence of a sugar moiety. Due to a correlation between the anomeric hydrogen signal (δ 5.24) and the anomeric carbon signal (δ 93.5) that was revealed by analysis of the heteronuclear multiple bond correlation (HMBC) spectral data obtained from other research(201), the position of the sugar moiety was assigned to the C-3 hydroxyl group. The methyl signal observed at δ 0.93 (3H, s) in the 1H NMR spectrum and at δ 17.19 in the 13C NMR spectrum indicated that the sugar moiety was rhamnose. Figures (3.29 a &b, 3.30 a&b).

Based on the accumulated data above, the compound (EJ1) was identified as kaempferol-3-O-rhamnoside:

150

Kaempferol-3-O-rhamnoside (C21H20O10, Mol wt. 432.38 g/mol) this is the second report of occurrence of this compound in the Echinops genus , the first report was in the Indian Echinops echinatus(135).

3- OH 2- 4-

8 - HO O - 5 9 1 1 7 2 6-

6 3 10 4 5 O = OH O C1 OH O = C2 = = C5 C3 HO = OH = C4 C6 OH

151

Figure (3.29a )- 13C-NMR analysis of the isolated EJ1 compound

152

153

Figure (3.29b )- Expansion of 13C-NMR analysis of the isolated EJ1 compound

154

3- OH 2- 4-

8 - HO O - 5 9 1 1 7 2 6-

6 3 10 4 5 O = OH O C1 OH O = C2 = = C5 C3 HO = OH = C4 C6 OH

155

Figure (3.30a )- 1H-NMR analysis of the isolated EJ1 compound

156

157

Figure (3.30b )- Expansion of 1H-NMR analysis of the isolated EJ1 compound

-־4 ,־13C and 1H-NMR for EJ2 showed identity with those reported for 5,7, 3 tetradydroxyflavonol glycoside with β- D – glucopyranoside and α -L – rahmnopyranoside moieties(199), and those reported for rutin isolated from Galium tortumense(202). The location of sugar moieties was deduced to be at C-3 position from the downfield shift of C-3 signal (δC 133.29) compared with that reported for the aglycone quercetin(203).

13C NMR (DMSO, 75 MHz): δC156.55 (C-2), 133.29 (C-3), 177.34 (C-4), 161.19 (C-5), 98.6 (C-6), 164.0 (C-7), 93.53 (C-8), 156.39 (C-9), 103.95 (C-10), 121.16 (C-1-), 116.24 (C-2-), 144.7 (C-3-), 148.36 (C-4-), 115.19 (C-5-), 121.54 (C-6-), 101.17 (C-1ʺ), 74.05 (C-2ʺ), 75.89 (C-3ʺ), 69.99 (C-4ʺ), 76.44 (C-5ʺ), 66.6 (C-6ʺ), 100.69 (C-1‴), 70.34 (C-2‴), 70.55 (C-3‴), 71.84 (C-4‴), 68.18 (C-5 ‴), 17.67 (C-6‴ ). Figure (3.31).

1H NMR (DMSO-d6-, 300 MHz): 12.6,10.8, 9.6, 9.1 for hydroxyl groups at (C-5, C-7,C-4′, C-3′),7.53 (1H, H-2′ ), 7.55 (1H, H-6′ ), 6.89(1H,H-5′), 6.38(1H, H- 8),6.198 (1H,H-6),) 5.23 (1H, d, H-1ʺ), 3.24-3.33 (4H, m, H-2ʺ ,H-3ʺ , H-4ʺ , H-5ʺ), 3.39 (1H, Ha-6ʺ ), 3.72 (1H, Hb-6ʺ), 4.47 (1H, H-1‴), 4.39 (1H, H-2‴), 4.34 (1H, H-3 ‴), 4.33 (1H, H-4‴ ), 4.29 (1H, H-5‴ ), 1.007 (3H, s, CH3-6‴ ). Figure (3.32). Based on the melting point and previous spectral analysis data (UV, FT-IR, CHN, 1HNMR and 13CNMR), the structure of this isolated compound was proposed as;

158

Rutin (C27H30O16, Mol wt. 610.53 g/mol), this is the first report of occurrence for rutin glycoside in the Echinops plant and specifically in the heterophyllus species.

3- OH - 2 4-

8 - B HO O 1 5- 9 1 2 - 7 C 6 A 3 6 10 4 5 O

OH O

O = C6 = H C5 O

OH = = C1 H C4 H

C /// OH 5 = = H O OH C3 C2 CH3 /// C6 /// OH /// C1 H C4

H /// /// H C3 C2 OH OH

159

Figure (3.31 )- 13 C-NMR analysis of the isolated EJ2 compound

160

3- OH - 2 4-

8 - B HO O 1 5- 9 1 2 - 7 C 6 A 3 6 10 4 5 O

OH O

O = C6 = H C5 O

OH = = C1 H C4 H

C /// OH 5 = = H O OH C3 C2 CH3 /// C6 /// OH /// C1 H C4

H /// /// H C3 C2 OH OH

161

Figure (3.32 )- 1H-NMR analysis of the isolated EJ2 compound

3.4.3.1.Isolation and purification of flavonoids as (aglycon) by preparative TLC :

Four grams (4 gm) of F-3 obtained from plant aerial parts (highest quantity) was dissolved in a minimum quantity of methanol and applied on a number of preparative TLC plates using S4f solvent system. The solvent was allowed to rise to 162

a height of 14cm from the base line. The separated bands of myricetin, quercetin and kaempferol were observed under UV light according to the references standard compounds. The three separated bands had been scrapped out, collected separately and crystallized out of hot methanol eluted to give yellow crystals from each band. (figure 3.33 ).

Figure (3.33 )- Chromatogram of preparative TLC for fraction-3 , using silica gel

GF254 as adsorbent and S4f as a mobile phase. Detection by UV-light at 254nm.

M : Myricetin Q : Quercetin K : Kaempferol 3.4.3.2- Characterization and identification of the isolated myricetin, quercetin and kaempferol

163

3.4.3.2.1- TLC:

The characterization of the isolated myricetin, quercetin and kaempferol was done by using TLC analysis. It was done by using (myricetin, quercetin , kaempferol) as standards reference and S4f as a mobile phase. The three isolated compounds appeared as a single spot having the same color and Rf value as that of reference standards as shown in figures (3.34 to 3.36).

3.4.3.2.2- M. P.:

The isolated compounds were identified to be myricetin, quercetin and kaempferol from their sharp melting point, Since the isolated myricetin had a C compared to myricetin standard melting pointهsharp melting point of 355-356 C compared هC(119). The other compound showed a melting point of 313 – 314ه357 C for standard quercetin(119), while 3rd one of theseه to melting point 316 C compared to kaempferol هcompounds showed a melting point of 274 – 275 .(C(119 هstandard melting point 276- 278

164

Figure (3.34)- TLC chromatogram of qualitative analysis of isolated myricetin, using silica gel GF254 as adsorbent and S4f as a mobile phase. Detection by UV- light at 254nm.

A: isolated myricetin S: reference standard

M: mixed spot of the isolated compound and the reference standard

165

Figure (3.35)- TLC chromatogram of qualitative analysis of isolated quercetin, using silica gel GF254 as adsorbent and S4f as a mobile phase. Detection by UV- light at 254nm.

A: isolated quercetin S: reference standard

M: mixed spot of the isolated compound and the reference standard

Figure (3.36)- TLC chromatogram of qualitative analysis of isolated kaempferol, using silica gel GF254 as adsorbent and S4f as a mobile phase. Detection by UV- light at 254nm.

A: isolated kaempferol S: reference standard

M: mixed spot of the isolated compound and the reference standard

166

3.4.3.2.3- U.V. spectra :

The isolated flavonoids show intense absorption band at 359 and 370nm which indicated the presence of flavonol structure. The first absorption maximum can be considered as originating from π-π* transitions in the ring A (aromatic system) and the second absorption maximum observed around 370nm, which may be assigned to transitions in ring B (cinnamayl system); this band appeared broad as a result of overlapping with LMCT band.(204,205) . Figure (3.37)

167

Figure(3.37)- UV spectrum of the isolated flavonoids (myricetin, quercetin, kaempferol)

3.4.3.2.4- FT- IR :

For further characterization of flavonoids (as aglycon) isolated from Iraqi Echinops plant , infrared – spectroscopy analysis was done for isolated compounds, using myricetin, quercetin and kaempferol standards as references .Figures(3.38 to 3.40).

The spectrum of the samples (isolated myricetin, quercetin and kaempferol) showed the significant group frequencies listed in table(3.12)

Table(3.12)- Characteristic FT-IR Absorption Band (cm-1) of the Isolated Flavonoids(199)

Functioal Isolated Isolated Isolated Assignment

168

group myricetin quercetin kaempferol O-H Broad band 3410-3321 Broad band O-H stretching of (3614-3101) (3414-3093) phenol central at central at 3317 3346 C=C-H 2979 2982 C-H stretching of aromatic ring C=O 1662 1664 1662 C=O stretching of keton conj. sys. C=C 1618 1610 1612 C=C stretching of conj. sys. O-H 1377 1381 1381 O-H bending of phenol C-O-C 1108 1132 1130 C-O-C stretching of ether C-H 854, 829, 864, 823, 885, 844, C-H of aromatic 769 792 796 group out of plane

169

170

Figure (3.38 ) FT-IR spectrum of the isolated myricetin

171

172

Figure (3.39) FT-IR spectrum of the isolated quercetin

173

174

Figure (3.40 ) FT-IR spectrum of the isolated kaempferol

175

3.4.3.2.5- HPLC analysis.

The isolated myricetin, quercetin and kaempferol were identified by HPLC method and compared with standard compounds using hyperclone ODCC C18 V-25cm column and a mixture of methanol: water (70:30 ratio) as a mobile phase with a flow rate of 0.5ml/min, and detected at 320 nm. In HPLC, qualitative identifications were made by comparison of retention times obtained at identical chromatographic conditions of analyzed samples and authentic standards. The information obtained from HPLC method of analysis reveal that myricetin and kaempferol were found in all plant parts while quercetin found in the aerial and roots part, and there was large differences in the percentage of these components between different plant parts, as shown in figures (from 3.41 to 3-49).

The percentage of these isolated compounds were calculated from each plant part extract relevance to the information given in section (2.4.4.7) and summarized in table (3.13).

Table (3.13)- Percentage of Flavonoids in the Different Plant Parts.

Plant part % of myricetin % of quercetin % of kaempferol Aerial parts 0.23 0.18 0.11 Roots 0.16 0.09 0.06 Seeds 0.015 --- 0.027

118

Generally, the percentage of myricetin, quercetin and kaempferol is higher in the aerial parts compared with roots and seeds. The percentage of these pharmacological active components in the Iraqi species could be considered a good percentage if compare with other species like Cameroonian species Echinops giganteus root which contained 0.27% flavonoids(62)and Egyptian Echinops spinosissimus, aerial parts only contained high percentage of flavonoids(50).

119

Figure(3.41)- HPLC of aerial parts

120

Figure(3.42)- HPLC of roots parts

121

Figure(3.43)- HPLC of seeds parts

122

Figure (3.44)-HPLC of myricetin standard.

123

Figure (3.45)- HPLC of isolated myricetin

124

Figure (3.46)-HPLC of quercetin standard.

Figure (3.47)- HPLC of isolated quercetin

125

Figure (3.48)-HPLC of kaempferol standard

126

Figure (3.49)- HPLC of isolated kaempferol 3.4.4-Identification of steroids by gas chromatography–mass spectrometry (GC-MS) analysis

Since the TLC does not give a clear idea about the content of steroidal compounds in fraction-4 obtained from both the aerial parts and roots , so GC- MS was used to identified steroidal compounds in these two parts.

The GC-MS spectrum of aerial plant parts (figures 3.50 a, b &c) exhibited a prominent molecular ion peak at m/z 413 [M]+ that correspond to molecular formula of stigmasterol (C29H48O). Ion peaks were also observed at m/z 380,

352, 303, 300,271,213, 199, 133, 97, 83, 43, which are in good agreement with reported values of the structure of stigmasterol(171,206,207), the ion peak m/z 271 due to the formation of carbocation by β bond cleavage of side chain leading to the loss of C10H21 that corresponds to the M‐141(176).

The same spectrum also showed strong peak appeared at m/z 415 [M]+ that correspond to molecular formula of β-sitosterol (C29H50O) and other prominent peak appeared at m/z 330 which is characteristic for sterols with C5-C6 double bond. Other peaks were also found in conformity with those reported for beta-sitosterol(171,176, 207,208).

127

β-Sitosterol Stigmasterol

(C29H50O; Mol.Wt. 414.71) (C29H48O; Mol.Wt: 412.69)

Figure-(3.50a) GC-MS analysis of aerial parts of Echinops plant

128

% 140

130

120

110

100 413

80 83 70 271 60

50 133 300 40 97

30 213 43 20 352 199 380 10

0 457 503 539 590 611 645 698 730 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750

Figure-(3.50b) GC-MS analysis of aerial parts of Echinops plant that exhibited a prominent molecular ion peak at m/z 413

129

150

140

130

120

110

100 415

50 330

43 95 40 81 213 397 145 255 30 303 178 20

0 475 503 534 584 598 648 673 750 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750

Figure-(3.50c) GC-MS analysis of aerial parts of Echinops plant that exhibited a prominent molecular ion peak at m/z 415

The GC-MS spectrum of plant roots (figures 3. 51 a, b &c ) exhibited the same results obtained from the aerial parts (i.e. a prominent molecular ion peak at m/z 413 [M]+ that correspond to molecular formula of stigmasterol and

130

other peak at m/z 415 [M]+ that correspond to molecular formula of β- sitosterol with a fragmentation pattern characteristic for sterols.

Figure-(3. 51a) GC-MS analysis of roots part of Echinops plant

131

140

130

120

110

100 413

70 83 60 271

50 133 300 40 97 30 213 20 43 351 199 380 10

0 448 503 546 579 604 646 673 714 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750

Figure-(3.51b) GC-MS analysis of root parts of Echinops plant that exhibited a prominent molecular ion peak at m/z 413

140

130

120

110

100 415

40 207 397 43 81 95 329 145 255 30 303 178 20

0 465 503 546 581 625 647 674 735 50 100 150 200 250 300 350 400 450 500 550 600 650 700 750

132

Figure-(3.51c) GC-MS analysis of root parts of Echinops plant that exhibited a prominent molecular ion peak at m/z 415.

The typical plant sterols, β-sitosterol and stigmasterol, appeared as main sterol components in the steroidal fraction of both aerial and roots part of Iraqi Echinops species, and from the high of peak of both compounds, they exist in a good quantity in the Iraqi heterophyllus species.

133

Chapter three Results & discussion 3.5- A relative assess on wound healing activity of crude Echinops extract and some of its bioactive fractions.

3.5.1- Visual remarks:

Group one ( untreated control group): normal healing took place at day

15 which involves continuous cell–cell and cell–matrix interactions that allow the process to proceed in three overlapping phases : inflammation (0–3 days), cellular proliferation (3–12 days) and remodeling (3–6 months) , so in this group there is some inflammatory signs seen from the first day with partial wound closure starting from the 4th day and some scar tissue at the 12th day. Figures(3.50-3.52).

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Chapter three Results & discussion

Figure (3.50)- Group-1 day-1. Figure(3.51) Group-1 day-6.

119

Chapter three Results & discussion

Figure (3.52)- Group-1 day-12

Group two ( rabbits surgically wounded and then treated with crude Echinops extract) : healing signs were very clear starting from day one. The complete fading of any inflammatory signs at day four and six. Finally, a complete wound closure at day twelve. Figures(3.53-3.55).

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Chapter three Results & discussion

Figure (3.53)- Group- 2 day-1 Figure (3.54)- Group-2 day-6

121

Chapter three Results & discussion

Figure (3.55)- Group 2-day-12

Group three ( rabbits surgically wounded and then treated with alkaloid fraction): remarkable wound healing signs from day one as there was no

122

Chapter three Results & discussion inflammatory signs. At day six wound edges started to convene. Lastly, an absolute wound closing at day twelve. Figures (3.56- 3.58).

Figure (3.56)- Group 3- day -1 Figure (3.57)- Group 3- day -6

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Chapter three Results & discussion

Figure (3.58)- Group -3 day-12

Group four ( rabbits surgically wounded and then treated with flavonoids fraction): mild inflammatory signs occurred at day one. A gradual healing appeared from day four till day twelve and few scar tissue at day 12th. Figures (3.59-3.61).

Figure (3.59)- Group -4 day-1 Figure (3.60)- Group-4 day-6

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Chapter three Results & discussion

Figure (3.61)- Group-4 day -12 3.5.2 Histology:

Results of histological examination demonstrated that the treatment group with the crude Echinops extract gave the best results, while the alkaloids bioactive fraction was more potent than flavonoids bioactive fraction. The following figures demonstrates these results:

Group one (untreated control rabbits): normal histological signs was observed at day 15. Figures(3.62-3.64).

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Chapter three Results & discussion

Figure (3.62)- Group one day one : ( X 200) a very clear appearance of inflammatory signs at the wound area during the first 24hrs, manifested by red arrows. Fibrous threads took place and new epidermal layer formed the marginal ends started to thicken.

Figure (3.63)- Group one day six: inflammatory signs are still obvious (red arrow). Collagen fibers formation still not organized.

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Chapter three Results & discussion

Figure (3.64)- Group one day twelve : there was no signs of inflammation (red arrow). There was also an increase in collagen proliferation. Wound is not healed yet.

Group two ( wounding with treatment with crude Echinops extract). Figures (3.65-3.67).

Figure (3.65)-Group two- day one: a selection of skin showing few amounts of inflammatory cells at the upper dermis layer and oedema (red arrow), a clear migration of epidermal cells.

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Chapter three Results & discussion

Figure (3.66)- Group two- day six: normal healing of dermis and epidermis layer (yellow arrow) .

Figure (3.67)- Group two- day twelve : there was a full thickness epidermal regeneration which covered completely the wound area (yellow arrow) .

Group three ( wounding with treatment with alkaloids fraction). Figures (3.68- 3.70).

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Figure (3.68)- Group three day one: some inflammatory cells at the upper dermis layer and oedema, (red arrow).

Figure (3.69)- Group three day six: marked infiltration of the inflammatory cells (red arrow) , increased blood vessel formation and enhanced proliferation of cells as a result of treatment with alkaloids fraction.

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Figure (3.70)- Group three day twelve: no inflammation, accumulation of granulation tissue (black arrow) , increase in the tensile strength. No scar formation.

Group four ( wounding with treatment with flavonoids fraction). Figures (3.71- 3.73).

Figure (3.78)- Group four day one: a very clear appearance of inflammatory signs (red arrow) with oedema .

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Figure (3.72)- Group four day six : almost complete healing. (yellow arrow)

Figure (3.73)- Group four day twelve: there was no signs of inflammation, accumulation of granulation tissue, increase in the tensile strength. (black arrow).

The present study was carried out to evaluate the effects of Echinops extract on the healing of experimentally induced wounds in rabbit . Collagenation, wound contraction and epithelization are crucial phases of wound healing. The phases of inflammation, macrophagia, fibroblasia and collagenation are intimately

131

Chapter three Results & discussion interlinked. Thus, intervention at any one of these phases using drugs could eventually either promote or inhibit one or all phases of healing . Herbal drugs have come to be increasingly used worldwide because of their effectiveness and safety(206). Echinops is a medicinally useful plant with many therapeutic properties like anti-oxidant , anti-inflammatory, anti-microbial and antifungal activities due to different secondary metabolites constituents like flavonoids, essential oil, alkaloids , sterols and others . The characteristic antioxidant properties of Echinops may serve to promote healing at the wound site. It was demonstrated that diverse mechanisms may be involved in the genesis of inflammatory reactions(207). Alkaloids showed also anti-inflammatory action which helps to accelerate wound healing , also antimicrobial effects of different constituents of Echinops plant constitute a further basis for wound healing activity. Indeed, β- sitosterol, a bioactive constituent found in Echinops, has been used in the wound healing and as anti scar agent since it(208) :

Inhibits hyperplasia of fibroblasts which results in scar formation. Promotes epithelial cell growth so as to maintain a normal, balanced ratio of fibroblasts to epithelial cells. Promotes remodeling of scar by enhancing microcirculation in the scar. Provides nutrients to promote regeneration of skin with normal physiological structure and function, such as restoration of hair follicles and the sebaceous gland. Inhibit enzymes associated with scarring. The results of this study showed that wound healing and repair was accelerated by applying crude extract of Echinops plant , which was highlighted by the full thickness coverage of the wound area by an organized epidermis. The enhanced capacity of wound healing with the plant could be explained on the basis of anti-inflammatory effects of the active constituents of the plant (quinoline

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Chapter three Results & discussion alkaloids, flavonoids, steroids and terpenoids), and since crude extract contained most of the active components ( alkaloids, flavonoids, sterols, terpenoids) , so it is more effective than the other fractions. So it can be concluded that this study is a good step to show that Echinops extract is effective in stimulating the enclosure of wounds and as anti-scar agent.

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Conclusion and Recommendation

Conclusion

1. Phytochemical investigation of a new wild Iraqi plant used traditionally for wound healing and snake bit named Echinops heterophyllus was done and the results revealed the presence of alkaloids, flavonoids, terpenoids and steroids in the different plant parts and in a different percentages, aerial parts contain the highest quantity of flavonoids, while seeds contain the highest amount of alkaloids. 2. Two chromatographic analysis were carried out to isolate in a pure form three alkaloids from seeds part (which contain highest quantity) : preparative HPLC and preparative TLC, where the quantity of compounds obtained by preparative HPLC was higher than that isolated by preparative TLC. 3. This is the first report of the occurrence of two quinoline alkaloids in the Iraqi Echinops plant which are : 1-methyl-2,3-dihydro-4(1H)-quinolinone and 3-methyl-4-amino-quinoline .

4. Two flavonoids glycoside "kaempferol-3-O-rhamnoside, "quercetin-3-O- rutinoside" and three flavonoids as free aglycon " myricetin, quercetin, kaempferol" were isolated from aerial part by column chromatography and preparative thin layer chromatography, respectively and identified by different physio-chemical and spectral analysis. 5. The quantities of flavonoids glycoside or as "free aglycon" was highest in the aerial part 6. This study demonstrates the positive effect of Echinops heterophyllus on the wound healing and provides a scientific support for the claimed ethenomedical uses of plant extracts in the treatment of wound, burn , snack bit and suggest its

potential as an antimicrobial and anti-scar agent that could be useful in the current search of such drugs from natural plants.

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Recommendation 1. Further chemical analysis is required to identify the exact chemical structure of the third alkaloid isolated from plant seeds like mass-spectroscopy and two- dimensional 1H and 13C NMR. 2. Investigation of other terpenoids and volatile oil content in the different parts of Iraqi Echinops plant. 3. Studying parameters affecting the production of biologically active compounds including season, environmental condition, chemical agent as well as genetic modification that could be achieved to enhance their production if possible. 4. Other studies are needed to determine the antimicrobial activity of crude extract and different fraction obtained from the plant at different concentration . 5. Further pharmacological and cytotoxicity studies specially on isolated alkaloids

are recommended. 6. The benefit of preparative HPLC to isolate the maximum amount of desirable products at a desired purity in a minimum of time from different Iraqi medicinal plants to use it as a standard reference or as lead structures for the design of useful drugs in the future studies .Preparative HPLC can be used in pharmaceutical development for troubleshooting purposes or as part of a

systematic scale-up process. 7. The application of plant tissue technique on this plant to increase the production

of therapeutically active compounds.

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الخالصة

َباث شٕك اندًم )شكشٔكت( ْٕ َباث بشي ٌُخًً انى انعائهت انًشكبت ًٌُٕ بظٕسة طبٍعٍت بشكم ٔاسع فً شًال انعشاق ٌسخخذيّ عايت انسكاٌ نعالج اندشٔذ ٔضذ نسعاث االفاعً. نظرا لعدم وجود دراسات حول هدا الجنس فً العراق ,لذلك اصبح من االهمٌة دراسة التركٌب الكٌمٌائً لبعض المركبات المهمة الموجودة فً هدا النبات التً قد تكون لها فعالٌة دوائٌة مهمة وراث يشدٔد اقخظادي . فً هذه الدراسة تم استخالص وكشف وفصل وتنقٌة بعض المركبات المهمة من الناحٌة االحٌائٌة ٔانخً حُخًً انى يدايٍع كًٍٍائٍت يخخهفت ) انقهٌٕذاث, الفالفٌنوٌدات ,انخشبٍُاث , ٔاالسخٍشٌٔذاث (فً اخضاء انُباث انًخخهفت ) انبزٔس ,االخضاءانٕٓائٍت, اندزٔس (. اٌ عًهٍت انكشف انُٕعً انخًٍٓذي نالٌضاث انثإٌَت انًخخهفت يٍ قبم كشٕفاث كًٍٍائٍت يحذدة قذ حًج عهى انًسخخهض االٌثإَنً الخضاء انُباث انًخخهفت ٔاشاسث انُخائح اٌ كم اخضاء انُباث يحخٌٕت عهى انقهٌٕذاث, انفالفٌٍُٕذاث , ٔانخشبٍُاث بُسب يخخهفت باالضافت انى ٔخٕد يشكباث االسخٍشٌٔذاث فً االخضاء انٕٓائٍت ٔ اندزٔس فقظ .

حى اسخخذاو انطشٌقت انعايت نهعانى خفشي ْاسبٕسٌ فً اسخخالص اخضاء انُباث انًخخهفت ٔ حدضئخٓا انى ٔ حى انحظٕل . soxhletاخضاء يخخهفت باسخعًال المذٌب العضوي اٌثإَل بنسبة 80% فً خٓاص عهى اخضاء يخخهفت:

انزي ٌحخٕي عهى انقهٌٕذاث. : االول الجزء

الثاني: انًحخٕي عهى الفالفٌنوٌدات فً انشابظ اندالٌكٕسٍذي. الجزء

الجزء الثالث: انًحخٕي عهى الفالفٌنوٌدات بدون رابط خالٌكٕسٍذي.

انًحخٕي عهى يشكباث سخٍشٌٔذٌت. : الجزء الرابع

تم البحث االولً عن المركبات المختلفة باستخدام تقنٌة كروماتوغرافٌا الطبقة الرقٌقة باستخدام مذٌبات مختلفة كوسٌط ناقل والكشف عنها اما باستخدام االشعة فوق البنفسجٌة او استخدام كشوفات كٌمٌائٌة ( يع اثٍٍُ يٍ E1,E2, E3 معٌنة وكانت النتائج كما ٌلً: احخٕاء انبذٔس عهى ثالثت قهٌٕذاث سًٍج ) فً االخضاء انٕٓائٍت ٔانخً حى فظهٓا بطشٌقخٍٍ: E1( ٔكًٍت قهٍهت يٍ E1,E2انقهٌٕذاث فً اندزٔس)

ولمعرفة تركٌبها الكٌمٌائً ووزنها Preparative TLC & preparative HPLC الجزٌئً استخدمت تقنٌات التحلٌل الطٌفً للمركبات وٌشمل مطٌاف االشعة فوق البنفسجٌة ،ومطٌاف االشعة تحت الحمراء وكذلك التحلٌل الطٌفً الكتلً واستخدام الرنٌن انزري المغناطٌسً نزرة الهٌدروجٌن1 والرنٌن انزري المغناطٌسً نررة الكاربون13 حٌث تم تحدٌد التركٌب الكٌمٌائً للمركبات وهو:E2, E3

1-methyl-2,3-dihydro-4(1H)-quinolinone(E2)

3-methyl-4-amino-quinoline (E3) ( على الرغم من اتمام جمٌع E1اجرٌت محاولة غٌر ناجحة لتحدٌد التركٌب الكٌمٌائً الدقٌق للمركب ) التحالٌل السابقة , لذا تم تركه لدراسة مستقبلٌة.

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تم فصل مركبٌن من الفالفٌنوٌدات فً انشابظ اندالٌكٕسٍذي بشكل نقً من األجزاء الهوائٌة باستخدام طرٌقة كروموتوغرافٌا العمود والتعرف على التركٌب الكٌمٌائً لهما عن طرٌق اجراء جمٌع التحالٌل rutin و rhamnoside-3-0 – kaempferolالسابقة وْران المركبان هما

كما تم الكشف عن الفالفٌنوٌدات )الكورستٌن ,الكامبفٌرول والماٌرستٌن( باستخدام تقنٌة كروماتوغرافٌا الطبقة الرقٌقة ، باستخدام مذٌبات مختلفة كوسٌط ناقل والكشف عنها باستخدام االشعة فوق البنفسجٌة ،وكذلك تقنٌة كروماتوغرافٌا االداء العالً السائلة فً االجزاء الهوائٌة وانخزور مع وجود الكامبفٌرول والماٌرستٌن فقط فً انبرور وبعدها تمت عملٌة الفصل والتنقٌة.

للجزء الرابع للكشف عن وجود سخٍشٌٔذاث فً TLCاستخدمت تقنٌة كروماتوغرافٌا الطبقة الرقٌقة الجزء الهوائً والجذور لكن لم ٌعط صورة واضحة عن هوٌة هذٌن المكونٌن لذا تم تركه لدراسة مستقبلٌة

Echinops heterophyllus تضمنت هذه الدراسة اٌضآ الكشف عن فعالٌة المستخلص الخام لنبتة العراقٌة المحلٌة وبعض اجزائها )الجزء الدي ٌحتوي على انقهٌٕذاث وجزء الفالفٌنوٌدات فً شفاء الجروح وكعامل مضاد للندوب . تم استخدام اربعة وعشرون ارنبا ذكرا بالغا تتراوح اعمارها بٌن ستة شهور الى سنة, وقد تم تقدٌر هذا التؤثٌر مرئٌا ومن خالل التغٌرات النسٌجٌة التً تصٌب النسج . تمت المعالجة ثالث مرات ٌومٌا بتركٌز 50% باستعمال مسحة قطنٌة. حٌث اظهرت النتائج ان مستخلص ٌعجل من عملٌة شفاء الجرح فً مجموعات المعالجة بالمقارنة مع مجموعات غٌر Echinops معالجة. اعطت المجموعة التً تم عالجها بالمستخلص الخام افضل النتائج. الجزء انقهٌٕذي كان اكثر فاعلٌة من جزء الفالفٌنوٌدات فً معالجة الجرح . لم تظهر كال المجموعتٌن المعالجة بالمستخلص الخام وانقهٌٕذاث تكون الندب لذا من الممكن ان نستنتج ان هذه الدراسة هً خطوة جٌدة لبرهنة ان مستخلص فعال فً تحفٌز التئام الجروح وكعامل مضاد لتكون الندب.Echinops

جمهورٌة العراق وزارة التعلٌم العالً والبحث العلمً جامعة بغداد- كلٌة الصٌدلة

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دراسة كٍمٍبئٍة واختببر فعبلٍةبعض المزكببت الفعبلة لنببت شوك الجمل الذي ٌنمو بزٌب فً العزاق على عملٍة التئبم الجزوح

أطروحة مقدمة الى فرع العقاقٌر والى لجنة الدراسات العلٌا فً كلٌة الصٌدلة - جامعة بغداد كجزء من متطلبات الحصول على درجة دكتوراه فً علوم الصٌدلة ) العقاقٌر( من قبل ايناس جواد كاظم

)ماجستٌر عقاقٌر 2001( اشراف األستاذ الدكتور عالء عبد الحسٌن عبد الرسول )مشرف اول ( األستاذ المساعد الدكتورة زٌنب جلٌل عواد )مشرف ثانً (

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