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 انشح انشحى

ن ٱ لله ٱشفع ﴿ ا ه ءاي ز

نز ٱ ى كه ي هى نع ٱ ا أحه

لله ٱ ج خ س د ا ح ب هه ع

﴾ ش ب خ

طذق هللا انعظى

۱۱سورة المجادلة : االة

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:

6

Dedication

TO

My Parents, Husband,

Sisters,Brothers,my sweet daughters

"Sarah & Rand

" and all my Friends with

respect

Enas

7

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.

8

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.

9

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

Allah.

Enas

10

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

11

1.3.4 The Folkloric uses 9

1.4

Pharmacological activities of different species of

Echinops extracts

11

1.4.1

Antibacterial activity 11

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-

induced prostatic hyperplasia

16

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

12

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

13

CHAPTER TWO : EXPERIMENTAL WORK

2.1 Reagents and Materials 39

2.2 Instruments 40

2.3 Plant material 41

2.4

2.4.1

Experimental work

Preliminary qualitative phytochemical analysis

41

42

2.4.2 Extraction and fractionation of different active

constituents 44

2.4.3 Isolation and purification of different active

constituents

48

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

compounds

52

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

14

2.4.4.6 One proton and thirteen carbon nuclear magnetic

resonance spectroscopy1H and 13C (NMR) analysis

53

2.4.4.7 Qualitative and quantitative estimation of isolated

compounds by HPLC

53

2.4.5 Investigation of some pharmacological activity of

the different isolated fractions

54

CHAPTER THREE : RESULTS and DISCUSSION

3.1 Preliminary qualitative phytochemical analysis 57

3.2 Extraction and fractionation of different active

constituents

58

3.3 Preliminary identification of different Echinops parts

by TLC

60

3.4 Isolation and purification of different active

constituents

78

3.4.1 Isolation and purification of alkaloids

78

3.4.1a Isolation and purification of alkaloids by preparative

HPLC

78

3.4.1b Isolation and purification of alkaloids by preparative

TLC

80

3.4.1.2 Characterization and identification of the isolated

alkaloids (E1, E2 and E3)

82

3.4.1.2.1 M. P. 82

15

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

column chromatography (CC)

93

3.4.2.2 Characterization and identification of the isolated

flavonoids glycoside (EJ1 and EJ2)

95

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)

by preparative TLC

109

3.4.3.2 Characterization and identification of the isolated

myricetin, quercetin and kaempferol

110

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

16

LIST

OF

FIGU

RES

3.4.3.2.5 HPLC analysis 118

3.5 A relative assess on wound healing activity of crude

Echinops extract and some of its bioactive fractions

124

3.5.1 Visual remarks 124

3.5.2 Histology 129

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

substitution may occur

27

17

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

alkaloids

49

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

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

adsorbent and S1a as a mobile phase.

61

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

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

adsorbent and S2a as a mobile phase.

62

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

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

adsorbent and S3a as a mobile phase.

62

18

3.4 TLC 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.

65

3.5 TLC 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.

66

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.

67

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.

70

3.8a 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

71

3.8b 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

72

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.

73

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.

75

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..

76

19

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..

77

3.13 Preparative HPLC analysis of fraction-1 obtained from seeds

plant

79

3.14 Co-TLC of three alkaloids (E1, E2, E3) isolated by

preparative HPLC .

80

3.15

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

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

81

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.

81

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

20

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

compound

103

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

silica gel GF254 as adsorbent and S4f as a mobile phase

109

3.34 TLC chromatogram of qualitative analysis of isolated

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

111

21

mobile phase

3.35 TLC chromatogram of qualitative analysis of isolated

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

mobile phase.

111

3.36 TLC chromatogram of qualitative analysis of isolated

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

mobile phase.

112

3.37 UV spectrum of the isolated flavonoids (myricetin,

quercetin, kaempferol)

113

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

22

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

23

LIST OF TABLES

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

24

NO. Table Page

1.1 Antibacterial, antimicrobial and antiviral activities of

flavonoids

31

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

Echinops heterophyllus

57

3.2 Percentage of different fractions obtained from different plant

parts (seeds, aerial parts, roots) 59

3.3 Rf values of alkaloids obtained from different plant parts in

different developing solvent systems in TLC 60

3.4 Rf values of flavonoids (as glycoside) obtained from different

plant parts in different developing solvent systems in TLC 64

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.

69

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

75

3.7 Characteristic FT-IR absorption bands( in cm-1

) of the

isolated alkaloids 84

3.8 Elemental microanalysis of the unknown isolated alkaloids 88

25

3.9 Major fractions obtained from column chromatography 94

3.10 Characteristic FT-IR absorption band (cm-1) of the isolated

EJ1 &EJ2

96

3.11 Elemental microanalysis of the unknown isolated flavonoids

glycoside

100

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

flavonoids

114

3.13 Percentage of flavonoids in the different plant

parts.

118

26

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

27

min Minutes

nm Nanometer

Rf value Retention factor (mobility relative to solvent

front)

silica gel

GF254nm

Silica gel with gypsum & fluorescence material

TLC Thin layer chromatography

U.V Ultra violet spectra

28

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.

29

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 (13

C-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 13

C-NMR.

Three flavonoids (myricetin, kaempferol , and quercetin,) were detected in

TLC of fraction-3 obtained from aerial parts and roots and the two former

30

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 .

31

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)

.

32

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

33

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

34

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"

35

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.

36

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

37

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,

38

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)

.

39

Figure (1.1) - Iraqi Echinops heterophyllus

40

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)

.

41

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)

.

42

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.

43

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

16 μg/ml, respectively against H37Ra and H37Rv(55)

.

44

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

antifungal activity against Kobresia fragilis (12 mm 50 μL-1

inhibition zone).

However methanol extracts of E.viscosus displayed antifungal activity against

Miniopterus pusilus with 10 mm 50 μL -1

inhibition zone while ethyl acetate and

45

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)

.

46

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

47

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

48

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.

49

grijisii and E. latifolius could correct the hepatocyte necrosis and functional

disorder induced by the CCl4 treatment(75)

. Another research evaluated hepato-

protective 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

50

against carrageenan-induced paw edema in mice and rats, and it selectively

inhibited prostaglandin E2 (PGE2) -induced inflammation(79)

.

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

51

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

52

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)

:-

53

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

54

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

55

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

56

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)

.

57

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)

.

N

N

O

CH2 CH2 OH

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-

58

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)

.

59

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)

:

Figure (1.9) (109)

- 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)

.

60

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)

.

61

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

62

(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

63

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 activity

Staphylococcus aureus

Staphylococcus albus

Streptococcus pyogenes

Streptococcus viridians

Streptococcus jaccalis

Streptococcus baris

Streptococcus pneumonia

Pseudomonas aeruginosa

Escherichia coli

Baccilus subtilis

Bacillus anthracis

Proteus vulgaris

Clostrium perfingens

Quercetin, Baicalin,

Hesperitin, Rutin.

Fisetin.

Apigenin.

Apigenin .

Chrysin.

Chrysin.

Chrysin.

Rutin,naringin,baicalin,

hydroxyethylrutosine.

Quercetin.

Quercetin.

Rutin.

Datisetin.

Hydroxyethylrutoside.

2. Antiviral

activity

Rabies virus

Herpes virus

Para influenza virus

Herpes simplex virus

Respiratory synctial virus

Auzesky virus

Polio virus

Mengo virus

Pseudorabies virus

Quercetin, quercetrin, rutin.

Quercetin.

Quercetin, rutin.

Galangin, quercetin,

kaempferol, apigenin.

Quercetin,naringin.

Quercetin.

Quercetin, apigenin.

Quercetin.

3. Antifungal

activity

Candida albicans

Candida tropicalis

Fusarium solani

Botrytis cinerea

Verticillum dahlia

Azotabacter vinelandii

Chloroflavonin.

Quercetin.

Chrysoeriol.

Chrysoeriol.

Chrysoeriol.

Quercetin, rutin, epicatechin.

64

Alternacia tennisima

Cladosporium herbarum Apigenin, echinacin.

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

65

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):

66

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

Flavonoids Sources References

Kaempferol, kaempferol 4'-methylether,

kaempferol 7-methylether,

kaempferol 3-O- alpha- L- rhamnoside,

myrecetin-3-O-alpha-L-rhamnoside

Echinops echinatus

135

Dihydroquercetin-4'-methyl ether,

5,7 -8,4 -dimethoxyflavanone-5-O- -L-

rhamnopyranosyl- 7-O- -D-arabinopyranosyl-

(1 4)-O- -D-glucopyranoside

Echinops echinatus

80

Silymarine Echinops tenuisectus 136

Quercetin Echinops tenuisectus 137

Apigenin (4',5,7-trihydroxyflavone, luteolin Echinops niveus 138

Kaempferol

Echinops galalensis and

Echinops hussoni

139

Apigenin, hispidulin, 5,4dihydroxy flavone and

apigenin 7-O- glucoside Echinops spinosissimus

140

Apigenin Echinops latifolius 141 Kaempferol, myricetin

Neoflavonoid nivetin

Apigenin, apigenin-7-O-glucoside, echinacin,

and echinaticin

Echinops spinosus

Echinops niveus

Echinops echinatus

79

142

143

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

67

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

involatile triterpenoids and sterols (C30) and carotenoid pigments (C40)(144)

.

The terpenoids group show significant pharmacological activities, such as anti-

viral, 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)

.

68

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

D3(156)

.

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)

.

69

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-

amyrin acetate

E. spinosissimus 158

4. methyl chavicol, 1,8-cineole , p-cymene

E.graecus&

E.ritro

159

5. dehydrocostus lactone, β-phellandrene,

germac-rene B, α-selinene, α and β-pinene and

caryophyllene oxide

E. kebericho 53

6. τ-cadinol . β-cubebene , β-patchoulene

longifolene and cyperene

E. kebericho 160

7. Sesquiterpenes:beta-selinene , beta-maaliene ,

caryophyllene oxide & cyperene

E. ellenbeckii 161

8. Sesquiterpenoids: Echinopines A and B E. spinosus

162

70

9. tricyclic sesquiterpenes: α and β-

caryophyllene, α and β-bisabolene,α and β-

santalene, guaiene

E. giganteus 163

10. sesquiterpene alcohols (e.g. (-)-nopsan-4-ol

and (+)-prenopsan-8-ol, silphiperfol-6-ene)

E. grijsii 164

11 sesquiterpene lactones (e.g.α and β-

caryophyllene epoxide

E. grijsii 165

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.

71

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.

72

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

chromatography

Sigma-Aldrich Chemie GmbH,

Germany

Potassium iodide Sigma-Aldrich, USA

Quercetin standard Chengdu Biopurify Phytochemicals

Rutin standard Chengdu Biopurify Phytochemicals

xylazine and Ketamine

haematoxylin and eosin

Bayer/ Germany

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

analyzer

IRMS/ Italy

Electrical sensitive balance Sartorius/ Germany

Fourier transforms infrared spectra (FT-IR) spectra

were scanned on Shimadzu FT-IR-8400S Infrared

Shimadzu /Japan

73

Spectrometer

Gas chromatography –mass spectroscopy GC-MS-QP

Ultra Shimadzu. Instrument model :AOC-2 Oi

Shimadzu /Japan

High Performance Liquid Chromatography (HPLC) Waters / Germany

Jobling Laboratory Division thin layer

chromatography TLC Coater.

Germany

Melting point: melting point was determined by

electro–thermal melting point

Stuart / UK

1H and

13C NMR was carried out in Al-Albayt

University, Al-Mafraq, Jordan (Euro-vectorEA

3000A)

Italy

Oven: Memmert 854 Buchi /Germany

Preparative HPLC (JASCO FC-2088-30) Jasco/ Japan

Rotatory evaporator: Buchi rotatory evaporator

attached to vacuum pump.

Buchi/ Germany

Ultraviolet light (DESAGA HEIDELBERG) of 254

nm and 366 nm wave lengths.

DESAGA/Germany

Ultra violet (UV) spectra were recorded in methanol

using computerized spectrophotometer Shimadzu

(UV-1700)

Japan

2.3-Plant material

74

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:

75

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

76

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

77

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

not exceeding 40 هC to give a dark greenish-yellow residue designated as a crude

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

exceeding 40 هC to give blackish residue designated as fraction 1(F-1) and aqueous

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

78

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

79

Ethyl acetate layer Aqueous layer

(neutral and acids components) (alkaloids and water soluble

components)

5%NaOH / CHCl3

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)

80

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

(MERCK) . The plates were activated at 110 هC for

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 :

S1a= Benzene : methanol: (80 : 20)(168)

S2a = Chloroform: acetone: diethylamine (50 : 40 :10)(168)

S3a = Toluene: ethylacetate: diethylamine (70 : 20 : 10)(169)

For fractions 2 and 3 :

S1f = Ethylacetate: formic acid : glacial acetic acid : water (100: 11: 11:27 )(169)

S2f = n-Butanol : glacial acetic acid : water (40: 10: 50 )(169)

S3f = Acetic acid: water (15:85) (168)

S4f = Chloroform: aceton : formic acid (75: 16.5 :8.5) (169)

S5f = Toluene: chloroform : aceton (40: 25: 35)(170)

S6f = Ethylacetate : methanol: formic acid (50: 50 :1)(169)

For fraction 4:

81

S1s = Chloroform : methanol (100 : 10 )(167)

S2s = Hexane : ethyl acetate (50: 50) (167)

S3s = Chloroform : ethyl acetate (80: 20)(171)

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.

82

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)

.

83

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 .

84

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

85

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.

86

2.4.4.5- Elemental microanalysis (CHN) :-

The CHN analysis for separated compounds was done.

2.4.4.6- 1H and 13

C 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

87

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)

88

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.

89

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

90

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

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

91

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

part

Traces + + - - - + - 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

92

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

93

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

F-1

27 gm

8.8 gm

7.4%

94

F-2

F-3

F-4

0.8 gm

5 gm

1.3 gm

0.7%

4.2%

1.1%

Aerial parts Crude extract

F-1

F-2

F-3

F-4

86 gm

2.5 gm

9.5 gm

30 gm

17 gm

0.5%

1.9%

6 %

3.4%

Roots Crude extract

F-1

F-2

F-3

F-4

30 gm

4 gm

6 gm

7.5 gm

5 gm

2%

3%

3.8 %

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.

95

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

96

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

97

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

98

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

99

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

100

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

101

102

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

103

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

104

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)

.

105

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,

106

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

107

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

108

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

109

110

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

111

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

112

K : Kaempferol standard

Q : Quercetin standard

S : Seeds A : Aerial parts R : Roots

113

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

114

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

part

0.75 Upper spot 0.93

Lower spot 0.8

Upper spot 0.89

Lower spot 0.88

Steroid isolated from root 0.75 Upper spot 0.93

Lower spot 0.79

one spot 0.86

115

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

10 mints at 105 هC

S : Stigmasterol standard B : Beta-sitosterol standard

A : Aerial parts R : Roots

116

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

mints at 105 هC

S : Stigmasterol standard B : Beta-sitosterol standard

A : Aerial parts R : Roots

117

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.

118

Visualization by Liebermann-Bur chard spray reagent, followed by heating for

10 mints at 105 هC

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

119

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).

120

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

121

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)

122

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.

123

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

method for purification tasks in the pharmaceutical industry(197)

. 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-

146 ه C, while E2 had a melting point 160-162هC and E3 of 123-125 ه C.

124

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.

125

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)

Functional

group

Group

frequency

wave number

( in cm-1

)

Assignment

For E1: N-H

C-H

3302

3090

N-H stretching(2 amine, one &very weak band)

C-H aromatic

126

C-H

C=O

N-H

C=C

2976, 2943

1681

1645

1593,1510,1491

Asymmetric and symmetric stretching of CH3

C=O of quinolone

N-H bending

C=C stretching of aromatic

For E2: =C-H

C=O

C-N

C-H

2875, 2773,

2713

1660

1329

943, 862, 765

C-H stretching

C=O stretching

C-N stretching

C-H of aromatic group out of plane

For E3: N-H

C-H

N-H

C-N

C=C

C-H

3190, 3144

2910, 2852

1640

1333, 1336

1489, 1431

914, 815, 750

N-H stretching (two band for 1 amine)

Asymmetric and symmetric stretching of CH3

N-H bending

C-N stretching bands of tertiary amine

C=C aromatic stretching

C-H of aromatic group out of plane

127

128

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

129

130

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

131

132

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

(found)

H% calculator

(found)

O% calculator

(found)

N% calculator

(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 13

C 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).

133

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).

134

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

135

136

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

H

H H

H

H

H

H

137

Depending on the above results, the expected chemical structure for the isolated E2

compound is may be :

N

O

CH3

12

34

5

6

7

89

10

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 13

C 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),

138

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

139

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 :

H H

H

H

H

H

H

H

H

H

140

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

141

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

20ml each

No. of spots Weight (gm)

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)

142

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)

143

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.

144

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

&EJ2(199)

Functional

group

Group frequency

wave number (cm-1

)

Assignment

EJ1

O-H

broad band (3600-

3083) central at 3334

O-H stretching of phenol

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:

O-H

broad band (3600-

3070) central at 3334

O-H stretching of phenol

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

145

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

146

147

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

148

149

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

150

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:

151

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).

O

OOH

HO

OH

O

O

OH

OH

OHHO

12

34

5

6

7

8

9

10

1-

2-

3-

4-

5-

6-

C1=

C2=

C3=

C4=

C5=

C6=

152

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

153

154

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

155

O

OOH

HO

OH

O

O

OH

OH

OHHO

12

34

5

6

7

8

9

10

1-

2-

3-

4-

5-

6-

C1=

C2=

C3=

C4=

C5=

C6=

156

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

157

158

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

13C and

1H-NMR for EJ2 showed identity with those reported for 5,7, 3

־, 4

־-

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-2

ʺ), 75.89 (C-3

ʺ), 69.99 (C-4

ʺ), 76.44 (C-5

ʺ), 66.6 (C-6

ʺ),

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-2

ʺ ,H-3

ʺ , H-4

ʺ , H-5

ʺ),

3.39 (1H, Ha-6ʺ ), 3.72 (1H, Hb-6

ʺ), 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;

159

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.

OOOH

HO O

OH

OH

1

OHH

OH

OH

H O

O

OHOH

H

OH

H

2

34

5

6

7

8

9

10

1-

2-

3-

4-

5-

6-

C1=

C2=C3

=

C4=

C5=

C1///

C2///

C3///

C4///

C5///

A

B

C

H

H

C6=

O

CH3

C6///

H

160

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

161

OOOH

HO O

OH

OH

1

OHH

OH

OH

H O

O

OHOH

H

OH

H

2

34

5

6

7

8

9

10

1-

2-

3-

4-

5-

6-

C1=

C2=C3

=

C4=

C5=

C1///

C2///

C3///

C4///

C5///

A

B

C

H

H

C6=

O

CH3

C6///

H

162

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

163

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

164

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

sharp melting point of 355-356هC compared to myricetin standard melting point

C(119). The other compound showed a melting point of 313 – 314ه357 ه C compared

to melting point 316 هC for standard quercetin(119), while 3rd one of these

compounds showed a melting point of 274 – 275 ه C compared to kaempferol

standard melting point 276- 278 ه C(119).

165

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

166

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

167

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)

168

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

169

group myricetin quercetin kaempferol

O-H Broad band

(3614-3101)

central at

3346

3410-3321 Broad band

(3414-3093)

central at 3317

O-H stretching of

phenol

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,

769

864, 823,

792

885, 844,

796

C-H of aromatic

group out of plane

170

171

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

172

173

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

174

175

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

118

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

119

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)

.

120

Figure(3.41)- HPLC of aerial parts

121

Figure(3.42)- HPLC of roots parts

122

Figure(3.43)- HPLC of seeds parts

123

Figure (3.44)-HPLC of myricetin standard.

124

Figure (3.45)- HPLC of isolated myricetin

125

Figure (3.46)-HPLC of quercetin standard.

Figure (3.47)- HPLC of isolated quercetin

126

Figure (3.48)-HPLC of kaempferol standard

127

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)

.

128

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

129

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

exhibited a prominent molecular ion peak at m/z 413

50 100 150 200 250 300 350 400 450 500 550 600 650 700 7500

10

20

30

40

50

60

70

80

90

100

110

120

130

140

%

413

83

271

300133

97

21343 352

199380

503 590457 698645 730539 611

130

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

50 100 150 200 250 300 350 400 450 500 550 600 650 700 7500

10

20

30

40

50

60

70

80

90

100

110

120

130

140

150

%

415

330

9543 81 397213145 255303

178

503475 648 673584 598 750534

131

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

132

Figure-(3.51b) GC-MS analysis of root parts of Echinops plant that

exhibited a prominent molecular ion peak at m/z 413

50 100 150 200 250 300 350 400 450 500 550 600 650 700 7500

10

20

30

40

50

60

70

80

90

100

110

120

130

140

%

413

83

271

300133

97

213

35143199

380

503 604 646448 714546 673579

50 100 150 200 250 300 350 400 450 500 550 600 650 700 7500

10

20

30

40

50

60

70

80

90

100

110

120

130

140

%

415

207 39743 95 32981 145 255303

178

503 546 647465 625581 735674

133

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.

Chapter three Results &

discussion

118

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).

Chapter three Results &

discussion

119

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

Chapter three Results &

discussion

120

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).

Chapter three Results &

discussion

121

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

Chapter three Results &

discussion

122

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

Chapter three Results &

discussion

123

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

Chapter three Results &

discussion

124

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

Chapter three Results &

discussion

125

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).

Chapter three Results &

discussion

126

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.

Chapter three Results &

discussion

127

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.

Chapter three Results &

discussion

128

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).

Chapter three Results &

discussion

129

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.

Chapter three Results &

discussion

130

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 .

Chapter three Results &

discussion

131

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

Chapter three Results &

discussion

132

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

Chapter three Results &

discussion

133

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.

118

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.

119

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.

118

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118

الخالصة

شكم اسع طبعت ب باث بشي خ انى انعائهت انشكبت بظسة )شكشكت( باث شك اندم

. نظرا لعدم وجود دراسات عايت انسكا نعالج اندشذ ضذ نسعاث االفاع ف شال انعشاق سخخذي

لبعض المركبات المهمة حول هدا الجنس ف العراق ,لذلك اصبح من االهمة دراسة التركب الكمائ

ف هذه الدراسة . يشدد اقخظادي اثرالموجودة ف هدا النبات الت قد تكون لها فعالة دوائة مهمة و

انى خانخ حتم استخالص وكشف وفصل وتنقة بعض المركبات المهمة من الناحة االحائة

اخضاء انباث ف شذاث (سخاالشباث , انخ, الفالفنودات ث,ذاكائت يخخهفت ) انقه يدايع

انع انخذي نالضاث انثات انكشف ا عهت س ,االخضاءانائت, اندزس (.انخخهفت ) انبز

ن الخضاء انباث انخخهفت االثا هضكائت يحذدة قذ حج عهى انسخخ كشفاثانخخهفت ي قبم

انخشباث بسب يخخهفت , انفالفذاث ث,ذاانقهيحخت عهى باث اشاسث انخائح ا كم اخضاء ان

.شذاث ف االخضاء انائت اندزس فقظ سخباالضافت انى خد يشكباث اال

فت حدضئخا انىاخضاء انباث انخخه الصف اسخخ اسبسنهعانى خفشي انطشقت انعايت اسخخذاو حى

ف خاص % 80بنسبة ثالاالمذب العضوي يخخهفت باسخعال اخضاء soxhlet . انحظل حى

:يخخهفت اخضاء عهى

.ثذاانقهانزي حخي عهى : االول الجزء

الكسذي.ف انشابظ اند الفالفنوداتانحخي عهى : الثاني الجزء

خالكسذي. الفالفنودات بدون رابط انحخي عهى :الثالثالجزء

الرابع الجزء ت.انحخي عهى يشكباث سخشذ :

تم البحث االول عن المركبات المختلفة باستخدام تقنة كروماتوغرافا الطبقة الرققة باستخدام مذبات

مختلفة كوسط ناقل والكشف عنها اما باستخدام االشعة فوق البنفسجة او استخدام كشوفات كمائة

) سج ث ذاقهثالثت احخاء انبذس عهى : معنة وكانت النتائج كما ل E1,E2 , E3 اث ي يع (

ث ف اندزس)ذاانقه E1,E2 كت قههت ي ( E1 ف االخضاء انائت انخ حى فظها بطشقخ:

Preparative TLC & preparative HPLC لمعرفة تركبها الكمائ ووزنها و

لل الطف للمركبات وشمل مطاف االشعة فوق البنفسجة ،ومطاف الجزئ استخدمت تقنات التح

رة نزري المغناطس نزااالشعة تحت الحمراء وكذلك التحلل الطف الكتل واستخدام الرنن

حث تم تحدد التركب الكمائ للمركبات 13رة الكاربوننرالمغناطس رينزوالرنن ا 1الهدروجن

E2 , E3 وهو:

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

3-methyl-4-amino-quinoline (E3)

) اجرت محاولة غر ناجحة لتحدد التركب الكمائ الدقق للمركب E1 على الرغم من اتمام جمع (

قة , لذا تم تركه لدراسة مستقبلة.التحالل الساب

119

جمهورة العراق

وزارة التعلم العال والبحث العلم

كلة الصدلة -جامعة بغداد

بشكل نق من األجزاء الهوائة باستخدام الكسذيف انشابظ اند الفالفنوداتتم فصل مركبن من

طرقة كروموتوغرافا العمود والتعرف على التركب الكمائ لهما عن طرق اجراء جمع التحالل

ان المركبان هما رالسابقة و rhamnoside – 0-3- kaempferol و rutin

كما تم الكشف عن الفالفنودات )الكورستن ,الكامبفرول والمارستن( باستخدام تقنة كروماتوغرافا

الطبقة الرققة ، باستخدام مذبات مختلفة كوسط ناقل والكشف عنها باستخدام االشعة فوق البنفسجة

الكامبفرول ور مع وجودنخزالسائلة ف االجزاء الهوائة وا،وكذلك تقنة كروماتوغرافا االداء العال

ور وبعدها تمت عملة الفصل والتنقة. نبروالمارستن فقط ف ا

ف اثسخشذ للجزء الرابع للكشف عن وجود TLC تقنة كروماتوغرافا الطبقة الرققة استخدمت

هذن المكونن لذا تم تركه لدراسة الجزء الهوائ والجذور لكن لم عط صورة واضحة عن هوة

مستقبلة

المستخلص الخام لنبتة تضمنت هذه الدراسة اضآ الكشف عن فعالة Echinops heterophyllus

ف شفاء لفالفنودات وجزء ا ثذانقهالعراقة المحلة وبعض اجزائها )الجزء الدي حتوي على ا

ربعة وعشرون ارنبا ذكرا بالغا تتراوح اعمارها بن ستة الجروح وكعامل مضاد للندوب . تم استخدام ا

شهور الى سنة, وقد تم تقدر هذا التؤثر مرئا ومن خالل التغرات النسجة الت تصب النسج . تمت

% باستعمال مسحة قطنة. حث اظهرت النتائج ان مستخلص 50المعالجة ثالث مرات وما بتركز

Echinops عجل من عملة شفاء الجرح ف مجموعات المعالجة بالمقارنة مع مجموعات غر

كان اكثر ذينقهمعالجة. اعطت المجموعة الت تم عالجها بالمستخلص الخام افضل النتائج. الجزء ا

م ف معالجة الجرح . لم تظهر كال المجموعتن المعالجة بالمستخلص الخا لفالفنوداتفاعلة من جزء ا

تكون الندب لذا من الممكن ان نستنتج ان هذه الدراسة ه خطوة جدة لبرهنة ان مستخلص ثذانقهوا

Echinops فعال ف تحفز التئام الجروح وكعامل مضاد لتكون الندب.

120

دراسة كمبئة واختببر فعبلةبعض المزكببت

ف ي نمو بزبلذالفعبلة لنببت شوك الجمل ا

لى عملة التئبم الجزوح العزاق ع

والى لجنة الدراسات العلا ف كلة العقاقرمقدمة الى فرع أطروحة

دكتوراه جامعة بغداد كجزء من متطلبات الحصول على درجة -الصدلة

(العقاقر) علوم الصدلة ف

قبلمن

ايناس جواد كاظم

( 2001 عقاقر)ماجستر

اشراف

عبد الحسن عبد الرسول )مشرف اول ( األستاذ الدكتور عالء

األستاذ المساعد الدكتورة زنب جلل عواد )مشرف ثان (

م 2013هجري 1434