ISOLATION and CHARACTERIZATION of SAPONINS from ASTRAGALUS Halicacabus and ASTRAGALUS Melanocarpus SPECIES

EGE UNIVERSITY GRADUATE SCHOOL OF NATURAL AND APPLIED SCIENCES

(Ph.D. THESIS)

ISOLATION AND CHARACTERIZATION OF SAPONINS FROM ASTRAGALUS halicacabus AND ASTRAGALUS melanocarpus SPECIES

Basile-Jimmy DJIMTOMBAYE

Supervisor: Prof. Dr. Hüseyin ANIL

Co-Supervisor: Prof. Dr. Özgen ALANKUŞ ÇALIŞKAN

Department of Chemistry

Department Code: 405.02.01 Presentation Date: 11.09.2014

Bornova-İZMİR 2014

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Basile-Jimmy DJIMTOMBAYE tarafından Doktora tezi olarak sunulan “Isolation and Characterization of Saponins from Astragalus halicacabus and Astragalus melanocarpus species” başlıklı bu çalışma E.Ü. Lisansüstü Eğitim ve Öğretim Yönetmeliği ile E.Ü. Fen Bilimleri Enstitüsü Eğitim ve Öğretim Yönergesi’nin ilgili hükümleri uyarınca tarafımızdan değerlendirilerek savunmaya değer bulunmuş ve 11.09.2014 tarihinde yapılan tez savunma sınavında aday oybirliği/oyçokluğu ile başarılı bulunmuştur.

Jüri Üyeleri : İmza

Jüri Başkanı : Prof. Dr. Hüseyin ANIL …………………

Raportör : Prof. Dr. Erdal BEDİR …………………

Üye : Prof. Dr. Özgen ÇALIŞKAN …………………

Üye : Doç. Dr. Tamer KARAYILDIRIM …………………

Üye : Yrd. Doç. Dr. Kadir AY …………………

EGE ÜNİVERSİTESİ FEN BİLİMLERİ ENSTİTÜSÜ ETİK KURALLARA UYGUNLUK BEYANI

E.Ü. Lisansüstü Eğitim ve Öğretim Yönetmeliğinin ilgili hükümleri uyarınca Doktora Tezi olarak sunduğum “Isolation and Characterization of Saponins from Astragalus halicacabus and Astragalus melanocarpus Species.” başlıklı bu tezin kendi çalışmam olduğunu, sunduğum tüm sonuç, doküman, bilgi ve belgeleri bizzat ve bu tez çalışması kapsamında elde ettiğimi, bu tez çalışmasıyla elde edilmeyen bütün bilgi ve yorumlara atıf yaptığımı ve bunları kaynaklar listesinde usulüne uygun olarak verdiğimi, tez çalışması ve yazımı sırasında patent ve telif haklarını ihlal edici bir davranışımın olmadığını, bu tezin herhangi bir bölümünü bu üniversite veya diğer bir üniversitede başka bir tez çalışması içinde sunmadığımı, bu tezin planlanmasından yazımına kadar bütün safhalarda bilimsel etik kurallarına uygun olarak davrandığımı ve aksinin ortaya çıkması durumunda her türlü yasal sonucu kabul edeceğimi beyan ederim.

11.09.2014

Basile-Jimmy DJIMTOMBAYE

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ÖZET

ASTRAGALUS halicacabus ve ASTRAGALUS melanocarpus TÜRLERİNDEN SAPONİNLERİN İZOLASYONU ve KARAKTERİZASYONU

Basile-Jimmy DJIMTOMBAYE

Doktora Tezi, Kimya Anabilim Dalı

Tez Danışmanı: Prof. Dr. Hüseyin ANIL

İkinci Tez Danışmanı: Prof. Dr. Özgen ALANKUŞ ÇALIŞKAN

Eylül 2014, 166 sayfa

Astragalus L., yaklaşık 3000 türle Leguminosae familyasındaki en geniş cinstir. Türkiye florasında 224 tanesi endemik olmak üzere toplam 445 türle temsil edilmektedir (Davis, 1970; Aytaç, 2000).

Astragalus türlerinin kökleri çok eski yıllardan beri halk arasında terlemeyi önleyici, tonik ve diüretik olarak kullanılagelmektedir. Ayrıca yine şeker hastalığının, nefritin, löseminin ve rahim kanserinin tedavisinde kullanım alanı bulmaktadır (Tang and Eisenbrand, 1992). Anadolu’da, Türkiye’nin Güney- Doğusu’nda Astragalus köklerinin sulu ekstreleri geleneksel olarak lösemiye karşı ve yara iyileştirici olarak kullanılmaktadır (Çaliş et al.,2008; Bedir et al., 2000).

Astragalus köklerinin bilinen biyolojik aktif bileşenleri, polisakkaritler ve saponinler olmak üzere iki temel kimyasal bileşik sınıfında toplanmıştır (Tang and Eisenbrand, 1992). Bugüne kadar 400’dan fazla sikloartan yapısında saponin tanımlanmış ve bunların arası yaklaşık 200 Astragalus cinsinden izole edilmiştir (Mamedova and Isaev, 2004; Li-Peng et al., 2013). Sikloartan yapısındaki saponinlerin en zengin kaynağını oluşturan Astragalus (Leguminosea) cinsine ait türler, bu türlerin ekstreleri ve bunlardan izole edilen bileşikler, bağışıklık uyarıcı, karaciğer koruyucu, antioksidan, antiviral, ateş düşürücü, kardiovasküler, iltihap giderici ve anti kanser etkiler göstermeleri nedeniyle giderek artan bir şekilde ilgi odağı haline gelmiştir (Rios and Waterman, 1997; Verotta El-Sebakhy, 2001).

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Türkiye Astragalus türlerinden biyolojik olarak aktif bileşiklerin izolasyonuna ilişkin devam eden çalışmalarımız kapsamında Astragalus halicacabus Lam.ve Astragalus melanocarpus Bunge. bitkileri çalışılmıştır.

Astragalus halicacabus bitkisinden 1 yeni sikloartan-tipi glikozit (halicacoside A, AHa1), 1 yeni maltol glikozit (halicacoside B, AHa2) ile birlikte 7 bilinen sikloartan-tipi glikozit (3-7) ve 1 bilinen maltol glikozit (10) izole edilmiştir. Astragalus melanocarpus bitkisinden 3 yeni saponin ile birlikte 7 bilinen saponin izole edilmiştir.. İzole edilen bu bileşiklerin yapıları, 1D- ve 2D- NMR teknikleri, ESIMS ve HRMS analizleri kullanılarak belirlenmiştir.

Anahtar Sözcükler: Leguminosae, Astragalus halicacabus, Astragalus melanocarpus, sikloartan, oleanan, maltol, saponin.

ABSTRACT

ISOLATION AND CHARACTERIZATION OF SAPONINS FROM ASTRAGALUS halicacabus AND ASTRAGALUS melanocarpus SPECIES Basile-Jimmy DJIMTOMBAYE

Ph.D. Thesis in Chemistry

Supervisor: Prof. Dr. Hüseyin ANIL

Co-Supervisor: Prof. Dr. Özgen ALANKUŞ ÇALIŞKAN

September 2014, 166 pages

Astragalus L. (Leguminosae), one of the largest genera of flowering plants with about 3000 species, is represented in the flora of Turkey by 445 species, 224 of which are endemic (Davis, 1970; Aytaç, 2000).

The roots of Astragalus species represent a very old and well-known drug in traditional medicine for their usage as an antiperspirant, tonic and diuretic. The have also been used in the treatment of diabetes mellitus, nephritis, leukemia and uterine cancer (Tang and Eisenbrand, 1992).

In the district of Anatolia, located in South Eastern Turkey, an aqueous extract of the roots of Astragalus is traditionally used against leukemia and for its wound-healing properties (Çaliş et al.,2008; Bedir et al., 2000).

Modern pharmacological studies of Astragalus species indicated the multiple biological activities, such as antioxidative, immunomodulation, cardiovascular, cerebrovascular diseases, neurodegenerative diseases, liver diseases, infectious diseases and antiviral properties, etc (Li-Peng et al., 2013).

Known biologically active constituents of Astragalus roots represent two major classes of chemical compounds, polysaccharides and saponins (Tang and Eisenbrand, 1992; Li-Peng et al., 2013). Up to now, more than 400 cycloartane- type saponins were determined and around 200 of them were isolated from Astragalus genus (Mamedova and Isaev, 2004; Li-Peng et al., 2013).

Furthermore, the species of Astragalus genus (Leguminosae) which are the richest source of cycloartane type saponins have attracted increasing interest because of their extract’s and isolated compound’s wide range of biological activities known as anti-inflammatory, hepatoprotective, antipyretic, immunostimulant and anti cancer (Rios and Waterman, 1997; Verotta El-Sebakhy, 2001) .

In our ongoing research for new bioactive compounds from Turkish Astragalus species, we carried out a study on two Astragalus species, namely Astragalus halicacabus.Lam. and Astragalus melanocarpus Bunge.

One new cycloartane-type triterpene glycoside (halicacoside A, AHa1) , one new maltol glycosides (halicacoside B, AHa2), were isolated from Astragalus halicacabus along with seven known cycloartane-type glycosides (3-7) and one known maltol glycosides (10). Moreover, three new saponins were isolated from Astragalus melanocarpus along with five known compounds. Their structures were established by the extensive use of 1D and 2D-NMR experiments along with ESIMS and HRMS analysis.

Keywords: Leguminosae, Astragalus halicacabus, Astragalus melanocarpus, cycloartane, oleanane, maltol, saponin.

ACKNOWLEDGMENT

This study was carried out at the Department of Chemistry, Ege University, Science Faculty, İzmir from 2008 to 2014

I am deeply grateful to Prof. Dr. Hüseyin ANIL, to Prof. Dr. Özgen ALANKUŞ ÇALIŞKAN and to Prof. Dr. Erdal BEDİR for their noble availability and precious suggestion, support, patience and for enlightening me via their deep knowledge during long years their experience, despite their heavy responsibility helped us in this study.

I am also very grateful to Assoc. Prof. Dr Tamer KARAYILDIRIM for his all valuable advice, help, suggestion and support during this study. Our discussions on the completion of the thesis have been of great value. All my gratitude to him.

The major credit for this scientific merit goes especially to them for their efficiency and help.

My thanks to the Turkish governmet for this fellowship which allowed me to complete this doctoral study.

I owe my special thanks to Prof. Dr. Sonia PIACENTE and her group for running my compounds spectra.

I am grateful to Assoc Prof. Dr. Fevzi Özgökçe for the identification of Astragalus halicacabus and Astragalus melanocarpus.

This project was granted by TUBITAK (109T425), Ege University Science and Technology Center (EBİLTEM) (2010 Bil 008) and Ege University Research Foundation (2009 FEN 090). I would like to thank them for their financial support. I also would like to thank TUBITAK for ( Ph.D). fellowship.

My deep gratitude and special thanks goes to Mireille and Richard PARIENTE to whom I dedicate this scientific work to testify their noble friendship during my studies and my stay in Turkey.

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I am grateful to Juan Gabriel FERRONE, Priest of Göztepe for his help, supports and the long time of sharing with all parishioners during my study.

I’m very thankful to my unforgettable constant companion ATILE and DJIBATI for showing patience and tolerance supported my long absence from them (during my working-times).

Finally, I would thank all my family, all my teachers and friends in Ege University for their good intention. Specially my colleagues Derya Gülcemal and Ibrahim Horro for their regular help during this study.

I dedicated this work in memory of my parents who gave me life, basic education but didn’t attend the culmination of their efforts. My deep gratitude to them and peace to their souls.

I also would like to dedicate this work in memory of Ichame KAMACH (Founder of Kamach group), for his gratitude. Rest in peace

Basile-Jimmy DJIMTOMBAYE

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CONTENTS

Page ÖZET……………………………………………………………………………VII

ABSTRACT……………………………………………………………………..IX

ACKNOWLEDGMENT………………………………………………………..XI

CONTENTS …………………………………………………………………. XIII

LIST OF FIGURES…………………………………………………………. XVII

LIST OF TABLES…………………………………………………………….XIX

LIST OF SCHEMES…………………………………………………………..XXI

LIST OF SPECTRA……………………………………………………….. XXIII

ABBREVIATIONS …………………………………………………………..XXV

1 INTRODUCTION ...... 1

1.1 Historical Origin of Natural Products ...... 4

1.2 Astragalus Genus ...... 7

1.2.1 Astragalus halicacabus Lam., (Section: Halicacabus) ...... 8

1.2.2 Astragalus melanocarpus Bunge., (Section: Hypoglottis) ...... 9

1.2.3 Chemical constituents of Astragalus species ...... 9

1.3 Terpenoids ...... 10

1.3.1 Monoterpenes (C10) ...... 11

1.3.2 Sesquiterpenes (C15) ...... 12

1.3.3 Diterpenes (C20) ...... 13

1.3.4 Triterpenes (C30) ...... 14

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CONTENTS (continued)

Page

1.3.5 Tetracyclic Triterpenes ...... 15

1.3.6 Triterpenoid Saponins ...... 15

1.4 Biosynthesis of Saponins ...... 20

1.4.1 Cycloartane Saponins ...... 26

1.4.2 Cycloartane-Type Glycoside ...... 33

1.5 Astragalus as Species Poisonous Plants ...... 65

1.6 Astragalus as Medicinal Plants ...... 68

1.6.1 Hepatoprotective and Antioxidative Effects of Astagalus ...... 69

1.6.2 Astragalus as Immunostimulant Agent ...... 70

1.6.3 Antiviral Properties of Astragalus species ...... 72

1.6.4 Cardiovascular Effects of Astragalus Species ...... 74

1.6.5 Effect of Astragalus Species on Diabetes and Diabetes Related Diseases 75

1.6.6 Antitumor Effects of Astragalus Species ...... 79

1.6.7 Other Pharmacological Effects of Astragalus Species ...... 81

1.7 Structure Identification by Chromatographic and Spectroscopic Methods 86

1.7.1 Infra-Red (IR) Spectroscopy ...... 87

1.7.2 Ultraviolet (UV) Spectroscopy ...... 88

1.7.3 Mass Spectrometry (MS) ...... 88

1.7.4 X-ray Spectroscopy ...... 88

1.7.5 Nuclear Magnetic Resonance (NMR) Spectroscopy ...... 89

CONTENTS (continued)

Page

2 MATERIALS AND METHODS ...... 94

2.1 General ...... 94

2.2 Plant Materials ...... 95

2.2.1 Astragalus halicacabus Lam...... 95

2.2.2 Astragalus melanocarpus Bunge...... 95

2.3 Isolation and Purification ...... 96

2.3.1 Astragalus halicacabus Lam...... 96

2.3.2 Astragalus melanocarpus...... 98

3 RESULTS AND DISCUSSION...... 100

3.1 Astragalus halicacabus Lam...... 100

3.1.1 Structural Identification of AHa1 ...... 100

3.1.2 Structural Identification of AHa2 ...... 111

3.1.3 Acidic Hydrolysis of Compounds from Astragalus halicacabus ...... 116

3.2 Astragalus melanocarpus. Bunge ...... 117

3.2.1 Structural Identification of AMJ5 ...... 117

3.2.2 Structural Identification of AMJ8 ...... 126

3.2.3 Structural Identification of AMJ18 ...... 133

REFERENCES ...... 148

CURRICULUM VITAE………………………………………………………. 165

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XVII

LIST OF FIGURES

Figure Page

1.1 Astragalus halicacabus ...... 8

1.2 Astragalus melanocarpus ...... 9

1.3 Classification of terpenes based on isoprene units ...... 11

1.4 Structures of monoterpenes ...... 11

1.5 Monoterpene iridoids ...... 12

1.6 Monoterpene skeletons ...... 12

1 7 Common structures of sesquiterpene ...... 13

1.8 Diterpenes of abietane and pimarane ...... 13

1.9 Steviol glycosides from Stevia rebaudiana ...... 14

1.10 Cyclization of main triterpene correlation skeletones ...... 18

1.11 Basic steroidal saponin skeletons ...... 19

1.12 Monodesmosidic and bidesmosidic saponins ...... 20

1.13 Cephalotoside A, a tridesmosidic cycloartane type glycoside...... 20

1.14 The cyclization of oxidosqualene to the various saponin skeletons ...... 22

1.15 Structure of toxic compounds found in Astragalus species ...... 66

1.16 Indolizidine alkaloids glycosidase inhibitors occuring in Astragalus species ...... 67

1.17 Polysaccharide chain ...... 79

1.18 COSY correlations ...... 90

1.19 TOCSY correlations ...... 91

1.20 NOESY correlations ...... 91

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LIST OF FIGURES (continued)

Figure ...... Page

1.21 NOESY correlations between sugar and aglycone ...... 92

1.22 HMQC correlations ...... 92

1.23 HMBC correlations ...... 93

3.1. Structure of AHa1 ...... 101

3.2 HMBC key correlation of AHa1 ...... 103

3.3 Structures of AHa2 ...... 112

3.4 Key correlations of AHa2 ...... 116

3.5. Structure of AMJ5 ...... 118

3.6 Structure of AMJ8 ...... 126

3.7 Structure of AMJ18 ...... 134

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LIST OF TABLES

Table Page

1 13 3.1 H- and C-NMR Data of the Aglycon Moieties of AHa1 in CD3OD at 500 MHz; δ in ppm, J in Hz ...... 102

1 13 3.2 H- and C-NMR Data of the Sugar Moieties of AHa1 in CD3OD at 600 MHz;  in ppm, J in Hz ...... 103

3.3 1H- and 13C-NMR Data of the Aglycon and Sugar Moieties of AHa2 in

CD3OD at 500 MHz; δ in ppm, J in Hz ...... 112

1 13 3.4 H- and C-NMR Assignments of (150/600 MHz,  ppm, in CD3OD) AMJ5 ...... 119

1 13 3.5 H- and C-NMR Assignments of (150/600 MHz,  ppm, in CD3OD) AMJ5 ...... 120

1 13 3.6 H- and C-NMR Assignments of AMJ8 (150/600 MHz,  ppm, in CD3OD) ...... 127

1 13 3.7 H- and C-NMR Assignments of AMJ18 (150/600 MHz,  ppm, in CD3OD) ...... 135

XXI

LIST OF SCHEMES

Scheme Page

2.1 Isolation of the new triterpene (AHa1) and maltol (AHa2) from Astragalus halicacabus Lam...... 97

2.2 Isolation of the new cycloartanes (AMJ5, AMJ8 and AMJ18) from Astragalus melanocarpus ...... 99

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XXIII

LIST OF SPECTRA

Spectrum Page

3.1. 1H-NMR Spectrum of AHa1 ...... 104

3.2 13C-NMR Spectrum of AHa1 ...... 105

3.3. HMQC Spectrum of AHa1 ...... 106

3.4. HMQC Spectrum of AHa1 ...... 107

3.5. HMQC Spectrum of AHa1 ...... 108

3.6. HMBC Spectrum of AHa1 ...... 109

3.7. HMBC Spectrum of AHa1 ...... 110

3.8. 1H-NMR Spectrum of AHa2 ...... 113

3.9 13C-NMR Spectrum of AHa2 ...... 114

3.10 DEPT spectrum of AHa2 ...... 115

3.11. 1H NMR Spectrum of AMJ5 ...... 121

3.12. HSQC Spectrum of AMJ5 ...... 122

3.13. HSQC Spectrum of AMJ5 ...... 123

3.14. HMBC Spectrum of AMJ5 ...... 124

3.15. HMBC Spectrum of AMJ5 ...... 125

3.16. 1H NMR Spectrum of AMJ8 ...... 128

3.17. HSQC Spectrum of AMJ8 ...... 129

3.18 . HSQC Spectrum of AMJ8 ...... 130

3.19 . HMBC Spectrum of AMJ8 ...... 131

3.20. HMBC Spectrum of AMJ8 ...... 132

3.21. 1H NMR Spectrum of AMJ18 ...... 137

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LIST OF SPECTRA (continued)

Spectrum ...... Page

3.22. HSQC Spectrum of AMJ18 ...... 138

3.23. HSQC Spectrum of AMJ18 ...... 139

3.24. HMBC Spectrum of AMJ18 ...... 140

3.25. HMBC Spectrum of AMJ8 ...... 141

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ABBREVIATIONS

Abbreviation Explanation

BuOH Butanol

CHCl3 Chloroform

CH2Cl2 Dichloromethane

EtOAc Ethylacetate

CH3OH Methanol

H2O Water

CD3OD Deuterated methanol

DMSO Dimethylsulfoxide

UV Ultraviolet

HPLC High Pressure Liquid Chromatography

MPLC Medium Pressure Liquid Chromatography

LC-MS Liquid Chromatography-Mass Spectrometry

FAB-MS Fast Atom Bombardment Mass Spectrometry

MALDITOF Matrix-assisted Laser Desorption Ionization Time-of- flight DQF-COSY Double Quantum Filtered Correlation Spectroscopy

CC Column Chromatography

GC Gas Chromatography

FT-IR Fourier Transform Infrared

TLC Thin Layer Chromatography

RP-VLC Reverse Phase Vacuum Liquid Chromatography

NMR Nuclear Magnetic Resonance

1D-NMR One-Dimensional Nuclear Magnetic Resonance

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2D-NMR Two-Dimensional Nuclear Magnetic Resonance

HSQC Heteronuclear Single Quantum Coherence

HMBC Heteronuclear Multiple Bond Coherence

1D-TOCSY One Dimensional Total Correlation Spectroscopy

HOHAHA 2D Homonuclear Hartman-Hahn Spectroscopy

HETCOR Heteronuclear Correlation Spectroscopy

S Singlet

D Doublet

T Triplet

Q Quartet

M Multiplet

1 INTRODUCTION

The use of medicinal plants in the treatment of disease dates back several millennia. Plants are for that purpose the major sources used in the treatment of pathologies

The Pharmacopoeia, being traditionally known one of processing pathways by people worldwide of treating diseases is the fastest way to access to care at lower cost.

Whereas, despite extraordinary advances in science and technology and their application in the field of modern medicine, the use of plants as an alternative or traditional medicine in the health system seems to take over.

Moreover, several alarming reports from the World Health Organization (WHO, 2002, 2003, 2005) has indicated that the main source and route of access to health care for people in less developing countries in regions without or remote health centers and / or victims of wars, natural catastrophes whose more than 60% in Africa and Asia are used exclusively traditional medicine-based plants. It means that herbal medicine seems until today become one of the main remedies for treating pathological signs of disease by humanity. This situation pushed laboratories in chemistry, biochemistry, biology and other fields of applied research worldwide to max out a traditional medicine in search of new active principles whose application becomes increasingly growing even in developed countries.

However, the main objective of these laboratories and scientific being to find new active molecules that can help to treat a range of diseases such as cancer, leukemia, Alzheimer, neurodegenerative diseases, malaria, HIV / AIDS that constituted a significant and major challenge for researchers today. Natural substances of vegetable or marine origin, are an inexhaustible reservoir of new secondary metabolites with little or no exploration activities that may pose a prerequisite for the development of new sources of drugs.

In the last few decades, natural products research has advanced tremendously in the field of chemistry, life sciences, food sciences and material sciences. Comparisons of natural products from microorganisms, lower eucaryotes, animals, higher plants and marine organisms are well documented.

Natural products are present in our everyday life. Some are active constituents of many medicines, vitamins, food additives, flavour and fragrances, agrochemicals and pesticides used for plants protection. Indeed the essential components of our life itself include proteins, carbohydrates, nucleic acids, lipids, vitamins, hormones, steroids, etc (Sujata et. al., 2005).

However, The term “natural product” derived from organic compounds originally found in animals, plants, fungi or microorganisms known as main precursor factories for the biosynthesis of both primary and secondary metabolites.

All living organisms need to survive; a certain number of organic materials drawn from their environments allowed them to change, grow, reproduce, live and interconvert. However, occurring compounds can be classified into 3 main categories by origin of the compounds. Compounds breeding in all cells, having a central role in the metabolism and reproduction of these cells (nucleic acids, amino acids and sugars) are known as primary metabolites. Moreover, the high molecular weight compounds such as cellulose, lignin and proteins forming cell structures. Polysaccharides are sugar units of compounds that the proteins are constituted from the amino acid and nucleic acid. In addition to minor variations and various characteristics of living organisms, biosynthetic pathways and modification of carbohydrates, proteins, fats and nucleic acids are found to be the same in all these organisms. These processes collectively described, demonstrate the basic unit of living matter known as primary metabolism.

Unlike the primary metabolic pathways, there are compounds which have a much more limited distribution in nature called secondary metabolites; which are small molecules (mol weight <2000 amu approximately) produced by an organism but are not strictly necessary for the survival of the organism. Secondary metabolites are found only in specific organisms or groups of organisms, and are an expression of the individuality of species. The plants generally produce a variety of secondary metabolites which have no apparent role in the physiological or biochemical processes.(Sujata et., 2005)

These compounds are important in mediating interactions between plants and their biotic environment (Berenbaum 1989, 2002, Kessler and Baldwin, 2002). Secondary metabolites are not necessarily produced in all conditions and in

most cases, the function of these compounds and their benefits effect on organism is not fully known.

Recent studies have shown that many plant species produce and accumulate a large variety of secondary metabolites which provide protection against insects (Berenbaum, 1989, 2002), such as volatile attractants to themselves or other species, or as coloring agents. Early studies led to the discovery of the purple pigment of Viola as an indicator of natural pH (Boyle, 1664). But it is logical to assume that all play vital roles for the welfare of the producer

For more details, they are supposed to play ecologically significant role in how the living organisms deal with their surroundings and therefore are important for their ultimate survival (Sujata et.al 2005).

Furthermore, natural products have important biological activities and play various functions in nature, such as plant hormones, have a regulatory role, while others function as chemical defense against pests. The role of certain compounds is to act as chemical messengers, such as sex-attractants (pheromones) in insects, terrestrial and marine animals and humans. Various extracts of flowers, plants and insects have been used for isolating compounds whose task, color and odor could be used for various purposes by mankind since his existences’ origin.

The purpose of this study is to search new saponin molecules from polar extracts of one of widespread plants Worldwide; Astragalus species (Leguminosae).

The present work is therefore to isolate and characterize saponins from Astragalus halicacabus and Astragalus melanocarpus species and will focus on:

Previous reviews on Natural Products and Astragalus Genus

Materials and Methods used in this work

Results and Discussions

Conclusion and perspectives.

1.1 Historical Origin of Natural Products

Throughout the ages humans have relied on nature for their basic needs for the production of foodstuffs, shelters, clothing, means of transportation, fertilizers, flavors and fragrances and, not least, medicines. Plants have formed the basis of sophisticated traditional medicine systems that have been in existence for thousands of years.

For centuries, China has led the world in the use of natural products for healing. One of the earliest health science anthologies in China is the Nei Ching, whose authorship is attributed to the legendary Yellow Emperor (30th century BC), although it is said that the dates were backdated from the 3rd century by compilers. Excavation of a Han Dynasty (206 BC-220 AD 220) tomb in Hunan Province in 1974 unearthed decayed books, written on silk, bamboo and wood, which filled a critical gap between the dawn of medicine up to the classic Nei Ching; book 5 of these excavated documents lists 151 medical materials of plant origin. Generally regarded as the oldest compilation of Chinese herbs is Shen Nung Pen Ts’ao Ching (Catalog of Herbs by Shen Nung), which is believed to have been revised during the Han Dynasty; it lists 365 materials. Numerous revisions and enlargements of Pen Ts’ao (herb) were undertaken by physicians in subsequent dynasties, the ultimate being the Pen Ts’ao Kang Mu (General Catalog of Herbs) written by Li Shih-Chen over a period of 27 years during the Ming Dynasty (1573-1620), which records 1898 herbal drugs and 8,160 prescriptions. This was circulated in Japan around 1620 and translated, and has made a huge impact on subsequent herbal studies in Japan; however, it has not been translated into English. The number of medicinal herbs used in 1979 in China numbered 5,267(Mechoulam et. al., 1967).

One of the most famous of the Chinese folk herbs is the ginseng root Panax ginseng, used for health maintenance and treatment of various diseases. The active principles were thought to be the saponins called ginsenosides but this is now doubtful; the effects could well be synergistic between saponins, flavonoids, etc. Another popular folk drug, the extract of the Ginkgo tree, Ginkgo biloba L., the only surviving species of the Paleozoic era (250 million years ago) family which became extinct during the last few million years, is mentioned in the Chinese Materia Medica to have an effect in improving memory and sharpening mental alertness. The main constituents responsible for this are now understood to be ginkgolides 1 and flavonoids, but again not much else is known. Clarifying the

active constituents and mode of (synergistic) bioactivity of Chinese herbs is a challenging task that has yet to be fully addressed (Mechoulam and Gaoni, 1967).

The Assyrians peoples left 660 clay tablets describing 1000 medicinal plants used around 1900-400 BC, but the best insight into ancient pharmacy is provided by the two scripts left by the ancient Egyptians who were masters of human anatomy and surgery because of their extensive mummification practices(Bown, 2001).

The Edwin Smith Surgical Papyrus purchased by Smith in 1862 in Luxor (now in the New York Academy of Sciences collection), is one of the most important medicinal documents of the ancient Nile Valley, and describes the healer’s involvement in surgery, prescription and healing practices using plants, animals and minerals. The Ebers Papyrus, also purchased by Edwin Smith in 1862, and then acquired by Egyptologist George Ebers in 1872, describes 800 remedies using plants, animals, minerals and, etc. (Thompson, 1949)

Indian medicine also has a long history, possibly dating back to the 2nd millennium BC. The Indian materia medica consisted mainly of vegetable drugs prepared from plants but also used animals, bones, and minerals. (Rosen, 1979).

Ancient Greece inherited much from Egypt, India and China, and underwent a gradual transition from magic to science. Pythagoras (580-500 BC) influenced the medical thinkers of his time, including Aristotle (384-322 BC), who in turn affected the medical practices of another influential Greek physician Galen (129- 216).

The Iranian physician Avicenna (980-1037) is noted for his contributions to Aristotelian philosophy and medicine, while the German-Swiss physician and alchemist Paracelsus (1493-1541) was an early champion who established the role of chemistry in medicine. The rain-forests in Central and South America and Africa are known to be particularly abundant in various organisms of interest to our lives because of their rich biodiversity, intense competition, and the necessity for self-defense. However, since folk-treatments are transmitted verbally to the next generation via shamans who naturally have a tendency to keep their plant and animal sources confidential, the recipes tend to get lost, particularly with deforestation of rain forests and the encroachment of ‘‘civilization.’’ Studies on folk medicine, hallucinogens and shamanism of the Central and South American

Indians conducted by Richard Schultes (Harvard Botanical Museum, emeritus) have led to renewed recent activity by ethnobotanists, recording the knowledge of shamans, assembling herbaria, and transmitting the record learning to the village (Bown, 2001).

Extracts of toxic plants and animals have been used throughout the world for thousands of years for hunting and murder. These include the various arrow poisons used all over the world. Strychnos and Chondrodendron (containing strychnine was used in South America and called ‘‘curare’’, Strophanthus (strophantidine was used in Africa, the latex of the upas tree Antiaris toxicaria (cardiac glycosides) was used in Java, while Aconitum napellus, which appears in Greek mythology (aconitine) was used in medieval Europe and Hokkaido (by the Ainus). The Colombian arrow poison is from frogs (batrachotoxins; 200 toxins have been isolated from frogs by B. Witkop, J. Daly at NIH). Extracts of Hyoscyamus niger and Atropa belladonna contain the toxic tropane alkaloids, e.g., hyoscyamine , belladonnine and atropine. The belladonna berry juice (atropine) which dilates the eye pupils was used during the Renaissance by ladies to produce doe-like eyes (belladona means beautiful woman). When rye is infected by the fungus, Claviceps porpurea, the toxin ergotamine and a number of ergot alkaloids are produced.

In 1952, Bloch and Woodward suggested a mechanism for the cyclization of squalene to cholesterol.

The Efik people in Calabar, southeastern Nigeria, used extracts of the calabar bean known as esere (physostigmine ) for unmasking witches.

The ancient Egyptians and Chinese knew of the toxic effect of the puffer fish, fugu, which contains the neurotoxin tetrodotoxin 10 (Y. Hirata, K.Tsuda, R.B. Woodward). Robert B. Woodward was involved in the structural determinations of penicillin, strychnine, patalin, terramycin, aureomycin and the synthesis of Vitamin B12. Among the recent outstanding contributions to the chemistry of natural products is the conformational analysis designed by Derek Barton. He used it for the structural determinations of many complex molecules such as β-amyrin and cycloartenol. Other famous investigators regarding the biosynthetic studies were A. Birch and R. Robinson who studied the biosynthesis of polyketides having C6-C3-C6 backbones such as plant phenolics, polyene macrolides, terpenoids and alkaloids, sterols, fatty acids and prostaglandins

(discovered in seminal fluids). Otto Wallach (1847–1931) proposed the “isoprene rule” and many scientists were engaged in isoprenoid studies; among them was Wieland, Windaus, Karrer, Kuhn, Butenandt and Ruzicka. Paul Karrer (1889– 1971) established the foundation of carotenoid chemistry which was proceeded by Otto Isler and Hans Eugster.

Furthermore, our study will focus on the Astragalus, one of the most widespread plant genus of the world, including Turkey and known to be rich in saponins that our current research is realized.

1.2 Astragalus Genus

The Fabaceae (Leguminosae) is a family of flowering plants comprising around 269 genera and 5100 species (Davis, 1970). It is generally considered as the largest genus of vascular plants (Podlech, 1986; Lock and Simpson, 1991). Astragalus L. (Leguminosae) is a genus widely distributed throughout the temperate regions of the Northern Hemisphere. The greatest numbers of species are found in the arid, continental regions of Western North America (400 species) and Central Asia (2000±2500 species). An additional 150 species are known from temperate South America and one species extends along the East Africa mountains to Transvaal, South Africa (Liston and Wheeler, 1994). More than 2000 species have been described, 372 of them in North America and 133 in Europe with 42 species located, specially in Iberian peninsula (Davis, 1982; Infante et al., 1991). A total of 93 sections are recognized for the North America species (Barneby, 1964). Astragalus is represented by 445 species in the flora of Turkey, of which 224 are endemic (Davis, 1970; Aytaç, 2000).

Astragalus refer to two Greek words "Astron" means star and "Gala" means milk, from the beliefs’ that the presence of Astragalus plants in grass-land improved milk yield of livestock (Plowden, 1968). Astragalus is generally considered to be the largest genus in the Angiosperms; most of the 445 Turkish species are in the Irano-Turanian region and classified in 62 sections (Özüdoğru et al., 2011; Aytaç et al., 2000; Davis, 1970). They are Annuals, herbaceous perennials, unarmed or spiny shrubs. Leaves paripinnate or imparipinnate, rarely l-3-foliolate; leaflets simple or bifurcate-hairy; stipules herbaceous or glumaccous, conspicuous. Inflorescence a sessile or pedunculate spike or raceme, or flowers sessile in upper leaf axils forming a dense compound inflorescence, rarely flowers solitary. Calyx campanulate to tubular or lobed to the base,

glabrous to densely simple- or bifurcate-hairy, equally or unequally 5-dentate. Corolla 3-50 mm, colour usually white, pink, purple or yellow; wings and obtuse keel usually shorter than standard. Stamens diadclphous. Fruit a variously shaped, longitudinally septate legume (Davis, 1989). The most common use of Astragalus is as forage for livestock and wild animals, although 32 have been recognized as of use in foods, medicines, cosmetics, as substitutes for tea or coffee, or as sources of vegetable gums (Uphof, 1968). However, a number of species are toxic for livestock and in many cases the toxins could be transferred to humans through meat or milk (Panter and James, 1990; James et al., 1990). Astragalus genus is one of the richest sources of cycloartane saponins; Oleanane type saponins are also found in Astragalus sp., but their occurrence is limited to structures common to Leguminosae (Isaev et al., 1989).

Nevertheless, our study on Astragalus halicacabus lead to one new maltol type-glycoside, the first found in Astragalus genus (Djimtombaye et al., 2013).

1.2.1 Astragalus halicacabus Lam., (Section: Halicacabus)

Figure 1.1 Astragalus halicacabus

Astragalus halicacabus Lam., (Section: halicacabus ) is a Scapose, often prostrate perennial with woody caudex (Fig. 1.1). Leaves 10-35 cm; leaflets narrowly elliptic, acute, mucronulate, ± glabrous or with short sparse simple adpressed hairs; stipules 8-10 mm, lanceolate to linear, glabrous or white-ciliate. Peduncles 10-30 cm, glabrous or sparsely adpressed-hairy. Flowers in lax 8-25- flowered racemes. Bracts 5-8 mm, ovate to lanceolate, sparsely black-and white- hairy. Bracteoles 1-2 mm, lanceolate. Calyx 12-20 mm, tubular at first, becoming

globose-inflated, reticulate-veined, glabrous or with sparse adpressed black and white simple hairs; teeth 0.5-1 mm, very broadly triangular, densely hairy. Corolla pink or lilac; standard 25-30 mm. Ovary glabrous or hairy. Legume unknown. Fl. 5-7. Steppe, rocky slopes, 900-1900 m.

1.2.2 Astragalus melanocarpus Bunge., (Section: Hypoglottis)

Figure 1.2 Astragalus melanocarpus

Caulescent, the stems and caudex-branches together 7-20 cm. long; stipules 2-11.5 mm. long, all fully amplexicaul and connate; bracts submembranous, lance-acuminate or -caudate, 4-10 mm. long; calyx 7.8-10 mm. long; banner 19- 20.5 mm. long; pod oblong-ellipsoid, straight, about 1.7-2 cm. long, glabrous or very sparsely pubescent along the ventral suture and on the beak; ovules 33-40. Collections: Bethel, Willey & Clokey 4181 (WS); Ripley & Barneby 7585/ (RSA) (Fig. 1.2).

1.2.3 Chemical constituents of Astragalus species

The genus Astragalus appears highly uniform from a chemical point of view, with two kinds of pharmacologically active principles and three different kinds of toxic compounds. In the former group the polysaccharides and the saponins stand out, and in the second, the indolizidine alkaloids (swainsonine and its N-oxide derivative, and lentiginosine), the nitro compounds endecaphyllins (nitropropionic acid-glucose derivatives) and 3-nitropropylglucosides, and the seleniferous derivatives (selenocysteine, cystathionine, cystine, and methionine). There are other interesting compounds, such as flavonoids (flavonols, flavones, isoflavones, and flavylions) in free and glycosidic forms; pterocarpans free and as

glucosides; and organic acid derivatives (homopilosinic and phaseic acids). Triterpenes and saponins are the most widely studied secondary metabolite. About 40 saponins mainly derivatives of the 20R, 24S form of cycloastragenol, named astragalosides, and more rarely the 20S, 24R form, named astramembrainnins.

1.3 Terpenoids

Terpenoids are a large and structurally diverse group of secondary metabolites. The term terpenes originates from turpentine (lat. balsamum terebinthinae). Turpentine, the so-called "resin of pine trees", is the viscous pleasantly smelling balsam which flows upon cutting or carving the bark and the new wood of several pine tree species (Pinaceae). Turpentine contains the "resin acids" and some hydrocarbons, which were originally referred to as terpenes. Traditionally, all natural compounds built up from isoprene subunits and for the most part originating from plants are denoted as terpenes. Their basic structure follows a general principle: 2-Methylbutane residues, isoprene rule (C5)n found by Ruzicka and Wallach. In nature, terpenoids occur predominantly as hydrocarbons, alcohols and their glycosides, ethers, aldehydes, ketones, carboxylic acids and esters.

Terpenoids are classified basing on five-carbon (isoprene) units as their building blocks, numbering more than 55,000 molecules having been discovered (Ramawat et al., 2013), containing wide assortment of structural types. The terpenes are classified depending on the number of 2-methylbutane (isoprene) subunit (C5).

Hemiterpenes (C5), monoterpenes (C10) (Dewick, 1999), sesquiterpenes (C15) (Fraga, 2006), diterpenes (C20) (Hanson, 2000) and triterpenes (C30) (Connolly and Hill, 2005), tetraterpenes (C40) and polyterpenes (C5)n with n > 8 according to Fig. 1.3 (Breitmaier, 2006). The isopropyl part of 2-methylbutane is defined as the head, and the ethyl residue as the tail. In mono-, sesqui-, di- and sesterterpenes the isoprene units are linked to each other from head-to-tail; tri- and tetraterpenes contain one tail-to-tail connection in the center.

Figure 1.3 Classification of terpenes based on isoprene units

1.3.1 Monoterpenes (C10)

Monoterpenes are major components of the aromatic plants (Guenther et al., 1995) (Fig. 1.4). These volatile compounds known as essential oils, like geraniol; the major component of geranium oil (Pelar, qoililint grnveoltvis). Its isomer, linalool found in the oil of a garden herb. The other isomer citral, a constituent of lemon oil was obtained commercially from lemon grass oil.

Figure 1.4 Structures of monoterpenes

The more highly oxygenated monoterpenoides are a family of bicyclic of piran and cyclopentane known as the iridoids, like piscrosin D and piscrosin E, isolated from Neopicrorhiza scrophularii flawers (Fig. 1.5).

Figure 1.5 Monoterpene iridoids

Structures of common monoterpene skeletons are shown in Figure 1.6.

Figure 1.6 Monoterpene skeletons

1.3.2 Sesquiterpenes (C15)

Sesquiterpene lactones are common biologically active constituents of plants. Sesquiterpenoids are the most common secondary metabolites produced in families such as Cactaceae, Solanaceae, Araceae, and the Euphorbiaceae. Natural

sesquiterpenoids are found as more than 30 different structural types. The most common ones are the bisabolanes, cadinanes, chamigranes, cuparanes and lauranes. It is also known that representatives of the genus Centaurea L. are sources of biologically active sesquiterpene lactones (Fig. 1.7).

Figure 1 7 Common structures of sesquiterpene

1.3.3 Diterpenes (C20)

Diterpenes compose the main components of resins colophony (roughly 90%) i.e. abietane and pimarane tricyclic diterpenes. It has been widely used since antiquity as protective varnishes and binding agents in painting (Mayer, 1991; Doerner, 1998) (Fig. 1.8).

Figure 1.8 Diterpenes of abietane and pimarane

Another well known diterpenoid is steviol. Their glycosides are the major compounds in the leaves of Stevia plants (Brandle et al., 1998; Chaturvedula et al.

2011). Steviol glycosides are found in high concentration in the leaves of Stevia rebaudiana. Their intense sweetness, as well as high concentration in Stevia leaf tissue, has made them the subject for research and interest for over 100 years (Brandle et al., 2007) (Fig. 1.9).

Figure 1.9 Steviol glycosides from Stevia rebaudiana

1.3.4 Triterpenes (C30)

Triterpenoids are a large group of natural products derived from C30 units. Nearly 200 different triterpene skeletons reported (Xu et al., 2004) from natural sources. These advancements have stimulated the classification of natural products based on the biosynthesis of their carbon skeletons, structurally consistent with being cyclization products of squalene, oxidosqualene or bis- oxidosqualene. Triterpenes biosynthesis proceeds via isoprenoid pathway which 3 isoprene are first linked in a head-to-tail to give the C15 molecule farnesyl pyrophosphate. Two farnesyl pyrophosphates linked in a tail-to-tail manner to give a compound called squalene. Squalene is oxidized to oxidosqualene (1). Oxidosqualene (1) is the precursor of most 3-OH triterpenoids, which is the common starting point for cyclization reactions in triterpenoid biosynthesis. Enzyme mediated cyclization of squalene or oxidosqualene under the biogenetic rule is the most credible origin of these triterpenoids. Cyclization of oxidosqualene to saponins can proceed in two ways, either via the ‘chair–chair–

chair’ (C-C-C) (1b) or via the ‘chair–boat–chair’ (C-B-C) (1a) conformation. An important difference between the two resulting skeletons lies in the stereochemistry, which is most clearly shown by the configurations of the C8 and the C14 atoms. After cyclization of the ‘chair–chair–chair’ conformation, the methyl group at the C8 atom is pointing upwards and the one at the C14 atom is pointing downwards, whereas the opposite is the case after cyclization of the ‘chair–boat–chair’ conformation. Most triterpenoids are 6-6-6-5 tetracycles (Lanostane, steroid, cucurbitane, cycloartane, tirucallane, dammarane), 6-6-6-6-5 pentacycles (Lupane, hupane) or 6-6-6-6-6 pentacycles (Oleanane, taraxastene, ursane). An unusually complex and flexible reaction mechanism generates these ring systems.

1.3.5 Tetracyclic Triterpenes

Compounds that retain the C–B–C ring system, result from direct deprotonation of the protosteryl cation either without rearrangement or with only a 17 hydride shift. 1,2-hydride and methyl shifts generates the lanosteryl cation, which undergoes deprotonation at C8, C11 or C19 to form the sterol precursors lanosterol (Curtis et al., 1952), parkeol (Schreiber and Osske, 1964) and cycloartenol (Bentley et al., 1953). Lanosterol is the initial carbocyclic intermediate in the biosynthesis of cholesterol in mammals.

1.3.6 Triterpenoid Saponins

Saponins are secondary compounds with high-molecular-weight found primarily in plants kingdom (Lasztity et al., 1998; Oleszek, 2002; Hostettmann and Marston, 2005) but also by lower marine animals and some bacteria (Riguera, 1997; Yoshild et al. 1998). The term ‘saponin’ refer to the Latin word ‘sapo’ which means ‘soap’ because saponin molecules form soap-like foams when shaken with water, in the other words saponins forms a stable foam in aqueous solutions such as soap, hence the name. In the Orient, these compounds were used as soap and many trivial names of saponin-rich species are derived from this feature, e.g. soapwort (Saponaria of.cinalis), soaproot (Chlorogalum pomeridianum), soapbark (Quillaja saponaria), soapberry (Sapindus saponaria) or soap- nut (Sapindus mukurossi) . Some of them find a commercial application as drugs and medicines, adjuvants, taste modifiers, precursors of hormone synthesis (Oleszek et al., 2006).

Indeed, other known saponins properties are reported such as sweetness and bitterness (Grenby, 1991; Kitagawa, 2002; Heng et al., 2006b), foaming and emulsifying properties (Price et al., 1987), pharmalogical and medicinal properties (Attele et al., 1999), haemolytic properties (Oda et al., 2000; Sparg et al., 2004), as well as antimicrobial, insecticidal, and molluscicidal activities (Sparg et al., 2004) are well known and have found wide applications in beverages, confectionery as in cosmetics (Price et al., 1987; Petit et al., 1987; Uematsu et al., 2000), and pharmaceutical products (Sparg et al., 2004). Furthermore, the research on biological and pharmacological activities of saponins have been shown during the last decade, both in vitro and in vivo experimental test systems to possess a broad spectrum with interesting results such as cancer-related activity, immunostimulating, immunoadjuvant, antihepatoxic, antiplogistic, antiallergic, molluscicidal, hemolytic, antifungal, antiviral, and hypoglycemic activities (Lacaille-Dubois et al., 2000).

Chemically, saponins as a group include compounds that are glycosylated steroids, triterpenoids, and steroid alkaloids (Wina et al., 2005; Abe et al., 1993). Two main types of steroid aglycones are known, spirostan and furostan derivatives. The main triterpene aglycone is a derivative of oleanane. The carbohydrate part consists of one or more sugar moieties containing glucose, galactose, xylose, arabinose, rhamnose, or glucuronic acid glycosidically linked to a sapogenin (aglycone or non-saccharide portion of the saponin molecule called also genin). Saponins that have one sugar molecule attached at the C3 position are called monodesmoside saponins (desmos in Greek means chain), and those that have a minimum of two sugars, one attached to the C3 and one at C22, are called bidesmoside saponins (Wulff, 1968). There are two main types of triterpenoid saponins: neutral, when a normal sugar is attached to sapogenin, and acidic, when the sugar moiety contains uronic acid or one or more carboxylic groups attached to the sapogenin (K.A. Hostettman, 1995). Most of them, however, deal with either specific sources or specific properties or biological effects on different animals. Hostettmann and Marston (1995), are reported based on the type of genin present that the saponins can be divided into three major classes:

1) Triterpene glycosides

2) Steroid glycosides

3) Steroid alkaloid glycosides

Triterpene glycosides have either oleanane (β-amyrin) or dammarane skeletons, with ursanes, hopanes, lanostanes or lupanes assuming secondary importance with respect to distribution. The schematic correlation of main triterpene skeletones were given in Figure 1.10. (Nakanishi et al., 1974).

Figure.1.10 Cyclization of main triterpene correlation skeletones

Steroidal saponins (C27 based aglycones) fall into two major and one minor classification: the spirostanol glycosides, the furostanol glycosides, and the steriodal alkaloids, Figure 1.11 (Berger, 2001).

Figure.1.11 Basic steroidal saponin skeletons

The aglycones are normally hydroxylated at C-3 and certain methyl groups are frequently oxidized to hydroxymethyl, aldehyde or carboxyl functionalities. When an acid moiety is esterified to the aglycone, the term ester saponin is often used for the respective glycosides. All saponins have in common the attachment of one or more sugar chains to the aglycone. Monodesmosidic saponins have a single sugar chain, normally attached at C-3, bidesmosidic saponins have two sugar chains, often with one attached through an ether linkage at C-3 and one attached through an ester linkage (acyl glycoside) at C-28 (triterpene saponins) (Fig. 1.12) or and ether linkage at C-26 (furostanol saponins). (Hostettmann, and Marston, 1995). Tridesmosidic saponins such as Cephalotoside A, have three sugar chains and are seldom found (Fig. 1.13, Calis et al., 1999).

Figure 1.12 Monodesmosidic and bidesmosidic saponins

Bidesmosidic saponins are easily transformed into monodesmosidic saponins by, for example, hydrolysis of the esterified sugar at C-28 in triterpene saponins; they lack many of the characteristic properties and activities of monodesmosidic saponins (Hostettmann, and Marston, 1995). The carbohydrate part consists of one or more sugar moieties containing glucose, galactose, xylose, arabinose, rhamnose, or glucuronic acid glycosidically linked to a sapogenin (Wina et al., 2005). The great complexity of saponin structure arises from the variability of the aglycone structure, the nature of the side chains and the position of the attachment of these moieties on the aglycone. Experiments demonstrating the physiological, immunological and pharmacological properties of saponins have provoked considerable clinical interest in these substances (Francis et al.,2002).

Figure.1.13 Cephalotoside A, a tridesmosidic cycloartane type glycoside

1.4 Biosynthesis of Saponins

Cyclization of the saponin skeletons from oxidosqualene

Saponin biosynthesis proceeds via the isoprenoid pathway in which 3 isoprene units (molecules containing 5 C-atoms) are first linked in a head-to-tail manner to each other, resulting in the 15 C-atom molecule farnesyl pyrophosphate. Two farnesyl pyrophosphates are subsequently linked in a tail-to- tail manner to give a compound of 30 carbon atoms, called squalene (Holstein and Hohl, 2004). Squalene is oxidized to oxidosqualene, which is the common starting

point for cyclization reactions in triterpenoid biosynthesis (Fig. 1.14) (Abe et al., 1993; Haralampidis et al., 2002). Oxidosqualene is converted to cyclic derivatives via protonation and epoxide ring opening, which creates a carbocation that can undergo several types of cyclization reactions. After these cyclizations, subsequent rearrangements can proceed in different ways by a series of hydride shifts and/or methyl migrations, which lead to the formation of new carbocations. Finally, the carbocations are neutralized by proton elimination to give a double bond or a cyclopropanyl ring, or by reaction with water to give a hydroxyl group. In Figure 1.14, the main cyclization and rearrangement reactions are shown that lead to the triterpenoid and steroid skeletons that have been found to occur in the saponins.

The type of cyclase that is involved in the cyclization reaction primarily determines the skeleton that is formed (Fig. 1.14). Many different kinds of cyclases (e.g. cycloartenol synthase, lanosterol synthase, β-amyrin synthase) have been described, and their mechanisms of action are well documented (Abe et al., 1993; Wendt et al., 2000; Wendt, 2005; Haralampidis et al., 2002; Thoma et al., 2004).

Cyclization of oxidosqualene to saponins can proceed in two ways, either via the ‘chair–chair–chair’ (C-C-C) or via the ‘chair–boat–chair’ (C-B-C) conformation. An important difference between the two resulting skeletons lies in the stereochemistry, which is most clearly shown by the configurations of the C8 and the C14 atoms. After cyclization of the ‘chair–chair–chair’conformation, the methyl group at the C8 atom is pointing upwards and the one at the C14 atom is pointing downwards, whereas the opposite is the case after cyclization of the ‘chair–boat–chair’ conformation (see the dammarenyl and protosteryl carbocations, respectively, in Fig.1.14).

Figure 1.14 The cyclization of oxidosqualene to the various saponin skeletons

A proton-initiated cyclization of the ‘chair–chair–chair’ conformation results in the tetracyclic dammarenyl C20 carbocation, and all saponins derived

from this carbocation are known as dammarane type saponins (Ryu et al., 1997; Ma et al., 1999; Chakravarty et al., 2001).

A series of hydride and methyl shifts in the dammarenyl carbocation leads to the tirucallenyl C8 carbocation and the saponins derived from are tirucallane type saponins (Teng et al., 2003).

The 5-membered ring next to the C20 dammarenyl carbocation can expand either by a shift of the C16-C17 bond, or by a shift of the C13-C17 bond. A shift of the C16-C17 bond leads to the tetracyclic C17 baccharenyl carbocation and can be followed by a reaction with the C24-C25 double bond to produce the pentacyclic C25 lupenyl carbocation to lead lupane type saponins (Pambou Tchivounda et al., 1990; Elgamal et al., 1998; Xiang et al., 2000; Yook et al., 2002).

The lupenyl carbocation can be rearranged further, first to the C18 germanicenyl carbocation, and then via a series of hydride shifts to the C13 oleanyl carbocation. All saponins derived from this oleanyl carbocation are oleanane type saponins (Sparg et al., 2004). Oleanane type saponins have been isolated from a wide array of plants (Osbourn, 1996, 2003; Woldemichael and Wink, 2002; Treyvaud et al., 2000; Voutquenne et al., 2003; Wandji et al., 2003),

and this skeleton is also referred to as the β-amyrin skeleton (Haralampidis et al., 2002).

A shift of the a methyl group in the germanicenyl carbocation produces the C20 taraxasterenyl carbocation, which can be deprotonated to yield taraxasterane type saponins (Yahara et al., 1997; Cheng et al., 2002).

A methyl shift in the germanicenyl carbocation, followed by several hydride shifts, ultimately produces the C13 carbocation, which can be deprotonated to ursane type saponins (Babady-Bila et al., 1991; Amimoto et al., 1993; Zhao et al., 1997; Sanoko et al., 1999; Sahpaz et al., 2000).

The ursane skeleton is also called the α-amyrin skeleton. The α-amyrin and β-amyrin skeletons are the cyclization products of distinct cyclases, α- amyrin synthase and β -amyrin synthase, respectively (Haralampidis et al., 2002). A shift of the C13-C17 bond in the C20 dammarenyl carbocation leads to a C17 carbocation, which can be cyclized by a reaction with the double bond in the side

chain to form the C25 pentacyclic hopenyl carbocation. All saponins derived from this carbocation are classified as hopane type saponins (Hamed et al., 1996; Meselhy and Aboutabl, 1997; Meselhy, 1998; Hamed and El-Emary, 1999; Sahu et al., 2001; Biswas et al., 2005). It has been shown that, in bacteria, hopanes are cyclized from squalene by squalene-hopene cyclase (reviewed by Wendt, 2005). We have found no information in the literature, whether hopanes in plants are formed from oxidosqualene (as is indicated in Fig.1.13) or from squalene. All hopane type saponins described in plants (Fig.1.13) contain a hydroxyl group at the C3 atom. From the proton-initiated cyclization of the ‘chair–boat–chair’ conformation of oxidosqualene, a tetracyclic protosteryl C20 carbocation is obtained, which undergoes a series of hydride and methyl shifts ultimately leading to the intermediate C9 lanosteryl carbocation. This carbocation can undergo further shifts of a methyl group and a hydride to the C5 cucurbitanyl carbocation. All saponins derived from this carbocation are classified as cucurbitane type saponins (Oobayashi et al., 1992).

The lanosteryl carbocation may also undergo deprotonation of the C19 methyl group leading to formation of a cyclopropane ring as is found in cycloartenol. All saponins derived from cycloartenol are classified as cycloartane type saponins (Choi et al., 1989; Xu et al., 1992; Xu and Xu, 1992; Kennelly et al., 1996; Sun and Chen, 1997; Zhao et al., 1997; Verotta et al., 1998, 2001; Radwan et al., 2004).

Deprotonation of the lanosteryl carbocation gives lanosterol and all saponins derived from lanosterol are classified as lanostane type saponins (Pires et al., 2002; Mamedova et al., 2003). Lanosterol can also undergo demethylation and isomerisation of the double bond, leading to cholesterol. The saponins derived from this skeleton are classified as steroid type saponins (Yahara et al., 1996a; Corea et al., 2005). The difference in deprotonation, to a cyclopropane ring or a double bond, indicates that the cyclization is catalysed by different cyclases (cycloartenol and lanosterol synthase, respectively).

After cyclization, rearrangement and degradation, additional modifications of the different carbon skeletons follow, ultimately yielding the saponins that are found in nature. These modifications include mostly oxidation and glycosylation reactions.Very little is known about the enzymes and the biological pathways mediating these modifications (Haralampidis et al., 2002). The diversity of saponin aglycones can be seen from several reviews (Agarwal and Rastogi, 1974; Mahato et al., 1988; Mahato and Nandy, 1991a). Besides the carbon skeleton, the aglycones differ in their number and/or type of substituents attached (e.g. -OH, =O, -CH3, -CH2OH, ACHO, -COOH, and or glycosyl residues), the number and position of double bonds, lactones, and spirocompounds.

1.4.1 Cycloartane Saponins

Cycloartane triterpenoids were first discovered in Astragalus plants. Plants of this genus drew attention of researcher after it was established that they produce cycloartane triterpenoids. This discovery had caused special attention opening an opportunity and a new page in the study of Astragalus plants. Since then, their content of cycloartane methylsteroids and glycosides have come under intense scrutiny in many scientific centers of the world. Cycloartanes are derivatives of 9,19-cyclolanost-24-ene-3-ol (cycloartenol) through oxidation at C-6, C-16, C-20, C-23, C-24 (Agzamova et al., 1986), followed by possible ring closures and exclusively produced by photosynthetic eukaryotes. All of the available hydroxyl (OH) groups are subject to glycosidation in this class of

compounds (C-3, C-6, C-16, C-24 or C-25). Cycloartanes dominate the known triterpenoids in plants of Astragalus genus (Mamedova and. Isaev, 2004). In the most reviews, cycloartanes described come from 8 families:

Leguminosae (Astragalus)

Ranunculaceae (Thalictrum, Beesia, Souliea, Cimicifuga)

Meliaceae (Heynea, Aglaia)

Orchidaceae (Cirrhopetalum, Pholidata, Otochilus)

Passifloraceae (Passiflora)

Combretaceae (Combretum)

Asteraceae (Balsamorhiza)

Anacardiaceae (Mangifera)

Since their discovery in early 70’s, the cycloartanes require special attention of researchers and progressively until today several cyloartanes have been described. It should be noted that most cycloartanes describeted are largely belong to astragalus genus, and this demonstrates how these species are rich in cycloartanes.

According to the structural arrangement of cycloartanes, a total of 30 structurally distinct genins have been found in the plants and the substances were classified into six based on structural features of the side chains (Mamedova and Isaev, 2004).

A. Cycloartanes with an acyclic side chain (1-11),

B. 20,24-epoxycycloartanes (12-22),

C. 16β,23;16,24-diepoxycycloartanes(23-26),

D. 16β,24;20,24-diepoxycycloartanes (27),

E. 20,25-epoxycycloartanes (28),

F. 24-nor-16β,23-epoxycycloartanes (29,30).

A. Cycloartanes with an acyclic side-chain

B. Cycloartanes with an acyclic side-chain (20,24-epoxycycloartanes)

20,24-Epoxycycloartanes are the most common groups represented by four possible side-chain stereoisomers and two pairs of enantiomers: 20R,24S and 20S,24R (1); 20R,24R and 20S,24S (2), which only the first pair has been found in Astragalus plants. The Side chains of 10 genins have identical stereochemistry (20R, 24S), while just cyclogalegigenin is enantiomeric (20S, 24R). Furthermore, 20,24-epoxy side-chain cycloartanes, with oxygen bearing centers are generally C-3, C-6, C-7, C-11, C-12, C-16 and C-25 but can undergo oxidation resulting keto compounds often encountered in C-3 position (cycloasgenin A, cyclopycnanthogenin), C-6 position (huangqiyenin I), C-13position (cycloalpigenin A) and C-16 position (cycloadsurgenin).

C. Cycloartanes which have acyclic side-chain: 16β,23;16,24 diepoxycycloartanes and 16β,24;20,24-diepoxycycloartanes, 20,25- epoxycycloartanes, 24-nor-16β,23-epoxycycloartanes

Early in 1986, Agzamova et al., were first reported that this compound 16,23;16,24-diepoxycycloartane belong only to Astragalus orbiculatus.The cycloorbigenin is an acid hydrolysis product of glycosides obtained from Astragalus orbiculatus. Mamedova et al., (2002) reported that three different skeletons present in this class of compound such as dihydrocycloorbigenin A, cycloorbigenin B and Cycloorbigenin A; is a glycosidic forms isolated from the aerial parts of Astragalus orbiculatus.

The structures of 1-30 show that the side chains of the compounds differ substantially. Only genin 1 retains the intact side chain of cycloartenol. The side chain of the remaining compounds is oxidized to one degree or another. Most of the compounds contain in the acyclic side chain an -diol on C-24–C-25 and a 16β-hydroxyl. A common structural element for cycloartanes with an acyclic side chain, 16,23;16,24-diepoxycycloartanes, and 24-nor-16,23-epoxycycloartanes is a secondary CH3-21 methyl. 20,24-Epoxycycloartanes (12-22) represent the most numerous group of the four possible side-chain stereoisomers [two pairs of enantiomers: 20R,24S and 20S,24R (1) and 20R,24R and 20S,24S (2)], until 2004, only the first pair has been found in Astragalus plants. Side chains in 10 genins (12-21) have identical stereochemistry (20R,24S) whereas that of 22 is enantiomeric. Cyclosieversigenin (18) is the most widely distributed genin of Astragalus plant glycosides. Thus, it is not surprising that it was discovered first. Cyclosieversigenin is also known as cycloastragenol and astramembrangenin (Mamedova and Isaev, 2004).

D. Cycloartanes which have 16β,24;20,24-diepoxy side-chain

Several reviews have shown that this class of cycloartane are very unsual compounds occuring in plant kingdom. Cycloalpigenin (20R, 24R)-16,24:20,24- diepoxycycloartane-3,7,25-triol have been isolated for the first time from the aerial part of Astragalus alopecurus (Agzamova and Isaev, 1995). Other research have reported that the 16,24;20,24-diepoxycycloartane-type derivatives have been isolated olso from Astragalus alopecurus (Agzamova and Isaev, 1995),

Astragalus campylosema (Calis et al., 2008), Souliea vaginata and Beesia calthaefolia (Sakurai et al., 1990).

E. Cycloartanes which have 20,25-epoxy side-chain

The class of this compound cyclocephalogenin, was first established as cyclocepholoside I; a cycloartane-type glycoside from the roots of Astragalus microcephalus Willd (Bedir et al., 1998b). 20,25-epoxycycloartane genin is only represented by cyclocephalogenin found in free state in roots of Astragalus zahlbruckneri (Calis et al., 2001) and Astragalus caucasicus (Alaniya et al., 2007).

3-O-[-L-Arabinopyranosyl-(1→2)--D-xylopyranosyl]-3,6,25-tetrahydroxy-20R,24R- 16,24;20,24-diepoxycycloartane

F. Cycloartanes which have 24-nor-16β,23-epoxy side-chain

The structural type, 24-nor-16β,23-epoxy cycloartane, includes four different genins. These compounds are rarely found in plant kingdom because of their unstable nature on the side chain. Up to now, 24-nor-16β,23- epoxy cycloartane have only been determined in four Astragalus species: Astragalus dasyanthus Pall., Astragalus tomentosus Lam., Astragalus bicuspis, and Astragalus eremophilus. Eremophilosides isolated from Astragalus eremophilus have a sugar sequence of 3-O-α-L arabinopyranosyl-(1→2)-β-D-xylopyranosyl. Only tomentoside IV contains glucose as a sugar moiety. As a rule, all of these compounds carry xylose moiety at C-3, so they can be called xylocycloartanes. Except glycosilation at C-3 (O) position, no different glycosilation sites have been obtained on sapogenol of 24-nor-16β,23-epoxy cycloartane up to present. They are monodesmosidic glycosides and only two 24-nor-16β,23-epoxy cycloartane (tomentoside III and IV) contain acylation at C-6 position on sapogenol moiety (Radwan et al., 2004). Compounds of this structural type are hemiacetals. Therefore, native genins can not be prepared by methods such as acid catalysis. Under acid catalysis conditions, hemiacetals are readily alkylated to form acetals. Conditions favorable for forming acetals arise often during isolation and seperation of plant substances. During isolation of acetals of type 31-34 genins (Figure 1.15), the question of the authenticity of the isolated substance must be resolved (Mamedova and Isaev, 2004).

1.4.2 Cycloartane-Type Glycoside

During the first decade of research on the triterpenoid content of Astragalus plants, more cycloartane were discovered (Isaev et al., 1986). Verotta and Ei- Sebakhy (2001) had reviewed the cycloartane from Astragalus up to 1999 and more new cycloartanes were identified.Other several researches on Astragalus genus reported the isolation of new cycloartanes and their glycosides form(Bedir et al., 2001; Kim et al., 2008; Polat et al., 2010)

Additionally, Mamedova and Isaev (2004) and Li-Peng et al., 2013 reviewed the chemical and biological properties of cycloartanes from Astragalus together with their spectral data.

In this paper we have decided to make a literature search for the cycloartanes appeared since 2008 to 2014.

3-O-[α-L-arabinopranosyl-(1→2)-β-D-xylopyranosyl]-25-O-β-D- glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20R,24S-epoxycycloartane (Çalış et al., 2008)

Plant Material : Astragalus campylosema Boiss. ssp. campylosema

Molecular Formula : C46H76O18

Molecular Weight : 939

25 Optical Rotation : [α] D +24 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-Dxylopyranosyl]-25- O-β-D-glucopyranosyl-3β,6α,16β,25-tetrahydroxy-20R,24Sepoxycycloartane

Chemical shifts C-1 32.9 C-13 45.8 C-25 79.8 α-L-Arap-1 106.2 2 30.2 14 46.9 26 23.0 2 73.3 3 89.4 15 46.3 27 25.2 3 73.8 4 43.2 16 74.3 28 28.4 4 69.3 5 54.6 17 58.9 29 16.1 5 66.9 6 69.1 18 22.0 30 20.2 β-D-Glup-1 98.2 7 38.8 19 31.8 β-D-Xylp-1 105.6 2 74.8 8 48.5 20 88.4 2 83.1 3 77.9 9 21.5 21 28.2 3 76.7 4 71.1 10 30.2 22 35.3 4 70.7 5 77.3 11 26.6 23 26.1 5 65.7 6 62.4 12 33.6 24 82.9

3-O-[α-L-arabinopranosyl-(1→2)-β-Dxylopyranosyl]-16-O- hydroxyacetoxy-23-O-acetoxy-3β,6α,16β,23α,25-pentahydroxy-20R,24S- epoxycycloartane (Çalış et al., 2008)

Plant Material : Astragalus campylosema Boiss. ssp. campylosema

Molecular Formula : C44H70O17

Molecular Weight : 893

25 Optical Rotation : [α] D +67 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-Dxylopyranosyl]-

16-O-hydroxyacetoxy-23-O-acetoxy-3β,6α,16β,23α,25-pentahydroxy-20R,24S- epoxycycloartane

Chemical shifts C-1 33.2 C-13 46.3 C-25 79.0 β-D-Glup-1 105.8 2 30.3 14 46.7 26 26.0 2 83.2 3 89.4 15 46.8 27 27.0 3 76.7 4 43.0 16 77.6 28 28.3 4 70.8 5 54.3 17 58.5 29 16.0 5 66.9 6 69.1 18 21.1 30 20.5 α-L-Arap-1 106.4

7 38.5 19 31.7 OCOCH3 172.1 2 73.4

8 48.3 20 86.0 OCOCH3 20.7 3 73.9

9 21.2 21 28.3 OCOCH2OH 173.8 4 69.3

10 30.1 22 44.8 OCOCH2OH 61.3 5 67.0 11 26.7 23 76.0 12 33.3 24 85.6

3-O-[-L-arabinopyranosyl-(1→2)--D-xylopyranosyl]-3,6,25- tetrahydroxy-20R,24R-16,24;20,24-diepoxycycloartane (Çalış et al., 2008).

Plant Material : Astragalus campylosema Boiss. ssp. campylosema

Molecular Formula : C40H64O14

Molecular Weight : 791

25 Optical Rotation : [α] D +20 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-Dxylopyranosyl]- 3β,6α,23α,25-tetrahydroxy-20R,24R-16β,24;20,24-diepoxycycloartane

Chemical shifts C-1 33.1 C-13 45.0 C-25 75.0 β-D-Glup-1 105.8 2 30.4 14 45.8 26 24.3 2 83.2 3 89.3 15 42.7 27 25.7 3 76.7 4 43.0 16 75.5 28 28.5 4 70.8 5 54.6 17 61.7 29 16.0 5 65.8 6 69.3 18 23.5 30 20.2 α-L-Arap-1 106.4 7 39.1 19 33.1 2 73.4 8 48.0 20 84.6 3 73.9 9 21.3 21 30.1 4 69.3 10 30.1 22 43.6 5 67.0 11 27.2 23 77.6 12 34.2 24 108.6

Cyclomacrogenin B (Iskenderov et al., 2008)

Plant Material : Astragalus macropus

Molecular Formula : C30H52O5

Molecular Weight : 492.37

13 C-NMR Solvent : CD3OD

Name : 24R-cycloartan-1α,3β,7β,24,25-pentaol

Chemical shifts C 13C 1H C 13C 1H C 13C 1H 1 72.65 3.87 11 26.47 1.52, 2.62 21 18.68 1.00 2 38.95 2.21, 2.40 12 33.30 1.65, 1.80 22 34.22 1.65, 1.80 3 72.96 4.40 13 45.99 - 23 28.20 1.83, 1.83 4 40.98 - 14 49.13 - 24 79.06 3.77 5 39.36 2.68 15 37.86 1.52, 1.99 25 72.72 - 6 32.31 1.42, 2.21 16 28.98 1.83, 1.99 26 25.94 1.50 7 70.14 4.00 17 52.38 1.63 27 26.19 1.53 8 55.21 1.95 18 17.91 1.12 28 19.02 1.25 9 21.00 - 19 28.98 0.43, 0.93 29 26.03 1.28 10 31.32 - 20 36.43 1.55 30 13.98 1.15

Cycloascauloside B (Alaniya et al., 2008)

Plant Material : Astragalus caucasicus

Molecular Formula : C42H70O14

Molecular Weight : 798

13 C-NMR Solvent : C5D5N

Name : 20R,25-epoxy-24S-cycloartan-3β,6α,16β,24-tetraol-3-O-[α-L- rhamnopyranosyl(1→2)]-β-D-glucopyranoside

Chemical shifts C-1 32.6 C-13 45.9 C-25 75.3 2 30.5 14 46.9 26 28.6 3 88.8 15 47.8 27 28.7 4 42.6 16 74 28 19.1 5 54.2 17 160.8 29 20.3 6 68.7 18 20.7 30 16.7 7 38.5 19 30.3 β-D-Glup-1 105.9 α-L-Rhap-1 101.8 8 46.7 20 79.9 2 79.0 2 72.1 9 21.0 21 28.0 3 78.0 3 72.5 10 28.7 22 26.6 4 72.5 4 73.9 11 26.3 23 24.0 5 78.1 5 69.6 12 34.2 24 68.0 6 62.8 6 18.7

Astramembranoside A (Kim et al., 2008)

Plant Material : Astragalus membranaceus

Molecular Formula : C42H69O15

Molecular Weight : 813

18 Optical Rotation : [α] D +23.5 (c 0.11, MeOH)

13 C-NMR Solvent : C5D5N

Name : Cycloastragenol 6,25-di-O-β-D-glucopyranoside

Chemical shifts

C-1 32.6 C-13 45.3 25 78.7 -D-Glcp2-1 99.0 2 31.3 14 46.2 26 22.9 2 75.2 3 78.3 15 45.8 27 25.7 3 78.2 4 42.5 16 76.6 28 29.1 4 71.4 5 52.5 17 58.1 29 16.1 5 78.1 6 79.6 18 21.3 30 19.9 6 62.8

7 34.5 19 29.4 -D-Glcp1-1 105.9 8 46.0 20 87.2 2 75.4 9 21.1 21 27.8 3 79.4 10 29.5 22 35.1 4 71.9 11 26.3 23 26.1 5 78.2 12 33.5 24 82.1 6 63.0

Eremophiloside A (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C55H92O22

Molecular Weight : 1127

25 Optical Rotation : [α] D +8.7 (c 0.2, MeOH)

13 C-NMR Solvent : CD3OD

MP : 264-266 °C

Name : 3-O-α-L-rhamnopranosyl-6-O-α-Lrhamnopranosyl-24-O-β-D- fucopyranosyl-25-O-β-D-xylopyranosyl-16-O-acetoxy3β,6α,16β,24R,25- pentahydroxycycloartane

Chemical shifts

C-1 32.5 C-16 76.8 OCOCH3 173.2 -D-Fucp-1 104.9

2 29.5 17 56.8 OCOCH3 21.8 2 73.3 3 89.7 18 18.3 3 75.7

4 42.6 19 30.2 α-L-Rhap1-1 104.0 4 73.7 5 53.0 20 32.0 2 72.0 5 71.2 6 80.6 21 18.2 3 72.3 6 16.4 7 34.8 22 34.4 4 73.0 8 47.5 23 30.2 5 69.8 -D-Xylp-1 98.7 9 21.4 24 86.8 6 17.6 2 74.8

10 29.4 25 83.4 α-L-Rhap2-1 103.9 3 77.4 11 26.6 26 19.5 2 72.4 4 71.o 12 33.2 27 24.3 3 72.2 5 66.4 13 46.4 28 28.9 4 73.5 6 14 47.8 29 16.4 5 69.5 15 46.1 30 20.2 6 17.6

Eremophiloside B (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C49H82O18

Molecular Weight : 981

25 Optical Rotation : [α] D -2.5 (c 0.2, MeOH)

13 C-NMR Solvent : CD3OD

MP : 270-272 °C

Name : 3-O-α-L-rhamnopranosyl-6-O-α-Lrhamnopranosyl-25- O-β-D-xylopyranosyl-16-Oacetoxy-3β,6α,16β,24(R),25-pentahydroxycycloartane

Chemical shifts C-1 32.4 C-11 26.3 C-21 27.9 -D-Xylp- 1 107.4 2 30.3 12 33.3 22 35.0 2 75.3 3 88.7 13 45.1 23 25.9 3 78.1 4 42.6 14 46.0 24 82.1 4 71.0 5 53.7 15 46.8 25 78.2 5 66.8 6 68.1 16 73.4 26 23.0 -D-Glcp-1 98.6 7 38.4 17 58.0 27 25.7 2 74.7 8 47.1 18 21.6 28 19.9 3 78.8 9 20.8 19 30.8 29 28.8 4 71.1 10 29.9 20 87.3 30 16.6 5 77.7 6 62.4

Eremophiloside C (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C40H68O14

Molecular Weight : 795

25 Optical Rotation : [α] D +23.7 (c 0.2, MeOH)

13 C-NMR Solvent : CD3OD

MP : 278-280 °C

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-Dxylopyranosyl]-

3β,6α,16β,23(R),24(R),25-hexahydroxycycloartane

Chemical shifts C-1 32.6 C12 33.4 C-23 27.1 4 70.9 2 30.0 13 45.9 24 83.0 5 66.5 3 89.9 14 47.4 25 72.4 -D-Glcp-1 104.7 4 42.9 15 45.3 26 25.5 2 75.1 5 52.8 16 77.6 27 25.8 3 78.1 6 79.2 17 58.4 28 20.1 4 70.4 7 33.6 18 20.3 29 28.1 5 77.6 8 45.4 19 28.0 30 16.3 6 62.7 9 22.4 20 87.0 -D-Xylp-1 107.1 Ac 20.6 10 29.5 21 27.8 2 75.1 Ac 171.2 11 26.8 22 37.1 3 77.8

Eremophiloside D (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C40H66O14

Molecular Weight : 793

25 Optical Rotation : [α] D +10.3 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

MP : 289-291 C

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-D-xylopyranosyl]-3β,6α,16β,23(R),24(R),25-pentahydroxycycloartan-6-one

Chemical shifts C-1 73.1 C-12 33.0 C-23 28.0 3 77.2a 2 36.4 13 45.9 24 77.0 4 70.8b 3 84.1 14 48.9 25 80.5 5 76.8 4 40.7 15 37.5 26 22.0 6 61.8

5 39.3 16 28.6 27 21.6 -D-Glcp-1 97.0 6 31.0 17 52.2 28 24.8 2 74.7 7 70.2 18 17.4 29 13.5 3 77.3c 8 55.1 19 28.4 30 18.2 4 70.6

9 21.0 20 36.2 -D-Glcp-1 105.7 5 77.0 10 30.4 21 17.9 74.3 62.1 11 26.0 22 33.7

Eremophiloside E (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C36H56O12

Molecular Weight : 703

25 Optical Rotation : [α] D -9.9 (c 0.15, MeOH)

13 C-NMR Solvent : CD3OD

MP : 275-278 C

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-D-xylopyranosyl]-3β,6α,16β-trihydroxy-24,25,26,27-tetranorcycloartan-23,16β-olide

Chemical shifts C-1 32.2 C-11 26.8 C21 26.3 -D-Xylp- 1 107.6 2 30.2 12 33.1 22 39.0 2 75.7 3 88.7 13 46.9 23 26.4 3 78.7 4 41.5 14 47.0 24 84.5 4 71.6 5 47.7 15 48.0 25 71.4 5 67.2

6 21.0 16 83.7 26 26.3 -D-Glcp-1 106.7 7 26.2 17 60.1 27 27.7 2 75.7 8 47.8 18 21.7 28 20.7 3 79.0 9 20.0 19 30.2 29 26.3 4 72.0 10 25.9 20 87.2 30 15.6 5 78.5 6 63.1

Eremophiloside F (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C36H54O12

Molecular Weight : 701

25 Optical Rotation : [α] D +11.1 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

MP : 285-288 0C

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-D-xylopyranosyl]-3β,16β-dihydroxy-24,25,26,27-tetranorcycloartan-6-on-23,16β-olide

Chemical shifts C-1 31.9 C-11 26.0 C-21 20.6 -D-Xylp-1 107.0 2 29.9 12 33.0 22 33.2 2 75.5 3 87.5 13 44.8 23 99.0 3 78.6 4 42.2 14 46.0 28 19.3 4 71.2 5 56.5 15 43.3 29 26.8 5 67.2 6 70.2 15 70.4 30 16.4 7 38.1 16 44.5 Ac 21.7 8 49.9 17 19.8 Ac 170. 3 9 20.9 18 27.8 Et 15.4 10 28.2 19 25.6 Et 62.7

Eremophiloside G (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C40H62O12

Molecular Weight : 757

25 Optical Rotation : [α] D -30.8 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

MP : 160-162 0C

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-D-xylopyranosyl]-3β,6α-dihydroxy-16β,23-epoxy-22,23-didehydro-25-hydrocycloartan-24- one

Chemical shifts C-1 32.11 C-11 26.70 C-21 28.84 O--D-Xylp-1 107.61 2 30.11 12 34.14 22 26.79 2 75.60 3 88.42 13 46.03 23 24.11 3 78.55a 4 42.60 14 46.80 24 68.77 4 71.26 5 52.35 15 47.26 25 75.25 5 67.05 6 78.55a 16 73.98 26 28.63 O--D-Xylp-1 105.74 7 34.24 17 60.76 27 28.16 2 75.36 8 44.91 18 20.43 28 19.96 3 78.29 9 21.19 19 29.59 29 28.16 4 71.10 10 28.16 20 79.00 30 16.71 5 66.99

Eremophiloside H (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C41H66O13

Molecular Weight : 789

25 Optical Rotation : [α] D -10.7 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

MP : 169-171 °C

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-D-xylopyranosyl]-3β,6α-dihydroxy-23α-methoxy-16β,23-epoxy-25-hydroxycycloartan-24-one

Chemical shifts C-1 32.3 C-13 46.0 C-25 75.2 -D-Xylp-1 105.4 -D-Glcp2-1 105.0 2 30.2 14 46.9 26 28.6 2 83.9 2 75.7 3 88.5 15 47.3 27 28.0 3 77.7 3 79.0 4 42.6 16 74.0 28 20.1 4 70.9 4 72.0 5 52.5 17 60.9 29 28.6 5 66.6 5 78.0 6 79.3 18 20.8 30 16.6 -D-Glcp1-1 106.2 6 63.2 7 34.6 19 29.0 2 77.0 8 45.7 20 79.0 3 78.0 9 21.2 21 28.9 4 72.0 10 29.0 22 26.7 5 78.1 11 26.4 23 24.1 6 63.0 12 34.2 24 68.8

Eremophiloside I (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C42H68O13

Molecular Weight : 803

25 Optical Rotation : [α] D -12.0 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-D-xylopyranosyl]-3β,6α-dihydroxy-23α-ethoxy-16β,23epoxy-25-hydroxycycloartan-24-one

MP : 170-173 °C

Chemical shifts C-1 33.2 C-13 45.4 C-25 -D-Xylp-1 105.1 2 30.2 14 47.3 26 2 83.1 3 89.4 15 44.1 27 3 76.6 4 42.8 16 73.6 28 4 70.7 5 54.4 17 57.4 29 5 65.8 6 68.9 18 20.7 30 α-L-Rhap-1 106.2

7 38.6 19 31.5 OCH2CH3 59.6 2 73.2

8 48.0 20 26.5 OCH2CH3 15.6 3 73.8 9 21.9 21 20.5 4 69.2 10 30.3 22 40.3 5 66.3 11 26.7 23 104.0 12 33.8 24 215.4

Eremophiloside J (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C40H64O14

Molecular Weight : 791

25 Optical Rotation : [α] D +13.9 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

Name : 3-O-[α-L-arabinopranosyl-(1→2) -D-xylopyranosyl]-3β,6α,23α,24β-tetrahydroxy-16β,23;22β,25-diepoxycycloartane

MP : 260-262 °C

Chemical shifts C-1 33.1 C-13 53.3 C-25 80.8 β-D-Xylp-1 105.1 2 30.4 14 47.2 26 28.8 2 83.1 3 89.4 15 43.7 27 21.1 3 76.6 4 42.7 16 72.8 28 28.3 4 70.7 5 54.5 17 52.5 29 15.9 5 65.8 6 69.1 18 20.6 30 19.7 α-L-Arap-1 106.2 7 38.7 19 31.9 2 73.2 8 48.2 20 36.5 3 73.8 9 21.3 21 16.8 4 79.2 10 29.8 22 85.4 5 66.8 11 26.6 23 103.5 12 34.1 24 81.9

Eremophiloside K (Perrone et al., 2008)

Plant Material : Astragalus eremophilus

Molecular Formula : C40H62O14

Molecular Weight : 789

Optical Rotation : [α] 25D +7.5 (c 0.1, MeOH)

13 C-NMR Solvent : CD3OD

Name : 3-O-[α-L-arabinopranosyl-(1→2)-β-D-xylopyranosyl]-3β,23α,24β-trihydroxy-16β,23;22β,25-diepoxy-cycloartan-6-one

MP : 269-271 °C

Chemical shifts C-1 33.1 C-13 46.1 C-25 73.9 α-L-Rhap-1 101.7 2 30.1 14 46.8 26 2 71.8 3 89.5 15 48.8 27 26.3 3 71.7 4 43.0 16 72.3 28 19.9 4 73.6 5 54.5 17 57.8 29 28.2 5 69.7 6 69.2 18 30 16.3 6 18.0 7 38.5 19 31.5 β-D-Xylp-1 105.9 β-D-Glcp-1 104.7 8 48.4 20 30.7 2 78.5 2 75.2 9 21.4 21 17.7 3 78.5 3 77.7 10 29.9 22 32.9 4 71.2 4 71.2 11 26.6 23 29.3 5 65.9 5 77.7 12 33.7 24 89.8 6 62.2

Cyclomacrogenin B (Iskenderov et al., 2008)

Plant Material :Astragalus macropus Bunge

Molecular Formula : C30H52O52

Molecular Weight : 209-211C

13 C-NMR Solvent : C5D5N

Name : 24R-cycloartan-1α,3β,7β,24,25-pentaol

Chemical shifts C-1 72.6 C-13 45.9 C-25 72.7 2 38.9 14 49.1 26 25.9 3 72.9 15 37.8 27 26.1 4 40.9 16 28.9 28 19.0 5 39.3 17 52.3 29 26.0 6 32.3 18 17.9 30 13.9 7 70.1 19 28.9 8 55.2 20 36.4 9 21.0 21 18.6 10 31.3 22 34.2 11 26.4 23 28.2 12 33.3 24 79.0

Bicusposide B (Choudhary et al., 2008)

Plant Material : Astragalus bicusp

Molecular Formula : C32H51O8

25 Optical Rotation : [α]D -4.4 (c 0.5, MeOH)

13 C-NMR Solvent : C5D5N

Name : 6α-hydroxy-23-methoxy-16β,23R-epoxy-24,25,26, 27-tetranor-9,19-cyclolanosta-3-O-β-xyloside

Chemical shifts C-1 32.4 C-13 46.1 29 16.6 2 30.3 14 44.8 30 20.2

3 88.6 15 43.5 OCH3 54.6 4 42.7 16 70.7 β-D-Xylp-1 107.6 5 53.9 17 56.6 2 75.6 6 67.4 18 19.5 3 78.5 7 37.9 19 29.6 4 71.3 8 46.2 20 25.6 5 67.1 9 21.2 21 20.5 10 29.2 22 38.5 11 26.2 23 100.4 12 33.3 28 28.7

Bicusposide C (Choudhary et al., 2008)

Plant Material : Astragalus bicuspis

Molecular Formula : C35H53O10

Optical Rotation : [α] 25D -4.8 (c 0.5, MeOH)

13 C-NMR Solvent : C5D5N

Name :23R,24S,25R,26S-16β/23,23/26,24/25-triepoxy- 6α,26-dihydroxy,9,19-cyclolanosta-3-O-β-xyloside

Chemical shifts C-1 32.4 C-13 46.2 25 63.7 2 30.3 14 44.7 26 97.7 3 88.6 15 43.7 27 13.2 4 42.6 16 74.5 28 28.7 5 53.7 17 56.4 29 16.6 6 67.4 18 19.5 30 20.2 7 38.2 19 29.7 β-D-Xylp-1 107.6 8 46.1 20 26.2 2 75.6 9 21.1 21 20.6 3 78.5 10 29.2 22 42.5 4 71.3 11 26.1 23 105.9 5 67.0 12 33.2 24 64.1

Astramembranoside B (Kim et al., 2008)

Plant Material : Astragalus membranaceus

Molecular Formula : C41H69O14

25 Optical Rotation : [α] D +32.1 (c 0.15, MeOH)

13 C-NMR Solvent : C5D5N

Name : Cyclocanthogenin 3-O-β-D-glucopranosyl(1→2)- β-D-xylopranoside

Chemical shifts C-1 32.5 C-13 45.7 25 72.5 β-D-Glup-1 106.2 2 30.4 14 46.8 26 25.8 2 77.1 3 88.6 15 48.3 27 26.5 3 78.3 4 42.8 16 72.0 28 28.8 4 71.7 5 54.0 17 57.3 29 16.6 5 78.0 6 67.7 18 18.8 30 20.1 6 62.8 7 38.3 19 29.6 β-D-Xylp-1 105.7 8 46.7 20 28.6 2 83.4 9 21.4 21 18.3 3 77.9 10 29.1 22 33.0 4 71.0 11 26.3 23 27.9 5 66.7 12 33.2 24 77.2

Cyclomacroside D (Iskenderov et al., 2009)

Plant Material : Astragalus macropus Bunge

Molecular Formula : C41H70O13

Molecular Weight : 770

13C-NMR Solvent : C5D5N

Name : 24R-cycloartan-1α,3β,7β,24,25-pentaol-3-O- α-L-rham-nopyranoside-24-O-β-D-xylopyranoside

M.P : 151-153 °C

Chemical shifts C-1 72.3 C-16 29.5 α-L-Rhap-1 104.8 2 37.0 17 52.1 2 72.3 3 84.1 18 17.9 3 72.8 4 40.7 19 28.9 4 74.0 5 39.1 20 36.7 5 69.7 6 32.5 21 19.0 6 18.3 7 69.9 22 33.5 8 55.5 23 28.2 β-D-Xylp-1 106.0 9 21.0 24 89.5 2 75.1 10 30.9 25 71.9 3 78.3 11 26.4 26 25.6 4 70.8 12 33.1 27 26.7 5 67.2 13 46.0 28 18.5 14 49.0 29 25.6 15 37.9 30 14.4

Moreover, we also in this study enriched our literature with chemical and biological investigations of a few new cycloartane-type triterpene and oleanane saponin Phytochemically isolated from Turkish Astragalus species up to 2009 until 2014.

In 2010, the Phytochemical study on Turkish Astragalus species reported by Polat et al (2010), indicated that three cycloartane-type triterpene glycosides were isolated from Astragalus wiedemannianus (Sect. Pterophorus) and their structures were established by using the combination of 1D and 2D-NMR techniques as 3- O-[-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl]-25-O-β-D- glucopyranosyl-20(R),24(S)-epoxy-3β,6,16β,25-tetrahydroxycycloartane (31), 3- O-[-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl- 24-O--(4’-O-acetoxy)-L-arabinopyranosyl-16-O-acetoxy-3β,6,16β,24(S),25- pentahydroxycycloartane (32), 3-O-[-L-rhamnopyranosyl-(1→2)-β-D- xylopyranosyl]-6-O-β-D-glucopyranosyl-24-O--L-arabinopyranosyl-16-O- acetoxy-3β,6,16β,24(S),25-pentahydroxycycloartane (33) together with eight secondary metabolites known as cycloastragenol, cycloascauloside B, astragaloside IV, astragaloside VIII, brachyoside B, astragaloside II, astrachrysoside A, and astrasieversianin X. The group reported for the first time the presence of an arabinose moiety on the acyclic side chain of cycloartane.

Another phytochemical study were accomplished on Astragalus icmadophilus (Sect. Acanthophace) leaded on isolation of six new cycloartane- type triterpene glycosides established as 3-O-[-L-arabinopyranosyl-(1→2)-O-3- acetoxy--L-arabinopyranosyl]-6-O-β-D-glucopyranosyl-3β,6,16β,24(S),25- pentahydroxycycloartane (34), 3-O-[-L-rhamnopyranosyl-(1→2)-O--L- arabinopyranosyl-(1→2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl- 3β,6,16β,24(S),25-pentahydroxy cycloartane (35), 3-O-[-L-arabinopyranosyl- (1→2)-O-3,4-diacetoxy--L-arabinopyranosyl]-6-O-β-D-glucopyranosyl- 3β,6,16β,24(S),25-pentahydroxycycloartane (36), 3-O-[-L-arabinopyranosyl- (1→2)-O-3-acetoxy--L-arabinopyranosyl]-6-O-β-D-glucopyranosyl- 3β,6,16β,25-tetrahydroxy-20(R),24(S)-epoxycycloartane (37), 3-O-[-L- arabinopyranosyl-(1→2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl- 3β,6,16β,24-tetrahydroxy-20(R),25-epoxycycloartane (38), 3-O-[-L- rhamnopyranosyl-(1→2)-O--L-arabinopyranosyl-(1→2)-O-β-D-xylopyranosyl]- 6-O-β-D-glucopyranosyl-3β,6,16β,24-tetrahydroxy-20(R),25-epoxycycloartane (39) by the extensive use of 1D- and 2D-NMR experiments along with ESIM, HRMS analysis together with two known cycloartane-type glycosides: oleifolioside B and astragaloside I and five known oleanane-type triterpene glycosides, azukisaponin V, azukisaponin V methyl ester , astragaloside VIII, astragaloside VIII methyl ester, 22-O-[β-D-glucopyranosyl-(1→2)-O--L- arabinopyranosyl]-3β,22β,24-trihydroxy-olean-12-ene and one known flavonol glycoside narcissin. The first four compounds are cyclocanthogenin and cycloastragenol glycosides, whereas the last two are based on cyclocephalogenin as aglycone, more unusual in the plant kingdom and reported only from Astragalus spp.(Polat et al.; 2010)

Phytochemical study was performed on Astragalus hareftae (Sect. Acanthophace) which resulted in isolation of four cycloartane-(hareftosides) and oleanane-type triterpenoids (hareftoside E) along with eleven known compounds. Structures of the new compounds were established as 3,6-di(O-β-D- xylopyranosyl)-3β,6,16β,24(S),25-pentahydroxycycloartane (40), 3,6,24-tri(O-β- D-xylopyranosyl)-3β,6,16β,24(S),25-pentahydroxycycloartane (41), 3-O-β-D- xylopyranosyl-3β,6,16β,25-tetra-hydroxy-20(R),25(S)-epoxycycloartane (42), 16-O-β-D-glucopyranosyl-3β,6,16β,25-tetrahydroxy-20(R),24(S)- epoxycycloartane (43), 3-O-[β-D-xylopyranosyl-(1→2)-O-β-D-glucopyranosyl- (1→2)-O-β-D-glucuronopyranosyl] soyasapogenol B by the extensive use of 1D- and 2D-NMR experiments along with ESI-MS and HR-MS analyses. The known compounds were identified as cyclocanthoside E, macrophyllosaponin B, 3-O-β- D-xylopyranosyl-6,25-di-O-β-D-glucopyranosyl-3β,6,16β,24(S),25-

pentahydroxycycloartane, oleifoloside B, cyclocephaloside I, astrasieversianin X, trojanoside B, cycloastragenol, astragaloside IV, brachyoside B, cyclodissectoside, and four known oleanane-type triterpene glycosides, azukisaponin V, dehydroazukisaponin V, wistariasaponin D , astragaloside VIII (Horo et al., 2012).

Three new cycloartane-type triterpene glycosides were isolated from the roots of Astragalus schottianus Boiss. (Sect. Rhacophorus). Their structures were established as 20(R),25-epoxy-3-O-β-D-xylopyranosyl-24-O-β-D-glucopyranosyl- 3β,6,16β,24-tetrahydroxycycloartane (45), 20(R),25-epoxy-3-O-[β-D- glucopyranosyl(1→2)]-β-D-xylopyranosyl-24-O-β-D-glucopyranosyl- 3β,6,16β,24-tetrahydroxycycloartane (46), 3-O-β-D-xylopyranosyl- 3β,6,16β,20(S),24(S),25-hexahydroxycycloartane (47) by the extensive use of 1D and 2D-NMR techniques and mass spectrometry (Karabey et al.,2012).

Phytochemical investigation of Astragalus erinaceus (Sect. Rhacophorus) was resulted in isolation of a new cycloartane-type saponin, 3-O-[β-D- xylopyranosyl-(1→2)-β-D-xylopyranosyl]-6-O-β-D-glucuronopyranosyl- 3β,6α,16β,24(S),25-pentahydroxycycloartane (48). Additionally, five known saponins cyclodissectoside, cycloastragenol,6-O-β-D-glucopyranosyl- 3β,6α,16β,24(S),25-pentahydroxycycloartane, oleifolioside B and 3,6-di-O-β-D- xylopyranosyl-3β,6α,16β,24(S),25-pentahydroxycycloartane were also identified (Savran et al., 2012).

Eight cycloartane-type triterpene glycosides were isolated from Astragalus aureus Willd along with ten known cycloartane-type glycosides such as 3-O-[- L-rhamnopyranosyl-(1→2)-O--L-arabinopyranosyl-(1→2)-O-β- Dxylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6,16β,24(S),25- pentahydroxycycloartane, (Horo et al., 2010), oleifolioside B (Ozipek et al., 2005), cyclocanthoside G, (Isaev et al., 1992),cyclocanthoside E (Isaev et al.,

1992), 3-O-[-L-rhamnopyranosyl-(1→2)-O--L-arabinopyranosyl-(1→2)-O-β- D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6,16β,24-tetrahydroxy- 20(R),25-epoxycycloartane (Horo et al., 2010), 3-O-[-L-arabinopyranosyl- (1→2)-O-β-D-xylopyranosyl]-6-O-β-D-glucopyranosyl-3β,6,16β,24- tetrahydroxy-20(R),25-epoxycycloartane (Horo et al., 2010), cyclocanthoside F, (Agzamova and Isaev, 1999),cyclocephaloside I (Bedir et al., 1998a), cyclotrisectoside (Sukhina et al., 2007) and macrophyllosaponin B (Calıs et al.,1996). The structures of new compounds were elucidated by the extensive use of 1D and 2D-NMR experiments along with ESIMS and HRMS analysis and were established. as 3-O-[-L-rhamnopyranosyl-(1→2)--Larabinopyranosyl-(1→2)- β-D-xylopyranosyl]-6-O-β-D-xylopyranosyl-3β,6,16β,24(S),25- pentahydroxycycloartane (49), 3,6-di-O-β-D-xylopyranosyl-3β,6,16β,24(S),25- pentahydroxycycloartane (50), 3,6-di-O-β-D-xylopyranosyl-25-O-β-D- glucopyranosyl-3β,6,16β,24(S),25-pentahydroxycycloartane (51), 3-O-β-D- xylopyranosyl-6,25-di-O-β-D-glucopyranosyl-3β,6,16β,24(S),25- pentahydroxycycloartane (52), 6-O-β-D-glucopyranosyl-3β,6,16β,24(S),25- pentahydroxycycloartane (53), 3-O-[a-L-arabinopyranosyl-(1→2)-β-D- xylopyranosyl]-3β,6,16β,24-tetrahydroxy-20(R),25-epoxycycloartane(54), 6- O-β-D-glucopyranosyl-3β,6,16β,24-tetrahydroxy-20(R),25-epoxycycloartane (55), 6-O-β-D-xylopyranosyl-3β,6,16β,24-tetrahydroxy-20(R),25- epoxycycloartane (56). The compounds 49-53 are cyclocanthogenin glycosides, whereas compounds are based on cyclocephalogenin as aglycon, more unusual in the plant kingdom, so far reported only from Astragalus spp (Bedir et al.,1998a; Sukhina et al., 2007; Agzamova and Isaev, 1999; Semmaret al., 2001). Moreover, for the first time monoglycosides of cyclocanthogenin and cyclocephalogenin are reported. All of the compounds tested for their cytotoxic activities against a number of cancer cell lines. Among the compounds, only exhibited activity versus human breast cancer (MCF7) at 45 lM concentration (Gülcemal et al.; 2011).

In 2012, another phytochemical investigation of Astragalus tauricolus has been carried out leading to twenty two oleananes-type triterpene glycosides, including ten compounds never reported before. Their structures were established by the extensive use of 1D and 2D-NMR experiments along with ESIMS and HRMS analysis.as 3-O-[-L-rhamnopyranosyl-(12)-β-D-xylopyranosyl-(1→2)-β- D-glucuronopyranosyl]-29-O-β-Dglucopyranosyl-3β,22β,24-trihydroxyolean-12- en-29-oic acid, 3-O-[-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β- D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl-3β,22β,24,29- tetrahydroxyolean-12-ene (57), 3-O-[-L-rhamnopyranosyl-(1→2)-β- Dglucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-29-O-β-D-glucopyranosyl- 3β,22β,24,-trihydroxyolean-12-en-29-oic acid (58), 3-O-[β-D-glucopyranosyl- (1→2)-β-D-glucuronopyranosyl]-29-O-β-Dglucopyranosyl-3β,22β,24,- trihydroxyolean-12-en-29-oic acid (59), 3-O-[β-D-xylopyranosyl-(1→2)-β-D- glucuronopyranosyl]-29-O-β-D-glucopyranosyl-3β,22 β,24,-trihydroxyolean-12-

en-29-oic acid, 3-O-[-L-rhamnopyranosyl-(1→2)-β-D-glucopyranosyl-(1→2)-β- D-glucuronopyranosyl]-3β,24-dihydroxyolean-12-ene-22-oxo-29-oic acid (60), 3- O-[-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl-(1→2)-β-D- glucuronopyranosyl]-21-O--L-rhamnopyranosyl-3β,21β,22,24- tetrahydroxyolean-12-ene (61), 3-O-[-L-rhamnopyranosyl-(1→2)-β-D- glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-21-O--L-rhamnopyranosyl-3 β,21β,22,24-tetrahydroxyolean-12-ene (80), 3-O-[-L-rhamnopyranosyl-(1→2)- β-D-glucopyranosyl-(1→2)-β-D-glucuronopyranosyl]-3β,21β,22,24,29- pentahydroxyolean-12-ene (62), 3-O-[-L-rhamnopyranosyl-(1→2)-β-D- xylopyranosyl-(1→2)-β-D-glucuronopyranosyl]-22-O--L-rhamnopyranosyl- 3β,22 β,24-trihydroxyolean-12-ene (63). Additionally, 12 known oleananes-type glycosides, astrojanoside A (Bedir et al., 1999a), melilotus-saponin O2 (Hirakawa et al., 2000), 3-O-[-L-rhamnopyranosyl-(1→2)-β-D-xylopyranosyl-(1→2)-β-D- glucuronopyranosyl]-3β,21β,22,24,29-pentahydroxyolean-12-ene (Gulcemal et al., 2012), azukisaponin V (Kitawaga et al., 1983a), wistariasaponin B2 (Konoshima et al., 1989), astragaloside VIII (Kitawaga et al., 1983b), wistariasaponin B1 (Konoshima et al., 1989), cloversaponin IV (Sakamato et al., 1992), azukisaponin II (Kitawaga et al.,1983c),wistariasaponin D (Konoshima et al., 1991), dehydroazukisaponin V (Mohamed et al., 1995), and 3-O-β-D- glucuronopyranosyl-soyasapogenin B (Udayama et al., 1998) were isolated. Noteworthy, cycloartane-type triterpene glycosides, the main constituents of Astragalus spp., were not found. This peculiar feature characterizes a very limited group of Astragalus spp. The antiproliferative activity of the isolated compounds was evaluated against a small panel of cancer cell lines. Only compound showed an IC50 of 22 lM against human leukemia cell line (U937). The other tested compounds, in a range of concentrations between 1 and 50 lM, did not cause any significant reduction of the cell number (D. Gülcemal et al.; 2012).

1.5 Astragalus as Species Poisonous Plants

The interesting researches on the toxicity of Astragalus have been published (James et al., 1990; Infante et al., 1991) and several additional papers refer to the toxicity of different species (James et al., 1992; Panter et al., 1992; Panter and James, 1990) are also reported.

Thus, poisonous Astragalus species can be classified into three general groups, according to the toxins and their effects on animals:

(1) species that synthesize aliphatic nitro compounds;

(2) species causing locoweed poisoning;

(3) species that can accumulate selenium.

Some Astragalus species can synthesize aliphatic nitro compounds (a group of toxins found in various milkvetches; Pass, 1994) and their glycosides are transformed in the rumen to the toxic principles 3-nitro-1-propanol (3-NPOH) and 3-nitropropionic acid (3-NPA) (Fig. 1.15). It seems that an individual species can synthesize 3-NPOH or 3-NPA derivatives, but not both (Williams, 1982). The 3- NPOH derivatives occur as β-glucosides (miserotoxin) and the 3-NPA compounds can be mono, -di, or -tri glucose esters (Infante et al., 1991). Among species with this kind of principle include A. miser, A. falcatus. A. glycyphyllos and A. hamosus (Williams and Davis, 1982). A. lusitanicus, a species widely distributed in Southwestern Spain, Portugal and Northern Africa, produces livestock poisoning. Tarazona and Sanz (1987) reported the presence of aliphatic nitro compounds and they hypothesize that these compounds are responsible for the toxicity. Furthermore, the results obtained about the toxicity of nitro compounds prouved that it has caused moderate-to-heavy losses of cattle and sheep on western rangelands of the United States (Cronin et al., 1981) and Canada (Majak and Cheng, 1983).

Figure 1.15 Structure of toxic compounds found in Astragalus species

The plants that cause locoweed poisoning (‘loco’ in Spanish means crazy) contain swainsonine, an indolizidine alkaloid (Fig. 1.16). All, to date, are species of sections Astragalus and Oxytropis. Livestock must graze these plants for a long period of time before signs of intoxication appear. Intoxication with these plants produces neurological disturbances, emaciation, habituation, reproductive alterations, and congestive right-heart failure. Swainsonine is known for its antimetastatic properties, and castanospermine, a related alkaloid, as an anti-HIV agent (James et al., 1990). Astragalus lentiginosus, A. pubentissimus, A. mollissimus, A. wootoni, A. thuseri, and A. nothrosys are species included in this group. Swainsonine is eliminated partially in milk and it can be fed to young

calves, and induces high mountain disease when the animals graze at high elevation (James et al., 1991). The alkaloid completely inhibits acidic a- mannosidases at different concentrations in various tissues, but it is less effective against β-mannosidase and some other enzymes. This effect alters the synthesis of glycoproteins, but does not appear to produce clinical effects, although the associated accumulation of lysosomal storage products produces the same symptoms as locoweed poisoning (Abraham et al., 1983). The accumulation of mannose-rich oligosaccharides in testis and epididymis induces biochemical abnormalities in the reproductive tissues and impacts on male reproduction (Tulsiani et al., 1990). The ingestion of locoweed by pregnant livestock may induce the fetal malformations, delayed placentation, reduced placental and uterine vascular development, hydrops amnii, hydrops allantois, abnormal cotyledonary development, interruption of fetal fluid balance and abortion (Bunch et al., 1992).

Figure 1.16 Indolizidine alkaloids glycosidase inhibitors occuring in Astragalus species

The third group includes plants growing on seleniferous soils that are able to accumulate several thousand p.p.m. of selenium. Chronic poisoning is characterized by rough hair coat, loss of hair, hoof lesions, lameness, decreased vitality, loss of appetite, emaciation, and a decrease in reproductive efficiency. In livestock, reduced reproductive performance is the most significant effect of chronic intoxication (James et al., 1990). The selenium compounds are toxic because of their pro-oxidant catalytic activity to produce superoxide, hydrogen peroxide, and probably other cascading oxyradicals. They can also have a carcinostatic effect (Spallholz, 1994). The species known to have seleniferous are A. bisulcatus, A. saurinus, A. praelongus, A. flavus and A. tenellus (Cowgill and Landenberger, 1992).

1.6 Astragalus as Medicinal Plants

Several Astragalus species are used as medicinal plants, especially A. membranaceus, which is the one most widely used and studied. It is officially listed in the Chinese Pharmacopoeia, as the species A. complanatus and A. mongholicus (A. membranaceus var. mongholicus). The Chinese, Huang qi translates as "yellow leader", referring to the yellow color of the root and its status as one of the most important tonic herbs. The primary medicinal parts of the herb are the roots. The pharmacologically active constituents of Astragalus belong to two different kinds of chemical compounds, polysaccharides and saponins, and the most interesting pharmacological properties are hepatoprotective, immunostimulant and antiviral.

Among the many pharmacological properties of Astragalus, the most interesting are their effects on the cardiovascular system. Griga (1975) described the general properties of A. cicer alkaloids and their acute toxicity (Griga et al., 1975). In later papers Griga (1977, 1983) reported the effect on blood pressure in rats with renal hypertension. At 50 mg/kg, the extract normalized the brain and heart oxygen consumption and decreased blood pressure (Griga et al., 1977; 1983).

In Turkish folk medicine, the aqueous extracts of some Astragalus species (declared by the healer) are used to treat leukemia as well as healing wounds (Çalış et al., 1997; Bedir et al., 2000). Roots of these plants are also used as antiperspirant, diuretic, tonic and for the treatment of nephritis, diabetes, leukemia and uterine cancer (Tang et al., 1992). Also the flowering parts of Astragalus lycius Boiss.; a part of this thesis, are in use for cough, asthma and urethritis expectorants (Tuzlacı and Şenkardeş, 2011).

The pharmacologically active constituents of Astragalus are incumbented on two different kinds of chemical compounds, polysaccharides and saponins that constitute the major class of chemical compounds isolated from Astragalus species. Otherwise, the most investigated constituents are saponins but cycloartane and oleanane-type glycosides from Astragalus species show interesting biological properties (Bedir et al., 2000; Yesilada et al., 2005; Nalbantsoy et al., 2011 and 2012), anti-protozoal (Özipek et al., 2005), wound healing (Sevimli-Gur et al., 2011) antiviral and cytotoxic activities (Tian et al., 2005).

1.6.1 Hepatoprotective and Antioxidative Effects of Astagalus

The hepatoprotective properties of Astragalus membranaceus extracts have been widely studied and, in some cases, the active principles have been described. Research has shown that its antioxidative properties can prevent liver damage.

Studies on animals with toxic hepatic injury induced by CCl4, indicates that A. membranaceus root extract prevents a decrease in hepatic glycogen content and raises the levels of total serum protein and albumin (Tang and Eisenbrand, 1992).

Han et al. (1989) studied the protective effect of A. membranaceus root extract against liver damage, using a model in which rats are exposed to a noise of 2 KHz/103 dB for one hour, and reported that an alteration in hepatic glycogen was a determining factor in live damage. They observed a decrease in values of hepatic glycogen in the pretreated group in relation to the control one.

Zang et al. (1990) assayed the protective effect of the same drug after oral administration in a stilbenemideinduced liver damage model. The ethanol extract of the roots reduced the alanine transaminase (SGPT) levels and the subacute toxicity of stilbenemide decreased. In 1992, S-200 column, and screened the activity of the different fractions by studying their effects on mononuclear cells derived from healthy normal donors, employing the local xenogeneic GVH reaction. Fraction 3 (F3), with an estimated molecular weight of 20 000 to 25 000, was the most active, with a potent immunorestorative activity in vitro. The activity was ratified in vivo, using cyclophosphamide-primed rats. After injection of 5.55 mg daily for 5 days, the cyclophosphamide-induced immunosuppression was completely reversed. The active fraction improved the response of lymphokine-activated killer (LAK) cell precursors, when it was applied together with the recombinant interleukin-2 (rIL-2). Finally the authors propose the use of A. membranaceus plus rIL-2 to reduce the dose of this compound alone, with the consequent reduction in toxicity.

Wang and Han (1992) studied a polysaccharide isolated from A. mongholicus as a possible hepatoprotector principle on E. coli endotoxin-induced liver damage in a mouse model. This polysaccharide increased the rate of survival in mice after endotoxin administration, and prevented the lowering of ATP levels, and adenylate energy change in mouse liver.

The water extract from A. membranaceus inhibited oxygen consumption and MDA production when it was assayed against the lipid peroxidation induced by ADP and FeSO4 on rat heart mitochondria (Hong et al. 1994).

1.6.2 Astragalus as Immunostimulant Agent

A modification of the immune response could be responsible for the antiviral and anticancer activity of A. membranaceus. Studies have shown that astragalan I and II, the main constituents of the water extract from this species, potentiated the immunological response in mice after i.p. administration. They increased the weight and cell number of mouse spleen, elevated the response of mouse spleen against sheep red blood cells, and stimulated the phagocytic activity of peritoneal macrophages. When the compounds were given i.v. or p.o., the phagocytic fraction of peritoneal macrophages did not change (Tang and Eisenbrand, 1992).

The immunostimulant activity of A. membranaceus was studied by Sun et al. (1983a, b) using the local graft-versushost (GVH) reaction as a test assay for T-cell function, with samples from cancer patients and healthy donors. In a second test they used mononuclear cells of normal subjects, cancer and immunodepressed patients to study the effects on lymphocyte blastogenic responses. The results obtained made it possible to hypothesize that A. membranaceus aqueous extract must contain immunomodulatory principles. Five years later, Chu et al. (1988 a, b, c) fractionated and purified the immunostimulant extract using a Sephacryl S- 200 column, and screened the activity of the different fractions by studying their effects on mononuclear cells derived from healthy normal donors, employing the local xenogeneic GVH reaction. Fraction 3 (F3), with an estimated molecular weight of 20 000 to 25 000, was the most active, with a potent immunorestorative activity in vitro. The activity was ratified in vivo, using cyclophosphamide-primed rats. After injection of 5.55 mg daily for 5 days, the cyclophosphamide-induced immunosuppression was completely reversed. The active fraction improved the response of lymphokine-activated killer (LAK) cell precursors, when it was applied together with the recombinant interleukin-2 (rIL-2). Finally the authors the dose of this compound alone, with the consequent reduction in toxicity.

Wang et al. (1992) studied the synergism A. membranaceus extract on the cytotoxicity of rIL-2 generated cells against murine renal cell carcinoma and reported the same results as Chu et al. (1988c). In this research the active fractions

were purified by high-performance liquid chromatography. Similar results were obtained by Lau et al. (1994) when the capacity of A. membranaceus extract to inhibit the growth of murine cell carcinoma in vivo was studied. An investigation of the mechanisms involved in this action showed that splenocytes from mice transplanted with murine renal cell carcinoma responded less favourably to IL-2 in generating LAK cells, but this depression was restored with A. membranaceus administration. In conclusion they suggest that this crude drug may exert the antitumour effect via augmentation of phagocyte and LAK activities.

The immunostimulant properties of the polysaccharide obtained from A. membranaceus has been studied by other researchers. D. C. Wang et al. (1989) used a local GVH reaction and the blastogenic response of lymphocytes in vitro as a test index for T-cell function. Samples were obtained from normal healthy donors and untreated patients with advanced cancer. Dose-dependent effects of polysaccharide were observed on the concavallin A and phytohaemagglutinin responses, and cell proliferation was inhibited at higher concentrations. They concluded that the mechanism of immunostimulation could be related to the suppressive T-cell activity. However, J. Wang et al. (1989) hypothesized that the immunostimulant activity of the A. membranaceus polysaccharide was related to the activity of the mouse complement C3. The effect in this case was investigated by immunofluorescence of the third component of the complement cleavage in mouse macrophages.

A. membranaceus extracts injected into normal, radationand cyclophosphamide-immunodepressed treated or aging mice were able to enhance the antibody response to a Tdependent antigen. The activity was dependent on the carbohydrate content of the extract (Zhao et al., 1990).

A. membranaceus and A. mongholicus extracts enhanced the phagocytic function of the endothelial reticula and stimulated the immunological activities in normal and immunodepressed mice (Kang et al., 1989). Tomoda et al. (1992) isolated a glycan from the hot water extract of A. membranaceus roots and studied its reticuloendothelial system potentiating activity in the carbon clearance system, where a net positive effect was demonstrated.

The polysaccharide from A. mongholicus administered i.p. to mice increased the immune response, and the amount of RNA in the spleen, but had no effect on thymus, heart or brain RNA, or on DNA metabolism (Tang and Eisenbrand,

1992). explained by immunological mechanisms on the basis of the results obtained in a study with urological neoplasm cells and bladder murine carcinomas, Rittenhouse et al. (1991) reported that A. membranaceus may exert its antitumour activity by abolishing tumour-associated macrophage suppression. The potentiation of the natural killer cytotoxicity of peripheral blood mononuclear cells in patients with systemic lupus erythematosus was demonstrated by Zhao (1992) using an enzyme-release assay. The activity was increased in the samples of healthy donors and patients with the pathology. The release of a natural killer cytotoxic factor by peripheral blood mononuclear cells was higher in the control group, and the levels of that factor correlated well with natural killer activity, and correlated negatively with the clinical effect.

1.6.3 Antiviral Properties of Astragalus species

Many researchers have tried to demonstrate the antiviral activity of Astragalus. The best-studied species is again A. membranaceus, particularly against Coxsackie viruses. In 1987, Yang et al. reported the effect of A. membranaceus on rat beating heart cell cultures infected with the Coxsackie B-2 virus. In similar research, Yuan et al. (1989, 1990) reported the effect of the same species on the electrical activities of neonatal rat myocardial cell cultures infected with the same virus. Evaluation of the cells in the post-infected period showed changes in the beating percentage and cytopathic effect. Alterations in the electrical activities were measured by standard microelectrode techniques. In the infected group the beating percentage had decreased by 24 h, cytopathic effects appeared quickly, and mthe action potential amplitude, duration and rate of uptake showed a significant decrease between 24 and 96 h. Premature beats, tachycardia and fibrillation were observed. In the treated group, the beating and the electrical activities were nearly normal, and a decrease in the cytopathic effect was observed in the myocardial cells treated with A. membranaceus extract. In conclusion, the authors confirm the protective effect of the drug in vitro against infection by Coxsackie B-2 virus and propose its possible use in prophylaxis and treatment of acute myocarditis caused by this virus.

In an in vivo experimental model, Yang et al. (1990a) evaluated the effectiveness of A. membranaceus in inhibiting Coxsackie B-3 virus propagation and in protecting the myocardium in mouse myocarditis. Four-week-old male BALB/c mice were used, and the parameters measured were gross, histopathologic and ultrastructural examinations of infected mice and those

treated with A. membranaceus groups. It was observed that the severity of the myocardial lesions and the area involved diminished, and virus titre was also smaller in the A. membranaceus administered mice. The antiviral effect of this species could be mediated by an immunological response according to Wu et al. (1992). These authors evaluated the effect of A. membranaceus on mice with Coxsackie B-3 myocarditis using an antigenspecific cytotoxic assay. On the basis of their results, they propose that the antiviral effect of A. membranaceus in acute viral myocarditis was mediated by immune reaction through the regulation of T lymphocytes.

Yang et al. (1990b) treated patients suffering from Coxsackie B viral myocarditis with A. membranaceus intramuscularly and compared with conventional therapy. They obtained a significant improvement in patients with A. membranaceus therapy and titres of IFN-a and IFN-g increased in comparison with those before therapy. In an in vitro study, Wei et al. (1992) reported the effect of A. membranaceus against Coxsackie B-3 and Echo-10 viruses, and the influence of the crude drug on the susceptibility of human heart cells to viral infection. The A. membranaceus extract had no direct activity against the virus, but the cells pretreated with the extract before inoculating and treated after viral infection both showed lower susceptibility to the viral infection. They hypothesized that the activity could be mediated by the capacity of A. membranaceus extract to induce an enhancement of IFN production in the human myocardial cells. These results seem to be in agreement with the clinical double blind trials done by Qian and Li (1987), who studied the synergism of A. membranaceus and IFN as antiviral agents. In further work, the same authors (Qian et al., 1990) reported the synergic effect of A. membranaceus and recombinant IFN-a1 in the treatment of chronic cervicitis produced by papillomavirus type 16, herpes simplex virus type 2 and cytomegalovirus infections. Wei et al. (1992) reported the effect of A. membranaceus against Echo- 19 virus in human heart cells, and they suggested that the activity could be mediated by IFN production.

Several other reports about the activity of A. membranaceus against different kinds of viral infections have been published.

Tang and Xiong (1990) studied the effect against epidemic haemorrhagic fever virus in mice, and they observed that the morbidity and infection rate of the treated mice were lower than in the control group. The effect seemed likely to be

mediated through the enhancement of the immunological mechanism of the host because the drug had no effect on the replication of EHF virus in Hero E6 cells.

Abdallah et al. (1993) studied the in vitro anticancer and antiviral activity of triterpene glycosides isolated from A. spinosus. One of them, astragaloside II, exhibited a cytoprotective effect on T-lymphocytes against HIV infection. However, Yao et al. (1992) screened several plant extracts against HIV virus, and obtained negative results in the case of A. membranaceus, the species from which Hirotani et al. (1994) isolated a series of astragalosides.

1.6.4 Cardiovascular Effects of Astragalus Species

Among the many pharmacological properties of Astragalus, the most interesting are the effects on the cardiovascular system. Griga (1975) described the general properties of A. cicer alkaloids and their acute toxicity. In later papers Griga (1977, 1983) reported the effect on blood pressure in rats with renal hypertension. At 50 mg/kg, the extract normalized the brain and heart oxygen consumption and decreased blood pressure.

The hypotensive effect of A. membranaceus was described and its active principle isolated by Hikino et al. (1976). It was identified as gamma- aminobutyric acid (GABA), and these authors observed a good correlation between the GABA content of pharmaceutical preparations and hypotensive activity in rats.

Other researchers have studied the effects of different Astragalus compounds against hypertension. Astramembrainnin I, a saponin isolated from A. membranaceus roots, produced a hypotensive effect after i.v. administration to anaesthesized cats and rats (Tang and Eisenbrand, 1992). Castillo et al. (1993a, b) described the antihypertensive properties of 3-NPA isolated from some toxic species. This compound elicited a dose-dependent relaxation of precontracted rabbit aortic rings, which was not affected by the presence of antagonist drugs such as atropine, propranolol or brompheniramine, but was inhibited by methylene blue. Aortic rings precontracted with KCl were less sensitive to relaxation. These results suggest that at least part of the mechanism of vasodilatation is not related to an inhibition of calcium influx through the voltage- dependent Ca2+ -channel, nor to blocking a specific set of receptors. In vivo experiments demonstrated the hypotensive effect of 3-NPA in normotensive rats

(i.v. acute administration) and renal hypertensive dogs (chronic oral administration). In both cases 3-NPA decreased the blood pressure and provoked bradycardia. The hypotensive effect was independent of the animal species and the administration route employed. On the other hand, 3-NPA had a negative effect on the inotropic and chronotropic properties of isolated guinea-pig auricles and inhibited the increase in contractile force and heart rate elicited by isoproterenol. The hypotensive effect of 3-NPA is probably a consequence of both the vasodilator and cardiodepressor activity. The mechanism of the former could be based on a guanylate cyclase stimulation (methylene blue inhibition), and the cardiac effects may be related to an inhibition of badrenergic mediated responses (isoproterenol antagonism).

From A. membranaceus, Wang (1992) obtained a saponin- enriched extract and studied its effect on isolated heart of rats. At doses of more than 50 mg/mL the extract showed a positive inotropic effect, but this turned negative at 30 mg/ mL. The mechanism could be related to the cardiotonic glycosides, because the extract‘s behaviour was similar to that of strophantidin K. Kudrin et al. (1987) studied the effects of A. dasyanthus extract on myocardial infarction in experiments with rats after coronary arterial ligation, and observed a moderate decrease in the damaged area in permanent and transient ischaemia.

1.6.5 Effect of Astragalus Species on Diabetes and Diabetes Related Diseases

In a systematic screening of wild Egyptian plants, Shabana et al. (1990) studied the hypoglycaemic effects on alloxanized diabetic rats of some extracts of Astragalus species. Saponin astramembrainnin I, decreased carrageenan induced oedema in rats (p.o.), and inhibited the increase in vascular permeability induced by serotonin and histamine after i.v. or oral administration (Tang and Eisenbrand, 1992).

APS was proved to have the preventive effects on type 1 diabetes in non obese diabetic (NOD) mice. The APS group has lower incidence of diabetes, higher serum C-P levels, decreased degree of the lymphocytic inflammation of pancreatic islets, stronger proliferation of CD8 T subsets, and lower ratio of CD4/CD8 subgroup in spleocytes than those of the normal solution group (Chen et al, 2001; Liu.et al., 2011).

Astragalus polysaccharides have an effect to two-dimensionally regulate the level of blood glucose, which can increase the blood glucose of hypoglycemic animals or humans to normal level, and significantly lower the level of blood glucose, triglyceride and myocardial calcium, improve the abnormalities of myocardial ultrastructure and the metabolism of diabetic rats and mice, and inhibit the onset of type 1 diabetes in non-obese diabetic mice. A. membranaceus was reported to have an effect for diabetic complications, such as protecting the myocardium in diabetic nephropathy by inhibiting lipid peroxidation, prolonging the incubation period of late diabetic neuropathy by decreasing the motion nerve conduction velocity as an aldose reductase inhibitor, and exerting a beneficial effect on experimentaldiabetic nephropathy by suppressing the renal hypertrophy and micro albuminuria (Li et al., 2004).

It was reported that APS could reduce fasting plasma glucose (FPG) and blood lipids on insulin resistance of rats with type 2 diabetes. APS could significantly decrease the blood sugar and increase the content of the blood serum HDL of 2-DM IR rats (Liu et al, 2007).

A crude extract of AR inhibits the formation of Nε- (carboxymethyl) lysine (CML) and pentosidine during the incubation of bovine serum albumin with ribose. Astragalosides significantly inhibited the formation of both CML and pentosidine, and astragaloside IV had the strongest inhibitory effect among all the isolated compounds. That suggested that AR and astragalosides might be a potentially useful strategy for the prevention of clinical diabetic complications by inhibiting AGE inhibitors (Motomura et al, 2009).

Astragalus mongolicus (root) was able to reverse hyperglycemia and protect pancreatic islets in type 1 DM in NOD mice by changing the predominant form of lymphocytes/cytokines from Th1 to Th2, thus preventing lymphocyte infiltration of the pancreas. In addition, astragaloside IV (saponin extract of AM root) can ameliorate high glucose-induced podocyte adhesion by up regulating -3--1 integrin and inhibiting the activation and overexpression of integrin-linked kinase. In diabetic rats treated with a preparations containing AM (root) and Angelica sinensis, attenuation of transforming growth factor (TGF)-mRNA in the renal cortex was observed. The inhibitory effects on TGF- were further substantiated by the down-regulation of reactive oxygen species NF-kappa B signal pathway by a combination of AM (root) and Arctium lappa (fruit) in diabetic mice. Not only can AM (root) effectively treat diabetic nephropathy, it can also improve other

renal diseases in animal models. AM (root) was observed to have significant anti fibrotic effects in rats with unilateral ureteral obstruction (UUO) by inhibiting myofibroblast activation and TGF-1 expression, and by inducing hepatocyte growth factor. AM (root) can also alleviate hyperlipidemia and proteinuria in rat models of nephritic syndrome by upregulating the expression of the hepatic low- density lipoprotein (LDL) receptor gene (Liet al., 2000) and reducing elevated levels of messenger RNA expression of arginine vasopressin (AVP) V2 receptor and AVP-dependent aquaporin-2 (Zhang et al., 2009).

Astragaloside II and isoastragaloside I isolated from AR selectively were reported to increase adiponectin secretion in primary adipocytes without any obvious effect on a panel of other adipokines. Adiponectin is an adipocyte-derived insulin-sensitizing hormone with antidiabetic, anti-inflammatory, and anti- atherosclerotic properties. Chronic administration of astragaloside II and isoastragaloside I in both dietary and genetic obese mice significantly elevated serum levels of total adiponectin and selectively increased the composition of its high molecular weight oligomeric complex. These results suggested that the two natural compounds might provide the lead as a novel class of therapeutics for obesityrelated diseases (Xu et al, 2009).

Liu M. et al. (2010) were examined whether treatment with Astragalus polysaccharide (APS) improves insulin sensitivity in insulin-resistant mice and whether this is associated with an improvement of dysregulated protein kinase B and glucose transporter 4 expressions in skeletal muscle. APS (700 mgkg−1 day−1) or vehicle was administered to 12-week-old diabetic KKAy and nondiabetic C57BL/6J mice for 8 weeks. Changes in body weight, blood glucose level, insulin resistance index, and oral glucose tolerance were routinely evaluated. The expressions of protein kinase B and glucose transporter 4 in skeletal muscle tissues were determined with Western blot. KKAy mice developed persistent hyperglycemia, impaired glucose tolerance and insulin resistance. Insulin-stimulated protein kinase B phosphorylation and glucose transporter 4 translocation were significantly decreased in KKAy compared to age-matched C57BL/6J mice. APS treatment ameliorated hyperglycemia and insulin resistance. Although the content of protein kinase B and glucose transporter 4 in KKAy skeletal muscle were not affected by APS, insulin-induced protein kinase B Ser-473 phosphorylation and glucose transporter 4 translocation in skeletal muscle were partially restored by APS treatment. In contrast, APS did not have any effect on C57BL/6J mice. These results indicate that APS can

regulate part of the insulin signaling in insulin-resistant skeletal muscle, and that APS could be a potential insulin sensitizer for the treatment of type 2 diabetes

Chen et al. (2012) were examined the effect of Astragalus polysaccharide (APS) on myocardial glucose and lipid metabolism in diabetes (DM) hamster and to explore its mechanism in intervention of DM cardiomyopathy. Low-dose- streptozotocin-induced hamsters (STZ, 40 mg/kg 93 days, i.p.) with blood glucose [13.9 mmol/L were considered as type 2 diabetic models. Blood glucose, serum lipid, insulin, C-peptide, myocardial enzyme levels, myocardial glycogen staining, myocardial ultrastructure, fluorescence quantitative RT–PCR detection of myocardial PPAR- and the target genes (FATP, ACS) and GLUT4mRNA expression in normal control group, DM group and APS treatment group hamsters were measured. The results showed that there was significant glycolipid metabolic disorders in DM group compared with normal group. Glucose, glycosylated serum protein, myocardial enzymes and lipid levels in APS treatment group decreased significantly than DM group, but insulin and C-peptide levels was no difference. Myocardial glycogen staining and abnormal myocardial ultrastructure in APS treatment group were significantly improved than in DM group. Gene expression of myocardial PPAR- and its target genes (FATP, ACS) in APS group were significantly lower than in DM group, while gene expression of GLUT4 in APS group was higher than DM group. It was concluded that APS can partially improve myocardial glucose and lipid metabolism disorders in diabetic hamsters and protect myocardium in some extent.

Among the polysaccharides usually used in medicinal practice, pectins, resins, and mucilages are the major. Pectins serve as emulsifiers and stabilizers, constitute the main components of ointments with salicylic and boric acids, and enter the composition of drugs decreasing blood cholesterol content and modulating bile acid metabolism. Resins are used for the production of oil emulsions, mucilaginous solutions, and blood substitute liquids. Aqueous and mucilaginous extracts prepared from mucilage-containing plants, flax and marsh mallow, are used to treat catarrhs and gastrointestinal tract lesions and to counteract the irritant effects of other drugs (Lovkova et al., 2001).

The polysaccharides from A. membranaceus are the well known, several research groups have isolated and purified them.

Tang and Eisenbrand (1992) cited astragalan I, II and III from A. mongholicus (A. membranaceus var. mongholicus). Astragalan I is a polysaccharide composed of D-glucose, D-galactose and D-arabinose in a molar ratio of 1.75:1.63:1, with a molecular weight of 36 300, while astragalan II and III are composed of D-glucose only, with molecular weights of 122 300 and 34 000 respectively.

Tomoda et al. (1992) purified a glycan from the hot water extract of the roots of A. membranaceus, constituted mainly of L-arabinoside, D-galactose, L- rhamnose and D-galacturonic acid in a molar ratio of 6:9:8:30.

Shimizu et al. (1991) isolated an acidic polysaccharide from A. mongholicus, composed of L-arabinoside, D-galactose, D-galacturonic acid, D- glucuronic acid in a molar ratio of 18:18:1:1, plus small amounts of acetyl groups and peptide moiety (Fig. 1.17, Rios and Waterman, 1997).

Figure 1.17 Polysaccharide chain

1.6.6 Antitumor Effects of Astragalus Species

The extract of Astragalus membranaceus roots (AR) prevented the cytotoxic activity of lymphocytes against YAC-1 cells from the depression by a carcinogen, N-butyl-N'-butanolnitrosoamine (BBN). It also protected the production of interleukin-2 and γ-interferon of lymphocytes from the depression by BBN. The results showed that the extract of AR exerted an anticarcinogenic effect in carcinogen-treated mice through cytotoxic activity and the production of cytokines (Kurashige et al., 1999; Liu et al., 2011). The inhibition rates of AR on the different cancer cell lines were detected by trypan blue exclusion, MTS (3- (4,5-159-dimethylthiazol-2-yl)-5-(3-carboxymethoxyphenyl)-2-(4-sulfophenyl)-

2H-tetrazolium) method, and tritium thymidine incorporation assay. Apoptosis was detected by DNA ladder method. The results indicated AR specifically inhibited gastric cancer cells growth in vitro and the mechanism was mainly cytostatic but not cytotoxic or inducing apoptosis (Lin et al, 2003; Liu et al., 2011). The water extract of AR inhibited the proliferation of human hepatocarcinoma cells, SMMC-7721, and their mitochondria metabolic activity in vitro. Studies also showed this water extract decreased the weight of S180 tumor in tumor-bearing mice, and increased the T/B-lymphocyte ratio and the activity of peritoneal macrophages (Xiao et al, 2004; Liu et al., 2011).

The in vitro and in vivo anti-tumor effects of A. membranaceus were investigated. Five bioactive fractions were isolated from the root of A. membranaceus, the fraction designated as AI was found to be the most potent among the five fractions with respect to its mitogenicity on murine splenocytes. Besides investigating the cytostatic effect of AI, its activities on macrophage function, tumor necrosis factor production, induction of lymphokine-activated killer cell and tumor cell differentiation were also examined. The macrophage-like tumors and the myeloid tumors were found to be more sensitive to the cytostatic activity of AI, whereas the fibroblast-like tumors and the mouse Ehrlich ascites tumor appeared to be relatively resistant. Moreover, AI could effectively suppress the in vivo growth of syngeneic tumor in mice. Results showed that murine macrophage pretreated with AI had increased in vitro and in vivo cytostatic activities towards MBL-2 tumor. AI could also act as a priming agent for tumor necrosis factor production in tumor-bearing mice. Preincubation of mouse splenocytes with AI could induce in vitro lymphokine-activated killer-like activity towards WEHI-164 cell. Furthermore, AI was able to induce monocytic differentiation of both human and murine cells in vitro. AI administered in vivo could even partially restore the depressed mitogenic response in tumor-bearing mice. Collectively, the results showed that A. membranaceus could exhibit both in vitro and in vivo anti-tumor effects, which might be achieved through activating the anti-tumor immune mechanism of the host (Cho and Leung, 2007).

Upon the treatment with astragaloside IV (ASI), human hepatocellular carcinoma HepG2 cells were evaluated for the colonogenic survival and anchorage independent growth. The cellular proteins of treated and untreated cells were resolved by 2D polyacrylamide gel electrophoresis. The protein spots mostly altered by drug treatment were identified by mass spectrometry and subsequently verified by Western blotting using specific antibodies and RT-PCR technique

using specific DNA primers. The results showed astragaloside IV attenuated the colonogenic survival and anchorage-independent growth of cancer cells (Qi et al, 2010; Liu et al., 2011).

Auyeung et al. (2012) had reported that Astragalus saponins (AST) exert promising anti-tumorigenic effects by suppressing the growth of HT-29 human colon cancer cells and tumor xenograft. Viability of AGS cells was measured by using the MTT reduction method. Western blotting was performed to examine the effect of AST on apoptotic- and cell growth-related protein expression. Effect of AST on cell cycle progression was also evaluated using PI staining. A Matrigel invasion assay was then employed to demonstrate the effect of AST on the invasiveness of gastric cancer cells. The expression of invasion-associated proteins (VEGF and MMPs) was also investigated. The results showed that AST could induce apoptosis in AGS cells by activating caspase 3 with subsequent cleavage of poly(ADP-ribose) polymerase. Besides, cell cycle arrest at the G2/M phase had been observed in AST-treated cells, leading to substantial growth inhibition. The anti-proliferative effect of AST was associated with the regulation of cyclin B1, p21 and c-myc. Results indicate that the number of AGS cells invaded through the Matrigel membrane was significantly reduced upon AST treatment, with concominant down-regulation of the pro-angiogenic protein vascular endothelial growth factor (VEGF) as well as the metastatic proteins metalloproteinase (MMP)-2 and MMP-9. It was concluded that AST derived from the medicinal plant Astragalus membranaceus could modulate the invasiveness and angiogenesis of AGS cells besides its pro-apoptotic and anti-proliferative activities. These findings also suggest that AST has the potential to be further developed into an effective chemotherapeutic agent in treating advanced and metastatic gastric cancers.

1.6.7 Other Pharmacological Effects of Astragalus Species

Wong et al. (1992) demonstrated the antimutagenic activity of A. membranaceus aqueous extract on aflatoxin B1-induced mutagenesis, using Salmonella typhimurium as a test strain and rat liver ultra-supernatant as the activating system. The results reinforce the use of this Chinese medicinal plant as a cancer chemopreventive agent, because the extract produced a concentration- dependent inhibition of histidine-independent reversant colonies induced by aflatoxin B1.

Eighteen Chinese medicinal plants were screened as stimulants of sperm motility (Hong et al., 1992), and A. membranaceus aqueous extract was active. However, other swainsonine-containing Astragalus can have adverse effects on male reproduction (Tulsiani et al., 1990).

Anti-inflammatory effect of Astragalus ssp. were also investigated. The effects of Astragalus membranaceous root (AR) extract on interleukin (IL-6) and tumor necrosis factor (TNF-α) production, prostaglandin E2 (PGE2) biosynthesis, and leukotriene C4 (LTC4) production from lipopolysaccharide (LPS)-stimulated human amnion cells were investigated. The data suggested that AR extract might play a role in inhibiting bacterial infection-associated preterm labor by suppressing the productions of IL-6, PGE2, and LTC4 by human amnion cells (Shon et al., 2002).

Studies investigated the effect of AR extract on IL-6 and TNF-α production, PGE2 and LTC4 released from IL-1β-stimulated human amnion. The results indicated that AR had a broad anti-inflammatory effect in human amnion and might be considered a promising agent to protect preterm labor (Shon and Nam, 2003).

It was reported that AR displayed antiinflammation in zymosan air-pouch mice by reducing the expression of iNOS, COX-2, IL-6, IL-1β, and TNF-α, and by decreasing the production of nitric oxide (NO). In a similar manner, AR reduced the expression of IL-6, iNOS, and COX-2 in LPS-treated Raw 264.7 cells. The data revealed that AR had an anti-inflammatory effect that was mediated by the MKP-1-dependent inactivation of p38 and Erk1/2 and inhibition of NF kappaB-mediated transcription (Ryu et al, 2008).

In conclusion, Astragalus species are a group of medicinal plants, whose active principles are principally saponins and polysaccharides. Some toxic principles have medicinal interest, for example swainsonine in cancer chemotherapy and 3-NPA as an antihypertensive principle. The use of A. membranaceus in Chinese traditional medicine seems to be justified as demonstrated by numerous studies. The potential use of polysaccharides from Astragalus species as immunostimulant agents is suggested and justified in several clinical studies. The effect on virus infections and cancer is related to its immunorestorative properties.

Two saponins, Astragaloside II and isoastragaloside I isolated from A. membranaceus selectively increased adiponectin secretion in primary adipocytes without any obvious effects on a panel of other adipokines. Furthermore, an additive effect on induction of adiponectin production was observed between these two compounds and rosiglitazone, a thiazolidinedione class of insulin- sensitizing drugs. Chronic administration of astragaloside II and isoastragaloside I in both dietary and genetic obese mice significantly elevated serum levels of total adiponectin and selectively increased the composition of its high molecular weight oligomeric complex. These changes were associated with an alleviation of hyperglycemia, glucose intolerance, and insulin resistance. By contrast, the beneficial effects of these two compounds on insulin sensitivity and glucose metabolism were diminished in adiponectin knockout mice. In conclusion, obtained results suggest that pharmacological elevation of circulating adiponectin alone is sufficient to ameliorate insulin resistance and diabetes and support the use of adiponectin as a biomarker for future drug discovery. The two natural compounds might provide the lead as a novel class of therapeutics for obesityrelated diseases (Xu et al., 2009).

A lectin (AMML) from the roots of Astragalus mongholicus was extracted and purified by affinity chromatography technique. Human cervical carcinoma cell line (HeLa), human osteoblast-like cell line(MG63) and human leukemia cell line (K562) were used to check the effects of AMML on cell proliferation, apoptosis and cell cycle. Maximum growth inhibition (92%) was observed with HeLa cells, followed by K562 cells (84%) and MG63 (48%) cells. Morphological observation showed that AMML-treated HeLa cells displayed outstanding apoptosis characteristics, such as nuclear fragmentation and appearance of membrane-enclosed apoptotic bodies. The apoptosis of HeLa cells was confirmed by flow cytometry using Annexin V/FITC and propidiumiodide (PI) staining technique (Yan et al., 2009b).

AI (Astragalus membranaceous injection) combined with routine therapy has the therapeutic effect on acute attack of bronchial asthma. One hundred and eight patients with acute attack of bronchial asthma were randomly divided into two groups. The patients in routine treatment group were treated by conventional therapy as control group, while the patients in treatment group were treated with AI on the basis of routine treatment. After treatment, the levels of forced vital capacity (FVC), 1 s forced expiratory volume (FEV1), and peak expiratory flow

(PEF) in treatment group were significantly higher than those in control group (Zhang and Li, 2009; Liu et al., 2011).

The study was designed to investigate efficacy and safety of Astragalus membranaceus (AM) in the treatment of patients with seasonal allergic rhinitis (SAR). AM is an active component in the herbal and mineral complex (HMC) registered in Croatia as a food supplement Lectranal®. The study was designed as a 6-weeks, doubleblind, placebo-controlled clinical trial and conducted in 48 adult patients with a moderate to severe SAR. The treatment efficacy was evaluated by the mean change in the symptom score (TSS), quality of life (QoL), specific serum IgE and IgG, nasal eosinophils, and physicians’ and patients’ global evaluation. Compared to placebo, HMC significantly decreased the intensity of rhinorrhea while for other primary efficacy variables the treatment groups did not differ. In contrast, investigators and patients equally judged the treatment with HMC as more efficacious. In addition, the analysis of changes from baseline inside the groups for TSS, QoL, and 4 main symptoms of SAR were strikingly in favor of the active treatment. In patients with SAR due to weed pollen allergy, HMC significantly improved primary variables, reflective TSS and QoL. The study revealed a significant number of positive signals indicating the therapeutic effectiveness of the HMC in patients with SAR which should be further tested in larger, multicentre trials with more patients (Matkovic et al., 2010).

Yin et al. (2010b) were evaluated the protective effect of Astragaloside IV on Alzheimer’s disease (AD) in rats induced by amyloid-β peptide (Aβ1-42) and its potential therapeutic mechanism. 50 Male Sprague Dawley rats were divided into five groups (10 rats for each): control group, model group, treatment groups 1~3. 10μg Aβ1-42 was injected bilaterally into the dorsal dentate gyrus of the hippocampus of rats in the model and treatment groups to prepare the AD models. 24h after modeling, Astragaloside IV administration, with different drug dosages of 20 mg/(kg•day), 40 mg/(kg•day) and 60 mg/(kg•day), was performed by gastric perfusion for rats in the treatment group 1~3. Later on, the cognitive ability of rats was examined by a series of behavioral tests, and the expression of Bcl-2 and Bcl- xl in the hippocampus of rats was detected by the fluorescein based Quantitative RT-PCR. The spontaneous alternation test in a Y maze and Morris water maze task have demonstrated that the repeated daily administration of Astragaloside IV at the doses of 20 mg/kg bw/day) (p<0.05), 40 mg/kg bw/day) (p<0.01), and 60mg/kg bw/day) (p<0.01) significantly ameliorated the impairment of performance caused by Aβ1-42. Furthermore, Astragaloside IV also enhanced the

expression of Bcl-2 and Bcl-xl in hippocampal neurons of rats in a dosage- dependent manner. In conclusion; these findings suggested that Astragaloside IV could alleviate cognitive impairment and enhance the expression of Bcl-2 and Bcl-xl in hippocampus of rat models with AD.

Zhang et al. (2011) were investigated whether astragaloside IV is able to prevent the development of hypertension and endothelial dysfunction in fructose- fed rats. Rats were fed with 10% fructose in their drinking water for 8 weeks. From the beginning of week 5, two groups of fructose-fed rats were treated with 0.5 or 2 mg/kg, i.p., astragaloside IV. Another group of fructose-fed rats, injected with the same volume of vehicle (dimethylsulfoxide, DMSO) from week 5, served as the control group. At the end of the treatment period, blood pressure, blood glucose, glucose tolerance, blood insulin and lipids were determined. In addition, in vitro experiments were conducted at the end of the eight week treatment period to evaluate endothelium-dependent aortic vasorelaxation, as well as myocardial and aortic tissue levels of nitrate and nitrite (NOx) and cGMP. Fructose-fed rats developed clustering signs of metabolic syndrome, such as increased bodyweight, mild hypertension, hyperinsulinaemia, hypertriglyceridaemia, impaired glucose tolerance and impaired endothelium-dependent vasorelaxation. Administration of astragaloside IV reduced blood pressure and triglyceride levels in fructose-fed rats and high dose of astragaloside IV also improved glucose tolerance and endothelium-dependent vasorelaxation. The astragaloside IV-induced improvement in vasorelaxation was associated with increased levels of aortic NOx and cGMP and was abrogated by blockade of nitric oxide synthase with NG-nitro- l-arginine methyl ester (l-NAME). On the basis of its favourable effects on lipid metabolism, endothelium-dependent vasorelaxation and the nitric oxide–cGMP- related pathway, it was concluded that astragaloside IV may be useful in ameliorating food-induced metabolic syndrome.

Yan et al. (2012) evaluated the role of Astragalus mongholicus aqueous extract AMAE on spinal cord lipid peroxidation (LPO), antioxidant status, apoptosis index and inducible nitric oxide synthases (iNOS) positive expression rate in rats. Male albino rats of Wistar strain were divided into four groups: Group I, normal control rats; Group II, model rats suffering from spinal cord injury (SCI); Group III and V, AMAE rats supplemented with AMAE. Results showed that increased spinal cord LPO, apoptosis index, iNOS positive expression rate and decreased antioxidant enzymes activities were observed in model rats (Group II). Administration of AMAE significantly dose-dependently decreased spinal

cord LPO, apoptosis index, iNOS positive expression rate and increased antioxidant enzymes activities in AMAE rats. These findings demonstrated that AMAE can enhance the antioxidant status and decrease the incidence of free radical-induced LPO, and display strong neuroprotective activity in the experimental rats. (Neuroprotective action of Astragalus mongholicus aqueous extract in experimental rats suffering from spinal cord injury.

In another research, Xiong et al. designed a study to compare hearing gains from hearing impairment in 46 ears treated with Radix astragali (RA) with 46 ears treated with non-RA. RA was given intravenously daily for 10 days. There were no significant differences in clinical or audiological data between RA and non-RA groups. The results showed that the hearing gain at 250, 500, 1000, 2000, and 4000 Hz in RA group was much higher than that of non-RA group correspondingly (P b<01). Also, the hearing gain at PTA (pure-tone average of 250, 500, 1000, 2000, and 4000 Hz) in RA group was significantly higher than that of non-RA group (P b<01). It was concluded that the recovery of hearing was significantly better after treatment of RA than non-treatment of RA. RA can be valuable concurrent therapy for patients with SD (Xiong et al., 2012).

1.7 Structure Identification by Chromatographic and Spectroscopic Methods

Recently, natural products chemistry has undergone explosive growth due to advances in isolation techniques, synthetic and biosynthetic approaches as well as spectroscopic and chromatographic methods. The advent of computers and Fourier transform completely revolutionized the detection and identification of organic compounds. Modern automated instruments allow very small samples in the nanogram (10−9 g) range to be characterized in a very short time. The application of Fourier transform nuclear magnetic resonance (FTNMR) and Fourier transform infrared (FTIR) allows recovery of the sample in contrast to mass spectrometric (MS) determination which is a destructive but quite often a necessary technique. Modern methods used to separate complex organic mixtures utilizing gas-liquid chromatography (GLC), high-pressure liquid chromatography (HPLC), and droplet counter-current (DCC) chromatography can separate samples rapidly and efficiently in the picogram range. This has been impossible until recently. Coupling the chromatographic instruments to spectrometers enables a partially automated analysis in an even shorter period of time. The following coupling of chromatographic instruments has been performed: GC-MS, GC-FTIR,

GC-MI-FTIR, GC-UV-VIS, HPLC-MS, HPLC-FTIR, HPLC-FTNMR and MS- MS.

These semiautomated systems of analyzing and characterizing small samples are vital to the natural product organic chemist and biochemist for the detection of highly active substances in extremely low concentrations in living organisms.

The separation of a mixture of saponins in different components is a difficult task which requires the combined application of several chromatographic technics. The thin layer chromatography (TLC) and high performance thin layer chromatography (HPTLC) are often used for qualitative analysis routine (Mangle and Jolly, 1998). Purification is then carried out, can involve chromatography liquid at atmospheric pressure on a silica gel column (CC), Flash chromatography, the Medium pressure liquid chromatography (MPLC) or High pressure liquid chromatography (HPLC), which became the most powerful technic and most commonly employee. In most cases, some of these steps must be repeated using a new support or eluent to achieve a high level of purity. Three types of support are often used, including normal phase silica, reverse phase silica (RP-18) and the gel Sephadex LH-20. Various solvents involve mostly mixtures of chloroform- methanol-water on normal silica, methanol-water mixtures on reverse phase silica and acetonitrile-water mixtures in HPLC.

1.7.1 Infra-Red (IR) Spectroscopy

IR spectroscopy provide information about the presence or absence of functional groups (COR, COOR, CN, NO2, etc.), but they are not as useful or information-rich as NMR or mass spectra. The characteristic IR vibrations are influenced strongly by small changes in molecular structure, thus making it difficult to identify structural fragments from IR data alone. In 2008, (Kareru et al.,) reported that the detection of saponins in medicinal plants is performed directly on crude sample powders using Fourier Transform Infrared (FTIR) spectroscopy as simple, fast and economical method. The spectra of saponins generally show -OH, -C=O, C-H and C=C absorptions bonds together with C-O-C absorptions derived from glycosidic linkages.

1.7.2 Ultraviolet (UV) Spectroscopy

The main application of UV spectroscopy, which depends on transitions between electronic energy levels in identifying conjugated (π) electron systems. The use of UV spectroscopy is very limited for triterpenes especially for cycloartanes due to the absence of conjugated (π) electron systems. 2,3-Dihydro- 2,5-dihydroxy-6-methyl-4H-pyran-4-one (DDMP), a conjugated soyasaponins, is an exceptional representation of UV active triterpenoids showing absorption maximum at (274 nm) (Kudou et al., 1993).

1.7.3 Mass Spectrometry (MS)

Mass Spectrometry is an important tool for the identification and structural elucidation of natural products. It gives information regarding molecular weight, or the formula weight of a compound. The mass spectrum shows the mass of the molecule and its portion masses. In 2006, Cabrera reported that mass Spectrometry can be used to determine the number and type of monosaccharide units of the glycoside (mono-, di-, trisaccharide, pentose, hexose, deoxyhexose).

Saponins are very polar compounds, thermally unstable, and non volatile to provide directly required ions that are suitable for analysis. The saponins are not suitable to be analyzed by classical mass techniques (EI or CI). Therefore, new techniques of soft ionization have emerged which are proved to be useful in the analysis of saponins, Field Desorption (FD) (Ganzera et al., 2004), Fast Atom Bombardment (FAB) (Melek et al., 2004), Matrix‐Assisted Laser Desorption Ionization Time‐of‐Flight Mass Spectrometry (MALDI‐TOF-MS) (Horo et al., 2010; Polat et al., 2010) and Electron spray ionization (ESI-MS) (De Leo et al., 2006).

1.7.4 X-ray Spectroscopy

X-ray spectroscopy is an excellent method to determine the structure of a compound but this technique requires the availability of a compound as a single crystal. Most chemists find this process very tedious and time consuming. It also requires a skillful hand. However, in the events, when other spectral methods fail to reveal a compound's identity, especially 3D-structure. X-ray spectroscopy is the method of choice for structural determination where the other parameters such as bond lengths and bond angles are also determined.

1.7.5 Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR spectroscopy is a useful technique for identifying and analyzing organic compounds. This extremely important experimental technique is based on magnetic nuclear spin of 1H, 13C, 15N, 19F, 31P. NMR is therefore contained many advantages in using.

Non-selective, non-destructive, detection of low molecular weight molecules in solution, both endogenous and xenobiotic metabolites can be monitored simultaneously in complex biofluids, in many cases quantitative data may be obtained, NMR provides uniquely rich structural content, such as chemical shifts, multiplicity (the interaction between neighboring nuclei), integrals, intermolecular relationships and a wide range of dynamic processes, including molecular motions in solution, chemical exchange and ligand binding, NMR requires minimal sample preparation, and the sample can be recovered for further analysis, it works with radiofrequency waves; therefore, it is not hazardous humeny.

Proton nuclear magnetic resonance (1H-NMR) spectrum gives information about the environments of the different hydrogens in a molecule. 13C-NMR spectroscopy informs us about the number of different kinds of carbons, and their chemical shifts relates to particular chemical environments. However, unlike 1H, which is the most abundant of the hydrogen isotopes (99.985%), only 1.1% of the carbon atoms in a sample are 13C. Moreover, the intensity of the signal produced by 13C nuclei is far weaker than the signal produced by the same number of 1H nuclei. A 13C NMR is useful technique in structure determination, but a vast increase in the signal-to-noise ratio is required.

There are few modified techniques that can be very useful for distinguishing the different types of carbons within a molecule and determining the relationship between nuclei. Of these, we have found the DEPT (Distortionless Enhancement by Polarization Transfer) the most useful technique to determine the nature of carbon. In this experiment, the quaternary carbons are edited out of the spectrum, while methyl and methine protons naturally phase at 180◦ relative to the methylene carbons and the spectra are usually plotted with methyls and methines positive.

With more complex organic structures, the 1D-NMR spectra will not be enough to determine structures. 2D NMR techniques now allow us to acquire direct structural information such as connectivity and proximity more efficiently than ever before.

The term 2D-NMR stands for two dimensional NMR refers to spectroscopic data that are collected as a function of tow time scales, F1 (the Fourier transform of the t1 time domain) and F2 (the Fourier transform of the t2 time domain).

COSY (Correlated Spectroscopy)

COSY spectra show direct couplings (geminal and vicinal) of protons in the same spin system. These are scalar couplings 2JH, H et 3JH, H (Fig. 1.18). This method can find the sequence of protons in a skeleton molecule and therefore its nature. This experiment provides information on the chemical shifts osidic protons and magnitude of coupling constants between proton (interproton) (Gunther1996; Massiot et al., 1995). The two-dimensional spectrum obtained from COSY experiment shows the frequencies for a single isotope, most commonly hydrogen (1H) along both axes. COSY spectra show two types of peaks. Diagonal peaks have the same frequency coordinate on each axis and appear along the diagonal of the plot; while cross peaks have different values for each frequency coordinate and appear of the diagonal. Diagonal peaks correspond to the peaks in a 1D-NMR experiment, while the cross peaks indicate couplings between pairs of nuclei.

Figure 1.18 COSY correlations

TOCSY (Total Correlation Spectroscopy)

From a TOCSY spectrum, the J connexion can be established. J connexion is defined as a group of protons which are connected in series by a scalar coupling. For example, all protons of a single saccharide unit belong to the same connexion-J (Agrawal, 1992). A spin system can be matched, if there is at least one resonance in the spin system as the anomeric proton is well insulated and has a large coupling with neighboring spins (Fig. 1.19). More practically a sugar, it Just observe a resonance to identify the other protons of the saccharide unit. For deoxy-6-hexoses, methyl characteristics often serve as a starting point.

Figure 1.19 TOCSY correlations

NOESY (Nuclear Overhauser Effect Spectroscopy)

NOE (Nuclear Overhauser Enhancement) highlights the close protons. The correlation signals are observed in the NOESY spectrum between pairs protons that are close in space. In general, 1,3-diaxial correlations observed and equatorial-axial between pairs of remote protons least 4.5 Å (Fig. 1.20).

Figure 1.20 NOESY correlations

If the intramolecular NOESY is an important tool in the allocation of signals from a saccharide residue, the intermolecular NOESY is used primarily to determine the sugars and their sequence junctions. When we observe a correlation NOESY between the anomeric proton of a sugar and a proton belonging to another sugar, these two protons define the connection between the two sugars. NOE correlations are most often observed between the anomeric proton and proton connected to the carbon atom binding with aglycone (Fig. 1.21)

Figure 1.21 NOESY correlations between sugar and aglycone

HMQC (Heteronuclear Multiple-bond Quantum Correlation)

HMQC detects correlations between nuclei of two different types which are separated by one bond. It correlates 1H signals with 13C signals. This method gives one peak per pair of coupled nuclei, whose two coordinates are the chemical shifts of the two coupled atoms (Fig. 1.22).

Figure 1.22 HMQC correlations

HMBC (Heteronuclear Multiple-Bond Correlation Spectroscopy)

HMBC experiment, correlations are obtained between 13C and 1H atoms that are separated by two and three bonds to give information about structural C-C connectivity (Fig. 1.23).

Figure 1.23 HMBC correlations

2 MATERIALS AND METHODS

2.1 General

Optical rotations were measured on a JASCO DIP 1000 polarimeter. IR measurements were obtained on a Bruker IFS-48 spectrometer. NMR experiments were performed on a Bruker DRX-600 spectrometer (Bruker Bio Spin GmBH, Rheinstetten, Germany) equipped with a Bruker 5 mm TCI Cryo Probeat 300 K.

All 2D-NMR spectra were acquired in CD3OD (99.95%, Sigma Aldrich) and standard pulse sequences and phase cycling were used for DQF-COSY, HSQC, and HMBC spectra. The NMR data were processed using UXNMR software. Exact masses were measured by a Voyager DE mass spectrometer. Samples were analyzed by matrix-assisted laser desorption ionization time-of-flight (MALDITOF) mass spectrometry. A mixture of analyze solution and α-cyano-4- hydroxycinnamic acid (Sigma) was applied to the metallic sample plate and dried. Mass calibration was performed with the ions from ACTH (fragment 18-39) at 2465.1989 Da and angiotensin III at 931.5154 Da as internal standard. ESIMS analyses were performed using a ThermoFinnigan LCQ Deca XP Max iontrap mass spectrometer equipped with Xcalibur software. GC analysis was performed on a Termo Finnigan Trace GC apparatus using a l-Chirasil-Val column (0.32 mm x 25 m). Column chromatography was carried out on Silica gel (JT Baker, 40 μm), Sephadex LH-20 (Amersham Biosciences, 17-0090-02) and RP (C-18, 40 μm) (Merck). TLC analyses were carried out on Silica gel 60 F254 (Merck) and RP-18 F254s (Merck) plates. Compounds were detected by UV and 20% o H2SO4/water spraying reagent followed by heating at 110 C for 1-2 min.

During chromatographic studies the following solvent systems were used;

I CHCl3:MeOH:H2O 80:20:2

II CHCl3:MeOH:H2O 80:20:1

III CHCl3:MeOH 80:20

V CHCl3:MeOH:H2O 70:30:3

V CHCl3:MeOH:H2O 61:32:7

VI CHCl3:MeOH:H2O 64:50:10

VII MeOH:H2O 8:2

VIII MeOH:H2O 7:3

IX MeOH:H2O 4:6

X MeOH:H2O 3:7

XI MeOH:H2O 2:8

XII EtOAc:MeOH:H2O 100:25:20

2.2 Plant Materials

2.2.1 Astragalus halicacabus Lam.

A. halicacabus Lam. was collected from the altitude of 2198 m in Çatak (Van, Eastern Anatolia, Turkey, on June 5, 2010. Samples of plant material were identified by Assoc. Prof. Dr. Fevzi. Özgökçe (Department of Biology, Faculty of Science & Art, Yüzüncü Yıl University, Van, Turkey). Voucher specimen has been deposited in the Herbarium of Yüzüncü Yıl University, Van, Turkey (VANF13709).

2.2.2 Astragalus melanocarpus Bunge.

Astragalus melanocarpus Bunge. (whole plant) was collected from Muş in district of Malazgirt, from altitude of 1600 m (Aktuzlar village), Van Turkey in July 2010. Samples of plant material were identified by Assoc. Prof. Dr. Fevzi Özgökçe (Deparment of Biology, Faculty of Sciences and Art, Yüzüncü Yıl University, Van, Turkey). Voucher specimen has been deposited in the Herbarium of Yüzüncü Yıl University, Van, Turkey (VANF 13838).

2.3 Isolation and Purification

2.3.1 Astragalus halicacabus Lam.

The air-dried and powdered whole plant (1150 g) of Astragalus halicacabus was extracted with MeOH (3x4 l) at 60ºC for 5 h each. The MeOH solvent was then evaporated to dryness under reduced pressure to give 43.0 g of a dark residue. The residue was dissolved in H2O and then partitioned successively with hexane (2x200 ml), CH2Cl2 (2x200 ml), and BuOH saturated with H2O (4x200 ml). The BuOH extract (21.0 g) was subjected to medium pressure liquid chromatography (MPLC) using reversed-phase (RP) material (300.0 g) employing

H2O (500 ml), H2O/MeOH (8:2, 500 ml; 6:4, 800 ml; 4:6, 500 ml; 2:8, 500 ml), and MeOH (300 ml) to give thirteen fractions (Frs.) A–M. Fr. L (200 mg) was subjected to CC (SiO2 (30.0 g); CHCl3:MeOH:H2O 80:20:2, 500 ml; 70:30:3, 750 ml) to yield compound AHa3, cyclocanthoside D (41.0 mg), compound AHa4, askendoside G (19.5 mg) and compound AHa9, askendoside F (10.0 mg). Fr. M (34.0 mg) was chromatographed on a reversed-phase material (20.0 g) with

MeOH:H2O 6:4 (500 ml) to give compound AHa1, cyclostipuloside A (6.0 mg).

Fr. B (1.6 g) was submitted to open CC (SiO2 (95.0 g); CHCl3:MeOH:H2O 80:20:2, 1200 ml; 70:30:3, 1000 ml) to afford compound AHa7, halicacoside A (17.5 mg). Fr. I (118.0 mg) was chromatographed on reversed-phase material

(30.0 g) with MeOH:H2O 6:4 (500 ml) to give compound AHa10, 3-O--D glucopyranosylmaltol (32.5 mg). Fr. D (60.8 mg) was subjected to open CC (SiO2

(30.0 g); CH2Cl2:MeOH:H2O 80:20:2, 500 ml; 70:30:3, 700 ml) to give compound AHa8, elongatoside (11.2 mg). Fr. K (172.0 mg) was purified by CC

(SiO2 (25.0 g); CH2Cl2:MeOH:H2O 90:10:1, 250 ml; 80:20:2, 400 ml; 70:30:3, 400 ml) to give compound AHa5, askendoside D (2.0 mg). Fr. J (47.0 mg) was submitted to open CC (SiO2 (10.0 g); CH2Cl2:MeOH:H2O 80:20:2, 200 ml; 70:30, 250 ml) to give compound AHa6, cyclosieversioside G (17.0 mg) and AHa2, (20R,24S)-3-O-[α-L-Arabinopyranosyl-(1→2)--D-xylopyranosyl]-20,24-epoxy- 16-O--D-glucopyranosyl-3b,6α,16,25-tetrahydroxycycloartane (3,6α,9,16,20R,24S)-3-{[2-O-(α-L-Arabinopyranosyl)--D- xylopyranosyl]oxy}-20,24-epoxy-6,25-dihydroxy-9,19-cyclolanostan-16-yl--D- Glucopyranoside (6.0 mg) (Scheme 2.1).

Scheme 2.1 Isolation of the new triterpene (AHa1) and maltol (AHa2) from Astragalus halicacabus Lam.

2.3.2 Astragalus melanocarpus

The air-dried and powdered plant material (Astragalus melanocarpus, 420 g) was extracted with MeOH (3x4 L) for 6 days, under reflux. After filtration, the solvent was removed by rotary evaporation to afford 45 g of crude extract. The

MeOH extract was dissolved in H2O (400 mL), and successively partitioned with n-hexane (2x250 mL), CH2Cl2 (2x250 mL), and n-BuOH saturated with H2O (4x250mL). The crude BuOH extract (8 g) was subjected to vacuum liquid chromatography (VLC) on reversed-phase material (Lichroprep RP-18, 25-40 μm,

300 g) employing H2O (950 mL), H2O-MeOH (80:20, 1000 mL; 70:30, 700mL; 6:4, 1500 mL; 50:50, 1050 mL), and MeOH (700 mL) to give six main fractions

(1-6). After TLC controls, fractions 3, 4, 5, and 6 eluted with H2O-MeOH (20:80) were supposed to be rich in saponins. Fraction 5 (1000 mg) was applied to an open column chromatography using silica gel (120 g) as stationary phase. Elution was carried out with CHCl3-MeOH (90:10, 1500 mL), CHCl3-MeOH (85:15,

1000 mL), CHCl3-MeOH-H2O (80:20:1, 1500 mL), CHCl3-MeOH-H2O (80:20:2,

800 mL), CHCl3-MeOH-H2O (70:30:3, 1300 mL), and CHCl3-MeOH-H2O (61:32:7, 900 mL) yielding 1 (25 mg), and the subfraction 5.1. Then subfraction 5.1 (120 g) was chromatographed over a reversed phase column (Lichroprep RP-

18, 25-40 μm, 55 g) using H2O-MeOH (70:30, 450 mL; 60:40, 500 mL; 50:50, 1000 mL; ) to give compound 4 (9.7 mg) and compound 6 (1 mg). Fractions 4 (400 mg) obtained from VLC column chromatography were further purified on an open column chromatography using reversed-phase material (Lichroprep RP-18,

25-40 μm, 80g) and eluted with H2O-MeOH (40:60, 1100 mL) to give 7 (2.2 mg), 8 (4.2 mg) and 11(1 mg). Finally, fractions 3 (1700 mg) was subjected to medium pressure liquid chromatography using a reversed-phase (Lichroprep RP-18, 25-40

μm, 140g) using H2O-MeOH (70:30, 600mL; 60:40, 400 mL; 50:50, 400 mL;) as mobile phase to afford 5 (24.5 mg), 14 (3 mg) and 18 (1.7 mg). Fractions 6 (31 mg) was subjected to silica gel column (20 g). Elution was started with CHCl3-

MeOH (90:10, 500 mL), and followed by CHCl3-MeOH (85:15, 650 mL), and

CHCl3-MeOH-H2O (80:20:2, 600 mL) to afford 32 (1 mg) (Scheme 2.2).

Scheme 2.2 Isolation of the new cycloartanes (AMJ5, AMJ8 and AMJ18) from Astragalus melanocarpus

100

3 RESULTS AND DISCUSSION.

3.1 Astragalus halicacabus Lam.

3.1.1 Structural Identification of AHa1

The LC/ESI-TOF mass spectrum of AHa1 (m/z 939.4991 ([M+Na+], + C46H76NaO18 ; calc. 939.4990) provided the molecular formula C46H76O18. The 1H-NMR spectrum displayed the aglycone moiety signals due to a cyclopropane

CH2 at δ (H) 0.43 and 0.20 (d, J=4.2, each 1 H), seven tertiary Me groups at δ (H) 1.31, 1.30, 1.19, 1.03, 1.00, 0.88, and 0.85, and four O-CH signals at δ (H) 4.13 (ddd, J=8.0, 8.0, 5.2), 3.63 (dd, J=8.2, 6.0), 3.30 (ddd, J=9.5, 9.0, 4.5), and 3.04 (dd, J=11.3, 4.0) indicative of secondary alcohol functions (Table 3.1). Furthermore, the 1H-NMR spectrum of 1 showed three anomeric H-atom doublets at δ (H) 4.37 (J=5.9), 4.28 (J=7.0), and 4.07 (J=8.4) in the downfield region, indicative of two β-linked and one α-linked sugar units (cf. Table 3.2). These correlated to C-atom signals at δ (C) 106.2 and 105.3 (2 C), respectively, in the HSQC spectrum. The 13C-NMR spectrum exhibited 46 resonances; 30 of them, attributable to the sapogenol moiety, were in good agreement with those of cycloastragenol (Kitagawa et. Al., 1983). Full assignment of the 1H and 13C signals of the aglycone moiety of 1, based on DQF-COSY and HSQC spectra, showed glycosylation shifts for C(3) (δ (C) 87.7) and C(16) (δ (C) 82.7), suggesting that 1 was a bidesmosidic saponin (Fig. 3.1). All connectivities within 1 were also confirmed by an HMBC experiment. The DQF-COSY experiments allowed us to unambiguously assign all sugar H-atom signals and to identify one α-arabinopyranosyl (δ (H) 4.37), one β-xylopyranosyl (δ (H) 4.28), and one β- glucopyranosyl unit. The determination of the sequence and linkage sites was accomplished from the key HMBCs (Fig. 3.1) between the anomeric H-atom signal at δ (H) 4.37 (H-C(1)(Ara)) and the C-atom resonance at δ (C) 82.1 (C(2)(Xyl)), and between the anomeric H-atom signal at δ (H) 4.28 (H- C(1)(Xyl)), and the C-atom resonance at δ (C) 87.7 (C(3)). Moreover, the anomeric H-atom signal at δ (H) 4.07 (H-C(1)(Glc)) showed long-range correlation with the C-atom resonance at δ (C) 82.7 (C(16)), indicating the presence of a glucopyranose unit at C(16). The D-configuration of xylose and glucose units and the L-configuration of arabinose unit were established by the hydrolysis of 1, followed by GC analysis (S. De Marino et al., 2003). Thus, the structure of 1 was elucidated as (20R,24S)-3-O-[α-l-arabinopyranosyl-(1→2)-β-

101

D-xylopyranosyl]-20,24-epoxy-16-O-β-D-glucopyranosyl-3β,6α,16β,25- tetrahydroxycycloartane (Fig. 3.1).

25 Amorphous white solid. [] D +41.4 (c=0.1, MeOH). IR (KBr): 3480 (>OH), 3035 (cyclopropane 1332 ring), 2945 (>CH), 1260 and 1055 (C-O-C). 1H- and 13C-NMR: Tables 3.1 and 3.3, resp. HR-ESI-MS: 939.4991, [M+Na]+ + 1 13 C46H76NaO 18; calc. 939.4990). H-NMR (CD3OD, 600 MHz) and C-NMR

(CD3OD, 150 MHz) data: see Table.3.1

Figure 3.1. Structure of AHa1

102

1 13 Table 3.1 H- and C-NMR Data of the Aglycon Moieties of AHa1 in CD3OD at 500 MHz; δ in ppm, J in Hz

Position δH δC 1 1.73–1.75, 1.36–1.38 (2m) 32.0 2 1.77–1.79, 1.48–1.50 (2m) 30.2 3 3.04 (dd, J=11.3, 4.0) 87.7 4 - 42.0 5 1.22 (d, J=9.7) 53.3 6 3.30 (ddd, J=9.7, 9.7, 4.5) 67.0 7 1.45–1.47, 1.21–1.23 (m) 38.0 8 1.76 (dd, J=11.3, 4.0) 46.2 9 - 46.2 10 - 29.0 11 1.96–1.98, 1.77–1.79 (2m) 25.1 12 1.67–1.69 (m, 2 H) 32.4 13 - 46.2 14 - 46.4 15 1.92 (dd, J=13.7,8),1.56 (dd,J=12.9, 5.6) 47.3 16 4.13 (ddd, J=8.0, 8.0, 5.6) 82.7 17 2.16 (d, J=8.0) 59.5 18 1.30 (s) 21.1 19 0.43, 0.20 (2d, J=4.2) 29.8 20 - 87.5 21 1.31 (s) 25.5 22 1.96–1.98, 1.38–1.40 (2m) 39.4 23 1.82–1.84, 1.74–1.77 (2m) 26.2 24 3.63 (dd, J=8.2, 6.0) 83.9 25 - 71.0 26 1.03 (s) 25.1 27 1.00 (s) 28.0 28 1.19 (s) 28.1 29 0.88 (s) 15.9 30 0.85 (s) 20.3

103

1 13 Table 3.2 H- and C-NMR Data of the Sugar Moieties of AHa1 in CD3OD at 600 MHz;  in ppm, J in Hz

Position δH δC β-D-Xyl (at C(3)) 1 4.28 (d,J=7.0) 103.3 2 3.21 (dd,J=7.5, 9.2) 82.1 3 3.34 (t, J=9.2) 76.2 4 3.04–3.07 (m) 70.6 5 3.60 (dd, J=5.2, 11.7), 3.30 (t,J=11.7) 65.6 6 α-L-Ara (at C(2) at Xyl) 1 4.37 (d,J=5.9) 102.0 2 3.47 (dd, J=8.5, 3.7) 71.9 3 3.42 (dd, J=8.5, 3.0) 72.6 4 3.60–3.63 (m) 67.6 5 3.67 (dd, J=11.9, 2.0), 65.6 3.05 (dd, J=11.9, 3.0) β -D-Glc (at C(16)) 1 4.07 (d, J=8.4) 105.2 2 2.93 (dd, J=7.5, 9.0) 74.5 3 3.12 (t, J=9.0) 77.4 4 3.04 (t, J=9.0) 69.9 5 3.05 (ddd, J=3.5, 4.5, 9.0) 77.1 6 3.61 (dd, J=3.5, 12), 61.6 3.43 (dd, J=4.5, 12)

Figure 3.2 HMBC key correlation of AHa1

104

Spectrum 3.1. 1H-NMR Spectrum of AHa1

105

Spectrum 3.2 13C-NMR Spectrum of AHa1

106

Spectrum 3.3. HMQC Spectrum of AHa1

107

Spectrum 3.4. HMQC Spectrum of AHa1

108

Spectrum 3.5. HMQC Spectrum of AHa1

109

Spectrum 3.6. HMBC Spectrum of AHa1

110

Spectrum 3.7. HMBC Spectrum of AHa1

111

3.1.2 Structural Identification of AHa2

The LC/ESI-TOF mass spectrum of AHa2 (m/z 443.1199 ([MNa],

C17H24NaO12; calc. 443.1182) revealed the molecular formula C17H24O12 . The C- atom signals due to the aglycone moiety of 2 were associated with a C=O group (d(C) 173.7), four sp2 C-atom signals (160.7, 155.7, 141.4, and 117.1) and one Me C-atom signal (d(C) 15.5). These data, together with the appearance of AB-type doublets (J=5.6) at d(H) 6.32 and 8.03, and a vinyl Me signal at d(H) 2.28 in the 1H-NMR spectrum, indicated that the aglycone was a -pyrone such as maltol (Wada et al., 1983).

The 1H-NMR spectrum for the sugar portion showed two anomeric H-atom doublets at d(H) 5.31 (J=1.2) and 5.08 (J=7.6) (Table 2). In the HSQC spectrum, these H-atom signals correlated with the C-atom signals at d(C) 109.1 and 99.8, respectively (Table 1). Complete assignments of the 1H- and 13C-NMR signals of the sugar portion were accomplished by HSQC, HMBC, and DQF-COSY experiments which allowed the unambiguous assignment all sugar H-atom signals and to identify one apiofuranosyl (d(H) 5.31) and one β-glucopyranosyl (d(H) 5.08) moiety (Fig. 2). The site of glycosidation on the aglycone of AHa2 as well as the position of the interglycosidic linkage were determined by an HMBC experiment, which showed longrange correlations between the anomeric H-atom signal at d(H) 5.31 (H-C(1)(Api)) and the C-atom resonance at d(C) 77.4 (C(2)(Glc)), and between the anomeric H-atom signal at d(H) 5.08 (H-C(1)(Glc)) and the C-atom resonance at d(C) 141.4 (C(3)). The configuration of glucopyranosyl and apiofuranosyl units were established as D by hydrolysis of AHa2 with 1N HCl, trimethylsilylation, and determination of the retention times by GC (De Marino et. Al, 2003). Therefore, the structure of AHa2 was elucidated as 3-O-[β-D-apiofuranosyl-(1→2)- β -D-glucopyranosyl]maltol (Fig. 2).

25 Amorphous white solid. [] D = -78.9 (c= 0.1, MeOH).IR (KBr): 3376, 1650. 1H- and 13C-NMR: Tables 1 and 2, resp. HR-ESI-MS: 443.1199, [M+Na]+ + 1 13 C17H24NaO 12 ; calc. 443.1182. H-NMR (CD3OD, 600 MHz) and C-NMR

(CD3OD, 150 MHz) data: see Table 3.3.

112

1 13 Table 3.3 H- and C-NMR Data of the Aglycon and Sugar Moieties of AHa2 in CD3OD at 500 MHz; δ in ppm, J in Hz

Position δ (H) δ (C) 1 - - 2 - 160.7 3 - 141.4 4 - 173.7 5 6.32 (d,J=5.6) 117.1 6 8.03 (d,J=5.6) 155.7 7 2.28 (s) 15.5

β-D-Glc (at C(3)) 1 5.08.(d,J=7.6) (d,J=7.6) 99.8 2 2.93 (dd,J=7.5, 9.0) 77.4 3 3.12 (t,J=9.0) 76.6 4 3.04 (t,J=9.0) 70.5 5 3.05 (ddd,J=3.5, 4.5, 9.0) 77.3 6 3.61 (dd,J=3.5, 12), 61.2 3.43 (dd,J=4.5, 12) β-D-Api (at C(2) of Glc) 1 5.31 (d, J=1.2) 109.1 2 3.78 (br. s) 77.3 3 79.8 4 3.84 (d, J=9.2), 3.52 (d, J=9.2) 74.4 5 3.54 (s, 2 H) 64.9

Figure 3.3 Structures of AHa2

113

Spectrum 3.8. 1H-NMR Spectrum of AHa2

114

Spectrum 3.9 13C-NMR Spectrum of AHa2

115

Spectrum 3.10 DEPT spectrum of AHa2

116

Figure 3.4 Key correlations of AHa2

Additionally, seven known cycloartane-type glycosides, cyclocanthoside D (Fadaev et al., 1988), askendoside G (Isaev 1996), askendoside D (Isaev 1993), cyclosieversioside G (Swechnikova et al., 1983), cyclostipulosideA (Karimov et al., 1999;), elongatoside (Calis et al., 2008), askendoside F (Isaev 1995), and one known -pyrone derivative, 3-O-β-D-glucopyranosylmaltol (Sala et al., 2001) were isolated.

This is the first report on the isolation of a maltol glucoside from Astragalus genus, and even from Leguminosae family. Thus, 3-O-[β-D-apiofuranosyl-(1→2)- β-D-glucopyranosyl]-maltol (AHa2) together with the known compound 3-O-β- D-glucopyranosylmaltol represent the first members of this class and can be of assistance as chemotaxonomic markers to differentiate A. halicacabus in Astragalus genus.

3.1.3 Acidic Hydrolysis of Compounds from Astragalus halicacabus

The configurations of sugar unit were established by hydrolysis of 1 and 2 with 1N HCl, trimethylsilation, and determination of the retention times (tR values) by GC operated under the experimental conditions previously reported by De Marino et al.,. The peaks of the hydrolysate of 1 were detected at tR 8.93 and 9.80 min (L-arabinose), at tR 10.99 and 12.00 min (D-xylose), and at tR 14.72 min (D-glucose). The peaks of the hydrolysate of 2 were detected at tR 14.73 (D- glucose) and at 7.2 min (D-apiose). The tR values for authentic samples after treatment in the same manner with 1-(trimethylsilyl)-1H-imidazole in pyridine were 8.92 and 9.80 (L-arabinose), 10.98 and 12.00 min (D-xylose), and 14.71 min (D-glucose), and 7.1 min (D-apiose).

117

3.2 Astragalus melanocarpus. Bunge

3.2.1 Structural Identification of AMJ5

The 1H NMR spectrum of AMJ5 showed signals due to a cyclopropane methylene at  0.59 and 0.25 (each 1H, d, J = 4.2 Hz), six tertiary methyl groups at  1.31 (3H, s), 1.18 (3H, s), 1.18 (3H, s), 1.16 (3H, s), 1.03 (3H, s) and 0.97 (3H, s), a secondary methyl group at  0.96 (d, J = 6.5 Hz), and four methine proton signals at  4.23 (ddd, J = 8.0, 8.0, 5.2 Hz), 3.46 (ddd, J = 9.5, 9.5, 4.5 Hz), 3.20 (dd, J = 11.3, 4.0 Hz) and 3.17 (dd, J = 10.5, 2.4 Hz) and which were indicative of secondary alcoholic functions (Table 3.4). The NMR data of the aglycon moiety of AMJ5 were in good agreement with those reported for cycloasgenin C (Kucherbaev et al., 2002) with glycosidation shifts for C-3 ( 89.6) and C-16 ( 73.3) (Table 3.4). Additionally, for compound AMJ5 it was also evident signals for a further secondary methyl group at  1.26 (d, J = 6.5 Hz) along with three anomeric protons at  5.36 (d, J = 1.2 Hz), 4.40 (d, J = 7.5 Hz) and 4.29 (d, J = 7.5 Hz). The chemical shifts of all the individual protons of the three sugar units were ascertained from a combination of 1D-TOCSY and DQF- COSY spectral analysis, and the 13C chemical shifts of their relative attached carbons were assigned unambiguously from the HSQC spectrum (Table 3.5). These data showed the presence of one -rhamnopyranosyl unit ( 5.36), one - xylopyranosyl unit ( 4.40) and one -glucopyranosyl unit ( 4.29). An unambiguous determination of the sequence and linkage sites was obtained from the HMBC spectrum, which showed key correlation peaks between the proton Rha signal at  4.40 (H-1xyl) and the carbon resonance at  89.6 (C-3),  5.36 (H-1 ) and  78.9 (C-2Xyl), and and the proton signal at  4.29 (H-1Glc) and the carbon resonance at  84.4 (C-16). The D configuration of xylose and glucose units and the L configuration of rhamnose unit were established after hydrolysis of AMJ5 with 1 N HCl, trimethylsilation and determination of retention time by GC. On the basis of all these evidences, the structure of the new compound AMJ5 was established as 3-O-[-L-rhamnopyranosyl-(1→2)-O--D-xylopyranosyl]-16-O-- D-glucopyranosyl-3,6,16,24R,25-pentahydroxycycloartane.

118

Figure 3.5. Structure of AMJ5

119

1 13 Table 3.4 H- and C-NMR Assignments of AMJ5 (150/600 MHz,  ppm, in CD3OD)

Position C Position H (J in Hz)

1 33.5 H2-1 1.55, 1.23, m

2 30.3 H2-2 1.93, 1.67, m 3 89.6 H-3 3.20, dd (11.3, 4.0) 4 43.3 - - 5 54.9 H-5 1.36, d (9.5) 6 69.7 H-6 3.46, ddd (9.5, 9.5, 4.5)

7 38.8 H2-7 1.49, 1.35, m 8 48.7 H-8 1.80, dd (11.9, 4.2) 9 22.0 - - 10 30.2 - -

11 27.5 H2-11 1.99, 1.20, m

12 33.9 H2-12 1.68-1.53 (2H), m 13 46.8 - - 14 48.6 - - 2.11, dd (12.7, 8.0) 15 48.5 H -15 2 1.72, dd (12.7, 5.2) 16 73.2 H-16 4.48, ddd (8.0, 8.0, 5.2) 17 58.6 H-17 1.84, dd (9.9, 8.0)

18 25.6 H3-18 1.18, s 0.58, d (4.2) 19 27.7 H -19 2 0.24, d (4.2) 20 32.3 H-20 1.92, m

21 18.4 H3-21 0.96, d (6.5)

22 34.3 H2-22 2.35,

23 30.4 H2-23 1.90, 1.17, m 24 80.5 H-24 3.17, d (10.3, 2.2) 25 73.9 - -

26 24.9 H3-26 1.16, s

27 25.6 H3-27 1.18, s

28 28.6 H3-28 1.31, s

29 16.7 H3-29 1.03, s

30 20.5 H3-30 0.97, s

120

1 13 Table 3.5 H- and C-NMR Assignments of AMJ5 (150/600 MHz,  ppm, in CD3OD)

Position C Position H (J in Hz) 3-O-β-D-Xyl (at C-3) 1 106.2 H-1 4.40 d (7.5) 2 78.9 H-2 3.45 dd (9.2, 7.5) 3 77.0 H-3 3.54 t (9.2) 4 71.5 H-4 3.49 m 3.87 dd (11.7, 5.2) 5 66.4 H -5 2 3.20 t (11.7)

-L-Rha (at C-2Xyl) 1 102.0 H-1 5.36 d (7.5) 2 72.2 H-2 3.97 dd (9.2, 7.5) 3 72.2 H-3 3.77 t (9.2) 4 74.0 H-4 3.41 m 4.00 dd (11.7, 5.2) 5 70.1 H -5 2 1.26 t (11.7) 3-O- β -D-Glc (at C-16) 1 106.2 H-1 4.29,d (7.5) 2 75.7 H-2 3.16, dd (7.5, 9.0) 3 78.5 H-3 3.31, t (9.0) 4 71.6 H-4 3.31, t (9.0) 5 77.8 H-5 3.26, ddd (3.5, 4.5, 9.0) 6 62.8 H-6 3.84, dd (3.5, 12.0), 3.69, dd (4.5, 12.0)

121

Spectrum 3.11. 1H NMR Spectrum of AMJ5

122

Spectrum 3.12. HSQC Spectrum of AMJ5

123

Spectrum 3.13. HSQC Spectrum of AMJ5

124

Spectrum 3.14. HMBC Spectrum of AMJ5

125

Spectrum 3.15. HMBC Spectrum of AMJ5

126

3.2.2 Structural Identification of AMJ8

The 1H NMR spectrum of AMJ8 showed for the aglycon moiety signals due to a cyclopropane methylene at  0.58 and 0.42 (each 1H, d, J = 4.2 Hz), seven tertiary methyl groups at  1.46, 1.33, 1.30, 1.20, 1.20, 1.05 and 1.03, and five methine proton signals at  5.46 (ddd, J = 8.0, 8.0, 5.2 Hz), 4.21 (ddd, J = 9.3, 6.3, 3.4 Hz), 3.55 (d, J = 6.3 Hz), 3.45 (ddd, J = 9.5, 9.5, 4.5 Hz) and 3.22 (dd, J = 11.3, 4.0 Hz) which were indicative of secondary alcoholic functions (Table 3.6). On the basis of DQF-COSY, HSQC and HMBC spectra and by comparison of these data with those of cycloastragenol (Kitagawa et al., 1983), it was observed that the aglycon of compound AMJ8 differs from cycloastragenol only by the presence of an additional secondary alcoholic function at C-23 (H 4.21, C 73.4) and a COCH2OH group at C-16 (H 5.46, C 78.0). The HMBC correlations between the proton signal at  5.46 (H-16) and the carbon resonance at  173.9 (-

COCH2OH) confirmed the location of the –COCH2OH group at C-16. Therefore, the new structure of AMJ8 was assigned as 3-O-[α-L-arabinopyranosyl-(1→2)-β- D-xylopyranosyl]-16-O-hydroxyacetoxy-3β,6α,16β,23α,25-pentahydroxy- 20(R),24(S)-epoxycycloartane.

Figure 3.6 Structure of AMJ8

127

1 13 Table 3.6 H- and C-NMR Assignments of AMJ8 (150/600 MHz,  ppm, in CD3OD)

Position C H (J in Hz) 1 33.5 1.77, 1.24, m 2 30.6 1.93, 1.68, m 3 89.4 3.22, dd (11.3, 4.0) 4 43.0 - 5 54.5 1.37, d (9.7) 6 69.4 3.45, ddd (9.7, 9.7, 4.5) 7 38.9 1.44, 1.36, m 8 48.5 1.82, dd (11.3, 4.0) 9 21.2 - 10 29.9 - 11 27.1 2.03, 1.25, m 12 33.5 1.77, 1.54 m 13 46.4 - 14 48.0 - 15 46.8 2.25, dd (13.7, 8), 1.36 dd (12.9, 5.6) 16 78.0 5.46, ddd (8.0, 8.0, 5.6) 17 58.8 2.54, d (8.0) 18 21.4 1.33, s 19 31.8 0.58, brs, 0.39, d (4.8) 20 85.8 - 21 28.8 1.46, s 22 47.7 2.87, 1.60, m 23 73.4 4.21,ddd (8.8, 6.0, 3.5) 24 88.8 3.55 dd (8.2, 6.0) 25 71.6 - 26 26.9 1.20, s 27 26.9 1.20, s 28 28.1 1.30, s 29 16.7 1.03, s 30 20.5 1.05, s

COCH2OH 173.9 -

COCH2OH 61.5 4.13, 4.07, brs β-D-Xyl (at C-3)-1 106.0 4.47, d (7.5) 2 83.2 3.44, dd (9.2, 7.5) 3 76.7 3.54, t (9.2) 4 71.0 3.53, m 5 65.8 3.87, dd (11.7, 5.2), 3.20, t (11.7)

α-L-Ara (at C-2Xyl)-1 106.4 4.49, d (3.7) 2 73.4 3.67, dd (8.5, 3.7) 3 73.9 3.57, dd (8.5, 3.0) 4 69.4 3.81, m 5 66.7 3.89, dd (11.9, 2.0) 3.54, dd (11.9, 3.0)

128

Spectrum 3.16. 1H NMR Spectrum of AMJ8

129

Spectrum 3.17. HSQC Spectrum of AMJ8

130

Spectrum 3.18 . HSQC Spectrum of AMJ8

131

Spectrum 3.19 . HMBC Spectrum of AMJ8

132

Spectrum 3.20. HMBC Spectrum of AMJ8

133

3.2.3 Structural Identification of AMJ18

As concerning the aglycon moiety, the 1H NMR spectrum showed signals due to a cyclopropane methylene at  0.61 and 0.42 (each 1H, d, J = 4.2 Hz), seven tertiary methyl groups at  1.48, 1.36, 1.32, 1.30, 1.29, 1.03 and 0.98, and four methine proton signals at  4.32 (ddd, J = 8.0, 8.0, 5.2 Hz), 4.17 (d, J = 6.3 Hz), 3.44 (ddd, J = 9.5, 9.5, 4.5 Hz) and 3.23 (dd, J = 11.3, 4.0 Hz) (Table 3). Comparison of 1H and 13C NMR data of the aglycon region of AMJ18 with those of compound AMJ8 revealed that the aglycon of compound AMJ18 differs from that of AMJ8 by the absence of the signals of C-24 (H 3.55, d, J = 6.3 Hz, C = 88.8) and –COCH2OH group at C-16 and by the presence of a ketalic function at  108.9 (Tables 3.7). HMBC correlations between the proton signals at  1.30 (Me-26), 1.36 (Me-27), 4.17 (H-23) and  1.85 (H-22) and the carbon resonance at  108.6 suggested for the aglycon of compound AMJ18 the structure 3β,6α,23α,25-tetrahydroxy-16β,24;20,24-diepoxycycloartane. An examination of molecular models indicates that the heterocycles can be fused as the 20R, 24R- and 20S, 24S-configurations. Therefore, establishing the stereochemistry of one of these asymmetric centers defines the configuration of the other chiral atom (Agzamova and Isaev, 1995). Additionally, for compound AMJ18 it was also evident signals for a further three anomeric protons at  4.48 (d, J = 3.7 Hz), 4.47 (d, J = 7.5 Hz) and 4.40 (d, J = 7.5 Hz). The chemical shifts of all the individual protons of the three sugar units were ascertained from a combination of 1D- TOCSY and DQF-COSY spectral analysis, and the 13C chemical shifts of their relative attached carbons were assigned unambiguously from the HSQC spectrum (Table 3.7). These data showed the presence of one -arabinopyranosyl unit ( 4.48), one -xylopyranosyl unit ( 4.47) and one -glucopyranosyl unit ( 4.40). An unambiguous determination of the sequence and linkage sites was obtained from the HMBC spectrum, which showed key correlation peaks between the proton signal at  4.47 (H-1xyl) and the carbon resonance at  89.6 (C-3),  4.48

(H-1ara) and  83.2 (C-2xyl), and and the proton signal at  4.40 (H-1glc) and the carbon resonance at  88.0 (C-23). The D configuration of xylose and glucose units and the L configuration of arabinose unit were established after hydrolysis of AMJ18 with 1 N HCl, trimethylsilation and determination of retention time by GC. Thus, the new compound AMJ18 was identified as 3-O-[α-L- arabinopyranosyl-(1→2)-β-D-xylopyranosyl]-3β,6α,23α,25-tetrahydroxy- 20(R),24(R)-16β,24;20,24-diepoxycycloartane. Compounds of this class are very unusual in the plant kingdom: 16b,24;20,24-diepoxycycloartane- type derivatives have been isolated only from Astragalus campylosema Boiss. ssp. campylosema

134

(Calis et al., 2008). Astragalus alopecurus (Agzamova and Isaev, 1995), Souliea vaginata and Beesia calthaefolia (Sakurai et al., 1990).

Figure 3.7 Structure of AMJ18

135

1 13 Table 3.7 H- and C-NMR Assignments of AMJ18 (150/600 MHz,  ppm, in CD3OD)

Position C H (J in Hz) 1 33.5 1.55, 1.23, m 2 30.6 1.93, 1.68, m 3 89.6 3.22, dd (11.3, 4.0) 4 43.2 - 5 54.8 1.36, d (9.5) 6 69.5 3.44, ddd (9.5, 9.5, 4.5) 7 38.9 1.44, 1.36, m 8 48.3 1.75, dd (11.9, 4.2) 9 21.7 - 10 30.4 - 11 27.4 2.11, 1.20, m 12 34.6 1.69, 2.34, m 13 45.0 - 14 46.8 - 15 43.8 1.84, dd (12.7, 8.0) 1.43, dd (12.7, 5.2) 16 75.4 4.32, ddd (8.0, 8.0, 5.2) 17 61.7 2.55, dd (9.9, 8.0) 18 23.5 1.29, s 19 31.8 0.57, brs, 0.43, d (4.2) 20 86.0 - 21 29.8 1.48, s 22 42.3 3.07, 1.85, m 23 88.0 4.17, d (6.3) 24 108.9 25 75.4 - 26 24.5 1.30, s 27 27.2 1.26, s 28 28.5 1.32, s 29 16.3 1.03, s 30 20.1 0.98, s β-D-Xyl (at C-3)-1 106.3 4.47, d (7.5) 2 83.2 3.45, dd (9.2, 7.5) 3 76.8 3.54, t (9.2) 4 70.9 3.53, m 5 66.1 3.87, dd (11.7, 5.2), 3.21, t (11.7)

α-L-Ara (at C-2Xyl) 106.6 4.48, d (3.7)

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2 73.5 3.67, dd (8.5, 3.7) 3 74.0 3.58, dd (8.5, 3.0) 4 69.5 3.81, m 5 67.2 3.90, dd (11.9, 2.0) 3.54, dd (11.9, 3.0

137

Spectrum 3.21. 1H NMR Spectrum of AMJ18

138

Spectrum 3.22. HSQC Spectrum of AMJ18

139

Spectrum 3.23. HSQC Spectrum of AMJ18

140

Spectrum 3.24. HMBC Spectrum of AMJ18

141

Spectrum 3.25. HMBC Spectrum of AMJ8

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Additionally, five known cycloartane-type glycosides as Askendoside G (AMJ1) (Isaev and Mamedova, 2004), 3-O-[α-L-arabinopyranosyl-(1→2)-β-D- xylopyranosyl]-3β,6α,16β,20S,24R,25-hexahydroxycycloartane (AMJ7) (Yalçın et al., 2011), 3-O-[α-L-arabinopyranosyl-(1→2)-β-D-xylopyranosyl]- 3β,6α,16β,23α,25-pentahydroxy-20R,24S-epoxycycloartane (AMJ11) (Çalış et al., 2008), Elongatoside (AMJ12) (Çalış et al., 2008), Askendoside C (AMJ14) (Isaev et al., 1893b), and two oleanane-type glycosides as Azukisaponin V (AMJ4) (Kitagawa et al., 1983a) and Astragaloside VIII (AMJ 32) (Kitagawa et al., 1983) were isolated.

Figure 3.8 Structure of AMJ7

Figure 3.9.Structure of AMJ1 (Askendoside G)

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Figure 3.10 Structure of AMJ11

Figure 3.11 Structure of AMJ4

Figure 3.12.Structure of AMJ32

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Figure 3.13.Structure of AMJ 12 (Astragaloside IV)

Known as cosmopolitan and important, Astragalus genus (Leguminosae) is an annual and perennial herbs, or small shrubs. The genus Astragalus appears highly uniform, including polysaccharides, saponins, and flavonoids. Current progress indicates that these components are three major chemical classes contributing to the multiple bioactivities of Astragalus species. Radix Astragali, the dried roots of Astragalus membranaceus or A. membranaceus var. mongholicus, is a very old and well-known tonic herb in traditional Chinese medicine, pharmacologically indicated by several studies to have multiple biological activities, such as antioxidative, immunomodulation, antiviral properties and its extracts show broad potentials for the treatments of cardiovascular and cerebrovascular diseases, neurodegenerative diseases, liver diseases, infectious diseases, and carcinomas, etc. As example, Astragali complanati semens, the dry seeds of A. complanatus R.Br. ex Bunge, generally used for anti-aging therapy and improving the function of sexual performance officially listed in the Chinese Pharmacopoeia (2010).

Moreover, in Turkish folk medicine, aqueous Astragalus species are traditionally used to treat leukemia and for wound healing (Bedir et al., 2000). Up to now, great progress has been made in exploring the metabolites and bioactivities of active compounds from Astragalus.

Various phytochemical and biological studies on Astragalus have resulted in isolation and characterization of secondary metabolites including triterpenoid saponins and sapogenins, flavonoids, phenylpropanoids, steroids, alkaloids, and some other compounds (Li-Peng et al., 2013).

145

However, triterpenoid saponins are the most widely studied secondary metabolites in Astragalus genus with cycloartane and oleanane saponins as dominated constituents (Verotta and Ei-Sebakhy 2001). Cycloartane triterpenoids were first discovered in Astragalus plants. Various researches reported to dominate the known triterpenoids of the Astragalus genus, which derived from cycloartenol by oxidation at C(6), C(16), C(20), C(23), or C(24), followed by possible ring closures to form a 20,24-epoxide (cycloastragenol or cyclogalegenin), a 20,25-epoxide, a 16,24;20,24-diepoxide (cycloalpigenin), or a 16,23;16,24-diepoxide (cycloorbigenin B)(Li-Peng et al., 2013).

The rapid development of modern analytical techniques such as bioinformatics, are one of the major advantages favoring the interpretation for understanding of complex compounds can provide some clues for further explorations of chemical basis and bioactivities of Astragalus in the treatments of diseases.

As part of continuing studies on Turkish Astragalus species, we carried out a full phytochemical study on two Astragalus species, A. halicacabus and A. melanocarpus.

Our studies resulted in the isolation of one new cycloartane-type triterpene glycosides, one new maltol-type glycoside from Astragalus halicacabus along with seven known cycloartane-type glycosides and one known maltol-type glycoside. This is the first report on the isolation of a maltol glucoside from Astragalus genus, and even from Leguminosae family. Thus, 3-O-[β-D- apiofuranosyl-(1→2)-β-D-glucopyranosyl]-maltol (AHa2) together with the known compound 3-O-β-D-glucopyranosylmaltol represent the first members of this class and can be of assistance as chemotaxonomic markers to differentiate A. halicacabus in Astragalus genus.

However, three new cycloartanes-type glycosides were isolated from Astragalus melanocarpus along with six known compounds; 4 cycloartane-type glycosides and two oleanane type triterpenoids The structures of compounds were established by the extensive use of 1D and 2D-NMR experiments along with ESIMS and HRMS analyses. Among the three isolated molecules two of them, 8 and 18 are rarely found in Astragalus genus and unsual in plant kingdom The compounds of this class have been isolated from Astragalus alopecurus

146

(Agzamova and Isaev, 1995), Souliea vaginata and Beesia calthaefolia (Sakurai et al., 1990) and Astragalus campylosema Boiss (Calıs et al., 2008.)

Furthermore, A. halicacabus and A. melanocarpus contain cycloartane and oleanane type triterpenoids. Until now, 30 out of 445 Turkish Astragalus species, from 13 different sections, have been investigated for their secondary metabolite contents and structures of 92 new triterpene saponins and 6 new phenolic glycosides were identified besides known compounds. Some sections; Sect. Pterophorus (A. brachypterus, A. baibutensis, A. trojanus, A. ptilodes var. cariensis, A. wiedemannianus, A. tmoleus var. tmoleus), Sect. Rhacophorus (A. microcephalus, A. zahlbruckneri, A. cephalotes var. brevicalyx, A. amblolepis, A. pycnocephalus var. pycnocephalus, A. erinaceus, A. prusianus, A. schottianus), Sect. Macrophyllium (A. oleifolius), Sect. Hololeuce (A. campylosema ssp. campylosema), Sect. Stereocalyx (A. stereocalyx), Sect. Christiana (A. gilvus, A. melanophrurius) and Sect. Proselius (A. elongatus), provided only cycloartane saponins, whereas both cycloartane and oleanane saponins were encountered in a few sections; Sect. Halicacabus (A. halicacabus) and sect. Hypoglottis (A. melanocarpus) and Sect. Eustales (A. flavescens).

Based on earlier phytochemical studies, one could readily claim that the cycloartane group is a chemical marker for the genus Astragalus. Cycloastragenol, possessing 20,24-epoxy side chain, is the primary aglycone in the Astragalus genera. However, cyclocephalogenol, possessing 20,25-epoxy side chain, is more unusual in the plant kingdom, so far reported only from Astragalus spp. from 3 sections, viz, Rhacophorus, Adiaspastus and Acanthophace. From the Rhacoporus section, which is the largest section of Astragalus with about 100 members, three out of eight studied Turkish species gave cyclocephalogenol (A. microcephalus, Bedir et al., 1998a; A. zahlbruckneri, Calis et al., 2001; A. schottianus, Karabey et al., 2012; Astragalus aureus, Gulcemal et al., 2011), whereas A. aureus, single examined species of Adiaspastus section, provided the same aglycone (Gulcemal et al., 2011). Only two members of Acanthophace section, viz., A. hareftae and A. icmadophilus, have been studied so far, and it is noteworthy that cyclocephalogenol is encountered in both members. This suggests that the presence of cyclocephalogenol in Acanthophace section could be of taxonomic importance (Polat et al.; 2010; Horo et al., 2012 ).

However, phytochemical study on A. lycius from Sect. Malacothrix resulted in the isolation of 8 secondary metabolites. Noteworthy, no cycloartane-type

147

triterpene glycoside, the main constituents of Astragalus spp., were found. This peculiar feature characterizes a very limited group of Astragalus spp. [A. hamosus, A. complanatus, A. sinicus, A. corniculatus and A. tauricolus (Ionkova, 1991; Cui et al., 1992a, b; Krasteva et al., 2006, 2007; Gulcemal et al., 2013)]. Further studies are required to confirm the above assumptions, and our continuing studies will be of use in clarifying chemotaxonomical classification of the genus Astragalus.

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CURRICULUM VITAE

Name-surname : Basile-Jimmy DJIMTOMBAYE

Date/Place of Birth : 15.12.1976/ Kabo, Central African Rep.

Nationality : Centrafricaine

Marital state : Single

Language : French, English, Turkish

Others language : Sango (mother language)

Address : Ege University, Faculty of Science, Department of Chemistry, 35100, Bornova-Izmir/TURKEY

Phone Number : +90 232 388 4000 /2371 (work)

: +90 531 834 0845 (cell)

Fax : +90 232 388 82 64

E-mail : [email protected]

EDUCATION AND PROFESSIONAL KNOWLEDGE

B.Sc.: 2000-2004, 2005: University of Bangui Faculty of Sciences (C.A.R).

M.Sc.: 2004-2005, 2005: University of Bangui (C.A.R).

Ph.D.: 2008-2014, Ege University, Graduate School of Natural and Applied Sciences, Izmir

166

CURRICULUM VITAE (continued)

PUBLICATIONS

B. J. Djimtombaye, Ö. A. Çalişkan, D. Gülcemal, I. A. Khan, H. Anil, and E. Bedir. Unusual Secondary Metabolites from Astragalus halicacabus Lam. Chemistry & Biodiversity - Vol. 10 (2013)

SCIENTIFIC MEETINGS

1- 2011, Djimtombaye, BJ., Karayıldırım T., Masullo, M., Piacente, S., Alankuş-Çalışkan, Ö., Gulcemal, D., Şenol, S.G., Constituents of Verbascum reeseanum - 59th International Congress and Annual Meeting of the Society-for- Medicinal-Plant-and-Natural-Product-Research, September 04-09, Antalya, Turkey.

2- 2012, Djimtombaye, J., Alankuş-Çalışkan, Ö., Gülcemal, D., Anıl, H., Khan I.A., Bedir, E., Phytochemical Investigation on Astragalus halicacabus- 2012 Phytochemical Society of Europe Congress on Bio-communication- Semiochemicals involving plants-September 10-12, Cadiz, Spain.

RESEARCH PROJECTS

Prof. Dr. Özgen ALANKUŞ ÇALIŞKAN, “Bazı Astragalus Türlerinden Doğal Bileşiklerin İzolasyonu, Kimyasal Yapılararının Belirlenmesi ve Stotoksik Aktivitelerinin İncelenmesi.”, TÜBİTAK 109T425, 2009-2013.

Prof. Dr. Hüseyin ANIL, “Astragalus halicacabus ve Astragalus melanocarpus Türlerinden Saponinlerin İzolasyonu ve Karakterizasyonu” Ege Üniversitesi Bilimsel Araştırma Projesi 2011-Fen-049, 2011-2013.

FELLOWSHIPS

2007-2014; Ph.D. Fellowship Programme for International Students, Turkish government.