©2011 John Peter Munafo, Jr ALL RIGHTS RESERVED

©2011

John Peter Munafo, Jr

ALL RIGHTS RESERVED

NATURAL PRODUCTS CHEMISTRY OF LILIUM LONGIFLORUM:

STRUCTURAL ELUCIDATION, QUANTIFICATION, BIOLOGICAL ACTIVITY

AND FUNGAL METABOLISM OF STERODAL GLYCOSIDES

by

JOHN PETER MUNAFO JR

A Dissertation submitted to the

Graduate School-New Brunswick

Rutgers, The State University of New Jersey

in partial fulfillment of the requirements

for the degree of

Doctor of Philosophy

Graduate Program in Plant Biology

written under the direction of

Professor Thomas J. Gianfagna

and approved by

________________________

________________________

________________________

________________________

New Brunswick, New Jersey

May, 2011

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ABSTRACT OF THE DISSERTATION

Natural Products Chemistry of Lilium longiflorum: Structural Elucidation, Quantification,

Biological Activity and Fungal Metabolism of Steroidal Glycosides

By JOHN PETER MUNAFO JR

Dissertation Director:

Professor Thomas J. Gianfagna

The Easter lily (Lilium longiflorum Thunb., Liliaceae) has beautiful white flowers

and a delicate aroma and is appreciated worldwide as an attractive ornamental plant. In

addition to its economic importance and popularity in horticulture, lily bulbs are regularly

consumed in Asia, as both food and medicine. The Easter lily is a rich source of steroidal

glycosides, a group of compounds that may be responsible for some of the traditional

medicinal uses of lilies and may play a role in the pant-pathogen interaction. This

research project was designed to: 1) Isolate and characterize new steroidal glycosides

from the bulbs of L. longiflorum, 2) quantify their contents in all of the organs of L.

longiflorum, and 3) perform studies on the antifungal activity and fungal metabolism of

the compounds.

A phytochemical investigation conducted on the bulbs resulted in the discovery of

several novel steroidal glycosides. A novel acetylated steroidal glycoalkaloid and two

novel steroidal furostanol saponins, along with three other steroidal glycosides were

isolated from the bulbs of L. longiflorum for the first time. A LC-MS/MS method

performed in multiple reaction monitoring (MRM) mode was developed for the

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simultaneous quantitative analysis of the five steroidal glycosides in the different organs

of L. longiflorum. The highest concentrations of total steroidal glycosides were detected

in flower buds, lower stems, and leaves. The steroidal glycoalkaloids were detected in

higher concentrations as compared to the furostanol saponins in all of the plant organs

except for the fibrous and fleshy roots. The proportions of steroidal glycoalkaloids to

furostanol saponins were higher in the plant organs exposed to light and decreased in

proportion from the aboveground organs to the underground organs. The highest

concentrations of the steroidal glycoalkaloids were detected in flower buds, leaves, and

bulbs.

Purified steroidal glycosides were evaluated for fungal growth inhibition activity

against the plant pathogenic fungus, Botrytis cinerea. All of the compounds showed weak

fungal growth inhibition activity; however, the natural acetylation of C-6′′′ of the

terminal glucose in the acetylated steroidal glycoalkaloid, increased the antifungal

activity by inhibiting the rate of metabolism of the compound by the fungus. A model

system was developed to generate fungal metabolites of the steroidal glycoalkaloids and

this system led to the discovery of several new fungal metabolites. The fungal

metabolites characterized from the model system were subsequently identified by LC-MS

and found to naturally occur in Easter lily tissues infected with the fungus.

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ACKNOWLEDGEMENTS

I would acknowledge my major advisor, Professor Thomas Gianfagna, for his

guidance and helpful insight throughout this research. I would also like to thank my

faculty committee members, Professor Richard Merritt and Professor Chee-Kok Chin and

my outside member Dr. John Didzbalis. I would like to acknowledge my collaborators,

colleagues and friends; Professor Leslie Jimenez, Professor Edward Durner, Dr. Ahalya

Ramanthan, Dr. Marshall Bergen, Dr. Christopher Johnson, Dr. Mark Kelm, Dr.

Catherine Kwik-Uribe, Thomas Collins, Jeanne Peters, Nimmi Rajmohan, Dr. Mahdu

Aneja, Bob Carhart, Jadwiga Leonczak, Professor Ilya Raskin, Dr. Slavko Komarnytsky,

and Debroa Esposito. I would like to give a special thanks to my family and especially

my wife, Kristin, for their constant encouragement and support. Most of all, I would like

to thank God, for creating such a wonderful Universe for us to explore and ponder.

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TABLE OF CONTENTS

ABSTRACT OF THE DISSERTATION ...................................................................... II

ACKNOWLEDGEMENTS ........................................................................................... IV

TABLE OF CONTENTS ................................................................................................ V

LIST OF TABLES ........................................................................................................... X

LIST OF FIGURES ........................................................................................................ XI

CHAPTER 1: GENERAL INTRODUCTION ............................................................... 1

1.1. INTRODUCTION .......................................................................................................... 1

1.2. BOTANICAL CLASSIFICATION .................................................................................... 2

1.3. BOTANICAL DESCRIPTION ......................................................................................... 2

1.4. NATURAL PRODUCTS FROM LILIACEAE ..................................................................... 5

1.5. SAPONINS IN GENERAL .............................................................................................. 6

1.5.1. Steroidal Saponins ............................................................................................. 9

1.5.1.1. Commercially Important Steroidal Saponins ............................................ 12

1.5.1.2. Dietary Sources of Steroidal Saponins ..................................................... 16

1.5.1.3. Steroidal saponins isolated from Lilium ................................................... 18

1.6 STEROIDAL ALKALOIDS ........................................................................................... 27

1.6.1 Steroidal Alkaloids in Liliaceae ....................................................................... 27

1.6.1.1. Classification of Isosteroidal Alkaloids of Liliaceae ................................ 28

1.6.1.2. Classification of Steroidal Alkaloids of Liliaceae .................................... 30

1.6.1.3. Steroidal Alkaloids in Lilium .................................................................... 31

1.6.2. Steroidal Glycoalkaloids ................................................................................. 32

1.6.2.1. Dietary Sources of Steroidal Glycoalkaloids ............................................ 35

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1.6.2.2. Steroidal Glycoalkaloids in Lilium ........................................................... 37

1.7. PLANT ORGAN DISTRIBUTION OF STEROIDAL GLYCOSIDES ....................................... 40

1.8. STEROIDAL GLYCOSIDES IN PLANT DEFENSE ............................................................ 42

1.9 DETOXIFICATION OF STEROIDAL GLYCOSIDES .......................................................... 45

CHAPTER 2: ISOLATION AND STRUCTURAL DETERMINATION OF

STEROIDAL GLYCOSIDES FROM THE BULBS OF EASTER LILY (LILIUM

LONGIFLORUM THUNB.) ........................................................................................... 48

2.1. ABSTRACT ............................................................................................................... 48

2.2. INTRODUCTION ........................................................................................................ 49

2.3. MATERIALS AND METHODS ..................................................................................... 51

2.3.1. Plant Material.................................................................................................. 51

2.3.2. Chemicals. ....................................................................................................... 52

2.3.3. Isolation and Purification of Steroidal Glycosides 1 – 5 from L. longiflorum.53

2.3.3.1. Sequential Solvent Extraction of Lyophilized L. longiflorum Bulbs ........ 53

2.3.3.2. Gel Permeation Chromatography (GPC) .................................................. 54

2.3.3.3. Semipreparative Reverse-Phase High-Performance Liquid

Chromatography (RP-HPLC) ................................................................................ 56

2.3.4. Structural Elucidation ..................................................................................... 59

2.3.4.1. Acid Hydrolysis of Compounds 1 – 5. ..................................................... 61

2.3.4.2. Aglycone Analysis .................................................................................... 61

2.3.4.3. Sugar Composition Analysis .................................................................... 62

2.3.4.4. Determination of Sugar Absolute Configurations .................................... 62

2.3.4.5. Thin Layer Chromatography (TLC) ......................................................... 63

vii

2.4. RESULTS AND DISCUSSION ...................................................................................... 64

2.4.1 Structure Elucidation of Compounds 1 – 5. ..................................................... 64

2.3.4.1. Structure Elucidation of Compound 1 ...................................................... 67




2.3.4.4. Structure Elucidation of Compound 5 .................................................... 102

2.5. CONCLUSION ........................................................................................................ 109

CHAPTER 3: QUANTITATIVE ANALYSIS OF STEROIDAL GLYCOSIDES IN

DIFFERENT ORGANS OF EASTER LILY (LILIUM LONGIFLORUM THUNB.)

BY LC-MS/MS .............................................................................................................. 114

3.1. ABSTRACT ............................................................................................................. 114

3.3. MATERIALS AND METHODS ................................................................................... 120

3.3.1. Plant material. ............................................................................................... 120

3.3.2. Chemicals ...................................................................................................... 123

3.3.3. Histology and Microscopy. ............................................................................ 123

3.3.4. Purification and Confirmation of Analytical Standards................................ 124

3.3.4.1. Nuclear Magnetic Resonance Spectroscopy (NMR). ............................. 126

3.3.5. Quantitative Analysis of Steroidal Glycosides in Lilium longiflorum. .......... 126

3.3.5.1. Sample Preparation ................................................................................. 126

3.3.5.2. Analytical Standard Preparation ............................................................. 127

3.3.5.3. Liquid Chromatography-Mass Spectrometry (LC-MS/MS). .................. 127

3.3.5.4. Recovery ................................................................................................. 132

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3.3.5.5. Thin Layer Chromatography (TLC). ...................................................... 132

3.3.5.6. Statistical Analysis. ................................................................................. 133

3.4. RESULTS AND DISCUSSION .................................................................................... 133

3.4.1. Quantification of steroidal glycosides in the different organs of L. longiflorum.

................................................................................................................................. 133

3.4.2. Histological visualization of furostanol localization in bulb scale sections of L.

longiflorum. ............................................................................................................. 152

3.4.3. Quantification of steroidal glycosides within bulb scales of L. longiflorum. 156

3.5. CONCLUSION ......................................................................................................... 160

CHAPTER 4: ANTIFUNGAL ACTIVITY AND FUNGAL METABOLISM OF

STEROIDAL GLYCOSIDES OF EASTER LILY (LILIUM LONGIFLORUM) BY

THE PLANT PATHOGENIC FUNGUS, BOTRYTIS CINEREA ............................ 163

4.1. ABSTRACT ............................................................................................................. 163

4.2. INTRODUCTION ...................................................................................................... 164

4.3. MATERIALS AND METHODS ................................................................................... 168

4.3.1. Plant material. ............................................................................................... 168

4.3.2. Fungal cultures. ............................................................................................. 169

4.3.3. Chemicals. ..................................................................................................... 169

4.3.4. Isolation and Purification of Steroidal Glycosides 1 – 5 from Lilium

longiflorum. ............................................................................................................. 170

4.3.4.1. Nuclear Magnetic Resonance Spectroscopy (NMR). ............................. 171

4.3.4.2. Liquid Chromatography-Mass Spectrometry (LC-MS). ........................ 171

4.3.4.3. Partial acid hydrolysis of compound 1. .................................................. 172

ix

4.3.5. B. cinerea growth inhibition assay. ............................................................... 173

4.3.6. In vitro fungal metabolism of compounds 1 and 2. ....................................... 174

4.3.7. Scale-up fungal metabolism of compound 1. ................................................. 175

4.3.7.1. Semi-preparative RP-HPLC isolation of the fungal metabolites of

compound 1. ......................................................................................................... 180

4.3.8. Isolation and Purification of Compound 6 from Lilium longiflorum bulbs. . 183

4.3.8.1. Sequential Solvent Extraction of Lyophilized L. longiflorum Bulbs. ...... 183

4.3.8.2. Gel Permeation Chromatography (GPC). .............................................. 184

4.3.9. Infection of L. Longiflorum tissue and sample preparation for LC-MS analysis.

................................................................................................................................. 188

4.3.10. Statistical Analysis. ...................................................................................... 188

4.4. RESULTS AND DISCUSSION .................................................................................... 189

4.4.1. Fungal growth inhibition assay. .................................................................... 189

4.4.2. Metabolism of compound 1 and 2 by B. cinerea. .......................................... 193

4.4.3. In planta identification of compounds 6 – 10 by LC-MS. .............................. 208

4.4.4. Isolation and identification of compound 6 from L. Longiflorum bulbs. ...... 214

4.5. CONCLUSION ......................................................................................................... 215

SUMMARY AND CONCLUDING REMARKS ....................................................... 216

LITERATURE CITED ................................................................................................ 220

CURRICULUM VITA………………………………………………………………..238

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LIST OF TABLES

Table 1.1. Steroidal saponins found in Lilium…………………………………………..19

Table 2.1. 13

C NMR spectral data of compounds 1 – 5 in pryridine-d5…………….…..113

Table 3.1. ANOVA for concentrations of compounds 1 – 5 in the different

organs of L. longiflorum………………………………………………………………..140

Table 3.2. Concentrations of compounds 1 – 5 in the different

organs of L. longiflorum………………………………………………………………..141

Table 3.3. Concentrations of compounds 1 – 5 in whole bulb scale,

bulb epidermis, and bulb mesophyll……………………………………………….…...157

Table 4.1. ANOVA for treatment, rate, and the interaction between

treatment and rate…………………………………………………………………….…189

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LIST OF FIGURES

Figure 1.1. Image of L. longiflorum in full bloom…………………………………….4

Figure 1.2. Structures of 2,3-oxidosqualene, a triterpenoid sapogenin

(quillaic acid), and a steroidal sapogenin (diosgenin)…………………………….7

Figure 1.3. Examples of monodesmosidic and didesmosidic saponins……….............8

Figure 1.4. Structures of a basic steroidal backbone, a spirostane

backbone, and a furostane backbone………………………………………...…..10

Figure 1.5. Examples of different carbohydrate linkages……………………………11

Figure 1.6. Structures of Dioscin and Protodioscin………………………………….14

Figure 1.7. Molecular structures of steroidal saponins from Lilium……....................20

Figure 1.8. Isosteroidal alkaloids of Liliaceae: Representative

examples of cevanine type, veratramine type, and jervine type

isosteroidal alkaloids……………………………………………………………..29

Figure 1.9. Steroidal alkaloids of Liliaceae: Representative examples

of solanidine type and verazine type steroidal alkaloids………………………...30

Figure 1.10. Structures of the steroidal alkaloids etioline and

teiemine isolated from the bulbs of L. candidum………………………………...32

Figure 1.11. Structures of the most common aglycones of the

steroidal glycoalkaloids………………………………………………………….34

Figure 1.12. Structures of steroidal glycoalkaloids isolated from

C. cordatum, L. philippinense, L. brownii, and L. mackliniae…………..……….39

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Figure 2.1. Isolation scheme for compounds 1 – 5 purified

from the bulbs of L. longiflorum…………………………………………………54

Figure 2.2. RP-HPLC chromatogram of 1 – 5 isolated from

L. longiflorum……………………………………………………………………57

Figure 2.3. Total ion chromatogram of L. longiflorum extract

and of compounds 1 – 5 isolated by RP-HPLC……………………………….…58

Figure 2.4. Structures of compounds 1 – 5 isolated from

L. longiflorum bulbs……………………………………………………………...60

Figure 2.5. High resolution mass spectrum of compound 1…………………………68

Figure 2.6. GC-MS chromatogram of the TMSi derivatives of

(22R, 25R)-spirosol-5-en-3 -ol.…………………………………………………69

Figure 2.7. GCMS mass spectra of TMSi derivatives of

(22R, 25R)-spirosol-5-en-3 -l……………………………………………...……70

Figure 2.8. GCMS mass spectra of TMSi derivatives of the

aglycone of compound 1…………………………………………………………71

Figure 2.9. Total ion chromatogram of (22R, 25R)-spirosol-5-en-3 -ol

and the aglycone of compound 1 generated by LC-MS………………………....72

Figure 2.10. HMBC long-range correlations for the interglycosidic

linkages for the carbohydrate moiety of compound 1……………………….…..73

Figure 2.11. ESI+–MS mass spectrum of compound 1……………………………...74

Figure 2.12. 1H NMR spectrum and

13C NMR spectrum of compound 1…………..75

Figure 2.13. High resolution mass spectrum of compound 2……………………......78

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linkages for the carbohydrate moiety of compound 2…………………………...79

Figure 2.15. ESI+–MS mass spectrum of compound 2………………………………80


13C NMR spectrum of compound 2…………...82

Figure 2.17. High resolution mass spectrum of compound 3………………………..85

Figure 2.18. LRMS- mass spectrum of compound 3…………………………...........86

Figure 2.19. GCMS spectra of TMSi derivatives of the aglycone of

compound 3 and (25R)-spirost-5-en-3 -ol………………………………………88


linkages for the carbohydrate moiety of compound 3……………………….….89

Figure 2.21. ESI+–MS mass spectrum of compound 3…………………………...….90


13C NMR spectrum of compound 3………...…91

Figure 2.23. High resolution mass spectrum of compound 4………………………..94

Figure 2.24. LRMS- mass spectrum of compound 4………………………..……….95

Figure 2.25. Partial HMBC spectrum of compound 4……………………………….96


linkages for the carbohydrate moiety of compound 4……………………..……98

Figure 2.27. ESI+–MS mass spectrum of compound 4………………………….…..99


13C NMR spectrum of compound 4………….100

Figure 2.29. High resolution mass spectrum of compound 5………………………103

Figure 2.30. LRMS- mass spectrum of compound 5……………………………….104


linkages for the carbohydrate moiety of compound 5…………………………105

xiv

Figure 2.32. ESI+–MS mass spectrum of compound 5………………………..……106


13C NMR spectrum of compound 5………….107

Figure 2.34. ESI+–MS mass spectra of compounds 1 – 5………………………..…112

Figure 3.1. Plant organs of L. longiflorum analyzed in this study…………….……122

Figure 3.2. Structures of steroidal glycoalkaloids 1 – 2 and furostanol

saponins 3 – 5 quantified in the various L. longiflorum organs…………….…...126

Figure 3.3. MS2 product ion spectra of steroidal glycoalkaloids 1 – 2………..……129

Figure 3.4. MS2 product ion spectra of furostanol saponins 3 – 5………………….130

Figure 3.5. MS/MS chromatograms for the quantitative analysis

of compounds 1 – 5 in a L. longiflorum bulb scale…………………………..…131

Figure 3.6. Calibration equation for compound 1………………………………..…134

Figure 3.7. Calibration equation for compound 2……………………………….….135



Figure 3.10. Calibration equation for compound 5…………………………………138

Figure 3.11. Proportions of steroidal glycoalkaloids 1 – 2 to furostanol

saponins 3 – 5 in the different organs of L. longiflorum…………………….….142

Figure 3.12. Concentrations of steroidal glycoalkaloid 1 in the

different organs of L. longiflorum………………………………………………143

Figure 3.13. Concentrations of steroidal glycoalkaloid 2 in the

different organs of L. longiflorum………………………………………………144

Figure 3.14. Concentrations of compound 3 in the different

organs of L. longiflorum………………………………………………………..147

xv

Figure 3.15. Differences in saccharide composition and

interglycosidic linkages of compounds 3 – 5………………………………..…148

Figure 3.16. Concentrations of compound 4 in the different organs of L.

longiflorum……………………………………………………………………..150

Figure 3.17. Concentrations of compound 5 in the different

organs of L. longiflorum………………………………………………………..151

Figure 3.18. Histochemical staining of a bulb scale section…………………….…153

Figure 3.19. Histochemical analysis of bulb basal plate and bulb scale sections….154

Figure 3.20. Histochemical analysis of bulb basal plate and bulb scale sections….155

Figure 3.21. Proportions of compounds 1 – 5 in whole bulb scale,

bulb epidermis, and bulb mesophyll……………………………………………158

Figure 3.22. Proportions of compounds 1 – 5 in different organs

of L. longiflorum……………………………………………………………..…159

Figure 4.1. Total ion chromatogram of the partial acid-catalyzed

hydrolysis products of compound 1………………………………………….…173

Figure 4.2. Extracted ion chromatograms of m/z 885 taken every 24 hours

over the course of 96 hours………………………………………………….…176


over the course of 96 hours…………………………………………….…….….…177


over the course of 96 hours……………………………………………….……178

Figure 4.5. Extracted ion chromatograms of m/z 414.6 taken every 24 hours

over the course of 96 hours……………………………………………….……179

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Figure 4.6. RP-HPLC chromatogram of compounds 6, 7, and 10…………….……181

Figure 4.7. Total ion chromatograms of compounds 6, 7, and 10 isolated

by RP-HPLC……………………………………………………………………182

Figure 4.8. Isolation scheme for compound 6 purified from the bulbs of L.

longiflorum...........................................................................................................185

Figure 4.9. RP-HPLC chromatogram of compound 6 isolated from

L. longiflorum bulbs……………………………………………………….........186

Figure 4.10. Structures of compounds 1 – 10............................................................187

Figure 4.11. Growth inhibition activity of compounds 1 – 5 on the radial

mycelia growth of B. cinerea…………………………………………...………190

Figure 4.12. Growth inhibition activity of compounds 1 and 2 on the

radial mycelia growth of B. cinerea……………………………………….……192

Figure 4.13. ESI+–MS mass spectra of steroidal glycoalkaloids 1 and 2……..……193

Figure 4.14. Metabolism of compound 1 by B. cinerea……………………………194

Figure 4.15. ESI+–MS mass spectra of fungal metabolite 6…………………..……195

Figure 4.16. Molecular structure and fragmentation of compound 6………………196

Figure 4.17. ESI+–MS mass spectra of fungal metabolite 7………………………..196

Figure 4.18. Molecular structure and fragmentation of compound 7………………197

Figure 4.19. ESI+–MS mass spectra of fungal metabolite 10………………………198

Figure 4.20. Molecular structure and fragmentation of compound 10……….…….199

Figure 4.21. Metabolism of compound 2 by B. cinerea……………………………200

Figure 4.22. ESI+–MS mass spectra of fungal metabolite 8…………………….….201

Figure 4.23. Proposed molecular structure and fragmentation of compound 8….…202

xvii

Figure 4.24. ESI+–MS mass spectra of fungal metabolite 9…………………….….203

Figure 4.25. TIC of the metabolites of compound 2, extracted ion

chromatogram of m/z 738.8, and the TIC of the partial acid-catalyzed

hydrolysis products of compound 1………………………………………….…204

Figure 4.26. Proposed molecular structure and fragmentation of compound 9…….205

Figure 4.27. Proposed partial metabolic pathways for compounds 1 and 2………..207

Figure 4.28. Extracted ion chromatograms (EIC) for compound 8

(m/z 780.5) of control plant tissue and plant tissue infected with B. cinerea…..209









Figure 4.33. Total ion chromatogram (TIC) of compound 6

isolated by RP-HPLC from L. longiflorum bulbs……………………………..……214

1

Chapter 1: General Introduction

1.1. Introduction

The Easter Lily, (Lilium longiflorum Thunb., family Liliaceae), has beautiful white

flowers and a delicate aroma and is appreciated worldwide as an attractive ornamental

plant. Easter lilies are most commonly seen as indoor potted plants or floral arrangements

around the Easter holidays; however, they are also often planted outdoors as bedding

plants in flower gardens. In addition to their esthetic value, lily bulbs and flower buds are

regularly consumed as a food in Asia for their distinctive bitter taste and have long

historical use in traditional Chinese medicine.

L. longiflorum is native to the Ryuckyu archipelago of Japan and the islands of

eastern Taiwan (Wilson, 1925; Hiramatsu et al., 2001). The archipelago located between

Ryukyu and Taiwan consists of approximately 200 islands located between southwestern

mainland Japan and the southeastern China. Geological studies suggest that the

archipelago was formed from a section of the Asian continent and it has been proposed

that the biota native to the islands evolved from common ancestors located on the

adjacent mainland (Kizaki and Oshiro, 1977; Kimura, 1996). Consistent with the

hypothesis, L. longiflorum is genetically similar to other Lilium species in the geographic

region, such as L. formosanum, and is readily capable of producing fertile interspecific

hybrids with other members of the genus (Hiramatsu, et al., 2001; Preston, 1933). In

addition, cytological analysis of forty-six species revealed very little variation in the

2

karyotypes within the genus; thus, interspecific hybridization is widely employed for the

development of new Lilium cultivars (Stewart, 1947).

1.2. Botanical Classification

L. longiflorum is classified under the family Liliaceae in the order Liliales.

According to the Germplasm Resources Information Network (GRIN), the Liliaceae

family contains 16 accepted genera and the genus Lilium contains 110 accepted species

(GRIN, 2011). The most common vernacular names for L. longiflorum include Easter

lily, Bermuda Easter lily, Bermuda lily, trumpet lily, and white trumpet lily.

1.3. Botanical Description

The following botanical description for L. longiflorum is based on wild specimens

endemic to Taiwan. It is important to note that commercial varieties of L. longiflorum

have undergone extensive selection and hybridization and as a result the commonly

available cultivars are variable in morphological characteristics. Nevertheless, wild

specimens are the original breeding stock of today‘s modern cultivars. The bulbs of L.

longiflorum are pale yellow to white in color, globose to obovoid in shape and

approximately 4.0 – 6.0 cm in diameter. Bulb scales are imbricate in morphology. The

stem is green in color, erect to ascending in habit, and approximately 45.0 – 90.0 cm

long. The surface of the stem is scabrid and pubescent. The plant has entire alternate

leaves and are lanceolate to falcate-lanceolate in morphology. Typical leaves are

3

approximately 15.0 – 25.0 cm long and 1.0 – 2.5 cm wide. The leaf apex is acute and the

base is amplexicual. The leaves are chartaceous and glabrous on both surfaces. Leaves

have three veins that are slightly impressed on upper surface and are elevated on the

lower surface. Easter lily flowers are terminal and are pure white in appearance. Flowers

range from solitary to several, have a fragrant aroma, and are typically 12.0 – 17.0 cm

long and 5.0 – 7.0 cm in diameter. The flower habit is horizontal or nodding. Bracts are

lanceolate and are approximately 3.0 – 5.0 cm long with an acute apex. The pedicel is

green in appearance, 2.5 – 6.0 cm in length, and glabrous. Six tepals occur in two series.

The tepals are white in appearance and are slightly tinged green toward base abaxially.

The outer tepals are oblanceolate to broadly oblanceolate and 2.5 – 3.0 cm wide. The

inner tepals are somewhat broader, ranging from 3.5 – 4.5 cm in width, and have an

obtuse apex. Filaments are 7.0 – 9.0 cm long and glabrous. Anthers are cylindric and

range from 2.0 – 3.0 cm in length. The ovaries are green in appearance, elongate,

cylindric, and 2.5 – 4.0 cm in length. The ovaries are glabrous. The style is 7.0 – 9.0 cm

long and pale yellow in appearance. The stigma is dark green and trifid. The fruit is a

loculicidal capsule that is cylindric and 3.0 – 5.0 cm in length and 2.5 – 3.5 cm in width

(Ying, 2000; Wu and Raven, 2000).

4

Figure 1.1. Image of L. longiflorum in full bloom.

5

1.4. Natural products from Liliaceae

The Liliaceae family is a rich source of natural products displaying a vast range of

structural diversity. A multitude of natural products have been isolated and characterized

from Liliaceae including, dimeric ent-kaurane diterpenes (Kitajima et al., 1982; Wu et al.,

1995), flavonoid glycosides (Fattorusso et al., 2002; Francis et al., 2004), anthocyanins

(Takeda et al., 1986; Nørbæk and Kondo, 1999), stilbenes (Zhou et al., 1999), phenolics

(Tai et al. 1981), phenolic glucosides (Shoyama et al., 1987), phenolic amides (Park,

2009) carotenoids (Tsukida et al., 1965), sterols (Itoh, et al., 1977), alkaloids

(Shimomura, H, 1987), and sulfur-containing compounds (Lanzotti, 2006). Most

notably, there has been extensive work done on the isolation and characterization of

steroidal glycosides including steroidal saponins (Harmatha et al., 1987; Matsuura et al.,

1989; Yang, et al., 2004) and steroidal glycoalkaloids (Mimaki and Sashida, 1990;

Sashida et al., 1990) from within the Liliaceae family.

Steroidal glycosides have been reported to exhibit a wide range of biological

activities including antifungal (Sautour et al., 2005; Zhou et al., 2003), platelet

aggregation inhibition (Zhang et al., 1999; Huang et al., 2006), anti-cholinergic (Gilani et

al., 1997) anti-diabetic (Nakashima et al., 1993), anti-hypertensive (Oh et al., 2003),

cholesterol lowering (Matsuura et al., 2001), anti-inflammatory (Shao et al., 2007),

antiviral (Gosse et al., 2002), and anticancer (Acharya et al., 2009; Pettit et al., 2005;

Mimaki et al., 1999; Jiang et al., 2005). Additionally, steroidal glycosides have a wide

variety of commercial uses including surfactants (Yamanaka et al., 2008), foaming agents

6

(Singh et al., 2003), vaccine adjuvants (Rajput et al., 2007), and serve as precursors for

the industrial production of pharmaceutical steroids (Hansen, 2007).

Steroidal saponins have been found in over 100 plant families and in some marine

organisms such as starfish and sea cucumber (Güçlü-Üstündağ and Mazza, 2006). They

are characterized by a steroid type skeleton glycosidically linked to carbohydrate

moieties. Steroidal glycoalkaloids are characterized by a nitrogen containing steroid type

skeleton glycosidically linked to carbohydrate moieties. In contrast to steroidal saponins,

the occurrences of steroidal glycoalkaloids are, thus far, limited to the members of the

plant families Solanaceae and Liliaceae (Li et al., 2006; Ghisalberti, 2006).

1.5. Saponins in general

Saponins are a structurally diverse class of natural products that are characterized

by a non-polar sapogenin moiety glycosidically linked to one or more polar carbohydrate

moieties. Based on the composition of sapogenin skeleton, they are generally classified

into two major categories, triterpenoid saponins and steroidal saponins (Abe et al., 1993).

Triterpenoid saponins contain a thirty carbon aglycone and steroidal saponins contain a

twenty-seven carbon aglycone. Both classes are derived from the thirty carbon precursor

2,3-oxidosqualene (Haralampidis et al., 2000). Isopentenyl pyrophosphate synthesized

via the mevalonate pathway is the five carbon donor for the biosynthesis of terpenes in

plants. Triterpenoid and steroidal sapogenins are synthesized from the thirty carbon

hydrocarbon squalene, which is subsequently oxidized to squalene 2,3-epoxide, and then

7

converted to tetra- or pentacyclic triterpenes by a family of 2,3-oxidosqualene cyclases.

Following cyclization, the sapogenin moiety is subsequently mono- or

Figure 1.2. Structures of (A) 2,3-oxidosqualene, (B) a triterpenoid sapogenin (quillaic

acid), and (C) a steroidal sapogenin (diosgenin) (Haralampidis et al., 2000;

Kuljanabhagavad, et al. 2008; Espejo, et al., 1982).

RO

O

O

COOH

RO

OH

CHO

O

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

6

727

20

11

8

9

10

29

12

13

14

1516

17

18

19

30

21

22

24

25

1

2

3

4

5

A

B

C

poly- glycosylated by a wide variety of glycosyltransferase enzymes. In addition to the

structure of the sapogenin, saponins are also classified according to the number of

carbohydrate moieties that are glycosidically linked to the aglycone. Accordingly, they

8

are referred to as mono-, di-, or tridesmosidic, based on the number of carbohydrate

moieties linked to the sapogenin skeleton.

Figure 1.3. Examples of (A) monodesmosidic and (B) didesmosidic saponins isolated

from Quillaja Saponaria and Chenopodium quinoa, respectively (Kuljanabhagavad, et al.

2008; Guo, et al., 1998).

O

O

OH

HO

OH

O

OH

OH

OH

HOCOO

O

OH

CHO

O

HO

OH

OH

O

HO

HOOC

HO

O

COOH

O

OH

CHO

O

HO

OH

HO

OH

A

B

9

1.5.1. Steroidal Saponins

Steroidal saponins are widely found throughout the plant kingdom and have been

reported in a broad range of orders including Solanales (Ferro, et al., 2005), Ranunculales

(Braca, et al., 2004), Sapindales (Achenbach, et al., 1994), Fabales (Murakami et al.,

2000), Cyperales (Osbourn, et al., 2000), Liliales (Debella, et al., 1999), Dioscoreales

(Haraguchi et al., 1994), Aspargales (Sautour et al., 2007), and Zingiberales (Lin, et al.,

1996). In addition, steroidal saponins have been documented in over 100 plant families

and in many marine organisms (Güçlü-Üstündağ and Mazza, 2006). Steroidal saponins

are divided into two main classes, spirostanols and furostanols, based on structural

differences in the aglycone. Spirostanols have a six-ring structure (A – F rings), referred

to as a spirostane skeleton, and are monodesmosidic, typically having a carbohydrate

moiety -glycosodically attached by an ether linkage to the C-3 carbon of the aglycone.

Furostanols have a pentacyclic aglycone (A – E rings), referred to as a furostane skeleton,

and are bidesmosidic with one carbohydrate moiety attached through an ether linkage at

C-3 carbon and a second carbohydrate moiety attached by an ether linkage at the C-26

carbon. The most common furostanols have a single glucose linked at the C-26 position;

however, multiple sugars can be attached, but this is less common.

10

Figure 1.4. Structures of (A) a basic steroidal backbone (A-F rings), (B) a spirostane

backbone, and (C) a furostane backbone.

A B

C D

F

RO

O

OHOR2

RO

O

O

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

2223

2425

26

27

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

A

B

C

11

Figure 1.5. Examples of different carbohydrate linkages: (A) linear arrangement ( -D-

glu-(1→4)- -D-glu), (B) branched arrangement ( -L-rha-(1→2)- -L-xyl-(1→3)- -D-

glu), and (C) branched arrangement ( -L-rha-(1→2)- -D-glu-(1→4)- -D-glu).

O

HOO

OH

O

OH3CHO

OH OH

O

R

O

HOHO

OH

O

HO

OH

HO

OH 2'

4'

1''

-L-Rha

O

HOO

OH

O

OH

RO

HO

OH

HO

OH

4'

O

OO

OH

HO

O

R

2'3'

OH3CHO

OH OH

1''

-D-Glc-D-Glc 4

-D-Glc3

-D-Xly

2

-L-Rha

-D-Glc4

2

-D-Glc

A

B

C

The sugar composition of the carbohydrate moiety of steroidal saponins most commonly

include, D-glucose, D-galactose, D-glucuronic acid, D-galacturonic acid, L-rhamnose,

L-arabinose, D-xylose, and D-fucose. All dextrorotatory form sugars are linked via a -

glycosidic linkage and all levorotatory form sugars are linked via an -glycosidic

linkage. The composition of the carbohydrate moiety can range from one sugar to

multiple sugars and can be linked in a linear or branched arrangement. In addition, sugars

can be attached by different interglycosidic linkages resulting in a vast number of

12

possible structural arrangements. Structural differences in the carbohydrate moiety have

been shown to play a role in the biological activity of the molecules and differential

biological activity of steroidal saponins containing the same aglycone but differing only

in carbohydrate composition has been previously reported (Mimaki et al., 2001).

1.5.1.1. Commercially Important Steroidal Saponins

Steroidal saponins have been reported to exhibit a wide range of biological

activities including antifungal (Sautour et al., 2005), platelet aggregation inhibition

(Zhang et al., 1999; Huang et al., 2006), anti-diabetic (Nakashima et al., 1993),

cholesterol lowering (Matsuura, 2001), anti-inflammatory (Shao et al., 2007), antiviral

(Gosse et al., 2002) and anticancer (Acharya et al., 2009; Pettit et al., 2005; Mimaki et al.,

1999). Additionally, steroidal saponins have wide a variety of commercial uses including

surfactants (Yamanaka et al., 2008), foaming agents (Singh et al., 2003), vaccine

adjuvants (Rajput et al., 2007), and serve as precursors for the industrial production of

pharmaceutical steroids (Hansen, 2007).

The pioneering work published by Russell Marker in the 1940s resulted in the

semi-synthetic preparation of progesterone from the sapogenin, diosgenin, obtained by

the hydrolysis of steroidal saponins extracted from the Japanese yam, Dioscorea tokoro

(Marker, et al., 1940). The Dioscoreaceae family is a rich source of steroidal saponins,

and through further investigations with the goal of discovering an even richer source of

precursors for steroid synthesis, other species such as D. Mexicana, D. composita, and D.

13

floribunda were identified. Diosgenin and similar steroid-based sapogenins are still an

important intermediate for the industrial preparation of pharmaceutical steroids including

anti-inflammatory, androgenic, estrogenic, and contraceptive drugs. In addition to a

source of pharmaceutical precursors, steroidal saponin rich tubers and roots from

members of the Dioscorea genus are commonly consumed as both food and medicine in

much of Africa, Asia, and Tropical America (Sautour, et al. 2007).

Steroidal saponins have been isolated and characterized from many Dioscorea

species including D. bulbifera var. sativa (Teponno, et al., 2006), D. cayenensis

(Sautour, et al. 2004), D. collettii var. hypoglauca (Hu, et al., 2003), D. composita

(Espejo, et al., 1982), D. deltoidea var. orbiculata (Shen, et al., 2002), D. futschauensis

(Liu, et al., 2003), D. nipponica (Cui, et al., 2004), D. olfersiana (Haraguchi, et al.,

1994), D. panthaica (Dong, et al., 2004), D. parviflora (Yang, et al., 2005), D.

polygonoides (Osorio, et al. 2003), D. prazeri (Wij, et al., 1977), D. pseudojaponica

(Yang, et al., 2003), D. spongiosa (Yin, et al., 2003), D. villosa (Sautour, et al., 2006), D.

zingiberensis (Sun, et al., 2003). The high levels of steroidal saponins in these species

may contribute to the reported medicinal properties of these plants. The tubers and roots

several Dioscorea species including D. colletii var. hypoglauca, D. panthaica , D.

nipponica, and D. futschauensis have been traditionally used in China for various

medicinal uses including anticancer, cardiovascular, rheumatism, and a general tonic

(Lacaille-Dubois, 2002; Li and Zhou, 1994; Li, et al., 2000). In fact, a crude drug used in

traditional Chinese medicine that is prepared with D. panthaica is regularly used for the

prevention and treatment of cardiovascular diseases in China today (Li and Zhou, 1994).

14

In addition, another crude drug made from a mixture of steroidal saponins extracted from

D. nipponica is used to treat rheumatism (Li, et al., 2000).

Steroidal saponins isolated from Dioscorea have shown various biological

activities including cytotoxic activity, immunomodulating activity, antimicrobial activity,

hormonal activity (Lacaille-Dubois, 2002), anti-osteoporotic activity (Yin et al., 2003),

anti-inflammatory activity (Tewtrakul et al., 2007), and anti-allergic activity (Tewtrakul

et al., 2006).

Figure 1.6. Structures of Dioscin and Protodioscin isolated from D. collettii var.

hypoglauca (Hu, et al., 2003)

O

HOO

OH

O

OH3CHO OHOH

O

O

OH

O

HO

OH

HO

OH

O

O

HOO

OH

O

OH3CHO

OH OH

O

O

O

O

HO

H3C

HO

OH

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

1''''

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

O

HO

H3C

HO

OH

1'''

1'''

Dioscin

Protodioscin

15

In addition to the commercial use of steroidal saponins as precursors for the

synthesis of pharmaceutical steroids, steroidal saponins serve as an important raw

material in the food, pharmaceutical, cosmetic and agricultural industries. The most

common commercial applications of steroidal saponins include surfactants (Yamanaka et

al., 2008), foaming agents (Singh et al., 2003), vaccine adjuvants (Rajput et al., 2007),

and feed additives (Anthony et al., 1994; Balog et al., 1994).

Yucca schidigera, a desert plant from the Agavaceae family, is one of the most

important commercial sources of steroidal saponins. Y. schidigera, commonly referred to

as yucca, is native to the southwestern United States and Mexico where it has a long

historical use as a medicine to treat ailments including inflammation, headaches,

gonorrhea, and arthritis (Cheeke, 1998). The primary commercial raw material use of

yucca extract is as a foaming agent for beverage manufactures, food manufacturers, and

cosmetic companies. The foaming activity of yucca extract is due to the high steroidal

saponin content (Oleszek et al., 2001).

In the agricultural feed industry, yucca extract is used as a livestock feed

supplement. It has been reported to increase livestock growth rates (Mader and Brumm,

1987; Anthony et al., 1994), increase feed efficiency (Mader and Brumm, 1987) and

improve general health of livestock (Anthony et al., 1994; Balog et al., 1994). In addition,

yucca extract utilized as an animal feed supplement has been reported to reduce

malodorous aromas associated livestock waste (Cheeke, 2000). Steroidal saponins have

also been isolated and characterized in other Yucca species including Y. aloifolia

(Bahuguna, et al., 1991), Y. elephantipes (Zhang, et al., 2008), Y. filamentosa (Dragalin,

et al., 1975), Y. glauca (Stohs and Obrist, 1975), and Y. gloriosa (Nakano, et al., 1991).

16

1.5.1.2. Dietary Sources of Steroidal Saponins

Plants from the Allium genus are of great agricultural importance and have a long

history of use as both food and medicine. In particular, garlic, A. sativum, and onion, A.

cepa, have been used in traditional medicine since ancient times (Block, 1985). The

Allium genus belongs to the Amaryllidaceae family which is closely related to the

Liliaceae family. The Allium genus is a rich source of steroidal saponins, a group of

compounds that may play a role in the traditional medicinal uses of Allium species.

Steroidal saponins isolated from Allium species exhibit various biological activities

including cytotoxicity (Mimaki et al., 1999a), antifungal activity (Morita et al., 1988),

anti-blood coagulation activity (Peng, et al., 1996), antispasmodic activity (Corea et al.,

2005), anti-tumor activity (Sang et al., 2003), anti-platelet aggregating activity (Peng, et

al., 1996), cholesterol lowering activity (Matsuura et al., 2001), and insecticidal activity

(Harmatha et al., 1987). Steroidal saponins with biological activity have been isolated

and characterized in over thirty species of the Allium genus (Lanzotti, 2005). Some other

Allium species that are commonly consumed as food and have steroidal saponins with

biological activity include shallots, A. ascalonicum (Fattorusso et al., 2002), leeks, A.

porrum (Harmatha et al., 1987; Fattorusso et al., 2000), and elephant garlic, A.

ampeloprasum (Morita et al., 1988; Mimaki et al., 1999b).

Plants from the Asparagus genus which are also high in steroidal saponins, are of

great agronomic importance and have a long history of use as both food and medicine. In

particular, garden asparagus, A. officinalis, is consumed worldwide and is a rich source of

steroidal saponins (Shimoyamada, et al., 1990; Shimoyamada, et al., 1996; Huang and

17

Kong, 2006). In addition to use as a food, many Asparagus species are used in traditional

medicine. In fact, the root of A. filicinus is used in traditional Chinese medicine as a

treatment for colds, coughs, and pneumonia (Zhou et al., 2007) and A. racemosus is used

in traditional Indian medicine for the treatment of spasm, chronic fevers, and rheumatism

(Hayes et al., 2008). The Asparagus genus belongs to the Asparagaceae family which is

closely related to the Liliaceae family. Steroidal saponins isolated from Asparagus

species exhibit putative biological activities including antifungal (Shimoyamada et al.,

1996), antiprotozoal (Oketch-Rabah and Dossaji, 1997) and cytotoxic activity (Zhang et

al., 2004). In addition, steroidal saponins with biological activities have been identified

and characterized in other members of the genus including A. acutifolius (Sautour, et al.,

2007), A. adscendens (Sharma, et al., 1982), A. africanus (Debella, et al.,1999), A.

cochinchinensis (Zhang, et al., 2004), A. dumosus (Ahmad, et al., 1998), A. filicinus

(Sharma, et al., 1996), A. gobicus (Yang, et al., 2004), A. oligoclonos (Kim, et al.,

2005), and A. plumosus (Sati, et al., 1985).

18

1.5.1.3. Steroidal saponins isolated from Lilium

Extensive work has been done on the isolation and characterization of steroidal

saponins in Lilium. Steroidal saponins have been reported in L. brownii (Mimaki and

Sashida 1990a; Mimaki and Sashida 1990b; Hou and Chen, 1998), L. candidum (Mimaki

et al., 1998; Mimaki et al., 1999; Eisenreichova, et al., 2000), L. hansonii (Ori et al.,

1992), L. henryi Baker (Mimaki et al., 1993), L. longiflorum (Mimaki et al., 1994), L.

mackliniae Sealy (Sashida et al., 1991), L. martagon L. (Satou et al., 1996), L.

pardalinum Kellogg (Shimomura et al., 1989), L. pensylvanicum (synonym: L. dauricum)

(Mimaki et al., 1992), L. pumilum (synonym: L. tenuiflolium) (Mimaki et al., 1989), L.

regale E. H. Wilson (Mimaki et al., 1993; Gur'eva et al., 1996; Kintya et al., 1996), L.

speciosum var. speciosum (Mimaki and Sashida, 1991), and L. speciosum x L.

nobilissimum (Makino) Makino (Nakamura et al., 1994). Steroidal saponins that have

been characterized in the Lilium genus are summarized in Table 1.1 and the molecular

structures are shown in Figure 1.7.

19

Table 1.1. Steroidal saponins isolated from Lilium.

species compound reference

L. candidum 5, 10, 23, 33, 50, 51, 52, 53, 55 Mimaki et al., 1998

57, 58, 59, 60, 61, 62 Mimaki, et al., 1999

56 Eisenreichova, et al., 2000

L. regale 5, 28, 29, 30, 31 Mimaki et al., 1993

32, 33, 34 ,35, 36 Gur'eva et al., 1996

3, 5, 37, 38, 39, 40 Kintya et al., 1996

L. longiflorum 4, 23, 24, 42, 43, 44, 45, 46, 47 Mimaki et al., 1994

L. pensylvanicum 10, 22, 23, 24, 25, 26, 27 Mimaki et al., 1992

L. pardarinum 11, 12, 13, 14, 15, 16, 17 Shimomura et al., 1989

L. speciosum x

nobilissum 4, 5, 10, 23, 41 Nakamura et al., 1994

L. hansonii 18, 19, 20, 21 Ori et al., 1992

L. brownii 3a, 4b, 5b, 6a, 7b Mimaki and Sashida, 1990 a, b

8, 9 Hou and Chen, 1998

L. speciosum 3, 5, 10 Mimaki ans Sashida, 1991

L. martagon 48, 49 Satou et al., 1996

L. henryi 4, 5 Mimaki et al., 1993

L. pumilum 1, 2 Mimaki et al., 1989

L. macklineae 4, 5 Sashida et al., 1991

20

RO

OHOH

OH

O

R

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19-D-Glcp1

2 -D-Allp

O

HOO

OH

R2O

OH3CHO

OH OH

O

O

O OR1

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

1''

O

HOO

OH

R3O

OH3CHO

OH OH

O

O

O

O

HO

OH

HO

OH

O

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

1'2'

1''

1'''

R3

H6

7 -D-Glcp

R1 R2

HH

H

-D-Glcp

3

4

5

HMG

HMG

O

HOO

OH

HO

OH3CHO OHOH

O

O

O

O

HO

OH

HO

OH

O

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

23

24

2526

27

1'2'

4'

1''

1''''

8

9 5 -H

O

HOO

OH

HO

OH3CHO

OH OH

O

O

OOCH3

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

27

1'2'

1''

10

Figure 1.7. Molecular structures of steroidal saponins from Lilium.

21

O

R4OO

OH

R3O

O

O

R2 OCH3

O

OH3CHO

OH OH

O

HO

R1

H

R1 R2 R3

H

H

H

H

OH H

OH

H

OH

H

CH3

CH2OH

CH3

CH3

CH3

CH3

CH3

R4

H

H

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

2022

24

25

27

1'2'

4'

-D-Glcp

11

12

13

14

15

-D-Glcp

-L-Arap

-L-Arap

16

17

-L-Arap

O

HOO

OH

O

OH3CHO

OH OH

O

O

O

O

HO

OH

HO

OH

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

O

HOO

OH

O

OH3CHO OHOH

O

O

OH

O

HO

OH

HO

OH

O

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

1''''

O

HO

OH

HO

OH

OH

19

18

5 -H

21

20

5 -H

O

R1OO

OH

R2O

OH3CHO

OH OH

O

O

OOCH3

R1 R2

H

H -D-Glcp

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

23

24

27

1'2'

1''

-L-Arap22

23

26

25

O

OO

OH

HO

OH3CHO

OH OH

O

O

O

O

HO

OH

OH

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

24

Figure 1.7. continued

22

O

R3OO

OH

HO

OH3CHO

OH OH

O

O

O

OH

HO

R3

H1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

252

627

1'2'

4'

1''

25

26 -L-Arap

O

HOO

OH

HO

OH3CHO

OH OH

O

OH

OH

OH

OH

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

26

27

1'2'

4'

1''

27

O

OO

OH

HO

OH3CHO

OH OH

O

O

O

O

HO

OH

HO

OH

O

O

OCH3

O

HO

OH

HO

OH

O

O

O

OH

HO

OH3CHO

OH OH

O

O

HO

OH

HO

OH

O

R2OO

OH

R1O

OH

O

OH

O

OH3CHO

OH OH

O

O

O O

R1 R2

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

3'

1''

-D-Glcp

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1''''

30

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

1''

28

29

4'

3'

1'2'

3'

1''

H


23

O

OO

OH

HO

OH3CHO

OH OH

O

O

O OHHO

O

HO

OH

HO

O

O

HO

OH

HO

OH

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

1''

1'''2'''

1''''

3'

31

O

O

O

O

R2O

OH

HO

OR1

O

R2OO

OH

HO

OR1

O

OH

O

HO

OH

HO

OH

O

R1 R2

H

H

H

R1 R2

H

H

H1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1'2'

3 '

1''''

35

36

34

-D-Glcp

-L-Rhap

-L-Rhap

1'2'

3 '

33

32

-L-Rhap

O

R2OO

OH

HO

OR1

O

O

OH

R1 R2

HH1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

3'

38

37

-D-Glcp-L-Rhap

O

HOO

OH

HO

OH

O

OH

O

OH

O

O O

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

3'

39


24

O

R1OO

OH

HO

OH

O

OH

O

OH3CHO

OH OH

O

O

O

O

R1

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

1''

3'

40 -D-Glcp

O

HOO

OH

O

OH3CHO

OH OH

O

O

OOCH3

O

HO

O

HO

OH

O

R1 R2

H

H

H

O

O

OCH3

O

HO

OH

HO

OH

O

O

R2O

OH

R1O

OH3CHO OHOH

O

O-HMG

O

R1OO

OH

R2O

OH3CHO

OH OH

O

O

O R

R R1

OH

R2

HOH

O

HOO

OH

O

OH

O

O OHHO

O

HO

OH

HO

O

O

HO

OH

HO

OH

43

44

42 -D-Glcp

-L-Arap

-D-Xlyp

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1''''

41

5 -H

1'2'

4'

1''

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

1''

4'

3'

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

23

24

27

1'2'

1''

26

25

1'''

4'

46

45

-D-Glcp

-L-Arap

-L-Arap

47

49

48

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'

1'''2'''

1''''

3'


25

O

HOO

OH

O

OH3CHO

OH OH

O

O

OOCH3

O

HO

OR2

HO

O

R1

HOH

OH -D-Glcp

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

23

24

27

1'2'

1''

50

51

26

25

1'2'

O

HOO

OH

HO

OH3CHO

OH OH

O

O

OR3

R2

R1

R1 R2

H H

R3

OH

CH3

OCH3 CH3

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

23

24

27

1'2'

1''

26

25

52

53

O

HOO

OH

O

OH3CHO

OOH

O

O

O OH

O

HO

OH

HO

OH

O

HO

OH

HO

OH

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

23

24

27

1'2'

1''

26

25

1'''

1''''

3''

54

R1

HO

O

OCH3

O

HO

OH

HO

OH

O

O

HO

OH

R1O

OH3CHO OHOH

O55

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1''''

1'2'

4'

1''

O

HOO

OH

O

OH3CHO

OH OH

O

O

O

OC2H5

O

HO

OH

HO

OH

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

23

24

27

1'2'

1''

26

25

1'''

56


26

O

HOO

O

HO

OH3CHO

OH OH

O

O

OR3

R4

R1

R2

O

HO

OH

HO

OH

R1 R2

H

H H

R3

H

H OH

CH3

CH2OH

OCH3

CH3

OCH3

CH3

R4

CH3

H

H

H

H H

OH H

O

O

OCH3

O

HO

OH

HO

OH

O

O

HO

O

HO

OH3CHO

OH OH

O

O

HO

OH

HO

OH 1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1''''

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

23

24

27

1'2'

1''

26

25

57

58

59

60

1'''

61

1'2'

1''

1'''

62


27

1.6 Steroidal Alkaloids

Steroidal alkaloids are a structurally diverse class of natural products that have

been isolated and characterized in a wide range of organisms including plants, marine

animals, and terrestrial animals (Atta-ur-Rahman and Choudhary, 1998). Structurally,

steroidal alkaloids contain a steroid-type backbone and with a nitrogen atom incorporated

into the molecule. The biosynthesis of steroidal alkaloids in plants differs from other

plant alkaloids due to the fact that the carbon atoms in the molecule are derived from the

melavonic acid pathway, whereas the carbon backbones of other alkaloids are derived

from amino acids (Atta-ur-Rahman and Choudhary, 1998). Steroidal alkaloids are most

commonly found in the plant families of Apocynanceae, Buxaceae, Liliaceae and

Solanaceae. Interestingly, highly cytotoxic steroidal alkaloids have also been isolated and

characterized from marine organisms such as the truncate Ritterea tokiokal (Fukuzawa et

al., 1994), and amphibians such as Salamandra sp. and Phyllobates sp. (Daly et al.,

2005).

1.6.1 Steroidal Alkaloids in Liliaceae

The occurrence of steroidal alkaloids in the Liliaceae family is well documented

(Li et al., 2006). Fritillaria, a genus in the Liliaceae family, and Veratrum, a genus in the

closely related Melanthiaceae family, have been recognized for centuries for their

pharmacological activities and have a long history of use in traditional medicine. In fact,

V. viride has been documented to be used by Native Americans for the treatment of

28

catarrah, the treatment of rheumatism, and as an insecticide against lice (Rahman and

Choudhary, 1998). Steroidal alkaloids isolated from the Liliaceae family have been of

great interest in pharmacology and have been documented to exhibit various putative

biological activities including antihypertensive (Oh et al., 2003), anticholinergic (Gilani

et al., 1997), antifungal (Zhou et al., 2003), and anticancer (Jiang et al., 2005). In

particular, the genera Veratrum and Fritillaria, have been the subject of extensive

chemical characterization and pharmacological investigations. Hundreds of new steroidal

alkaloids have been isolated from these plants and over 100 steroidal alkaloids have been

isolated between the years of 1980 and 2005 (Li et al., 2006). The steroidal alkaloids

isolated from the Liliaceae have been classified into main two groups, isosteroidal

alkaloids and steroidal alkaloids, on the basis of connectivity of the carbon skeleton.

1.6.1.1. Classification of Isosteroidal Alkaloids of Liliaceae

Isosteroidal alkaloids, also referred to as Veratrum steroidal alkaloids, are

characterized by a C-nor-D-homo-[14(13→12)-abeo] ring system (Li et al., 2006).

Isosteroidal alkaloids are further sub-divided into three groups according to the linkage

patterns between rings E and F into cevanine type, veratramine type, and jervine type.

The cevanine type, structurally characterized by the hexacyclic benzo [7,8] fluoreno [2,1-

b] quinolizine nucleus, constitutes the largest class. The veratramine type is characterized

by the absence of ring E and the presence of an aromatic ring D. Analogues with an

unaromatized ring D are also placed in this class. The jervine type contain hexacyclic

29

compounds that have the furan ring E fused onto a piperidine ring system forming an

ether bridge between carbon atoms at C17 and C23.

Figure 1.8. Isosteroidal alkaloids of Liliaceae: Representative examples of cevanine type

(A), veratramine type (B), and jervine type (C) isosteroidal alkaloids, isolated from F.

imperialis, F. ningguoensis, and F. camtschatcensis, respectively (Li et al., 2006).

N

HN

HN

O

OH

OH

HO

HO

O

HO

HO

OH

HO

A

B

C

30

1.6.1.2. Classification of Steroidal Alkaloids of Liliaceae

The steroidal alkaloids of Liliaceae, also referred to as Solanum steroidal

alkaloids, are characterized by a six membered C-ring and a five-membered D-ring. The

steroidal alkaloids are further sub-divided to two groups on the basis of the position of

the nitrogen atom. Steroidal alkaloids with the nitrogen atom incorporated into an

indolizidine ring are referred to as solanidine type. The solanidine type is derived from

epiminocholestanes with the amino group incorporated into an indolizidine ring, resulting

in a hexacyclic carbon framework. If the nitrogen atom is incorporated into a piperidine

ring, they are of verazine type. The verazine type is based on the 22/23,26-

epiminocholestane heterocyclic skeleton.

Figure 1.9. Steroidal alkaloids of Liliaceae: Representative examples of solanidine type

(A) and verazine type (B) steroidal alkaloids, isolated from F. delavayi and F. ebeiensis

var. purpurea, respectively (Li et al., 2006).

N

N

HO

OHHO

HO

OOH

A

B

31

1.6.1.3. Steroidal Alkaloids in Lilium

Although extensive work has been done on the characterization of steroidal

alkaloids in the Liliacece family, less is known on the steroidal alkaloids in the Lilium

genus. In 1996 Noor-e-Ain reported on the identification of two steroidal alkaloids from

L. candidum, commonly known as the Madonna lily (Noor-e-Ain, 1996). Two 22, 26-

epiminocholestane-type steroidal alkaloids, named etioline and teinemine, that were

previously found in several Solanum and Veratrum species were isolated from L.

candidum bulbs (Lin et al., 1986; Atta-ur-Rahman and Choudhary, 1998). In 2001,

Erdoğan et al. also reported on the isolation of etioline from L. candidum bulbs (Erdoğan

et al., 2001). Although these compounds were isolated as free form steroidal alkaloids,

the isolation procedure was performed over several weeks under acidic conditions and it

is unclear whether the conditions caused glycosidic cleavage, resulting in the formation

of artifacts during the isolation process. Thus far, the glycosylated forms of these two

steroidal alkaloids have not been identified L. candidum. Nevertheless, these compounds

are the only free form steroidal alkaloids that have been reported from the Lilium genus.

32

Figure 1.10. Structures of the steroidal alkaloids (A) etioline and (B) teiemine isolated

from the bulbs of L. candidum (Noor-e-Ain, 1996; Erdoğan et al., 2001)

HO

N

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

2223

24

2526

27

OH A

B

HO

HN

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

2223

24

2526

27

OH

1.6.2. Steroidal Glycoalkaloids

In contrast to the widespread distribution of steroidal alkaloids in plants and

animals, steroidal glycoalkaloids, thus far, have only been identified in the Solanaceae

and Liliaceae families (Ghisalberti, 2006). Steroidal glycoalkaloids are characterized by a

steroidal alkaloid type aglycone glycosidically linked to carbohydrate moieties. The most

common aglycones of the steroidal glycoalkaloids can be classified based upon their

structural features into six major groups. The first group is referred to as (1) spirosolanes

and are characterized by an oxazaspirodecane ring system. The second group, the (2) 22,

26-epiminocholestanes, are characterized by a 22/23, 26-epiminocholestane heterocyclic

skeleton. The third group, the (3) solanidanes, are characterized by a fused indolizidine

ring system. The fourth group, the (4) epiminocyclohemiketals, are characterized the

33

presence of an epiminocyclohemiketal functionality. The fifth group, the (5) 3-

aminospirostanes, is characterized by an amino group at the C3-position. The sixth group,

the (6) leptines, are characterized by a fused indolizidine ring system with 23-hydroxy or

23-acetoxy moieties. The most common steroidal glycoalkaloids belong to the solanidane

and the spirosolane groups (Ghisalberti, 2006). The carbohydrate moiety of steroidal

glycoalkaloids is -glycosidically linked at the C-3 hydroxy position of the steroidal

alkaloid backbone. The most common sugars are D-glucose, D-galactose, D-xylose, L-

rhamnose, and L-arabinose. Similar to the steroidal saponins, all dextrorotatory form

sugars are linked via a -glycosidic linkage and all levorotatory form sugars are linked

via an -glycosidic linkage. The composition of the carbohydrate moiety can range from

one sugar to multiple sugars, linked in a linear or branched arrangement.

34

Figure 1.11. Structures of the most common aglycones of the steroidal glycoalkaloids:

(A) spirosolanes, (B) 22, 26-epiminocholestanes, (C) solanidanes, (D)

epiminocyclohemiketals, (E) 3-aminospirostanes, and (F) leptines

N

HO H2N

O

HN

OH

O

H2N

O

N

HO

R

HO

O

HN

HO

N

OH

R

OH

OAc

A B

C D

F1

2

E

35

1.6.2.1. Dietary Sources of Steroidal Glycoalkaloids

Steroidal glycoalkaloids from the Solanaceae family are found in many

agriculturally important foods crops such as tomato, S. lycopersicum, potato, S.

tuberosum, eggplant, S. melongena, and pepper, Capsicum annuum. -Solasonine and -

solamargine are the two predominant steroidal glycoalkaloids found in the common

cultivated eggplant, S. melongena (Blankemeyer et al. 1998). -Solasonine and -

solamargine share the same aglycone, solasodine, but differ only in the carbohydrate

moiety. The carbohydrate moiety of -solasonine contains a trisaccharide moiety, 3-O- -

L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→3)- -D-galactopyranoside, whereas

-solamargine contains a disaccharide moiety, 3-O- -L-rhamnopyranosyl-(1→2)- -D-

galactopyranoside. Solasodine is a common aglycone and has been identified in over 200

Solanum species alone (Dinan et al. 2001). -Solanine and -chaconine are the most

prevalent glycoalkaloids found in cultivated potato, S. tuberosum. Similar to -solasonine

and -solamargine, -solanine and -chaconine share the same aglycone, solanidine, but

differ only in the carbohydrate moiety. The carbohydrate moiety of -solanine contains a

trisaccharide, 3-O- -L-rhamnopyranosyl-(1→2)- -D-galactopyranosyl-(1→3)- -D-

glucopyranoside, whereas -chaconine contains a disaccharide moiety, 3-O- -L-

rhamnopyranosyl-(1→2)- -D-galactopyranoside. -Tomatine and dehyrotomatine are

the most abundant steroidal glycoalkaloids in the leaves and unripe fruit of tomato, S.

lycopersicum. In the case of -tomatine and dehyrotomatine, they only differ in the

degree of saturation between the C5 and C6 carbon of the aglycone and share the same

36

carbohydrate moiety, namely 3-O- -D-glucopyranosyl-(1→2)- -D-xylopyranosyl-

(1→3)- -D-glucopyranosyl-(1→4)- -D-galactopyranoside.

Steroidal glycoalkaloids are toxic to many organisms including bacteria, fungi,

and animals. The literature suggests that their toxicological effects are based on the

disruption of cellular membranes, anticholinesterase activity, and their effect on the

active transport of ions through membranes (Friedman et al., 1992a; Keukens et al., 1995;

Blankemeyer et al., 1992; Blankemeyer et al., 1995). Steroidal glycoalkaloids are

amphiphilic in nature due to the lipophilic steroidal aglycone and the hydrophilic

carbohydrate moiety. Recent evidence suggests that this structural characteristic

contributes to their biological activity. Accordingly, free aglycones are less active as

compared to their glycosylated forms (Roddick, 1989). Interestingly, chaconine and

solanine share the same aglycone and only differ in the carbohydrate moiety; however,

chaconine has been shown to be the more teratogenic as compared to solanine

(Blankemeyer et al., 1997; Blankemeyer et al., 1998). Furthermore, spirosolanes have

been shown to be less teratogenic as compared to solanidanes. Differences in the

carbohydrate moiety, the absolute configuration of the F ring, and saturation between C5

and C6 has been shown to play a role in biological activity (Gaffield and Keeler, 1996).

In addition, different forms of steroidal glycosides that occur in the same plant have been

shown to act synergistically, thus the importance of investigating the biological activity

of the compounds both individually and in mixtures (Rayburn et al., 1994; Smith et al.,

2001). Due to the structural diversity of steroidal glycoalkaloids, differential biological

activities have been observed. For example, some steroidal glycoalkaloids are highly

toxic whereas others may potentially have beneficial properties, in particular the

37

spirosolanes. Beneficial biological activities that have been observed include cholesterol

and triglyceride lowering, anti-inflammatory activity, anti-viral activity, and anti-cancer

activity (Friedman et al. 2000; Kuo et al., 2000; Ikeda et al., 2003; Carter and Lake,

2004).

1.6.2.2. Steroidal Glycoalkaloids in Lilium

Although steroidal alkaloids are well documented in the Solanaceae and Liliaceae

families, a very small number of steroidal glycoalkaloids have been isolated from the

Lilium genus. In Lilium, steroidal glycoalkaloids have been identified in L. philippinense

(Espeso et al., 1990), L. mackliniae (Sashida et al., 1991), and L. brownii (Mimaki and

Sashida, 1990a; Mimaki and Sashida, 1990b). In 1990, Espeso et al. reported the isolation

of a steroidal glycoalkaloid from the aerial parts of the L. philippinense, commonly

known as the Banquet lily. Interestingly, the compound was a veratramine type

isosteroidal alkaloid glucoside, containing an unaromatized D ring. Prior to this work, the

only steroidal glycoalkaloid previously isolated from the genus was from L. cordatum;

however, this plant was taxonomically moved into the closely related genus

Cardiocrinum (Endl.) Lindl. Nevertheless, in 1987 Nakano et al. reported the isolation

and structural elucidation of (25R)- and (25S)-22,26-epimino-5α-cholest-22(N)-en-3β,6β-

diol O(3)-β-D-glucopyranoside from C. cordatum (Nakano et al., 1987). Also in 1990,

Mimaki et al. reported on the isolation a structural elucidation of two steroidal

glycoalkaloids from L. brownii, (22R, 25R)-spirosol-5-en-3 -yl O- -L-

rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside and (22,R

38

25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranoside. Both

alkaloids were solasodine-based and differed only in the number of sugars in the

carbohydrate moiety. In 1991, Sashida et al. reported on the isolation of a solanidine-

based steroidal glycoalkaloid, solanidine O- -L-rhamnopyranosyl-(1→2)- -D-

glucopyranosyl-(1→4)- -D-glucopyranoside, from the bulbs of L. mackliniae.

Interestingly, this compound was isolated previously from two Fritillaria species, F.

thunbergii and F. camtschatcensis (Sashida et al., 1991). In contrast to the steroidal

saponins which have been extensively isolated and characterized in the genus, there are a

very limited number of reports on the occurrence of steroidal glycoalkaloids in the Lilium

genus.

39

Figure 1.12. Structures of steroidal glycoalkaloids isolated from C. cordatum (1, 2), L.

philippinense (3), L. brownii (4, 5), and L. mackliniae (6).

O

HOO

OH

HO

OH

1

2

3

4

56

7

8

910

11 12

13

14 15

16

17

18

19

O

O

HOO

OH

R1O

OH3CHO

OH OH

O

O

HN

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

20

21

22

23 24

25

26

27

HN

O

HOO

OH

O

OH3CHO

OH OH

O

N

O

HO

OH

HO

OH

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

1'2'

1''

23

24

26

27

3

4

R1

-D-Glcp

H

O

NR2

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

2223

24

2526

OH

R1

R2R1

HCH31

2 H CH3

O

HO

OH

HO

OH

5

6

40

1.7. Plant organ distribution of steroidal glycosides

Although steroidal glycosides are widespread in plants and have been identified in

almost all plant organ types, there are only a few investigations comparing the

concentration of these natural products in the different organs of the same plant species.

Even less is known about tissue specific localization and cellular and sub-cellular

synthesis and storage of these compounds. This is due, in part, to the complexity of

performing quantitative analysis of this diverse class of natural products.

Steroidal glycosides lack a strong chromophore; therefore, quantitative methods

using non-specific short wavelength ultraviolet (UV) detection (200 – 210nm) is a

challenge due to interference from phytochemicals with strong chromophores that occur

in the same plant matrix. Analytical methods using evaporative light scattering detection

(ELSD) have been developed to help overcome this obstacle; however, laborious sample

preparation and sensitivity issues persist (Oleszek and Bialy, 2006). Recently, analytical

methods utilizing liquid chromatography - mass spectrometry (LC-MS) operating in

selected ion monitoring (SIM) mode and liquid chromatography–tandem mass

spectrometry (LC-MS/MS) have been developed with increased sensitivity and

specificity over other methods (Oleszek and Bialy, 2006). Due to recent advances in

analytical chemistry, including better chromatography and analytical instrumentation,

more studies on the organ distribution of steroidal glycosides in plants will become

increasingly available.

41

Although reports on the organ distribution of steroidal glycosides in plants are not

common, there have been some reports on the concentrations of these compounds within

the different plant organs. The organ distribution of steroidal glycosides in plants such as

Solanum nigrum, Solanum incanun (Eltayeb et al., 1997), Asparagus officinalis L. (Wang

et al., 2003) and Dioscorea pseudojaponica Yamamoto (Lin et al., 2008) have been

reported. Steroidal glycoalkaloids are generally found in all plant organs, with the highest

concentrations occurring in the metabolically active parts including in flowers, sprouts,

immature fruits, young leaves and shoots. In 1997, Eltayeb et al. reported on the

concentrations of steroidal glycoalkaloid, solasodine, in the different organs of S. nigrum

and S. incanun throughout maturation using a colorimetric assay (Eltayeb et al., 1997).

Interestingly, all of the plant organs tested contained detectable levels of solasodine with

the highest concentrations occurring in small actively growing leaves. In addition, the

concentration in the roots was observed to be higher than in the stems. In 2003, Wang et

al. developed a LC-MS method and quantified the concentration of the furostanol

saponin, protodioscin, in common garden asparagus, A. officinalis (Wang et al., 2003).

The distribution of protodioscin within the shoots was found to vary in concentration,

with the highest concentration observed in the lower stem tissue adjacent to the rhizome.

In 2008, Lin and Yang reported on the concentration of several steroidal saponins in the

different organs of the yam, D. pseudojaponica, using HPLC-ESLD (Lin et al., 2008).

Saponins were found occur in increasing concentrations in the vine, leaf, rhizophor, tuber

flesh and then tuber cortex. Although there are some reports on the distribution of

steroidal glycoside in the different plant organs, reports on tissue specific localization are

even less common.

42

Although scarce, there are some reports on the tissue specific localization and cellular

localization of steroidal glycosides in plants. In potato, S. tuberosum, the concentration of

the steroidal glycoalkaloid, -solanine, has been observed to increase in potato tubers in

response to mechanical wounding (McKee, 1955; Ishizaka and Tomiyama, 1972). When

potato tubers are wounded, the development of meristematic tissue near the site of the

wounded tissue occurs. This area develops a suberized wound periderm that is believed

provide a protective structural barrier from fungi and bacteria. It has been shown that the

tissues adjacent to the wound periderm accumulate -solanine, suggesting a role in

wound healing (McKee, 1955). In addition, in D. pseudojaponica, differential

concentrations of steroidal saponins were observed in the inner yam tuber cortex as

compared to the tuber flesh (Lin et al., 2008). Furthermore, histological observations of

the cellular localization of furostanol saponins in D. caucasia have been reported with

furostanol accumulation in specialized idioblasts in the upper and lower leaf epidermis as

compared to the leaf mesophyll where no furostanols were detected (Gurielidze et al.,

2004). Although researchers have reported on the occurrences of steroidal glycosides in

the different plant organs, plant tissues, and plant cell types, considering the widespread

abundance of these compounds in the plant kingdom, investigations this area of plant

biology is surprisingly lacking.

1.8. Steroidal glycosides in plant defense

Plants have multiple protection strategies from plant pathogens and herbivory.

The strategies include structural barriers, constitutive chemical defenses and inducible

43

chemical defenses. The first line of plant defense is the morphological structure of the

plant‘s surface. Physical structures such as the waxy cuticle and epidermal cell wall serve

as a protective barrier from pathogens. Specialized structures such as thorns or spines

serve as protection from herbivory. In addition structural features, it has been well

established that plant derived secondary metabolites can increase plant resistance to

pathogens and herbivory. Plant chemical defenses are broadly classified as constitutive

chemical defenses, known as phytoanticipins, or inducible chemical defenses, known as

phytoalexins (VanEtten et al, 1994; Müller and Börger, 1940). These definitions are

based on how the compounds are regulated rather than by chemical structure.

Accordingly, the definitions are plant species specific and in some case even tissue

specific within the same species.

Phytoalexins are inducible secondary metabolites that are produced in response to

infection or by chemical or mechanical injury (Müller and Börger, 1940). The synthesis

of phytoalexins occur in healthy cells adjacent to wounded or infected cells and are

induced by chemical elicitors released from damaged cells. Chemical elicitors trigger the

expression of phytoalexin biosynthetic genes resulting in de novo synthesis. Phytoalexins

are toxic to many organisms including fungi, bacteria, and animals. Some well known

examples of phytoalexins include resveratrol in grape, pisatin in pea, and capsidiol in

pepper.

Phytoanticipins are secondary metabolites that are constitutively present in plant

cells prior to infection (VanEtten et al, 1994). Some phytoanticipins are present in their

biologically active forms and in some cases present as biologically inactive precursors.

Upon cellular disruption by infection or mechanical damage, the biologically inactive

44

precursors are rapidly converted to biologically active forms. Phytoanticipin distribution

within plants is often tissue specific and in some plants the compounds are localized in

the outer cell layers, creating a defensive barrier to invading pathogens. For example, the

plant pathogenic fungus Colletotricum circinans infects white onions but does not infect

pigmented onions. In pigmented onions, phenolic compounds that are inhibitory to C.

circinans are preferentially accumulated in the bulb skin, thus providing a protective

barrier to infection (Link and Walker, 1933).

Steroidal glycosides are a class of compounds that in many plant species are

considered phytoanticipins due to the fact that they are constitutively present in healthy

plant tissues and are inhibitory or toxic to some organisms (Osbourn, 1996). The role of

steroidal glycosides in plant defense, including antifungal and antiherbivory, has been

studied extensively (Zullo et al., 1984; Nozzolillo et al., 1997; Adel et al., 2000; Bowyer

et al., 1995; Osbourn, 1996; Morrissey and Osbourn, 1999; Osbourn, 1999;

Papadopoulou et al., 1999; Morrissey et al., 2000; Trojanowska et al., 2000; Osbourn et

al., 2003; Osbourn, 2003; Hughes et al., 2004; Choi et al., 2005). For example, some

steroidal glycosides are toxic to insects such as the European corn borer, Ostrinia

nubialis, and army worm, Spodoptera littoralis (Nozzolillo et al., 1997; Adel et al.,

2000). In oats, Avena sativa, biologically inactive steroidal saponins are converted into an

antifungal form in response to tissue damage, suggesting a role in the plant-pathogen

interaction (Osbourn, 1996; Morrissey et al., 2000; Osbourn, 2003; Hughes et al., 2004).

In addition, the steroidal glycoalkaloids -tomatine and -chaconine play a role in fungal

resistance of tomato, Solanum lycopersicum, and potato, Solanum tuberosum,

45

respectively (Morrissey and Osbourn, 1999); however, the exact mechanism of resistance

to the pathogens has not been fully elucidated.

In general, the molecular mechanism for the antifungal activity of steroidal

glycosides is not well characterized; however, interaction with cellular membranes has

been proposed to play a role. Due to the amphipathic nature of the molecules, steroidal

glycosides have been shown to disrupt cell membranes both in vitro and in vivo (Steel

and Drysdale, 1988; Roddick et al, 2001). Some studies suggest that membrane

disruption may be due either to the interaction of the aglycone with membrane bound

sterols, resulting in the formation of membrane pores (Armah et al., 1999) or the

extraction of membrane bound sterols, causing loss of lipid bilayer integrity and

membrane leakage (Keukens et al, 1992; Keukens et al, 1995). Despite the fact that

steroidal glycosides have antifungal properties and may play a protective role against

potential pathogens, in the case of successful pathogens this is not sufficient and infection

occurs. Some mechanisms that fungal pathogens utilize to overcome host defense

strategies are avoidance, tolerance, and enzymatic metabolism of plant defense

compounds.

1.9 Detoxification of steroidal glycosides

Some successful fungal pathogens have the ability to overcome plant defenses by the

metabolism of plant defense compounds. The plant pathogen Botrytis cinerea has been

shown to produce enzymes that can metabolize a variety of plant defense compounds

from active forms to inactive forms (Staples and Mayer, 1995). For example, B. cinerea

46

produces laccases that metabolize plant defense compounds induced during infection and

reduces lignification by the host (Bar-Nun et al., 1988). In addition, fungal produced

lactases have been shown to detoxify phytoalexins from a wide variety of plants by cross-

linking host produced phenols (Van Etten et al., 1989; Pezet et al., 1992). B. cinerea

produced lactases are capable of degrading the grape phytoalexins, pterostilbene and

resveratrol, that are up regulated by the plant in response to infection, thus overcoming

the plant‘s defense response (Pezet et al., 1992).

The enzymatic detoxification of steroidal glycosides by fungal pathogens is well

documented (Arneson et al., 1967; Verhoeff and Liem, 1975; Ford et al., 1977; Bowyer

et al., 1995; Morrissey et al., 2000). Plant pathogens such as Gaeumannomyces graminis

and Stagonospora avanae have the ability to enzymatically detoxify host plant saponins

by sugar cleavage, resulting in loss of antifungal activity of the compounds (Bowyer et

al., 1995; Morrissey et al., 2000). In tomato, B. cinerea metabolizes the antifungal

steroidal glycoalkaloid, -tomatine, to an inactive form by enzymatic cleavage of the

entire carbohydrate moiety, or by the cleavage of the terminal xylose by a -xylosidase

enzyme (Verhoeff and Liem, 1975; Quidde and Osbourn, 1998). In addition, other plant

pathogenic fungi such as Septoria lycopersici and Fusarium oxysporum f.sp. lycopersici

detoxify -tomatine by cleavage of sugar residues through two separate independent

metabolic pathways (Arneson et al., 1967; Ford et al., 1977). The ability to efficiently

detoxify host plant chemical defenses may play a role in the virulence and host range of

some fungal pathogens (Bowyer et al, 1995). Pedras et al. suggested that a better

understanding the detoxification pathways utilized by plant pathogenic fungi could

potentially lead to new approaches to control plant pathogens (Pedras et al., 2011).

47

Plants produce both constitutively expressed and inducible plant defense compounds

as a means of protection from plant pathogens. Some compounds are stored in an active

form and are preferentially localized, creating a protective barrier to potential plant

pathogens. In other cases, precursors are stored and rapidly activated upon infection.

Inducible plant defense compounds are produced in healthy cells by signaling molecules

that originate in damaged cells. Despite the chemical diversity of plant defense

compounds and the differential mechanisms of expression, successful plant pathogens

evolved multiple means by which they overcome plant defenses and thus cause infection.

Friedman and McDonald speculated that in response to plant pathogen‘s ability to

overcome host plant defenses, some plants may have adapted structural modifications to

plant defense compounds, thus increasing their biological activity (Friedman and

McDonald, 1997). For example, in many Solanaceous species ―paired‖ glycoalkaloids

occur that share the same aglycone, only differ in the carbohydrate moiety, and express

differential biological activity (Friedman and McDonald, 1997; Roddick et al., 2001). A

plant defense strategy aimed at inhibiting the pathogens ability of to detoxify plant

defense compounds may be an alternative strategy in plant defense. The structural

modification of plant defense compounds as an adaptive response to plant pathogens is an

interesting hypothesis and needs further exploration.

48

CHAPTER 2: Isolation and Structural Determination of Steroidal Glycosides from

the Bulbs of Easter Lily (Lilium longiflorum Thunb.)

2.1. Abstract

The bulbs of Easter Lily (Lilium longiflorum Thunb.) are used as a food and

medicine in several Asian cultures and they are cultivated as an ornamental plant

throughout the world. A new steroidal glycoalkaloid and two new furostanol saponins,

along with two known steroidal glycosides, were isolated from the bulbs of L.

longiflorum. The new steroidal glycoalkaloid was identified as (22R, 25R)-spirosol-5-en-

3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-glucopyranosyl-(1→4)]- -D-

glucopyranoside. The new furostanol saponins were identified as (25R)-26-O-( -D-

glucopyranosyl)-furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -L-

arabinopyranosyl-(1→3)- -D-glucopyranoside and (25R)-26-O-( -D-glucopyranosyl)-

furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -L-xylopyranosyl-

(1→3)- -D-glucopyranoside. The previously known steroidal glycosides, (22R, 25R)-

spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-

glucopyranoside and (25R)-26-O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol 3-

O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside were

identified in L. longiflorum for the first time. These new compounds from L. longiflorum

and the isolation methodologies employed can be used for studies on the biological role

49

of steroidal glycosides in plant development and plant-pathogen interactions, as well as

for studies in food and human health, for which little is known.

2.2. Introduction

The Easter lily (Lilium longiflorum Thunb., family Liliaceae) has beautiful white

flowers and a delicate aroma and is appreciated worldwide as an attractive ornamental. In

addition to its economic importance and popularity in horticulture, lily bulbs are regularly

consumed in Asia, as both food and medicine. The bulbs of several Lilium species,

including L. longiflorum, L. brownii, L. pensylvanicum, and L. pumilum, have been used

traditionally in China as sedatives, anti-inflammatory and antitussive agents, and a

general tonic (Su, 1979; Mimaki et al., 1990; Mimaki et al., 1992, Varrier, 2002). The

crude drug ―Bai-he‖, used in traditional Chinese medicine, is prepared from the bulbs of

Lilium sp. and is regularly used for lung ailments in China today. Although the medicinal

use of L. longiflorum is well documented, the compounds responsible for the reported

properties are not known.

Bulbs of the genus Lilium are a rich source of secondary metabolites, including

bitter phenylpropanoid glycosides identified in the bulbs of L. speciosum Thunb.

(Shimomura et al., 1986), antitumor alkaloids identified in L. hansonii Leichtlin ex D. D.

T. Moore (Shimomura et al., 1987), and steroidal glycoalkaloids identified in L. brownii

(Mimaki and Sashida, 1990b). Extensive work has been done on the isolation and

characterization of steroidal saponins in Lilium. Steroidal saponins have been reported in

50

L. brownii (Mimaki and Sashida, 1990a; Mimaki and Sashida, 1990b; Sashida et al.,

1990; Hou and Chen, 1998; Ji et al., 2001), L. candidum (Mimaki et al., 1998; Mimaki et

al., 1999; Eisenreichova et al., 2000), L. hansonii (Ori et al., 1992), L. henryi (Mimaki et

al, 1993), L. longiflorum (Mimaki et al, 1994), L. mackliniae (Sashida et al., 1991), L.

martagon (Satou et al., 1996), L. pardalinum (Shimomura et al, 1989), L. pensylvanicum

(Mimaki et al., 1992), L. pumilum (Mimaki et al, 1989), L. regale (Mimaki et al, 1993,

Gureva et al, 1996, Kintya et al, 1997), L. speciosum var. speciosum (Mimaki et al,

1991), and L. speciosum x L. nobilissimum (Nakamura et al, 1994).

Steroidal saponins have been reported to exhibit a wide range of biological

activities including antifungal (Sautour et al., 2005), platelet aggregation inhibition

(Zhang et al., 1999; Huang et al., 2006), antidiabetic (Nakashima et al, 1993), cholesterol

lowering (Matsuura et al., 2001), anti-inflammatory (Shao et al., 2207), antiviral (Gosse

et al., 2002), and anticancer (Mimaki et al., 1999; Pettit et al., 2005; Acharya et al.,

2009). Although the putative biological activities of steroidal saponins are well

documented, the biological role of these compounds within the plant is poorly

understood.

The occurrence of steroidal alkaloids in the Liliaceae family is also well

documented (Li et al, 2006). Steroidal alkaloids isolated from the Fritillaria genus and

the closely related Veratrum genus show various biological activities including

antihypertensive (Oh et al., 2003), anticholinergic (Gilani et al., 1997), antifungal (Zhou

et al, 2003), and anticancer (Jiang et al., 2005). In Lilium, steroidal alkaloids have been

identified in L. candidum L. (Erdoğan et al., 2001) and steroidal glycoalkaloids have been

identified in L. philippinense (Espeso and Guevara, 1990), L. mackliniae (Sashida et al.,

51

1991), and L. brownii (Sashida et al., 1990); however, no steroidal alkaloids or steroidal

glycoalkaloids have been previously reported from L. longiflorum.

With regard to the steroidal glycosides of L. longiflorum bulbs, six spirostanol

saponins and three furostanol saponins with antitumor activity have been reported

(Mimaki et al, 1994). To set the stage for biological activity studies on the role of

steroidal glycosides in plant development and plant-pathogen interactions, as well as for

studies in food and human health, this chapter reports the isolation and structural

determination of several new steroidal glycosides from the bulbs of L. longiflorum. The

structures of the steroidal glycosides were elucidated by a combination of spectroscopic

and chemical analysis.

2.3. Materials and Methods

2.3.1. Plant Material.

L. longiflorum, cultivar 7-4, bulbs were provided from the Rutgers University lily

breeding program. Bulbs were treated with Captan (Bayer CropScience AG, Monheim

am Rhein, Germany) fungicide prior to planting. Bulbs were planted in beds containing

Pro-Mix (Premier Horticulture Inc., Quakertown, PA) soil mix and were grown to mature

plants under greenhouse conditions for 9 months prior to harvest. The greenhouse

temperatures were set to provide a minimum day temperature of 24 ºC and a minimum

52

night temperature of 18 ºC. Plants were fertilized biweekly with a 100 mg L-1

solution of

NPK 15-15-15 fertilizer (J. R. Peters Inc., Allentown, PA). Each plant produced three to

five new bulbs, which were used for extraction. The new bulbs were full-sized, fresh, and

mature at harvest. Each plant was harvested by hand, and the bulbs were manually

separated, immediately frozen under liquid nitrogen, lyophilized on a VirTis AdVantage

laboratory freeze-dryer (SP Industries Inc.,Warminster, PA), and stored at -80 ºC until

analysis.

2.3.2. Chemicals.

The following compounds were obtained commercially: (25R)-spirost-5-en-3β-ol,

Sephadex LH-20, N,O-bis(trimethylsiyl)trifluoroacetamide with trimethylchlorosilane

(99:1) silylating reagent, Dragendorff reagent, p-(dimethylamino)benzaldehyde,

hydrochloric acid, sodium hydroxide, pyridine-d5 (0.3% v/v TMS), D-(+)-glucose, L-(-)-

glucose, D-(+)-rhamnose, L-(-)-rhamnose, D-(+)-arabinose, L-(-)-arabinose, D-(+)-

xylose, and L-(-)-xylose (Sigma-Aldrich, St. Louis, MO); and (22R,25R)-spirosol-5-ene-

3β-ol (Glycomix Ltd., Reading, U.K.). All solvents (acetonitrile, chloroform, ethanol,

ethyl acetate, formic acid, n-butanol, and n-pentane) were of chromatographic grade

(Thermo Fisher Scientific Inc., Fair Lawn, NJ). Water was deionized (18 MΩ cm) using a

Milli-Q water purification system (Millipore, Bedford, MA).

53

2.3.3. Isolation and Purification of Steroidal Glycosides 1 – 5 from L. longiflorum.

2.3.3.1. Sequential Solvent Extraction of Lyophilized L. longiflorum Bulbs

Lyophilized lily bulbs (100 g) were frozen in liquid nitrogen, ground into a fine

powder with a laboratory mill (IKA Labortechnik, Staufen, Germany), and extracted with

n-pentane (3 x 100 mL) on a wrist-action autoshaker (Burrell Scientific, Pittsburgh, PA)

at room temperature for 30 min. After centrifugation (5000 rpm for 10 min) (Sorvall RC-

3C Plus, Thermo Fisher Scientific Inc.), the organic layers were discarded and the pellet

was freed from residual solvent in a fume hood overnight. The residual defatted material

was then extracted with a mixture of ethanol and water (7:3, v/v; 2 x 150 mL) on an

autoshaker for 45 min at room temperature (25 ºC). After centrifugation (5000 rpm for 10

min) and vacuum filtration through a Whatman 114 filter paper (Whatman International

Ltd., Maidstone, U.K.), the supernatant was collected and the residue discarded. The

supernatant was then evaporated under reduced pressure (30 ºC; 1.0 x 10-3

bar) using a

Laborota 4003 rotary evaporator (Heidolph Brinkman LLC, Elk Grove Village, IL) and

lyophilized, yielding a crude bulb extract (13.7 g). The lyophilized crude bulb extract was

then dissolved in deionized water (100 mL) and washed with ethyl acetate (5 x 100 mL),

and the organic phase was discarded. The aqueous phase was then extracted with n-

butanol (5 x 100 mL) and the aqueous phase discarded. The organic phase was then

evaporated under reduced pressure (30 ºC; 1.0 x 10-3

bar) and lyophilized, yielding a

crude glycoside extract (2.42 g) (Figure 2.1).

54

Figure 2.1. Isolation scheme for compounds 1 – 5 isolated from the bulbs of L.

longiflorum. EtOH, ethanol; EtAC, ethyl acetate; n-BuOH, n-butanol; ACN, acetonitrile.

2.3.3.2. Gel Permeation Chromatography (GPC)

Crude glycoside extract (1.0 g) was dissolved in a solution of ethanol and water

(7:3, v/v; 5.0 mL), filtered with a 0.45 μm PTFE syringe filter (Thermo Fisher Scientific

Inc.), and then applied onto a standard threaded 4.8 cm x 60 cm glass column (Kimble

Chase Life Science and Research Products LLC, Vineland, NJ) packed with Sephadex

LH-20 (Amersham Pharmacia Biotech, Uppsala, Sweden) that was washed and

conditioned in the same solvent mixture overnight. Chromatography was performed with

55

isocratic ethanol/water (70:30, v/v) at a flow rate of 3.5 mL min-1

. The first 200 mL of

effluent was discarded, and 30 fractions (25 mL each) were collected and analyzed by

LC-MS. LC-MS analyses were performed on a HP 1100 series HPLC (Agilent

Technologies Inc., Santa Clara, CA) equipped with an autoinjector, a quaternary pump, a

column heater, and a diode array detector and interfaced to a Bruker 6300 series ion-trap

mass spectrometer equipped with an electrospray ionization chamber. HP ChemStation

and BrukerData Analysis software were used for data acquisition and data analysis.

Reverse phase separations were performed using a Prodigy C18 column (250 mm x 4.6

mm i.d.; 5.0 μm particle size) (Phenomenex, Torrance, CA). The flow rate was set to 1.0

mL min-1

, and the column temperature was at 23 ± 2 ºC. The binary mobile phase

composition consisted of (A) 0.1% formic acid in deionized water and (B) 0.1% formic

acid in acetonitrile. Chromatography was performed using a linear gradient of 15 – 43%

B over 40 min and then to 95% B over 5 min; thereafter, elution with 95% B was

performed for 10 min. The re-equilibration time was 10 min. All mass spectra were

acquired in positive ion mode over a scan range of m/z 100 – 2000. Ionization parameters

included capillary voltage, 3.5 kV; end plate offset, -500 V; nebulizer pressure, 50 psi;

drying gas flow, 10 mL min-1

; and drying gas temperature, 360 ºC. Trap parameters

included ion current control, 30000; maximum accumulation time, 200 ms; trap drive,

61.2; and averages, 12 spectra. On the basis of the LC-MS profile, GPC fractions 6 – 17

were combined, evaporated under reduced pressure (30 ºC; 1.0 x 10-3

bar), and

lyophilized, yielding GPC fraction A (180 mg).

56

2.3.3.3. Semipreparative Reverse-Phase High-Performance Liquid Chromatography (RP-

HPLC)

Fractionation of GPC fraction A was achieved by semipreparative RP-HPLC

performed on a Luna C18 column (250 mm x 21.2 mm i.d.; 10 μm particle size)

(Phenomenex) to afford 1 – 5 (Figure 2.2). Chromatography was performed on a

Shimadzu LC-6AD liquid chromatograph (Shimadzu Scientific Instruments Inc.,

Columbia, MD) using a UV-vis detector and a 2 mL injection loop. Mixtures of (A) 0.1%

formic acid in deionized water and (B) 0.1% formic acid in acetonitrile were used as the

mobile phase. The flow rate was set to 20 mL min-1

, the column temperature was 23±2

ºC, and UV detection was recorded at λ=210nm. GPC fraction A was dissolved in a

mixture of mobile phase A and mobile phase B (75:25, v/v) and filtered through a 0.45

μm PTFE syringe filter prior to injection. Chromatography was performed using a linear

gradient of 5 – 30% B over 45 min and then to 90% B over 10 min; thereafter, elution

with 90% B was performed for 10 min. The re-equilibration time was 10 min.

57

Figure 2.2. RP-HPLC chromatogram (λ = 210 nm) of 1 – 5 isolated from L. longiflorum

n-butanol extract fractionated by gel permeation chromatography (GPC).

The target compounds were collected, freed from solvent under reduced pressure (30 ºC;

1.0 x 10-3

bar), and lyophilized. Final purification of 1 and 2 was performed with an

isocratic separation using a mixture of 0.1% formic acid in deionized water and 0.1%

formic acid in acetonitrile (80:20, v/v). The target compounds were collected, freed from

solvent under reduced pressure (30 ºC; 1.0 x 10-3

bar), and lyophilized, yielding 1 (15mg)

and 2 (7 mg) as white amorphous powders in high purity (>98%), as determined by LC-

MS (Figure 2.3) and NMR. Final purification of 3 – 5 was performed with an isocratic

separation using a mixture of 0.1% formic acid in DI water and 0.1% formic acid in

acetonitrile (75:25, v/v). The target compounds were collected, freed from solvent under

reduced pressure (30 ºC; 1.0 x 10-3

bar), and lyophilized, yielding 3 (25 mg), 4 (7 mg),

and 5 (5 mg) as white amorphous powders in high purity (>98%), as determined by LC-

MS (Figure 2.3) and NMR.

5

10

15

32

20

34 36 38 40 42

Inte

nsity (

210)

time (min)

1

2

3

4

5

58

Figure 2.3. (A) Total ion chromatogram (TIC) of crude L. longiflorum n-butanol extract

(B – F) Total ion chromatograms (TIC) of 1 – 5 isolated by RP-HPLC.

1

2 3 4

5

A

B

C

D

E

F

1

3

2

4

5

5 10 15 20 25 30 35 Time [min]

0

1

2

3

0

1

2

0

2

4

0

2

4

8 x10

Intens.

0

2

4

0

1

2

3

59

2.3.4. Structural Elucidation

Compounds 1 – 5 (Figure 2.4) were identified by a combination of spectroscopic

data (1H NMR,

13C NMR, HMBC, HMQC, MS, IR), chromatographic data, and chemical

analysis. Melting points were obtained using a Thomas-Hoover Capillary Melting Point

Apparatus (Arthur H. Thomas Co., Philadelphia, PA) and by differential scanning

calorimetry using a Perkin-Elmer Diamond DSC (Perkin-Elmer, Waltham, MA). IR

spectra were recorded on a Nexus 670 FT-IR spectrophotometer. Observed rotations were

obtained on a Perkin-Elmer model 341LC polarimeter. High -resolution mass spectra

(HRMS) were recorded on a BioTOF II ESI under the following conditions: source

temperature, 150 ºC; acceleration voltage, 8500; mass resolution, 10000 fwhm; scan

range, m/z 100 – 1000; drying gas, N2. ESI+–MS spectra were recorded on a Bruker 6300

series ion-trap mass spectrometer under the conditions reported above.1D1H NMR and

13CNMR spectra and 2D heteronuclear multiple bond coherence (HMBC) and

heteronuclear multiple quantum coherence (HMQC) spectra were acquired on an AMX-

400 spectrometer and an AMX-500 spectrometer (Bruker, Rheinstetten, Germany).

Samples for NMR analysis were dissolved in pyridine-d5, and chemical shifts are given

as δ values with reference to tetramethylsilane (TMS).

60

Figure 2.4. Structures of compounds 1 – 5 isolated from L. longiflorum bulbs.

O

R3OO

OH

R2O

OH3CHO OHOH

O

O

OH

O

HO

OH

HO

OH

O

O

HOO

OH

R1O

OH3CHO

OH OH

O

O

HN

O

HO

O

HO

OH

O

O

HO

OH

OH

O

HOHO

OH

O

HO

OH

HO

OH

R1

R2 R3

H

H

H

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

1''''

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

6-Ac- -D-Glcp-D-Glcp

1

2

4

5

-L-Arap -D-Xlyp

3

-D-Glcp

6-Ac- -D-Glcp

-D-Glcp

-L-Arap

-D-Xlyp

61

2.3.4.1. Acid Hydrolysis of Compounds 1 – 5.

A solution of each compound (1 mg) in 1NHCl in methanol (0.5 mL) was

refluxed at 80 ºC for 2 h. After hydrolysis, the solution was adjusted to pH 7 with NaOH

(4 N) and evaporated to dryness under reduced pressure (30 ºC; 1.0 x 10-3

bar). The

residue was dissolved in water (1 mL) and extracted with n-pentane (2 mL). The n-

pentane phase was used for aglycone analysis, and the aqueous phase was used for sugar

analysis.

2.3.4.2. Aglycone Analysis

The n-pentane phase obtained after hydrolysis was evaporated to dryness under


bar), dissolved in a mixture of pyridine and BSTFA

with TMCS (99:1) silylating reagent (1:1, v/v, 100 μL), and refluxed in a sealed tube (60

ºC for 1 h). After cooling to room temperature, the solution was analyzed by GC-MS. An

Agilent 6890 series GC system coupled to an Agilent 5973 mass spec detector (Santa

Clara, CA) was used for GC-MS analysis. A capillary column (HP-5, 5% phenyl, 95%

dimethyl polysiloxane stationary phase, 30 m x 0.25 mm i.d. x 0.25 mm film thickness)

was used for the chromatographic separation. The temperature program was as follows:

70 ºC for 2 min, then increased 8 ºC min-1

to 240 ºC, and held for 10 min. The other

parameters used were splitless injector heated at 250 ºC and helium as the carrier gas with

a constant flow of 1 mL min-1

. MS parameters were as follows: operated in electron

62

impact (EI) ionization mode at 70 eV; scan range, m/z 50 – 550. The transfer line was

maintained at 250 ºC. Comparisons were made with retention times and mass spectra of

aglycone reference standards prepared according to the same procedure.

2.3.4.3. Sugar Composition Analysis

The aqueous fraction obtained after hydrolysis was evaporated to dryness under


bar), dissolved in a mixture of pyridine and BSTFA

with TMCS (99:1) silylating reagent (1:1, v/v, 100 μL), and refluxed in a sealed tube (60

ºC for 1 h). After cooling to room temperature, the solution was analyzed by GC-MS.

The temperature program was as follows: 50 ºC for 6 min, then increased at 4 ºC min-1

to

160 ºC, and held for 5 min. The other parameters used were a splitless injector heated to

200 ºC and helium as the carrier gas with a constant flow of 1 mL min-1

. MS parameters

were the same as reported above. Identifications were made based on retention times and

mass spectra of sugar standards prepared according to the same procedure.

2.3.4.4. Determination of Sugar Absolute Configurations

Absolute configuration of sugars was determined by enantioselective GC-FID. A

chiral RTBetaDEXsm capillary column (30m x 0.25mm x 0.25 μm; Restek Corp., State

College, PA) was used for chromatographic separation. The temperature program was as

63

follows: 60 ºC for 0 min, then increased at 4 ºC min-1

to 160 ºC, then increased at 15 ºC

min-1

to a final temperature 230 ºC, and maintained for 15 min. The other parameters

used were as follows: FID detector heated to 230 ºC; split injector with a 10:1 split ratio

maintained at 230 ºC; helium as the carrier gas with a constant flow of 1 mL min-1

.

Comparisons were made with retention times of optically pure sugar standards prepared

following the same procedure.

2.3.4.5. Thin Layer Chromatography (TLC)

Each compound (1 mg) was dissolved in methanol (0.5 mL), spotted on a 20 cm x

20 cm silica gel 60 F254 TLC plate (Merck & Co., Inc., Whitehouse Station, NJ), and

developed with chloroform/methanol/water (8:4:1, v/v/v). To detect furostanols, TLC

plates were developed with Ehrlich‘s reagent [3.2 g of p-(dimethylamino)benzaldehyde

in 60 mL of 95%ethanol and 60 mL of 12 N HCl] and heated to 110 ºC for 5 min. Bright

red spots were indicative of a positive reaction. To detect alkaloids, the TLC plates were

developed with Dragendorff‘s reagent and heated to 110 ºC for 5 min. Orange spots were

indicative of a positive reaction.

64

2.4. Results and Discussion

2.4.1 Structure Elucidation of Compounds 1 – 5.

Lyophilized lily bulbs were washed with n-pentane and extracted with ethanol

and water. After the removal of solvent, the extract was dissolved in deionized water,

washed with ethyl acetate and extracted with n-butanol. The organic phase was

evaporated under reduced pressure and lyophilized, yielding a crude steroidal glycoside

extract. The crude glycoside extract was fractionated by gel permeation chromatography

(Sephadex L-H20) and repeated semi-prep RP-HPLC to yield compounds 1 – 5 (Figure

2.3). Based on 1H NMR,

13C NMR, 2D NMR (HMQC and HMBC), HRESI–TOFMS and

chemical analysis, including GC-MS analysis of the sugar and aglycone TMSi derivatives

after acid hydrolysis, 1 and 3 were identified as (22R, 25R)-spirosol-5-en-3 -yl O- -L-

rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside, previously

isolated from L. brownii (Mimaki and Sashida et al., 1990), and (25R)-26-O-( -D-

glucopyranosyl)-furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -D-

glucopyranosyl-(1→4)- -D-glucopyranoside, previously isolated from Dioscorea

deltoidea Wall. ex Griseb., Ophiopogon planiscapus Nakai, L. hansonii and Allium

nutans L. (Sviridov et al., 1975; Watanabe et al., 1983; Ori et al., 1992; Akhov et al.,

1999 ) (Figure 2.4). This is the first report of these compounds isolated from the bulbs of

L. longiflorum. Compound 2 was characterized as a new acetylated steroidal

glycoalkaloid. The structure of compound 2 was determined to be (22R, 25R)-spirosol-5-

65

en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-glucopyranosyl-(1→4)]- -D-

glucopyranoside. Compounds 4 and 5 were characterized as new furostanol saponins.

The structure of compound 4 and 5 were determined to be (25R)-26-O-( -D-


arabinopyranosyl-(1→3)- -D-glucopyranoside and (25R)-26-O-( -D-glucopyranosyl)-


(1→3)- -D-glucopyranoside, respectively. This is the first report of these new natural

products.

The 1H NMR spectra of steroidal glycoside are complex to analyze due to

overlapping proton resonances. For example, the proton resonances of the carbohydrate

moiety occur in a narrow spectral width of 3.0 – 4.2 ppm, making assignments difficult.

Despite overlapping signals, there are many diagnostic features in the 1H NMR spectrum.

The methyl peaks of the steroidal backbone are readily distinguishable and informative.

For example, resonances integrating for three protons which are indicative of tertiary

methyl groups and secondary methyl groups are diagnostic of the aglycone. Additionally,

the 13

C NMR spectra have many diagnostic characteristics. For example, olefinic carbon

resonances at approximately C 139.7 – 142.1 and 120.9 – 124.3 in the 13

C NMR

spectrum are indicative of unsaturation between C-5 and C-6. A quarternary carbon

signal at approximately C ~110 ppm suggests a furostane skeleton possessing a hydroxyl

group at the C-22 position whereas C ~113 ppm suggests the methoxyl furostane

derivative (Agrawal, 1995).

Resonances in the 1H NMR and

13C NMR spectra are useful for the determination

of both the number of saccharide residues and anomeric configurations of interglycosidic

66

linkages. Anomeric carbon resonances of the oligosaccharide moiety typically occur in

the C 92 – 108 region of the 13

C NMR spectra, thus allowing the number of individual

sugar residues to be determined. In the 1H NMR spectra, anomeric proton resonances

typically occur as broad singlets or doublets in the range of C 4.2 – 6.4 ppm.

Furthermore, coupling constants for anomeric proton resonances of the C-1 position of -

linked sugars have a coupling constant of approximately J = 1.0 – 3.0 Hz and -linked

sugars have a coupling constant of approximately J = 6.0 – 8.0 Hz. (Agrawal, 1995).

Although there are many distinguishable and diagnostic features of 1H NMR spectra,

overlapping resonances often inhibit complete assignments and do not provide sufficient

information for the determination of integlycosidic linkages. With 2-D NMR techniques

such as HMBC, often the integlycosidic linkages can be determined; however, the

absolute configurations of sugars can not be determined. One approach to determine the

absolute configurations of the monosaccharide residues is by acid-catalyzed hydrolysis,

derivatization and entantioslective GC-FID analysis. This approach allows for the

determination of the absolute configurations of the monosaccharide residues of the

carbohydrate moiety.

67

2.3.4.1. Structure Elucidation of Compound 1

Compound 1 was obtained as a white amorphous powder. The compound was

positive to the Dragendorff reaction, indicative of an alkaloid. The IR spectrum showed

absorption due to the presence hydroxyl groups at 3400 cm-1

. HRESI–TOFMS showed a

[M + H]+ ion at m/z: 884.5028 (calculated for C45H74NO16, 884.5002). Additionally,

[M+H+Na]++

ion was observed at m/z 453.7443 (calculated for C45H74NO16Na,

453.7447) (Figure 2.5). Thus, molecular formula was calculated as C45H73NO16,

suggestive of steroidal glycoalkaloid. The aglycone was readily deduced from 1H NMR,

13C NMR (Figure 2.12; Table 2.1), ESI

+–MS (Figure 2.33) and chemical analysis. The

1H NMR spectrum showed two singlets at 1.06 and 0.88 which is indicative of tertiary

methyl groups of the spirosolane skeleton. Furthermore, two doublet signals at 1.09

and 0.82 were assignable to secondary methyl groups. A quaternary carbon signal at C

98.4 and olefinic carbon signals at C 140.8 and 121.8 in the 13

C NMR spectrum were

consistent with a 5 spirosolane aglycone. The unsaturation between C-5 and C-6 is

further substantiated by a doublet at 5.3 in the 1H NMR spectrum for the H-6 signal.

68

Figure 2.5. (A) High resolution mass spectrum of compound 1 and (B) expanded view of

[M + H + Na]++

ion at m/z 453.7443 (calculated for C45H74NO16Na, 453.7447). The mass

spectrum was acquired on a BioTOF II.

12

9.0

49

4

15

4.1

54

7

22

7.2

42

6

36

9.1

75

2

38

7.1

91

6

43

8.2

85

1

45

3.7

44

34

67

.36

02

49

6.3

31

0

52

5.3

52

5

55

4.3

72

6

58

3.3

92

8

61

2.4

13

8

64

1.4

32

7

67

0.9

64

3

69

9.9

73

8

75

7.5

12

9

79

5.5

42

1

85

3.5

90

8

88

4.5

02

8

91

1.6

24

8

96

9.6

74

1

10

27

.71

72

200 400 600 800 1000 m/z0

250

500

750

1000

1250

1500

Intens.

45

3.7

44

3

45

4.2

45

0

45

4.7

45

2

450 451 452 453 454 455 456 457 458 459 m/z0

250

500

750

1000

1250

Intens.

A

B

69

Upon acid hydrolysis, derivatization and GC-MS analysis, the retention time and mass

spectrum of the TMSi derivative of the aglycone was consistent with that of the steroidal

alkaloid, (22R, 25R)-spirosol-5-en-3 -ol (solasodine), which was prepared following the

same procedure. In 2008, Eanes and Tek reported that during derivitazation of

solasodine, two products are formed (Eanes and Tek, 2008). Consistent with their

observations, two products were detected by GC-MS for both solasodine and the

aglycone of compound 1 (Figure 2.6; Figure 2.7; Figure 2.8). In addition to

derivitazation and GC-MS analysis, the retention time and mass spectrum the aglycone of

compound 1 was consistent with that of authentic (22R, 25R)-spirosol-5-en-3 -ol

analyzed by LC-MS (Figure 2.9).

Figure 2.6. GC-MS chromatogram of the TMSi derivatives of authentic (22R, 25R)-

spirosol-5-en-3 -ol standard. Two products are generated during derivatization, labeled

A and B.

16.00 17.00 18.00 19.00 20.00 21.00 22.00 23.00 24.00 25.00 26.00 27.00 28.00 29.00 Time-->

100000 200000 300000 400000 500000 600000 700000 800000 900000

1000000 1100000 1200000 1300000 1400000 1500000 1600000 1700000 1800000 1900000 2000000 2100000 2200000

Abundance

A

B

70

Figure 2.7. GCMS mass spectra of TMSi derivatives, A and B, generated from authentic

(22R, 25R)-spirosol-5-en-3 -ol standard.

50 100 150 200 250 300 350 400 450 500 550 0

5000

10000

15000

20000

25000

30000

35000

40000

45000

50000

55000

60000

65000

70000

75000

m/z-->

Abundance 111

73

557 138 452 162 190 412 347 238 281 41 484 528 384 214

A

50 100 150 200 250 300 350 400 450 500 550 0

100000

200000

300000

400000

500000

600000

700000

800000

900000

m/z-->

Abundance 125

73 452 238

148

96 542 484 41 181 207 362 394 308

B

71

Figure 2.8. GCMS mass spectra of TMSi derivatives, A and B, generated from the

aglycone of compound 1.

m/z--> 50 100 150 200 250 300 350 400 450 500 550 0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

30000

Abundance 111

73

557 138 452 162

190 412 238 281 41 484 528

50 100 150 200 250 300 350 400 450 500 550

0

220000

240000

260000

280000

300000

320000

340000

360000

380000

m/z-->

Abundance 125

73 452

238 148 96 542 484 41 208 394 309

72

Figure 2.9. Total ion chromatogram (TIC) of (A) authentic (22R, 25R)-spirosol-5-en-3 -

ol standard (Rt = 32.1 min) and (B) the aglycone of compound 1 (Rt = 32.1 min)

generated by LC-MS.

The structure of the oligosaccharide moiety was readily deduced from 1H NMR,

13C

NMR, ESI+–MS, and chemical analysis. Three anomeric protons were observed at

4.97, 6.27, and 5.15, which implied the presence of three saccharide residues. Coupling

constants of the anomeric proton resonances suggested -interglycosidic linkages.

5 10 15 20 25 30 35 0

1

2

3

8 x10 Intens.

Time [min]

A

0

1

2

3

4

5

8 x10 Intens.

5 10 15 20 25 30 35 Time [min]

B

73

Additionally, the 13

C NMR spectrum contained three anomeric carbon signals observed at

C 105.3, 1 and consistent with the presence of three saccharide residues.

The HMBC experiment showed long-range correlations between the anomeric proton

signal at 4.97 [H-1′] and the carbon signal at C 78.1 [C-3], between the anomeric

proton signal at 5.15 [H-1′′′] and the carbon signal at C 82.1 [C-4′], and the anomeric

proton signal at 6.27 [H-1′′] and the carbon signal at C 77.8 [C-2′] (Figure 2.10).

Figure 2.10. HMBC long-range correlations for the interglycosidic linkages for the

carbohydrate moiety of compound 1.

O

HO

O

OH

O

OH3CHO

OHOH

O

O

HO

OH

HO

OHH

H

H

H H 78.1

4.97 (7.2) 77.8

6.27

5.15 (7.6)

82.1

1

Upon acid hydrolysis, derivatization and GC-MS/entantioslective GC-FID analysis, the

sugars of the trisaccharide moiety were identified as D-(+)-glucose and L-(–)-rhamnose in

a 2 to 1 ratio.1H NMR spectrum showed a doublet integrating for three protons at 1.78,

which is indicative of the methyl group of rhamnose. The ESI+–MS mass spectrum

showed the molecular ion 884.7 [M+H]+

and the doublely charged sodium adduct at

453.8 [M+H+Na]++

. Additionally, ion fragments at m/z 738.5 [M–Rha+H]+, 576.4 [M–

Glu–Rha+H]+, and 414.4 [M–2Glu–Rha+H]

+ were observed and were consistent with a

74

trisaccharide moiety containing two D-(+)-glucoses and a L-(–)-rhamnose moiety (Figure

2.11).

Figure 2.11. ESI+-MS mass spectrum of compound 1.

Accordingly, the structure of 1 was determined to be (22R, 25R)-spirosol-5-en-3 -yl O-

-L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside,

previously reported in L. brownii (Mimaki and Sashida, 1990b).

253.1

414.4

453.8

576.4

738.5

884.7

0.0

0.5

1.0

1.5

6 x10

Intens.

200 400 600 800 1000 1200 1400 m/z

75

Figure 2.12. (A) 1H NMR spectrum and (B)

13C NMR spectrum of compound 1.

A

B

76

Compound 1, (22R, 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)- -D-

glucopyranosyl-(1→4)- -D-glucopyranoside: amorphous solid; [28

D - 76.9 (c 0.02;

MeOH); mp 303 ºC (dec); IR max (film) cm-1

: 3336 (OH), 2930 (CH), 1585, 1450, 1347,

1255, 1029, 984, 897, 811; HRESI–TOFMS, m/z 884.5028 [M + H]+ (calculated for

C45H74NO16, 884.5002); ESI+

– MS, m/z 884.7 (100, [M+H]+

), 738.5 (3, [M–Rha+H]+),

576.4 (19, [M–Glu–Rha+H]+), 453.8 (1.6, [M+H+Na]

++ ), 414.4 (15, [M–2Glu–Rha+H]

+

); 1H NMR (400 MHz) 0.82 [d, 3H, J = 5.2 Hz, 27-H], 0.88 [s, 3H, 18-H], 1.06 [s, 3H,

19-H], 1.09 [d, 3H, J = 6.8 Hz, 21-H], 1.78 [d, 3H, J = 6.4 Hz, 6′′-H], 2.75 [m, 2H, 26-

H], 3.88 [m, 1H, 3-H], 3.90 [m, 1H, 2′-H], 4.33 [1H, 6b′′′-H], 4.56-4.40 [1H, 6a′′′-H],

4.48 [m, 2H, 6′-H], 4.61 [dd, 1H, J =9.2, 3.2, 3′′-H], 4.97 [d, 1H, J = 7.2 Hz, 1′-H], 5.15

[d, 1H, J =7.6 Hz, 1′′′-H], 5.30 [d, 1H, J = 4.8 Hz, 6-H], 6.27 [s, 1H, 1′′-H]; 13

C NMR

(400 MHz, pyridine-d5) 37.5 [C-1], 30.2 [C-2], 78.1 [C-3], 38.9 [C-4], 140.8 [C-5],

121.8 [C-6], 32.4 [C-7], 31.6 [C-8], 50.3 [C-9], 37.2 [C-10], 21.2 [C-11], 40.1 [C-12],

40.7 [C-13], 56.7 [C-14], 32.6 [C-15], 78.8 [C-16], 63.6 [C-17], 16.5 [C-18], 19.4 [C-19],

41.6 [C-20], 15.7 [C-21], 98.4 [C-22], 34.7 [C-23], 31.1 [C-24], 31.7 [C-25], 48.1 [C-26],

19.9 [C-27], 100.0 [C-1′], 77.8 [C-2′], 76.2 [C-3′], 82.1 [C-4′], 77.3 [C-5′], 62.0 [C-6′],

101.8 [C-1′′], 72.5 [C-2′′], 72.8 [C-3′′], 74.2 [C-4′′], 69.5 [C-5′′], 18.7 [C-6′′], 105.3 [C-

1′′′], 75.0 [C-2′′′], 78.3 [C-3′′′], 71.2 [C-4′′′], 78.5 [C-5′′′], 62.1 [C-6′′′]; 1H NMR and

13C

NMR are consistent with the literature (Mimaki and Sashida, 1990b).

77



positive to the Dragendorff reaction, indicative of an alkaloid (Mimaki and Sashida,

1990). The IR spectrum showed absorption at 1732 cm-1

and 3353 cm-1

due were due to

the presence of an acetyl group and hydroxyl groups. HRESI–TOFMS showed a [M+H]+

ion at m/z: 926.5085 (calculated for C47H76NO17, 926.5108). Additionally, [M+H+Na]++

ion was observed at m/z 474.7520 (calculated for C47H76NO17Na, 474.7500) (Figure

2.13). Thus, the molecular formula was calculated as C47H75NO17, suggestive of an

acetylated steroidal glycoalkaloid. The aglycone was readily deduced from 1H NMR,

13C

NMR (Figure 2.16; Table 2.1), ESI+–MS (Figure 2.34), and chemical analysis. The

1H

NMR spectrum showed two singlets at 1.06 and 0.88 which is indicative of tertiary

methyl groups of the spirosolane skeleton. Furthermore, two doublet signals at 1.11

and 0.82 were assignable to secondary methyl groups. A quaternary carbon signal at

C 98.4 and olefinic carbon signals at C 140.8 and 121.9 in the 13

C NMR spectrum were

consistent with a 5 spirosolane aglycone. The unsaturation between C-5 and C-6 was

further substantiated by a doublet at 5.29 in the 1H NMR spectrum for the H-6 signal.

78

Figure 2.13. (A) High resolution mass spectrum of compound 2 and (B) expanded view

of [M + H + Na]++

ion at m/z 474.7520 (calculated for C47H76NO17Na, 474.7500). The

mass spectrum was acquired on a BioTOF II.

12

9.1

16

4

22

7.2

85

8

39

3.3

02

4

43

8.2

87

1

47

4.7

52

0

49

6.3

27

3

52

5.3

52

8

54

2.7

39

5 55

4.3

70

4

58

3.3

93

4

61

2.4

12

0

64

1.4

32

2

66

2.3

90

3

92

6.5

63

5

200 400 600 800 1000 m/z0

1000

2000

3000

4000

5000

Intens.

47

4.7

52

0

47

5.2

53

9

47

5.7

53

3

47

6.2

57

7

472 473 474 475 476 477 478 479 480 m/z0

1000

2000

3000

4000

5000

Intens.

A

B

79


spectrum of the TMSi derivative of the aglycone was consistent with that of (22R, 25R)-

spirosol-5-en-3 -ol, which was prepared following the same procedure. The structure of

the oligosaccharide moiety was readily deduced from 1H NMR,

13C NMR, HMBC, ESI

+–

MS and chemical analysis. Three anomeric protons were observed at 4.98, 6.24 and

5.09, which implied the presence of three saccharide residues. Coupling constants for the

anomeric proton resonances suggested -interglycosidic linkages. Additionally, the 13

C

NMR spectrum contained three anomeric carbon signals observed at C 105.6,

and consistent with the presence of three saccharide residues. The HMBC

experiment showed long-range correlations between the anomeric proton signal at

4.98 [H-1′] and the carbon signal at C 78.1 [C-3], between the anomeric proton signal at

5.09 [H-1′′′] and the carbon signal at C 83.3 [C-4′], and the anomeric proton signal

at 6.24 [H-1′′] and the carbon signal at C 77.6 [C-2′] (Figure 2.14).



O

HO

O

OH

O

OH3CHO

OHOH

O

O

HO

O

HO

OHH

H

H

H H 78.1

4.98 (7.2) 77.6

6.24

5.09 (8)

83.3

O

2

80

Upon acid hydrolysis, derivatization and GC-MS/entantioslective GC-FID analysis, the

sugars of the trisaccharide moiety were identified as D-(+)-glucose and L-(–)-rhamnose in

a 2 to 1 ratio. 1H NMR spectrum showed a doublet integrating for three protons at

1.78, indicative of the methyl group of rhamnose. The ESI+–MS mass spectrum showed

the protonated molecular ion 926.6 [M+H]+

and the protonated double charged sodium

adduct at 474.8 [M+H+Na]++

. Additionally, ion fragments at m/z 780.5 [M–Rha+H]+,

576.4 [M–Glu–Ac– Rha+H]+ and 414.3 [M–2Glu–Ac–Rha+H]

+ were observed and were

consistent with a trisaccharide moiety containing D-(+)-glucose, an acetylated D-(+)-

glucose and a L-(-)-rhamnose moiety (Figure 2.15).

Figure 2.15. ESI+-MS mass spectrum of compound 2.

253.2

414.3

474.8 576.4

780.5

926.7

0

2

4

6

8

6 x10

Intens.

200 400 600 800 1000 1200 m/z

81

The presence of an acetyl group was shown by the IR absorption at1732 cm-1

, H NMR

2.06 (s, 3H, CO-CH3) and 13

C NMR C 170.9 and 20.8. The carbon signals

corresponding to the saccharide moiety of 2 were similar to those reported for (25R,26R)-

26-methoxyspirost-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-

glucopyranosyl-(1→4)]- -D-glucopyranoside, isolated from L. speciosum x L.

nobilissimum (Nakamura et al., 1994). Alkaline hydrolysis of 2 with 1M sodium

hydroxide yielded 1. Upon comparison of the 13

C NMR spectra of 1 and 2, the C-6′′′ and

C-4′′′ signals were shifted from C 62.1 and 71.2, to C 64.8 and 71.9, respectively. The

signal for C-5′′′ was shifted from C 78.5 to 74.9 and all other carbon signals were

similar. Upon comparison of the 1H NMR spectra of 1 and 2, the signals assignable to H2-

6′′′ methylene protons of the terminal glucose were shifted to a lower field as compared

to those of 2. Thus, the acetyl moiety was linked to the C-6 hydroxy position of the

terminal glucose unit. Accordingly, the structure of 2 was determined to be (22R, 25R)-

spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-glucopyranosyl-

(1→4)]- -D-glucopyranoside (Figure 2.4).

82



A

B

83

Compound 2, (22R, 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-

acetyl- -D-glucopyranosyl-(1→4)]- -D-glucopyranoside: amorphous solid; [ D

28 -

32.0º (MeOH; c 0.05); mp 285 ºC (dec); IR max (film) cm-1

: 3353 (OH), 2932 (CH),

1732 (C=O), 1588, 1451, 1370, 1252, 1033, 983, 897, 813; HRESI–TOFMS m/z:

926.5085 [M+H]+ (calculated for C47H76NO17, 926.5108); ESI+–MS, m/z 926.6 (100,

[M+H]+), 780.5 (14, [M–Rha+H]

+), 576.4 (15, [M–Glu–Ac–Rha+H]

+), 474.8 (3,

[M+H+Na]++

), 414.3 (30, [M–2Glu–Ac–Rha+H]+);

1H NMR (400 MHz) 0.82 [d, 3H,

J = 4.8 Hz, 27-H], 0.88 [s, 3H, 18-H], 1.06 [s, 3H, 19-H], 1.11 [d, 3H, J = 6.4 Hz, 21-H],

1.78 [d, 3H, J = 6 Hz, 6′′-H], 2.06 [s, 3H, CO-CH3], 4.35 [t, 1H, J =9.4, 4′′-H], 4.60 [dd,

1H, J =8.8, 3.2, 3′′-H], 4.64 [d, 1H, J = 8.4 Hz, 6b′′′-H], 4.92 [dd, 1H, J =11.6, 2, 6a′′′-H],

4.98 [d, 1H, J = 7.2 Hz, 1′-H], 5.09 [d, 1H, J =8.1′′′-H], 5.29 [d, 1H, J = 4.4 Hz, 6-H],

6.24 [br s, 1H, 1′′-H]; 13

C NMR (500 MHz, pyridine-d5) 37.5 [C-1], 30.2 [C-2], 78.1 [C-

3], 39.0 [C-4], 140.8 [C-5], 121.9 [C-6], 32.4 [C-7], 31.6 [C-8], 50.4 [C-9], 37.2 [C-10],

21.2 [C-11], 40.1 [C-12], 40.7 [C-13], 56.7 [C-14], 32.6 [C-15], 79.0 [C-16], 63.5 [C-17],

16.5 [C-18], 19.4 [C-19], 41.6 [C-20], 15.7 [C-21], 98.4 [C-22], 34.7 [C-23], 31.0 [C-24],

31.7 [C-25], 48.0 [C-26], 19.8 [C-27], 99.9 [C-1‘], 77.6 [C-2‘], 76.1 [C-3‘], 83.3 [C-4‘],

77.4 [C-5‘], 62.0 [C-6‘], 102.0 [C-1‘‘], 72.5 [C-2‘‘], 72.8 [C-3‘‘], 74.2 [C-4‘‘], 69.6 [C-

5‘‘], 18.7 [C-6‘‘], 105.6 [C-1‘‘‘], 75.1 [C-2‘‘‘], 78.2 [C-3‘‘‘], 71.9 [C-4‘‘‘], 74.9 [C-5‘‘‘],

64.8 [C-6‘‘‘], 20.8 [Ac-CH3], 170.9 [AcC=O].

84


Compound 3 was obtained as a white an amorphous powder. The compound was

positive to the Ehrlich‘s reaction, indicative of a furostanol saponin (Yoshiaki et al.,


due to the presence of hydroxyl

groups. HRESI–TOFMS showed a [M + Na]+ ion at m/z: 1087.5307 (calculated for

C51H84O23Na, 1087.5296 ) (Figure 2.17). Additionally, [M–H]- ion was observed at m/z

1063.6 (Figure 2.18). Thus, the molecular formula was calculated as C51H84O23,

consistent with a furostanol saponin. The aglycone was readily deduced from 1H NMR,

13C NMR (Figure 2.22; Table 2.1), ESI

+–MS (Figure 2.34), HMBC, and chemical

analysis. The 1H NMR spectrum showed two singlets at 1.06 and 0.90 which is

indicative of tertiary methyl groups of the furostane skeleton. Furthermore, two doublet

signals at 1.35 and 1.0 were assignable to secondary methyl groups. A quarternary

carbon signal at C 110.7 suggests a furostane skeleton possessing a hydroxyl group at

the C-22 position. (25R)-26-O- -D-glucopyranosyl-22 -methoxy-furost-5-en-3 26-triol

3-O- -L-rhamnopyranosyl-(1→2- -D-glucopyranosyl-(1→4)- -D-glucopyranoside,

previously identified in L. longiflorum possessing antitumor activity, contains a methoxy

group at the C-22 position with a OCH3 signal at C 47.73 which is missing in 3. Also,

the quaternary carbon signal for C-22 had a minor upfield shift to C 110.7 instead of the

reported value of C 112.7 for the C-22 methoxy derivative (Mimaki et al., 1994).

85


of [M + Na]+ ion at m/z: 1087.5307 (calculated for C51H84O23Na, 1087.5296 ). The mass


22

9.1

71

5

30

9.2

26

1

37

1.1

13

73

93

.31

44

42

7.2

23

2

46

7.3

19

5

49

6.3

20

4

52

5.3

60

2

55

5.2

71

1

58

3.3

99

5

61

2.4

19

6

64

1.4

38

6

67

0.4

64

4

69

9.4

77

1

73

7.5

02

8

79

5.5

47

0

85

3.5

87

2

91

1.6

28

5

96

9.6

66

3

10

27

.71

22

10

87

.53

07

11

43

.79

58

200 400 600 800 1000 1200 1400 1600 1800 m/z0

200

400

600

800

Intens.

10

85

.75

54

10

86

.75

67

10

87

.53

07

10

88

.02

63

10

88

.53

43

10

89

.53

68

1084 1086 1088 1090 1092 1094 m/z0

200

400

600

Intens.

A

B

86

Figure 2.18. LRMS- mass spectrum of compound 3 acquired on BioTOF II.

14

5.0

17

1.1

21

2.1

53

1.3

10

63

.61

08

3.6

200 400 600 800 1000 1200 1400 1600 1800 m/z0

200

400

600

Intens.

C-22 hydroxy furostanols are readily converted to C-22 methoxy derivatives upon

refluxing in methanol (Watanabe et al., 1983; Wang et al., 2003). It has been suggested

that C-22 methoxy furostanol saponins identified in plants may be artifacts formed during

the extraction process when methanol is used as a solvent (Oleszek and Bialy, 2006).

The carbon peaks from C-5 to C-27 were similar to those reported for (25R,S)-26-

O- -D-glucopyranosyl-furost-5-en-3 22 -triol 3-O- -D-galactopyranosyl-(1→2)-O-

-D-glucopyranosyl-(1→4)- -D-galactopyranoside, isolated from Tribulus terrestris L.,

which suggests the presence of an α-hydroxy group at the C-22 position of the furostane

skeleton (Wang et al., 1997). Olefinic carbon signals at C 140.8 and 121.9 in the 13

C

87

NMR spectrum are consistent with a 5 furostane skeleton. The unsaturation between C-5

and C-6 is further substantiated by a doublet at 5.29 in the 1H NMR spectrum for the H-

6 signal. Upon acid hydrolysis, derivatization and GC-MS analysis, the retention time

and mass spectrum of the TMSi derivative of the aglycone was consistent with that of

(25R)-spirost-5-en-3 -ol, which was prepared following the same procedure (Figure

2.19). The structure of the oligosaccharide moiety was readily deduced from 1H NMR,

13C NMR, HMBC, ESI–MS

+ and chemical analysis. The

1H NMR spectrum contained

four anomeric proton signals observed at 6.26, 4.96, and . The coupling

constants of the anomeric proton resonances suggested -interglycosidic linkages. The

13C NMR spectrum contained four anomeric carbon signals observed at C 105.2, 105.0,

and consistent with the presence of four saccharide residues.

88

Figure 2.19. GCMS spectra of (A) TMSi derivative of the aglycone of compound 3 and

(B) TMSi derivative of (25R)-spirost-5-en-3 -ol standard.

50 100 150 200 250 300 350 400 450 0

2000

4000

6000

8000

10000

12000

14000

16000

18000

20000

22000

24000

26000

28000

m/z-->

Abundance

139

282

73 187

243 372 119 95

207 414 159 41 343 471

B

50 100 150 200 250 300 350 400 450 m/z--> 0

50000

100000

150000

200000

250000

300000

350000

400000

450000

500000

550000

600000

Abundance 139

282

187 73

243 372 119

93 159 414 213 343 41 471

A

89

The HMBC experiment showed long-range correlations between the anomeric proton

signal at 4.96 [H-1′] and the carbon signal at C 78.2 [C-3], between the anomeric

proton signal at 5.14 [H-1′′′] and the carbon signal at C 82.1 [C-4′], and the anomeric

proton signal at 6.26 [H-1′′] and the carbon signal at C 77.8 [C-2′] (Figure 2.20).



O

HO

O

OH

O

OH3CHO

OHOH

O

O

HO

OH

HO

OHH

H

H

H H 78.2

4.96 (7.2) 77.8

6.26

5.14 (8)

82.1

3

Upon acid hydrolysis, derivitazation and GC-MS/entantioslective GC-FID analysis, the

sugars were identified as D-(+)-glucose and L-(-)-rhamnose in a 3:1 ratio. 1H NMR

spectrum showed a doublet integrating for three protons at 1.77, which is indicative of

the methyl group of rhamnose. The ESI+–MS mass spectrum showed a base ion peak at

1047.7 [M-18+H]+ and the sodium adduct at 1087.7 [M + H+Na]

+. Additionally, ion

fragments at m/z 901.5 [M-18-Rha+H]+, 739.4 [ M-18-Glu-Rha+H]+, 577.4 [M-18-

2Glu-Rha+H]+, and 415.3 [M-18-3Glu-Rha+H]

+ were observed and are consistent with 3

being bidesmodic with the trisaccharide moiety at the C3 position containing two

90

glucoses and one rhamnose, and a glucose moiety at the C26 position, indicative of a

furostanol saponin (Figure 2.21).

Figure 2.21. ESI+–MS mass spectrum of compound 3.

Accordingly, the structure of 3 was determined to be (25R)-26-O-( -D-glucopyranosyl)-

furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-

(1→4)- -D-glucopyranoside, previously reported from D. deltoidea, O. planiscapus, L.

hansonii, and A. nutans (Sviridov et al., 1975; Watanabe et al., 1983; Ori et al., 1992;

Akhov et al., 1999) (Figure 2.4).

253.1 415.3 577.4 739.4 901.5

1047.7

1087.7

0

1

2

3

4

6 x10

Intens.

200 400 600 800 1000 1200 1400 m/z

91



A

B

92

Compound 3, (25R)-26-O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol 3-O- -L-

rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside: amorphous

solid; [28

D - 60.0 (c 0.05; MeOH); mp 207 ºC (dec): IR max (film) cm-1

: 3347 (OH),

2893 (CH), 1662, 1377, 1256, 1027, 909, 839, 812; HRESI–TOFMS m/z: 1087.5296

[M+Na]+ (calculated for C51H84O23Na, 1087.5307); ESI

+ – MS, m/z 1087.7 (20,

[M+Na]+), 1047.7 (100, [M–18+H]

+), 901.5 (9, [M–18–Rha+H]

+), 739.4 (17, [M–18–

Glu–Rha+H]+), 577.4 (11, [M–18–2Glu–Rha+H]

+), 415.3 (9, [M–18–3Glu–Rha+H]

+);

1H NMR (500 MHz) 0.91 [s, 3H, 18-H], 1.00 [d, 3H, J = 6.4 Hz, 27-H], 1.07 [s, 3H,

19-H], 1.35 [d, 3H, J = 6.8 Hz, 21-H], 1.76 [d, 3H, J = 6.5 Hz, 6′′-H], 4.56 [dd, 1H, J

=12, 2.5, 6′′′′-H], 4.60 [dd, 1H, J =9.5, 3.5, 3′′-H], 4.83 [d, 1H, J = 8 Hz, 1′′′′-H], 4.96 [d,

1H, J = 6.5 Hz, 1′-H], 5.14 [d, 1H, J =8, 1′′′-H], 5.29 [d, 1H, J = 4 Hz, 6-H], 6.26 [br s,

1H, 1′′-H]; 13

C NMR (400 MHz, pyridine-d5) 37.5 [C-1], 30.2 [C-2], 78.2 [C-3], 39.0 [C-

4], 140.8 [C-5], 121.9 [C-6], 32.5 [C-7], 31.7 [C-8], 50.4 [C-9], 37.2 [C-10], 21.2 [C-11],

39.9 [C-12], 40.8 [C-13], 56.6 [C-14], 32.4 [C-15], 81.1 [C-16], 63.9 [C-17], 16.5 [C-18],

19.4 [C-19], 40.7 [C-20], 16.5 [C-21], 110.7 [C-22], 37.1 [C-23], 28.4 [C-24], 34.3 [C-

25], 75.3 [C-26], 17.5 [C-27], 100.0 [C-1′], 77.8 [C-2′], 76.2 [C-3′], 82.1 [C-4′], 77.3 [C-

5′], 62.1 [C-6′], 101.8 [C-1′], 72.5 [C-2′′], 72.8 [C-3′′], 74.2 [C-4′′], 69.5 [C-5′′], 18.7 [C-

6′′], 105.2 [C-1′′′], 75.0 [C-2′′′], 78.5 [C-3′′′], 71.2 [C-4′′′], 78.3 [C-5′′′], 61.9 [C-6′′′],

105.0 [C-1′′′′], 75.2 [C-2′′′′], 78.6 [C-3′′′′], 71.7 [C-4′′′′], 78.2 [C-5′′′′], 62.8 [C-6′′′′]; 1H

NMR and 13

C NMR are consistent with the literature (51).

93



positive to the Ehrlich‘s reaction, indicative of a furostanol saponin (Yoshiaki et al.,


due to the presence of hydroxyl

groups. HRESI–TOFMS showed a [M+Na]+ ion at m/z: 1057.5211 (calculated for

C50H82O22Na, 1057.5190) (Figure 2.23). Additionally, [M–H]- ion was observed at m/z

1033.6 (Figure 2.24). Thus, the molecular formula was calculated as C50H82O22,

consistent with a furostanol saponin. The aglycone was readily deduced from 1H NMR,

13C NMR (Figure 2.28; Table 2.1), HMBC, ESI

+–MS (Figure 2.34), and chemical

analysis. The 1H NMR spectrum showed two singlets at 1.07 and 0.91 which is

indicative of the tertiary methyl groups of the furostane skeleton. Furthermore, two

doublet signals at 1.35 and 1.0 were assignable to secondary methyl groups. (25R)-26-

O- -D-glucopyranosyl-22 -methoxy-furost-5-en-3 26-diol 3-O- -L-rhamnopyranosyl-

(1→2)- -L-arabinopyranosyl-(1→3)- -D-glucopyranoside, previously identified in L.

longiflorum possessing antitumor activity, contains a methoxy group at the C-22 position

with a OCH3 signal at C 47.73 which is missing in 4. Also, the quaternary carbon signal

for C-22 had a minor upfield shift to C 110.7 instead of the reported value of C 112.7

for the C-22 methoxy derivative (Mimaki et al., 1994).

94


of [M + Na]+ ion at m/z: 1057.5211 (calculated for C50H82O22Na, 1057.5190). The mass


22

9.1

74

4

30

9.2

30

6

39

3.3

18

2

43

8.3

03

4 46

7.3

26

0 49

6.3

44

1

54

0.2

66

4

58

3.4

02

8

61

2.4

22

9

64

1.4

42

8

67

0.4

66

1

69

9.4

79

2

73

7.5

05

1

76

3.6

08

9 79

5.5

39

2

85

3.5

89

5

91

1.6

27

7

96

9.6

70

6

10

27

.71

28

10

57

.52

11

10

85

.73

76

11

43

.79

51

12

01

.83

76

200 400 600 800 1000 1200 1400 1600 1800 m/z0

250

500

750

1000

1250

1500

Intens.

10

57

.52

11

10

58

.01

41

10

58

.52

65

10

59

.52

73

1056 1058 1060 1062 1064 m/z0

200

400

600

Intens.

A

B

95


14

3.1

17

1.1

21

2.1

51

6.2

10

33

.6

200 400 600 800 1000 1200 1400 1600 1800 m/z0

250

500

750

1000

1250

1500

Intens.

Long range coupling was observed between the methyl proton signal at H-21]

and the carbon signals at C 40.7 [C-20], 63.9 [C-17] and 110.7 [C-22], supporting that

the shift seen in this region is consistent with a C-22 hydroxy compared to the C-22

methoxy derivative with the reported values of C 40.5 [C-20], 64.2 [C-17] and 112.7 [C-

22] (Mimaki et al., 1994) (Figure 2.25). The carbon peaks from C-5 to C-27 were similar

to those reported for (25R,S)-26-O- -D-glucopyranosyl-furost-5-en-3 22 -triol 3-O-

-D-galactopyranosyl-(1→2)-O- -D-glucopyranosyl-(1→4)- -D-galactopyranoside,

isolated from Tribulus terrestris L., which suggests the presence of an α-hydroxy group

at the C-22 position of the furostane skeleton (Wang et al., 1997).

96

Figure 2.25. Partial HMBC spectrum of compound 4, showing the correlation between

H-21 and the carbon signals of C-20, C17 and C-22.

O

OH

17

2022

1.35

40.7

63.9 110.7

H21

Consistent with (25R)-26-O- -D-glucopyranosyl-furost-5-en-3 22 triol 3-O- -D-

glucopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside, isolated from

the rhizomes of Tupistra chinensis Bak., the peak of C-25 at C 34.3 as compared to C

34.42, suggests an R configuration (Wang et al., 1992). Olefinic carbon signals at C

140.7 and 121.9 in the 13

C NMR spectrum were consistent with a 5 furostane skeleton.

C-22

C-17

C-20

0.80 1.0

0

1.2

0

1.60 1.40 1.8

0

2.00 PPM F2

0

10

20

30

40

50

60

70

80

90

100

110

PPM F1

97

The unsaturation between C-5 and C-6 is further substantiated by an olefinic proton

signal at 5.33 in the 1H NMR spectrum for H-6. Upon acid hydrolysis, derivatization

and GC-MS analysis, the retention time and mass spectrum of the TMSi derivative of the

aglycone was consistent with that of (25R)-spirost-5-en-3 -ol, which was prepared

following the same procedure. The structure of the oligosaccharide moiety was readily

deduced from 1H NMR,

13C NMR, HMBC, ESI

+–MS and chemical analysis. All four

sugars of 4 have similar carbon NMR values to those reported for (25R)-26-O- -D-

glucopyranosyl-22 -methoxy-furost-5-en-3 26-diol 3-O- -L-rhamnopyranosyl-(1→2)-

-L-arabinopyranosyl-(1→3)- -D-glucopyranoside (Mimaki et al., 1994). The 1H NMR

spectrum contained four anomeric proton signals observed at 6.30, 4.99, 4.92 and

4.84. The coupling constants of the anomeric proton resonances suggested -

interglycosidic linkages. The carbon signals were at C 105.6, 105.0, 102.5 and 99.9,

which is consistent with the presence of four saccharide residues. The HMBC experiment

showed long-range correlations between the anomeric proton signal at 4.99 [H-1′] and

the carbon signal at C 77.7 [C-3], between the anomeric proton signal at 4.92 [H-1′′′]

and the carbon signal at C 88 [C-3′], and the anomeric proton signal at 6.30 [H-1′′]

and the carbon signal at C 78 [C-2′] (Figure 2.26).

98



O

OO

OH

HO

OH3CHO OHOH

O

O

HO

OH

OH H

H

H

H 77.7

4.99 (7.2)

78

6.30

4.92 (7.6) 88

4

Upon acid hydrolysis, derivitazation and GC-MS/entantioslective GC-FID analysis, the

sugars were identified as D-(+)-glucose, L-(–)-rhamnose and L-(–)-arabinose in a 2:1:1

ratio. 1H NMR spectrum showed a doublet integrating for three protons at 1.76,

indicative of the methyl group of rhamnose. The ESI+–MS mass spectrum showed a base

ion peak at 1017.7 [M–18+H]+ and the sodium adduct at 1057.7 [M+Na]

+ . Additionally,

ion fragments at m/z 871.6 [M–18–Rha+H]+, 739.4 [ M–18–Ara–Rha+H]+, 577.2 [M–

18–Ara–Rha–Glu+H]+ and 415.2 [M–18–2Glu–Ara–Rha+H]

+ were observed and were

consistent with 4 being bidesmodic with the trisaccharide moiety at the C-3 position

containing D-(+)-glucose, L-(–)-arabinose and L-(–)-rhamnose, and a D-(+)-glucose

moiety at the C-26 position, indicative of a furostanol saponin (Figure 2.27).

99



furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -L-arabinopyranosyl-

(1→3)- -D-glucopyranoside (Figure 2.4).

271.1 415.3 577.2 739.4 871.6

1017.7

1057.7

0

1

2

3

4

5

6

6 x10

Intens.

200 400 600 800 1000 1200 m/z

100



A

B

101


rhamnopyranosyl-(1→2)- -L-arabinopyranosyl-(1→3)- -D-glucopyranoside:

amorphous solid; [ D

28 - 48.6º (MeOH; c 0.07); mp 200 ºC (dec); IR max (film) cm

-1:

3367 (OH), 2899 (CH), 1699, 1377, 1256, 1040, 912, 840, 811, 780; HRESI–TOFMS

m/z: 1057.5211 [M+Na]+ (calculated for C50H82O22Na, 1057.5190); ESI

+ – MS, m/z

1057.7 (5, [M+Na]+), 1017.7 (100, [M–18+H]

+), 871.6 (7, [M–18–Rha+H]

+), 739.4 (13,

[M–18–Ara–Rha+H]+), 577.2 (7, [M–18–Ara–Rha–Glu + H]

+), 415.2 (4, [M–18–2Glu–

Ara–Rha+H]+);

1H NMR (400 MHz) 0.91 [s, 3H, 18-H], 1.00 [d, 3H, J = 6.4 Hz, 27-

H], 1.07 [s, 3H, 19-H], 1.35 [d, 3H, J = 6.8 Hz, 21-H], 1.76 [d, 3H, J = 6 Hz, 6′′-H], 3.08

[d, 1H, J = 11.6, 5′′′-H], 4.05 [m, 1H, Hz, 2′′′′-H], 3.90-3.85 [m, 1H, 2′-H], 4.84 [d, 1H, J

= 7.6 Hz, 1′′′′-H], 4.91 [m, 1H, 16-H], 4.92 [d, 1H, J = 7.6, 1′′′-H], 4.99 [d, 1H, J = 7.2

Hz, 1′-H], 5.33 [d, 1H, J = 4 Hz, 6-H], 6.30 [br s, 1H, 1′′-H]; for 13

C NMR (400 MHz,

pyridine-d5) 37.5 [C-1], 30.1 [C-2], 77.7 [C-3], 38.7 [C-4], 140.7 [C-5], 121.9 [C-6], 32.4

[C-7], 31.7 [C-8], 50.3 [C-9], 37.2 [C-10], 21.1 [C-11], 39.9 [C-12], 40.8 [C-13], 56.6 [C-

14], 32.5 [C-15], 81.1 [C-16], 63.9 [C-17], 16.5 [C-18], 19.4 [C-19], 40.7 [C-20], 16.5

[C-21], 110.7 [C-22], 37.2 [C-23], 28.4 [C-24], 34.3 [C-25], 75.2 [C-26], 17.5 [C-27],

99.9 [C-1′], 78.0 [C-2′], 88.0 [C-3′], 69.7 [C-4′], 77.6 [C-5′], 62.4 [C-6′], 102.5 [C-1′′],

72.5 [C-2′′], 72.9 [C-3′′], 74.4 [C-4′′], 69.5 [C-5′′], 18.7 [C-6′′], 105.6 [C-1′′′], 72.3 [C-

2′′′], 74.6 [C-3′′′], 69.7 [C-4′′′], 67.8 [C-5′′′], 105.0 [C-1′′′′], 75.3 [C-2′′′′], 78.6 [C-3′′′′],

71.7 [C-4′′′′], 78.5 [C-5′′′′], 62.8 [C-6′′′′].

102



positive to the Ehrlich‘s reaction, indicative of a furostanol saponin. The IR spectrum

showed absorption at 3362 cm-1

due to the presence of hydroxyl groups. HRESI–TOFMS

showed a [M+Na] +

ion at m/z: 1057.5211 (calculated for C50H82O22Na, 1057.5190)

(Figure 2.29). Additionally, [M–H]- ion was observed at m/z 1033.6 (Figure 2.30). Thus,

the molecular formula was calculated as C50H82O22, consistent with a furostanol saponin.

The aglycone was readily deduced from 1H NMR,

13C NMR (Figure 2.33; Table 2.1),

HMBC, ESI+–MS (Figure 2.34), and chemical analysis. The

1H NMR spectrum showed

two singlets at 1.08 and 0.91 which is indicative of tertiary methyl groups of the

furostane skeleton. Furthermore, two doublets at 1.35 and 1.0 were assignable to

secondary methyl groups. The carbon signals for the two tertiary methyl groups were at

C 19.4 and 16.5 and secondary methyl groups at C 17.5 and 16.5, respectively. A

quaternary carbon signal at C 110.7 was observed, supporting a furostane skeleton

possessing a hydroxyl group at the C-22 position and the C-25 carbon signal at C 34.3

was indicative an R configuration. Similar to 4, long range coupling was observed

between the methyl proton signal at H-21 and the carbon signals at C 40.7 C-20,

63.9 C-17 and 110.7 C-22. Olefinic carbon signals at C 140.7 and 121.9 in the 13

C NMR

spectrum were consistent with a 5 furostane skeleton. The unsaturation between C-5 and

C-6 was further substantiated by a doublet at 5.33 in the 1H NMR spectrum for the H-

6 signal.

103


of [M + Na] +

ion at m/z: 1057.5211 (calculated for C50H82O22Na, 1057.5190). The mass


22

9.1

00

5

30

9.1

70

5

39

3.2

72

0

43

8.2

67

1 46

7.2

89

4

49

6.3

12

2

54

0.2

42

0

58

3.3

86

4

61

2.4

07

7

64

1.4

38

5

67

0.4

52

8

69

9.4

77

2

73

7.5

00

6

76

3.6

04

7

79

5.5

45

3

85

3.5

84

7

91

1.6

32

3

96

9.6

69

9

10

27

.71

28

10

57

.52

42

10

85

.74

82

11

43

.79

94

12

01

.83

74

200 400 600 800 1000 1200 1400 1600 1800 m/z0

500

1000

1500

2000

Intens.

10

57

.52

42

10

58

.02

74

10

58

.52

74

10

59

.52

29

1052 1054 1056 1058 1060 1062 1064 1066 1068 m/z0

200

400

600

800

1000

Intens.

A

B

104


14

5.0

17

1.1

21

2.1

51

6.2

10

33

.6

200 400 600 800 1000 1200 1400 1600 1800 m/z0

250

500

750

1000

1250

1500

Intens.


spectrum of the TMSi derivative of the aglycone was consistent with that of (25R)-

spirost-5-en-3 -ol, which was prepared following the same procedure. The structure of

the oligosaccharide moiety was readily deduced from 1H NMR,

13C NMR, HMBC, ESI

+–

MS and chemical analysis. 5 showed similar carbon NMR peaks to those of compound 3

and 4, except for the 3′′′ sugar peaks. This identifies 5 to be similar in structure,

connectivity and configuration to compound 3 and 4, except for the 3′′′sugar. The 1H

NMR spectrum contained four anomeric proton signals observed at 6.34, 5.01, 4.99

and 4.89. Coupling constants of the anomeric proton resonances suggested -

interglycosidic linkages. The 13

C NMR spectrum contained four anomeric carbon signals

105

observed at C 105.4, 105.0, 102.4 and 100.0, consistent with the presence of four

saccharide residues. The HMBC experiment showed long-range correlations between the

anomeric proton signal at 4.99 [H-1′] and the carbon signal at C 77.4 [C-3], between

the anomeric proton signal at 5.01 [H-1′′′] and the carbon signal at C 88.2 [C-3′], and

the anomeric proton signal at 6.34 [H-1′′] and the carbon signal at C 78 [C-2′]

(Figure 2.31).



5

O

OO

OH

HO

OH3CHO OHOH

O

O

HO

HO

OH H

H

H

H 77.4

4.99 (7)

78

6.34

4.01 (5.5) 88.2

Upon acid hydrolysis, derivatization and GC-MS analysis, the sugars were identified as

D-(+)-glucose, L-(-)-rhamnose and L-(-)-xylose in a 2:1:1 ratio. 1H NMR spectrum

showed a doublet integrating for three protons at 1.77, indicative of the methyl group

of rhamnose. The ESI+–MS mass spectrum showed a base ion peak at 1017.7 [M–18+H]

+

and the sodium adduct at 1057.7 [M+Na]+. Additionally, ion fragments at m/z 871.6 [M–

18–Rha+H]+, 739.4 [M–18–Xyl–Rha+H]

+, 577.2 [M–18–Xyl–Rha–Glu+H]

+, and 415.2

[M–18–2Glu–Xyl–Rha+H]+ were observed and were consistent with 5 being bidesmodic

with the trisaccharide moiety at the C-3 position containing D-(+)-glucose, L-(-)-xylose

106

and L-(-)-rhamnose, and a D-(+)-glucose moiety at the C-26 position, indicative of a

furostanol saponin (Figure 2.32).




(1→3)- -D-glucopyranoside (Figure 2.4).

271.1 415.3 577.2 739.4 871.6

1017.7

1057.7

0

1

2

3

4

5

6

6 x10

Intens.

200 400 600 800 1000 1200 m/z

107



A

B

108


rhamnopyranosyl-(1→2)- -L-xylopyranosyl-(1→3)- -D-glucopyranoside: amorphous

solid; [ D

28 - 46.4º (MeOH; c 0.03); mp 200 ºC (dec); IR max (film) cm

-1: 3362 (OH),

2898 (CH), 1636, 1377, 1256, 1035, 912, 838, 811; HRESI–TOFMS m/z: 1057.5242 [M

+ Na]+ (calculated for C50H82O22Na, 1057.5190); ESI+

– MS, m/z 1057.7 (2, [M+Na]+),

1017.7 (100, [M–18+H]+), 871.6 (7, [M–18–Rha+H]

+), 739.4 (9, [M–18–Xyl–Rha+H]

+),

577.2 (3, [M–18–Xyl–Rha–Glu+H]+), 415.2 (2, [M–18–2Glu–Xyl–Rha+H]

+);

1H NMR

(500 MHz) 0.91 [s, 3H, 18-H], 1.00 [d, 3H, J = 6.5 Hz, 27-H], 1.08 [s, 3H, 19-H], 1.35

[d, 3H, J = 7 Hz, 21-H], 1.77 [d, 3H, J = 6.5 Hz, 6′′-H], 4.83 [d, 1H, J = 8 Hz, 1′′′′-H],

4.89-4.88 [m, 1H, 16-H], 4.99 [d, 1H, J = 7 Hz, 1′-H], 5.01 [d, 1H, J = 5.5, 1′′′-H], 5.33

[d, 1H, J = 4 Hz, 6-H], 6.34 [br s, 1H, 1′′-H]; 13

C NMR (400 MHz, pyridine-d5) 37.5 [C-

1], 30.1 [C-2], 77.7 [C-3], 38.7 [C-4], 140.7 [C-5], 121.9 [C-6], 32.4 [C-7], 31.7 [C-8],

50.3 [C-9], 37.2 [C-10], 21.1 [C-11], 39.9 [C-12], 40.8 [C-13], 56.6 [C-14], 32.5 [C-15],

81.1 [C-16], 63.9 [C-17], 16.5 [C-18], 19.4 [C-19], 40.7 [C-20], 16.5 [C-21], 110.7 [C-

22], 37.2 [C-23], 28.4 [C-24], 34.3 [C-25], 75.2 [C-26], 17.5 [C-27], 100.0 [C-1′], 78.0

[C-2′], 88.2 [C-3′], 69.7 [C-4′], 77.7 [C-5′], 62.4 [C-6′], 102.4 [C-1′′], 72.5 [C-2′′], 72.9

[C-3′′], 74.4 [C-4′′], 69.5 [C-5′′], 18.7 [C-6′′], 105.5 [C-1′′′], 74.7 [C-2′′′], 78.4 [C-3′′′],

70.7 [C-4′′′], 67.3 [C-5′′′], 105.0 [C-1′′′′], 75.3 [C-2′′′′], 78.6 [C-3′′′′], 71.7 [C-4′′′′], 78.5

[C-5′′′′], 62.8 [C-6′′′′].

109

2.5. Conclusion

Although, saponins are widely distributed secondary metabolites and have been

identified in over 100 plant families and in marine organisms such as starfish and sea

cucumber (Güçlü-Üstündağ and Mazza, 2006), thus far, steroidal glycoalkaloids are

limited to members of the families Solanaceae and Liliaceae (Ghisalberti, 2006). In

addition, novel or ―alien‖ glycoalkaloids have been reported from interspecific hybrids of

Solananceous plants (Grassert and Lellbach, 1987). For example, a novel tomatidine

glycoalkaloid, not present in either parental species, was identified in the sexual hybrids

of S. acaule and Solanum x ajanhuiri (Osman et al., 1986). Interspecific hybridization is

widely employed for the development of new Lilium cultivars. Similar to Solananceous

plants, interspecific hybridization in the Liliaceae family may result in novel steroidal

glycosides. Several novel steroidal saponins including a steroidal saponin with an

acetylated glucose, (25R,26R)-26-methoxyspirost-5-en-3 -yl O- -L-rhamnopyranosyl-

(1→2)-[6-O-acetyl- -D-glucopyranosyl-(1→4)]- -D-glucopyranoside, has been isolated

from the interspecific hybrid L. speciosum x L. nobilissimum (Nakamura et al., 1994).

Steroidal saponins with acetylation of saccharide residues have been identified;

however, the biological significance of acetylation of these compounds is unclear. Some

genotypes of Solanum chacoense, a wild potato species that is resistant to Leptinotarsa

decemlineata, contain glycoalkaloids that are acetylated at the C-23 position of the

steroid aglycone (Sinden et al., 2005), but we are unaware of any steroidal glycoalkaloids

that contain naturally occurring acetylated saccharides. Interestingly, the presence or

absence of the acetyl moiety of the S. chacoense glycoalkaloids markedly affected

110

resistance to foliar feeding of both adults and larvae of L. decemlineata. Differences in

acetylation of the terminal glucose of the trisaccharide moiety of 1 and 2 may also play a

biological role in L. longiflorum.

The furostanol saponins 3 – 5, are similar in structure except for the terminal

monosaccharide residues and interglycosidic linkages. 3 has a hexose as the terminal

sugar linked via the C-4′ carbon of the inner glucose, whereas both 4 and 5 contain a

pentose as the terminal sugar linked via the C-3′ carbon of the inner glucose. In fact,

differences in oligosaccharide composition and interglycosidic linkages have been shown

to affect the biological activity of steroidal saponins possessing the same aglycone moiety

(Yang et al., 2006). Differences in oligosaccharide composition and interglycosidic

linkages in 3 – 5 may also play a role in the biology of these compounds in L.

longiflorum.

In this chapter, a new acetylated steroidal glycoalkaloid, (22R, 25R)-spirosol-5-

en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-glucopyranosyl-(1→4)]- -D-

glucopyranoside, and two new furostanol saponins, (25R)-26-O-( -D-glucopyranosyl)-


(1→3)- -D-glucopyranoside and (25R)-26-O-( -D-glucopyranosyl)-furost-5-en-

3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -L-xylopyranosyl-(1→3)- -D-

glucopyranoside, from the bulbs of L. longiflorum have been isolated and structures

elucidated. Additionally, a known steroidal glycoalkaloid, (22R, 25R)-spirosol-5-en-3 -

yl O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside,

and a known furostanol saponin, (25R)-26-O-( -D-glucopyranosyl)-furost-5-en-

111

3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-

glucopyranoside, were isolated for the first time from the bulbs of L. longiflorum. The

extraction and purification procedures reported in this chapter may be used for the

production of sufficient quantities of pure compounds for biological investigations. These

new compounds from L. longiflorum can be used for studies on the biological role of

steroidal glycosides in plant development and plant-pathogen interactions, as well as for

studies in food and human health, for which little is known.

112

Figure 2.34. ESI+–MS mass spectra of compounds 1 – 5.

[M + Na]+

[M – 18 + H]+

1057.7

[M + H]+

453.8

[M + Na]+

[M + Na]+

415.2 271.3

O

O

OH

O

HO

OH

HO

OH

O

871.6

415.2

739.4

577.2

O

O

OH

HO

OH3CHO

OH OH

O

O

HO

OH

OH

O

O

OH

O

HO

OH

HO

OH

O

871.6

415.2

739.4

577.2

O

O

OH

HO

OH3CHO

OH OH

O

O

HOHO

OH

1

2

3

4

5

[M – 18 + H]+

[M – 18 + H]+

[M + H + Na]++

[M + H + Na]++

414.6576.4

738.5

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

OH

HO

OH

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

O

HO

OH

414.3576.4

780.5

O

901.5

415.3

739.4

577.4

O

HO

OH

O

OH3CHO

OH OH

O

O

HO

OH

HO

OHO

O

OH

O

HO

OH

HO

OH

O

1087.7

414.6 576.4 738.5

884.7

271.2

414.3

474.8 576.4 780.5

926.6

271.3 415.3 577.4 739.4 901.5

1047.7

415.2 577.2 739.4 871.6

1017.7

1057.7

577.2 739.4

871.6

1017.7

1

2

3 7 x10

Intens

.

0.25

0.50

0.75

1.00

1.25 7 x10

0.2

0.4

0.6

0.8

1.0

7 x10

2

4

6

6 x10

0

1

2

3

4 6 x10

200 400 600 800 1000 1200 1400 m/z

[M + H]+

271.4

271.3

113

Table 2.1. 13

C NMR spectral data of compounds 1 – 5 in pryridine-d5.

compound

carbon 1 2 3 4 5

C-1 37.5 37.5 37.5 37.5 37.5

C-2 30.2 30.2 30.2 30.1 30.1

C-3 78.1a 78.1

a 78.2

a 77.7

a 77.4

a

C-4 38.9 39.0 39.0 38.7 38.7

C-5 140.8 140.8 140.8 140.7 140.7

C-6 121.8 121.9 121.9 121.9 121.9

C-7 32.4 32.4 32.5 32.4 32.4

C-8 31.6b 31.6

b 31.7 31.7 31.7

C-9 50.3 50.4 50.4 50.3 50.3

C-10 37.2 37.2 37.2 37.2 37.2

C-11 21.2 21.2 21.2 21.1 21.1

C-12 40.1 40.1 39.9 39.9 39.9

C-13 40.7 40.7 40.8b 40.8

b 40.8

b

C-14 56.7 56.7 56.6 56.6 56.6

C-15 32.6 32.6 32.4 32.5 32.5

C-16 78.8 79.0 81.1 81.1 81.1

C-17 63.6 63.5 63.9 63.9 63.9

C-18 16.5 16.5 16.5 16.5 16.5

C-19 19.4 19.4 19.4 19.4 19.4

C-20 41.6 41.7 40.7b 40.7

b 40.7

b

C-21 15.7 15.7 16.5 16.5 16.5

C-22 98.4 98.4 110.7 110.7 110.7

C-23 34.7 34.7 37.1 37.2 37.2

C-24 31.1 31.0 28.4 28.4 28.4

C-25 31.7b 31.7

b 34.3 34.3 34.3

C-26 48.1 48.0 75.3c 75.3

c 75.3

c

C-27 19.9 19.8 17.5 17.5 17.5

C-1' 100.0 99.9 100.0 99.9 100.0

C-2' 77.8 77.6 77.8 78.0 78.0

C-3' 76.2 76.1 76.2 88.0 88.2

C-4' 82.1 83.3 82.1 69.7 69.7

C-5' 77.3 77.4 77.3 77.6a 77.7

a

C-6' 62.0 62.0 62.1 62.4 62.4

C-1" 101.8 102.0 101.8 102.5 102.4

C-2" 72.5 72.5 72.5 72.5 72.5

C-3" 72.8 72.8 72.8 72.9 72.9

C-4" 74.2 74.2 74.2 74.1 74.1

C-5" 69.5 69.6 69.5 69.5 69.5

C-6" 18.7 18.7 18.7 18.7 18.7

C-1'" 105.3 105.6 105.2 105.6 105.5

C-2'" 75.0 75.1 75.0 72.3 74.7

C-3'" 78.3 78.2 78.5 74.6 78.4

C-4'" 71.2 71.9 71.2 69.7 70.7

C-5'" 78.5 74.9 78.3 67.8 67.3

C-6'" 62.1 64.8 61.9

C-1"" 105.0 105.0 105.0

C-2"" 75.2c 75.2

c 75.2

c

C-3"" 78.6 78.6d 78.6

d

C-4"" 71.7 71.7 71.7

C-5"" 78.3a 78.5

d 78.5

d

C-6"" 62.8 62.8 62.8

Ac-CH3 20.8

Ac-C=O 170.9 a-d

Assignments may be interchanged in each column

114

Chapter 3: Quantitative Analysis of Steroidal Glycosides in Different Organs of

Easter Lily (Lilium longiflorum Thunb.) by LC-MS/MS

3.1. Abstract

The bulbs of the Easter lily (Lilium longiflorum Thunb.) are regularly consumed

in Asia as both food and medicine, and the beautiful white flowers are appreciated

worldwide as an attractive ornamental. The Easter lily is a rich source of steroidal

glycosides, a group of compounds that may be responsible for some of the traditional

medicinal uses of lilies. Since the appearance of recent reports on the role steroidal

glycosides in animal and human health, there is increasing interest in the concentration of

these natural products in plant-derived foods. A LC-MS/MS method performed in

multiple reaction monitoring (MRM) mode was used for the quantitative analysis of two

steroidal glycoalkaloids and three furostanol saponins, in the different organs of L.

longiflorum. The highest concentrations of the total five steroidal glycosides were 12.02

± 0.36, 10.09 ± 0.23, and 9.36 ± 0.27 mg g-1

dry weight in flower buds, lower stems, and

leaves, respectively. The highest concentrations of the two steroidal glycoalkaloids were

8.49 ± 0.3, 6.91 ± 0.22, and 5.83 ± 0.15 mg g-1

dry weight in flower buds, leaves, and

bulbs, respectively. In contrast, the highest concentrations of the three furostanol

saponins were 4.87 ± 0.13, 4.37 ± 0.07, and 3.53 ± 0.06 mg g-1

dry weight in lower stems,

fleshy roots, and flower buds, respectively. The steroidal glycoalkaloids were detected in

higher concentrations as compared to the furostanol saponins in all of the plant organs

except the roots. The ratio of the steroidal glycoalkaloids to furostanol saponins was

115

higher in the plant organs exposed to light and decreased in proportion from the

aboveground organs to the underground organs. Additionally, histological staining of

bulb scales revealed differential furostanol accumulation in the basal plate, bulb scale

epidermal cells, and vascular bundles, with little or no staining in the mesophyll of the

bulb scale. An understanding of the distribution of steroidal glycosides in the different

organs of L. longiflorum is the first step in developing insight into the role these

compounds play in plant biology and chemical ecology and aids in the development of

extraction and purification methodologies for food, health, and industrial applications. In

this chapter, (22R, 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)- -D-

glucopyranosyl-(1→4)- -D-glucopyranoside, (22R, 25R)-spirosol-5-en-3 -yl O- -L-

rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-glucopyranosyl-(1→4)]- -D-glucopyranoside,

(25R)-26-O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol 3-O- -L-

rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside, (25R)-26-

O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)-

-L-arabinopyranosyl-(1→3)- -D-glucopyranoside, and (25R)-26-O-( -D-


xylopyranosyl-(1→3)- -D-glucopyranoside were quantified in the different organs of L.

longiflorum for the first time.

116

3.2. Introduction

The Easter lily (Lilium longiflorum Thunb., family Liliaceae), with its showy

white flowers and fragrant aroma, is enjoyed worldwide as an attractive ornamental plant.

Easter lilies are most commonly seen as indoor potted plants or floral arrangements

around the Easter holidays; however, they are also often planted outdoors as bedding

plants in flower gardens. In addition to their esthetic value, lily bulbs and flower buds are

regularly consumed as a food in Asia for their distinctive bitter taste and have a long

historical use in traditional Chinese medicine. In particular, a preparation of bulbs of

various Lilium species, referred to as ―Bai-he‖, is used as a treatment for inflammation

and lung ailments (Mimaki and Sashida, 1990; Mimaki et al., 1992). Among many other

secondary metabolites, L. longiflorum is a rich source of steroidal glycosides, a

structurally diverse class of natural products that includes steroidal saponins and steroidal

glycoalkaloids. Steroidal glycosides have been reported to exhibit a wide range of

biological activities including antifungal (Sautour et al., 2005; Zhou et al., 2003), platelet

aggregation inhibition (Zhang et al., 1999; Huang et al., 2006), anticholinergic (Gilani et

al., 1997), antidiabetic (Nakashima et al., 1993), antihypertensive (Oh et al., 2003),

cholesterol lowering (Matsuura, 2001), anti-inflammatory (Shao et al., 2007), antiviral

(Gosse et al., 2002), and anticancer (Acharya et al., 2009; Pettit et al., 2005; Mimaki et

al., 1999; Jiang et al., 2005). Additionally, steroidal glycosides have a wide variety of

commercial uses including as surfactants (Yamanaka et al., 2008), foaming agents (Singh

et al., 2003), and vaccine adjuvants (Rajput et al., 2007) and serve as precursors for the

industrial production of pharmaceutical steroids (Hansen, 2007). Steroidal saponins have

117

been found in over 100 plant families and in some marine organisms such as starfish and

sea cucumber (Güçlü-Üstündağ and Mazza, 2006). They are characterized by a steroid

type skeleton glycosidically linked to carbohydrate moieties. Steroidal glycoalkaloids are

characterized by a nitrogen-containing steroid type skeleton glycosidically linked to

carbohydrate moieties. In contrast to steroidal saponins, the occurrences of steroidal

glycoalkaloids are, thus far, limited to members of the plant families Solanaceae and

Liliaceae (Li et al., 2006; Ghisalberti, 2006). Some glycoalkaloids from solanaceaous

plants have been shown to play a role in plant defense and are toxic to animals and

humans. The potato glycoalkaloids, -solanine and -chaconine, are highly toxic to

animals due to their interaction with membrane sterols, disruption of cell membranes, and

inhibition of acetylcholinesterase, suggesting a biological role in antiherbivory (Sánchez-

Mata et al., 2010). In Lilium, steroidal glycoalkaloids have been identified in L.

philippinense (Espeso and Guevara, 1990), L. mackliniae (Sashida et al., 1991), and L.

brownii (Mimaki and Sashida, 1990), and in the previous chapter solasodine-based

glycoalkaloids were identified for the first time from L. longiflorum. Interestingly, both

the leaves and flowers of L. longiflorum have been reported to be highly nephrotoxic to

domesticated cats; however, the toxic compounds have yet to be identified (Rumbeiha et

al., 2004; Langston, 2002). Although it has been reported that solasodine-based

glycoalkaloids are less toxic then solanidine-based glycoalkaloids (Roddick et al., 2001),

the animal and human toxicity of the steroidal glycoalkaloids from L. longiflorum has yet

to be investigated. Although the putative biological activities of steroidal glycosides are

well documented, the biological role of these compounds in plant metabolism and

development is poorly understood. The role of steroidal glycosides in wound response

118

and plant defense, including antifungal and antiherbivory, has been studied extensively

(Zullo et al., 1984; Nozzolillo et al., 1997; Adel et al., 2000; Bowyer et al., 1995;

Osbourn, 1996; Morrissey and Osbourn, 1999; Osbourn, 1999; Papadopoulou et al.,

1999; Morrissey et al., 2000; Trojanowska et al., 2000; Osbourn et al., 2003; Osbourn,

2003; Hughes et al., 2004; Choi et al., 2005). In fact, some steroidal glycosides are toxic

to insects such as the European corn borer, Ostrinia nubialis, and army worm,

Spodoptera littoralis (Nozzolillo et al., 1997; Adel et al., 2000). In oats, Avena sativa,

biologically inactive steroidal saponins are converted into an antifungal form in response

to tissue damage, suggesting a role in the plant-pathogen interaction (Osbourn, 1996;

Morrissey et al., 2000; Osbourn, 2003; Hughes et al., 2004). In addition, the steroidal

glycoalkaloids -tomatine and -chaconine play a role in fungal resistance of tomato,

Solanum lycopersicum, and potato, Solanum tuberosum, respectively (Morrissey and

Osbourn, 1999). Although the literature suggests that steroidal glycosides are involved in

the plant pathogen interaction and antiherbivory in oats, tomato, and potato, there are no

reports on biological role of steroidal glycosides in L. longiflorum.

In the previous chapter, two steroidal glycoalkaloids, (22R, 25R)-spirosol-5-en-

3 -yl O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside

(1), (22R, 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-

glucopyranosyl-(1→4)]- -D-glucopyranoside (2), and three furostanol saponins, (25R)-

26-O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-

(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside (3), (25R)-26-O-( -D-


arabinopyranosyl-(1→3)- -D-glucopyranoside (4) and (25R)-26-O-( -D-

119


xylopyranosyl-(1→3)- -D-glucopyranoside (5) were identified for the first time in L.

longiflorum. Although steroidal glycosides have attracted scientific attention in recent

years for their medicinal and industrial uses, there are only a few studies that have

quantified these compounds within different plant organs throughout plant development.

The organ distribution of steroidal glycosides in various plants such as Solanum nigrum,

Solanum incanun (Eltayeb et al., 1997), Asparagus officinalis L. (Wang et al., 2003) and

Dioscorea pseudojaponica (Lin et al., 2008) have been reported, however, there are no

studies on the distribution of steroidal glycosides in L. longiflorum.

Steroidal glycosides lack a strong chromophore and occur in complex biological

matrices; therefore, nonspecific short-wavelength UV detection is often inadequate.

Analytical methods using evaporative light scattering detection (ELSD) are used to help

overcome this obstacle; however, laborious sample preparation and sensitivity issues

persist (Oleszek and Bialy, 2006). LC-MS methods operating in selected ion monitoring

(SIM) mode have been developed to increase sensitivity and specificity; however, the

separation of structurally similar compounds and shared ions still poses a challenge

(Ghisalberti, 2006; Oleszek and Bialy, 2006). LC-MS/MS in MRM mode overcomes

these obstacles, allowing for sensitive quantitative analysis in complex biological

matrices with increased specificity over SIM. The purpose of this investigation was to

utilize LC-MS/MS in MRM mode to quantify five steroidal glycosides in the different

organs of L. longiflorum. Additionally, histological techniques were employed to

qualitatively visualize tissue-specific localization of furostanols in bulb scales. An

understanding of the distribution and tissue specific localization of steroidal glycosides in

120

L. longiflorum is the first step to develop insight into the biological role these compounds

play in plant metabolism, plant development, and chemical ecology. Quantitative analysis

of steroidal glycosides in the different organs of L. longiflorum will aid in development

studies in animal and human health, toxicology, and optimization of extraction

methodologies for potential commercial applications including functional foods,

cosmetics, and pharmaceuticals.


3.3.1. Plant material.

Ten L. longiflorum cv. 7-4 plants were grown from tissue-cultured bulbs provided

by the Rutgers University lily breeding program. The young bulbs were treated with

Captan (Bayer CropScience AG, Monheim am Rhein, Germany) fungicide prior to

planting. Bulbs were planted in raised beds containing Pro-Mix (Premier Horticulture

Inc., Quakertown, PA) soil mix and were grown to mature plants, containing both flower

buds and flowers, under greenhouse conditions for 9 months prior to harvest. The

greenhouse temperatures were set to provide a minimum day temperature of 24 °C and a

minimum night temperature of 18 °C. Plants were fertilized biweekly with a 100mg L

min-1

solution of NPK 15-15-15 fertilizer (J. R. Peters Inc., Allentown, PA). Each plant

was harvested by hand and manually separated into the following plant organs: bulb

scales, fibrous roots, fleshy roots, leaves, lower stems, upper stems, flower buds, and

121

mature flowers. Bulb scales included both inner and outer bulb scales and ranged from

0.8 to 2.0 cm in width and from0.9 to 4.0 cm in length. Fibrous roots were 0.25 – 0.5mm

in diameter. Fleshy roots were 2 – 4 mm in diameter. Leaves ranged in size from 6 to 14

cm. Lower stems were defined as the underground portion of the stem, ranged in size

from 6 to 10 cm, and were from white to yellow in appearance. Upper stems were defined

as the aboveground portion of the stem, ranged in size from 19 to 31 cm, and were green

in appearance. Flower buds ranged in size from 3 to 6 cm. Mature flowers ranged in size

from 6 to 14 cm. All of the organs from 10 individual plants were pooled together by

organ type, immediately frozen under liquid nitrogen, lyophilized on a VirTis AdVantage

laboratory freeze-dryer (SP Industries Inc., Warminster, PA), and stored at -80 °C until

analyzed (Figure 3.1).

122

Figure 3.1. The different plant organs of L. longiflorum analyzed in this study.

123

3.3.2. Chemicals

The following compounds were obtained commercially: p-

(dimethylamino)benzaldehyde, hydrochloric acid, and pyridine-d5 (0.3%v/v TMS)

(Sigma-Aldrich, St. Louis, MO). All solvents (acetonitrile, chloroform, ethanol, ethyl

acetate, formic acid, n-butanol, and n-pentane) were of chromatographic grade (Thermo

Fisher Scientific Inc., Fair Lawn, NJ). Water was deionized (18 MΩ cm) using a Milli-Q

water purification system (Millipore, Bedford, MA).

3.3.3. Histology and Microscopy.

Histological detection of furostanols was modified from the method of Gurielidze

et al., 2004 (Gurielidze et al., 2004). Bulb scales were carefully cross-sectioned (∼0.5

mm) parallel to the basal plate, soaked for 2 min in a solution of Ehrlich‘s reagent [3.2 g

of p-(dimethylamino)-benzaldehyde in 60 mL of 95% ethanol and 60 mL of 12 N HCl],

and briefly heated on a microscope slide under an open flame. Transmitted light

microscopy was performed with an Axiovert 200 inverted microscope (Carl Zeiss

Microimaging Inc., Thornwood, NY) at magnifications of 10x, 20x, and 40x Axiovision

version 3.0 software was used for image acquisition. Furostanol localization was

visualized as dark red areas.

124

3.3.4. Purification and Confirmation of Analytical Standards.

Closely following the procedure recently reported in chapter two, the steroidal

glycosides 1-5 were isolated as analytical standards from lyophilized L. longiflorum bulbs

(Figure 3.2). Briefly, lyophilized lily bulb powder was washed with n-pentane and

extracted with ethanol and deionized water (7:3, v/v). After the removal of solvent, the

extract was dissolved in deionized water, washed with ethyl acetate, and extracted with n-

butanol. The organic phase was evaporated under reduced pressure and lyophilized,

yielding a crude steroidal glycoside extract. The crude glycoside extract was fractionated

by gel permeation chromatography and repeated semipreparative RP-HPLC to yield

compounds 1 – 5. The standards were obtained as white amorphous powders in high

purity, >98%, as determined by LC-MS and NMR.

125

O

R3OO

OH

R2O

OH3CHO OHOH

O

O

OH

O

HO

OH

HO

OH

O

O

HOO

OH

R1O

OH3CHO

OH OH

O

O

HN

O

HO

O

HO

OH

O

O

HO

OH

OH

O

HOHO

OH

O

HO

OH

HO

OH

R1

R2 R3

H

H

H

1

2

3

4

56

7

8

9

10

11

1213

14 15

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

1''''

1

2

3

4

56

7

8

9

10

11

12

13

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

6-Ac- -D-Glcp-D-Glcp

1

2

4

5

-L-Arap -D-Xlyp

3

-D-Glcp

6-Ac- -D-Glcp

-D-Glcp

-L-Arap

-D-Xlyp

Figure 3.2. Structures of steroidal glycoalkaloids 1 – 2 and furostanol saponins 3 – 5

quantified in the various L. longiflorum organs.

126

3.3.4.1. Nuclear Magnetic Resonance Spectroscopy (NMR).

1D1H NMR and

13C NMR spectra were acquired on an AMX-400 spectrometer

and an AMX-500 spectrometer (Bruker, Rheinstetten, Germany). Samples for NMR

analysis were dissolved in pyridine-d5 and chemical shifts were calculated as δ values

with reference to tetramethylsilane (TMS).

3.3.5. Quantitative Analysis of Steroidal Glycosides in Lilium longiflorum.

3.3.5.1. Sample Preparation

Lyophilized lily organ samples were removed from the freezer and allowed to

reach room temperature. The samples were ground to a fine powder with a laboratory

mill (IKA Labortechnik, Staufen, Germany) and passed through a sieve (pore size =

270mesh) (W. S. Tyler Inc., Mentor, OH). Each sample (125 mg except 250 mg for

fibrous roots and fleshy roots) was weighed separately and transferred into a 50 mL

volumetric flask, which was partially filled with ethanol and deionized water (7:3, v/v; 35

mL each). The samples were then extracted on a wrist-action autoshaker (45 min)

(Burrell Scientific, Pittsburgh, PA), sonicated in an ultrasonic water bath (30 min)

(B3500A-DTH ultrasonic bath, VWR International Inc., West Chester, PA), and filled to

full volume (50 mL) with ethanol and deionized water (7:3, v/v). The solution was

transferred to a centrifuge tube, centrifuged (5000 rpm for 10 min) (Sorvall RC-3C Plus,

127

Thermo Fisher Scientific Inc.), and filtered through a 0.45 μm PTFE syringe filter

(Thermo Fisher Scientific Inc.) prior to LC-MS/MS analysis.

3.3.5.2. Analytical Standard Preparation

Steroidal glycosides 1 – 5, isolated and purified as described in chapter two were

used as analytical standards. The analytical standards were accurately weighed into

volumetric flasks (10 mL) and partially filled with ethanol and deionized water (7:3, v/v;

7 mL each). Solutions were sonicated (5 min) and filled to full volume (10 mL) with

ethanol and deionized water (7:3, v/v). Solutions used for calibration curves were

prepared by dilution of the stock solutions. External calibration curves were established

over six data points covering a concentration range of 0.086 – 2.75 μg mL-1

for

compound 1 and 0.078 – 2.5 μg mL-1

for compounds 2 – 5. Mean areas (n = 3) generated

from the standard solutions were plotted against concentration to establish calibration

equations. Standard solutions were stored at 4 °C and were allowed to reach room

temperature prior to analysis.

3.3.5.3. Liquid Chromatography-Mass Spectrometry (LC-MS/MS).

LC-MS/MS analysis of L. longiflorum extracts was performed using an Agilent

1200 series HPLC system (Agilent Technologies Inc., Santa Clara, CA) equipped with a

FC/ALS Therm autosampler thermostat, a HiP-ALS SL autosampler, a BIN Pump SL

128

binary pump, a TCC SL thermostated column compartment, and a DADSL diode array

detector, interfaced to a 6410 triple-quadrupole LC-MS mass selective detector equipped

with an API-ESI ionization source. Chromatographic separations were performed on a

Prodigy C18 column (250 x 4.6 mm i.d.; 5.0 μm particle size) (Phenomenex, Torrance,

CA) operated at a flow rate of 1.0 mL min-1

, column temperature set to 25 °C, and an

injection volume of 10 μL. The binary mobile phase consisted of (A) 0.1% formic acid in

deionized water and (B) 0.1% formic acid in acetonitrile. Chromatographic separations

were performed using a linear gradient of 15 – 43%B over 40min and then to 95%B over

5 min; thereafter, elution with 95% B was performed for 10 min. The re-equilibration

time was 10 min. Mass Hunter Workstation Data Acquisition, Qualitative Analysis, and

Quantitative Analysis software were used for data acquisition and analysis. Quantitative

analysis was performed in positive ion mode. Ionization parameters included capillary

voltage, 3.5 kV; nebulizer pressure, 35 psi; drying gas flow, 10.0 mL min-1

; and drying

gas temperature, 350 °C. Full-scan mass data were collected for a mass range of m/z 100

– 1500. MS2 experiments were conducted for precursor and product ion selection for

steroidal glycoalkaloids 1 and 2 (Figure 3.3) and furostanol saponins 3 – 5 (Figure 3.4).

129

Figure 3.3. MS2 product ion spectra of steroidal glycoalkaloids 1 – 2.

x103

0

2

4

6

8

884.5

866.5

x104

0

1

2

926.5

908.5

850 900 950 Counts vs. Mass-to-Charge (m/z)

130

Figure 3.4. MS2 product ion spectra of furostanol saponins 3 – 5.

Flow injection analysis (FIA) experiments were performed to optimize

fragmentor voltages and collision energies. The fragmentor voltage was set at 120 V, and

collision energies were set to 60, 55, 30, 25, and 25 for compounds 1 – 5, respectively.

By means of the multiple reaction monitoring (MRM) mode, the individual steroidal

glycosides were analyzed using the following mass transitions given in parentheses: 1

(m/z 926.5 → 908.5), 2 (m/z 884.5 → 866.5), 3 (m/z 1047.5 → 885.5), 4 (m/z 1017.5 →

855.5), 5 (m/z 1017.5 → 855.5) (Figure 3.5).

x103

0

1

2 1047.5

885.5

x103

0

1

2

3 1017.5

855.5

x10

3

0

1

2

3

4

5 1017.5

855.5

800 900 1000 1100

Counts vs. Mass-to-Charge (m/z)

131

Figure 3.5. MS/MS chromatograms for the quantitative analysis of compounds 1 – 5 in a

L. longiflorum bulb scale using multiple reaction monitoring (MRM) mode.

x10 2

0

1

2

3

4

0

1

2

3

4

0

1

0

1

2

3

0

1

2

Counts vs. Acquisition Time (min) 5 10 15 20 25 30 35 40

x10 2

x10 3

x10 3

x10 3

m/z 1017.5 → 855.5

m/z 1047.5 → 885.5

m/z 926.5 → 908.5

m/z 884.5 → 866.5

TIC 1

2

3 4 5

1

2

3

4

5

132

3.3.5.4. Recovery

Recovery rates were calculated using the standard addition method (Skoog et al.,

1997). Lyophilized and finely ground lily organs were separately weighed, transferred

into volumetric flasks (50 mL), and spiked with three different concentrations (50, 100,

and 200 μg g-1

) of purified reference standards dissolved in ethanol and deionized water

(7:3, v/v). The volumetric flasks were then partially filled with ethanol and deionized

water (7:3, v/v; 35 mL each). After extraction on a laboratory shaker (45 min) and

sonication (30 min), they were filled to full volume (50 mL) with ethanol and deionized

water (7:3, v/v). Quantitative analysis was then performed as described above. The

recovery rate for each steroidal glycoside in the different plant organs was calculated by

comparing the amount of standard in the spiked sample with the content found in the lily

organ sample that was not spiked with additional standards (control). Each analysis was

performed in triplicate.

3.3.5.5. Thin Layer Chromatography (TLC).

L. longiflorum bulb extract, prepared as described above, was evaporated under

reduced pressure (30 °C; 1.0 x 10-3

bar) using a Laborota 4003 rotary evaporator

(Heidolph Brinkman LLC, Elk Grove Village, IL) and lyophilized. Lyophilized bulb

extract (1 mg) and compounds 3 – 5 (1 mg) were individually dissolved in methanol (0.5

mL), spotted on a 20 cm x 20 cm silica gel 60 F254 TLC plate (Merck & Co., Inc.,

Whitehouse Station, NJ), and developed with chloroform/methanol/water (8:4:1, v/v/v).

133

To detect furostanols, TLC plates were developed with Ehrlich‘s reagent [3.2 g of p-

(dimethylamino)benzaldehyde in 60 mL of 95% ethanol and 60 mL of 12NHCl] and

heated to 110 °C for 5 min. Bright red spots were indicative of a positive reaction.

3.3.5.6. Statistical Analysis.

To examine differences in concentrations of steroidal glycosides in the different

plant organs, data were subjected to analysis of variance (ANOVA) and means were

separated with Fisher‘s protected LSD (R = 0.05) using SAS version 9.2 for Windows

(SAS Institute Inc., Cary, NC).


3.4.1. Quantification of steroidal glycosides in the different organs of L. longiflorum.

In chapter two, five steroidal glycosides including two steroidal glycoalkaloids

and three furostanol saponins were identified for the first time in L. longiflorum. To

investigate the natural distribution of these compounds in the different organs of L.

longiflorum, compounds 1 – 5 were purified as analytical standards. To quantify

compounds 1 – 5 in different organs of L. longiflorum, extracts prepared from bulb

scales, fibrous roots, fleshy roots, leaves, lower stems, upper stems, flower buds, and

mature flowers were analyzed by LC-MS/MS operating in MRM mode. To assess

134

linearity, calibration curves were constructed over a range of six concentrations. Good

linearity was achieved over the concentration ranges of 0.086 – 2.75 μg mL-1

for

compound 1 and 0.078 – 2.50 μg mL-1

for compounds 2 – 5 (Figure 3.6; Figure 3.7;

Figure 3.8; Figure 3.9; Figure 3.10). The correlation coefficients for compounds 1 – 5

ranged from R2 = 0.9997 to R

2 = 0.9999.

Figure 3.6. Calibration equation for compound 1.

135


136


137


138


To assess the accuracy of the analytical method, recovery rates were calculated

for compounds 1 – 5 in each plant organ. Standards were added in defined amounts to

each plant organ sample prior to quantitative analysis and compared to a control with no

standard addition. Recovery rates were calculated in the bulb scales (98.7 – 100.2%),

fibrous roots (95.7 – 102.4%), fleshy roots (98.6 – 102.0%), leaves (95.8 – 103.0%),

lower stems (97.7 – 100.1%), upper stems (95.9 – 102.3%), flower buds (95.3 – 101.0%),

and mature flowers (96.3 – 101.9%). The recovery rates for compounds 1 – 5 in all

139

organs analyzed were within the range of 95.7 – 103.0%. The precision of the method

was tested by multiple injections of the same bulb scale sample (n = 6) and calculating

the relative standard deviation (RSD) of compounds 1 – 5. The RSD values for

compounds 1 – 5 were 3.24, 2.54, 4.51, 1.63, and 2.58%, respectively. These data clearly

demonstrate acceptable recovery rates, RSD, and linearity, suggesting that the LC-

MS/MS method operating in MRM mode is a reliable method for the accurate

quantitative determination of compounds 1 – 5 in the different organs of L. longiflorum.

Concentrations of compounds 1 – 5 were determined in bulb scales, fibrous roots,

fleshy roots, leaves, lower stems, upper stems, flower buds, and mature flowers of L.

longiflorum cv. 7-4. The concentration of compounds 1 – 5 in the different plant organs

were significantly different as determined by ANOVA, P < 0.0001 (Table 3.1). The

highest concentrations of the total five steroidal glycosides were 12.02 ± 0.36, 10.09 ±

0.23, and 9.36 ± 0.27 mg g-1

dry weight in flower buds, lower stems, and leaves,

respectively (Table 3.2). Interestingly, the proportions of the steroidal glycoalkaloids 1

and 2 to furostanol saponins 3 – 5 were variable and decreased from the aboveground

plant organs to the underground organs (Figure 3.11). The highest concentrations of the

two steroidal glycoalkaloids were 8.49 ± 0.3, 6.91 ± 0.22, and 5.83 ± 0.15 mg g-1

dry

weight in flower buds, leaves, and bulbs, respectively (Table 3.2).

140

Table 3.1. ANOVA for concentrations of compounds 1 – 5 in the different organs of L.

longiflorum.

Compound Source SS df MS F P-value

1 Between organs 55.0459958 7 7.86371369 813.84 < 0.0001

Error 0.1546 16 0.0096625

Total 55.2005958 23

2 Between organs 30.0797958 7 4.29711369 389.32 < 0.0001

Error 0.1766 16 0.0110375

Total 30.2563958 23

3 Between organs 11.5567167 7 1.65095952 406.81 < 0.0001

Error 0.06493333 16 0.00405833

Total 11.62165 23

4 Between organs 11.18485 7 1.59783571 1681.93 < 0.0001

Error 0.0152 16 0.00095

Total 11.20005 23

5 Between organs 3.81072917 7 0.54438988 706.24 < 0.0001

Error 0.01233333 16 0.00077083

Total 3.8230625 23

141

Table 3.2. Concentrations of compounds 1 – 5 in the different organs of L. longiflorum.

1

Values with the same letter in each row are not significantly different (p < 0.05). 2

Concentrations are means of triplicates ± SD, expressed on a dry weight basis (dw).

compounds

concentration (mg g-1 dw)1,2

bulb scale fibrous root fleshy root leaf lower stem upper stem bud flower

1 3.31c ± 0.12 0.30g ± 0.05 0.55f ± 0.02 4.23b ± 0.01 3.17c ± 0.05 2.87d ± 0.15 4.82a ± 0.19 1.96e ± 0.03

2 2.52bc ± 0.07 0.26f ± 0.04 0.44f ± 0.01 2.68b ± 0.22 2.05d ± 0.05 2.34c ± 0.12 3.67a ± 0.11 0.96e ± 0.05

Total 1-2 5.83c ± 0.15 0.56g ± 0.09 0.99f ± 0.01 6.91b ± 0.22 5.22d ± 0.1 5.21d ± 0.27 8.49a ± 0.30 2.92e ± 0.07

3 1.51c ± 0.08 0.47f ± 0.03 1.55c ± 0.01 2.01b ± 0.07 1.50c ± 0.12 1.15d ± 0.04 2.92a ± 0.06 0.88e ± 0.05

4 0.81c ± 0.04 0.26f ± 0.02 1.76b ± 0.04 0.29f ± 0.01 2.18a ± 0.04 0.67d ± 0.03 0.41e ± 0.01 0.35e ± 0.01

5 0.69c ± 0.05 0.13f ± 0.01 1.06b ± 0.03 0.15f ± 0.01 1.19a ± 0.02 0.37d ± 0.04 0.20e ± 0.01 0.22e ± 0.01

Total 3-5 3.01d ± 0.17 0.86h ± 0.07 4.37b ± 0.07 2.45e ± 0.09 4.87a ± 0.13 2.19f ± 0.05 3.53c ± 0.06 1.45g ± 0.05

Total 1-5 8.84d ± 0.3 1.42h ± 0.14 5.36f ± 0.08 9.36c ± 0.27 10.09b ± 0.23 7.4e ± 0.32 12.02a ± 0.36 4.38g ± 0.12

142

Figure 3.11. Proportions of steroidal glycoalkaloids 1 – 2 to furostanol saponins 3 – 5 in

the different organs of L. longiflorum. Proportions are based on mg g-1

dry weight basis.

The two steroidal glycoalkaloids, 1 and 2, had a similar pattern of distribution in the

various organs of L. longiflorum (Figure 3.12; Figure 3.13). Compound 1 occurred in

significantly different concentrations in all of the plant organs analyzed, except for the

concentration in the bulb scale compared to the lower stem, which were not significantly

different from each other. Compound 1 occurred in the highest concentration of 4.82 ±

0.19 mg g-1

in the flower buds followed by 4.23 ± 0.01 mg g-1

in the leaf tissue.

143

Figure 3.12. Concentrations of steroidal glycoalkaloid 1 in the different organs of L.

longiflorum. Bars with the same letter are not significantly different (p < 0.05).

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

OH

HO

OH

The concentration of 1 was significantly higher in the lower stem as compared to the

upper stem, at 3.17 ± 0.05 and 2.87 ± 0.15 mg g-1

, respectively. In the underground

organs, compound 1 occurred in the highest concentration of 3.31 ± 0.12 mg g-1

in the

bulb scale as compared to the lowest concentrations of 0.30 ± 0.05and 0.55 ± 0.02 mg g-1

in the fibrous roots and fleshy roots, respectively. Compound 2, the acetylated derivative

1

c

g

b

f

c d

a

e

144

of compound 1, was distributed similarly to compound 1; however, it generally occurred

in slightly lower concentrations.

Figure 3.13. Concentrations of steroidal glycoalkaloid 2 in the different organs of L.

longiflorum. Bars with the same letter are not significantly different (p < 0.05).

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

O

HO

OH

O

Compound 2 occurred in the highest concentration of 3.67 ± 0.11 mg g-1

in the flower

bud followed by 2.68 ± 0.22 mg g-1

in the leaf tissue and 2.52 ± 0.07 mg g-1

in the bulb

scale. In contrast to compound 1, compound 2 was significantly higher in the upper stem

2

bc

f

b

f

d c

a

e

145

as compared to the lower stem, at 2.34 ± 0.12 and 2.05 ± 0.05 mg g-1

, respectively. The

concentration of compound 2 was not significantly different between bulb scales and

leaves, 2.52 ± 0.07 and 2.68 ± 0.22 mg g-1

, bulbs scales and upper stem, 2.52 ± 0.07 and

2.34 ± 0.12 mg g-1

, and between fibrous roots and fleshy roots, 0.26 ± 0.04 and 0.44 ±

0.01 mg g-1

. Both steroidal glycoalkaloids occurred in the lowest concentration in the

fleshy roots and fibrous roots as compared to the other organs of the plant. Interestingly,

compounds 1 and 2 were lower in the flowers as compared to flower buds, which

contained the highest concentration of both compounds. Similar to solanaceous plants,

glycoalkaloid pairs that differed only in the composition of the carbohydrate moiety were

found (Sánchez-Mata et al., 2010; Roddick et al., 2001). Although glycoalkaloids are also

present in the edible parts of solanaceous plants, they can be toxic. Solanidine

glycoalkaloids found in potato tubers are generally considered to be safe at

concentrations of < 0.2 mg g-1

fresh weight (Sinden and Webb, 1972). Lily bulbs contain

solasodine glycoalkaloids similar to the glycoalkaloids found in eggplant, and these

compounds are less toxic than the solanidine based compounds (Roddick et al., 2001).

The content of steroidal glycoalkaloids, 1 and 2, in lily bulbs and flower buds is > 0.2 mg

g-1

fresh weight, but is similar to the content of solasonine and solamargine found in

Solanum macrocarpon, the Gboma eggplant, consumed in parts of Africa, Southeast

Asia, and the Caribbean. The glycoalkaloid levels in the fruits of Gboma eggplant are 5 –

10 times higher than the levels that are considered to be safe for human consumption

based on current standards (Sánchez-Mata et al., 2010). Feeding experiments are clearly

needed to determine the safe levels of L. longiflorum bulbs and flower buds for human

consumption and if the steroidal glycoalkaloids are the toxic compounds in flowers and

146

leaves that are responsible for poisoning in domesticated cats. Nevertheless, solasodine-

based glycoalkaloids have been used to treat human skin carcinomas and are of

commercial interest as a raw material for the production of pharmaceutical steroids

(Eltayeb et al., 1997).

The distribution of the furostanol saponins 3 – 5 was somewhat different from

that of the steroidal glycoalkaloids in the various plant organs of L. longiflorum (Figure

3.14; Figure 3.16; Figure 3.17).

147

Figure 3.14. Concentrations of compound 3 in the different organs of L. longiflorum.

Bars with the same letter are not significantly different (p < 0.05).

O

HOO

OH

O

OH3CHO OHOH

O

O

OH

O

HO

OH

HO

OH

O

O

HO

OH

HO

OH

The highest concentrations of the three furostanol saponins were 4.87 ± 0.13, 4.37

± 0.07, and 3.53 ± 0.06 mg g-1

dry weight in lower stems, fleshy roots, and flower buds,

respectively (Table 3.2). Structurally, compounds 3 – 5 are similar except for the

interglycosidic linkage and terminal saccharide residues of the C-3 trisaccharide moiety

(Figure 3.15). In compound 3, the terminal sugar is a (+)-D-glucose linked from the C-

1′′′ carbon of the terminal sugar to the C-4′ carbon of the inner glucose. In compound 4,

c

f

b

c c d

a

e

3

148

the terminal sugar is (-)-L-arabinose linked from the C-1′′′ carbon to the C-3′ carbon of

the inner glucose. Compound 5 has the same interglycosidic linkage as compound 4;

however, it contains a (+)-(D)-xylose as the terminal sugar.

Figure 3.15. Differences in saccharide composition and interglycosidic linkages of

compounds 3 – 5.

Compound 3 occurred in significantly different concentrations in all of the plant

organs except that the bulb scale, lower stem, and fleshy roots were not significantly

different. The concentrations in the bulb scale, lower stem, and fleshy roots were 1.51 ±

0.08, 1.50 ± 0.12, and 1.55 ± 0.01 mg g-1

, respectively. Compound 3 occurred in the

highest concentration of 2.92 ± 0.06 mg g-1

in the flower buds followed by 2.01 ± 0.07

mg g-1

in the leaf tissue. Interestingly, the fibrous roots had the lowest concentration, and

O

HOO

OH

O

OH3CHO

OH OH

O

O

HO

OH

HO

OH

O

HO

OH

OH

O

OO

OH

HO

OH3CHO OHOH

O

O

HOHO

OH

O

OO

OH

HO

OH3CHO OHOH

O

3''

4''

3''

3

4

5

-D-Glcp

-D-Glcp

-L-Arap

-D-Xlyp

-L-Rhap

-L-Rhap

-L-Rhap

-D-Glcp

-D-Glcp

4

2

3

2

2

3

3

4

5

149

similarly to the steroidal glycoalkaloids 1 and 2, the flower buds had a higher

concentration than the mature flowers.

Compounds 4 and 5 had a similar pattern of distribution in the various organs of

L. longiflorum; however, compound 5 was slightly lower than compound 4 in all plant

organs. The concentrations of both compounds 4 and 5 were not significantly different

between fibrous roots and leaves or between flower buds and mature flowers.

150



O

HO

OH

OH

O

OO

OH

HO

OH3CHO OHOH

O

O

OH

O

HO

OH

HO

OH

O

c

f f

b a

d e e

4

151



O

HOHO

OH

O

OO

OH

HO

OH3CHO OHOH

O

O

OH

O

HO

OH

HO

OH

O

c

f f

b a

d e e

5

152

3.4.2. Histological visualization of furostanol localization in bulb scale sections of L.

longiflorum.

The Ehrlich reagent color reaction was employed to visualize furostanols in bulb

scale sections. The furostanols gave a bright red positive reaction, whereas the steroidal

glycoalkaloids were not positive for the reaction. Additionally, a crude extract of bulb

scales was separated by TLC and only furostanol bands gave a positive reaction,

suggesting that staining bulb scales with Ehrlich reagent should not produce positive

reactions with non-target compounds. Macroscopically, bulb scale cross sections showed

accumulation of furostanols in the outermost layers of the bulb scale. Additionally, a

positive reaction was observed surrounding three vascular bundles located in the

mesophyll, suggesting furostanol localization is associated with vascular bundles and

closely adjacent cells (Figure 3.18).

153

Figure 3.18. Histochemical staining of a bulb scale section. Arrows indicate the

epidermis (A) and three vascular bundles (B). Red color indicates the presence of

furostanols.

Interestingly, a positive reaction was not observed in the mesophyll, suggesting

preferential accumulation and elevated levels of furostanols in the outermost layer of the

bulb scale and association with vascular bundles. Microscopically, furostanols were

visualized in the highest intensity within the cells of the basal plate, and vascular bundles

and preferential accumulation in the intercellular spaces between the mesophyll cells and

palisade cell layer were observed (Figure 3.19; Figure 3.20).

154

Figure 3.19. Histochemical analysis of bulb basal plate and bulb scale sections: (A) Bulb

basal plate and adjacent bulb scale section. Lettered boxes indicate topography of images

(B) and (C); (B) subepidermal intercellular furostanol accumulation along the palisade

parenchyma of the basal plate (red); (C) Intercellular furostanol accumulation between

spongy tissue cells and palisade parenchyma of the basal plate (red). Furostanol

localization is visualized as dark red areas. VB, vascular bundles; BP, basal plate; M,

bulb scale mesophyll; EP, epidermal cells.

B

C

C B

M

BP

VB

EP EP

VB

20x

40x 40x

A

155

Figure 3.20. Histochemical analysis of bulb basal plate and bulb scale sections: (D)

Epidermal (red) and mesophyll cells of a bulb scale section; (E) Bulb scale epidermis

(red), mesophyll and apical meristem (red); (F) Apical meristem (red) and mesophyll

cells; (G) Bulb scale epidermal cells (red) and mesophyll cells. Furostanol localization is

visualized as dark red areas. VB, vascular bundles; BP, basal plate; M, bulb scale

mesophyll; EP, epidermal cells; AP, apical meristem.

D E

F G

EP

EP

AP

AP

M

M M

40x

10x

10x

10x

M

156

These observations were consistent with a histochemical analysis of mature leaf slices of

Achyranthus bidentata that showed triterpenoid saponin accumulation in palisade tissue

and phloem cells of the main vein vascular bundles and in phloem cells of the normal and

medullary vascular bundles of stem sections (Li and Hu, 2008). Interestingly, in oats, A.

sativa, the fluorescent steroidal saponin, avenacin A-1, has been visualized under UV

light and found to be localized in the epidermal cell layer of roots (Osbourn, 1999).

Similarly, in a histological study of Dioscorea caucasia, furostanol accumulation was

observed in specialized idioblasts in the upper and lower leaf epidermis and was not

observed in the leaf mesophyll (Gurielidze et al., 2004). Consistent with the observations

in D. caucasia and A. bidentata, there was no positive staining reaction in the mesophyll

of the bulb scale of L. longiflorum, suggesting lower levels of furostanols in these tissues.

3.4.3. Quantification of steroidal glycosides within bulb scales of L. longiflorum.

LC-MS/MS operating in MRM mode was employed to quantitatively determine

the levels of compounds 1 – 5 within whole bulb scales, the inner portion of bulb scales,

and the outermost portion of bulb scales. The outermost layers of intact lyophilized bulb

scales were carefully excised with a scalpel. The excised outermost cell layer was

approximately 15% of the average mass of the whole intact bulb scale. Quantitative

analysis was performed on whole bulb scale, the outermost layer of bulb scale (mostly

epidermal and subepidermal cells), and the innermost layer of bulb scale (mostly

mesophyll and vascular bundles) (Table 3.3).

157

Table 3.3. Concentrations of compounds 1 – 5 in whole bulb scale, bulb epidermis, and

bulb mesophyll.

1 Values with the same letter in each row are not significantly different (p < 0.05). 2 Concentrations are means of triplicates ± SD, expressed on a dry weight basis (dw).

Consistent with what was observed from the histological experiment, the concentrations

of furostanols 3 – 5 were higher in the outermost portion of the bulb scale verses the

inner bulb scale tissue. These quantitative data confirm the qualitative histological

observations made for localization of furostanols in bulb scale sections. Interestingly, the

steroidal glycoalkaloids 1 and 2 had the same organ distribution pattern as the furostanols

and occurred in elevated levels in the outermost portion of the bulb. The relative portions

of compounds 1 – 5 in the outermost layer of bulb scales were 38, 31, 20, 6, and 5%,

respectively (Figure 3.21).

compounds

concentration (mg g-1 dw)

1,2

whole bulb

scale

bulb

epidermis

bulb

mesophyll

1 3.31b ± 0.12 13.76

a ± 0.5 0.06

c ± 0.01

2 2.52b ± 0.07 11.22

a ± 0.3 0.07

c ± 0.01

3 1.51 b ± 0.08 7.27

a ± 0.4 0.10

c ± 0.01

4 0.81b ± 0.04 1.91

a ± 0.1 0.20

c ± 0.02

5 0.69b ± 0.05 1.90

a ± 0.1 0.17

c ± 0.02

158

Figure 3.21. Proportions of compounds 1 – 5 in (A) whole bulb scale, (B) bulb epidermis,

and (C) bulb mesophyll. Proportions are based on mg g-1

dry weight basis.

The relative portions of compounds 1 – 5 in the innermost portion of bulb scales

were 9, 12, 17, 34, and 28%, respectively. The relative proportions of compounds 1 – 5 in

the outermost layer of the bulb scale were similar to that of the whole bulb scale. The

relative proportions of compounds 1 – 5 in the innermost section of the bulb scale were

most similar to those of the fleshy roots, suggesting that the vascular bundles are most

likely contributing to the elevated levels of compound 3 – 5 in this tissue, which is

consistent with the histological visualizations (Figure 3.22).

159

Figure 3.22. Proportions of compounds 1 – 5 in different organs of L. longiflorum.

Proportions are based on mg g-1

dry weight basis.

In summary, the outermost cell layer of bulb scales that are associated with the

bulb epidermis had elevated levels of both steroidal glycoalkaloids and furostanols. The

innermost section of bulb scales had lower levels; however, the proportions of furostanols

to steroidal glycoalkaloids were different, demonstrating that the cells associated with the

vascular bundles have proportions of compounds 1 – 5 similar to those of the fleshy roots

as compared to the bulb epidermis. Elevated levels and preferential accumulation of

steroidal glycosides in the outermost cell layer of bulb scales and the cells associated with

vascular bundles may play a role in wound response and plant-pathogen interaction of L.

longiflorum.

160

3.5. Conclusion

In this chapter, two steroidal glycoalkaloids, (22R, 25R)-spirosol-5-en-3 -yl O- -

L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside and (22R,

25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-

glucopyranosyl-(1→4)]- -D-glucopyranoside, and three furostanol saponins, (25R)-26-

O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)-

-D-glucopyranosyl-(1→4)- -D-glucopyranoside, (25R)-26-O-( -D-glucopyranosyl)-


(1→3)- -D-glucopyranoside and (25R)-26-O-( -D-glucopyranosyl)-furost-5-en-

3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -L-xylopyranosyl-(1→3)- -D-

glucopyranoside, were quantified in the different organs of L. longiflorum for the first

time. The highest concentrations of steroidal glycosides were detected in flower buds,

lower stems, and leaves. The steroidal glycoalkaloids were detected in higher

concentrations as compared to the furostanol saponins in all of the plant organs except for

the fibrous and fleshy roots. The proportions of steroidal glycoalkaloids to furostanol

saponins were higher in the plant organs exposed to light and decreased from the

aboveground organs to the underground organs. The highest concentrations of the

steroidal glycoalkaloids were detected in flower buds, leaves, and bulbs. Both steroidal

glycoalkaloids had a similar pattern of distribution in the various plant organs; however,

the acetylated derivative occurred at lower levels. The furostanol saponins were detected

in the highest concentrations in the lower stems, fleshy roots, and flower buds.

Interestingly, the flower buds contained the highest concentrations of compounds 1 – 3,

161

and the fleshy roots contained the highest levels of compounds 4 and 5. In addition,

differential accumulation of steroidal glycosides was observed in the basal plate, bulb

scale epidermal cells, and vascular bundles.

Steroidal saponins and steroidal glycoalkaloids have been shown to play a role in

host defense in several plant species (Osbourn, 1996; Morrissey and Osbourn, 1999;

Osbourn, 1999; Papadopoulou et al, 1999; Osbourn et al., 2003; Osbourn, 2003; Hughes

et al., 2004). It has been recognized that pathogen infection is often dependent upon the

developmental stage of the plant and is tissue or organ specific (Straathof and Löffler,

1994). It is possible that location specific infection or resistance is related to the level of

specific steroidal glycosides present at a developmental stage or tissue specific location.

A correlation between total saponin content and resistance to the plant pathogenic fungus

Fusarium oxysporum f. sp. lilii has been observed in several hybrid Lilium cultivars;

however, only the total saponin content was measured and specific compounds were not

discriminated in the analysis (Curir et al, 2003). The results in this chapter show that the

levels of the two steroidal glycoalkaloids and three furostanol saponins varied in the

different organs and are preferentially accumulated in different tissues. Thus, it is

possible that the levels of specific steroidal glycosides present at a developmental stage

or tissue specific location may play a role in pathogen resistance (e.g. the high levels of

compounds 1 – 3 in the developing flower buds). This concept may be investigated by

observing whether there is a correlation between resistance or susceptibility in a specific

plant tissue and the presence or absence of a specific steroidal glycoside or steroidal

glycoside profile exists. If resistance to the pathogen is found to be associated with a

specific steroidal glycoside concentration or steroidal glycoside profile, an investigation

162

can be conducted on the exogenous application of isolated steroidal glycosides on other

plant species susceptible to the pathogen. This work can also be extended to plant

pathogenic fungi that are not pathogenic to L. longiflorum.

Quantitative analysis of steroidal glycosides in the different organs of L.

longiflorum is the first step to developing insight into the biological role these

compounds play in plant metabolism, plant development, and plant-pathogen

interactions. The results of this study will aid in the development of future studies in

animal and human health and toxicology and of commercial applications such as

functional foods, cosmetics, and pharmaceuticals.

163

Chapter 4: Antifungal Activity and Fungal Metabolism of Steroidal Glycosides of

Easter Lily (Lilium longiflorum) by the Plant Pathogenic Fungus, Botrytis cinerea

4.1. Abstract

Botrytis cinerea Pers. Fr. is a plant pathogenic fungus and the causal organism of

blossom blight of Easter lily (Lilium longiflorum Thunb.). Easter lily is a rich source of

steroidal glycosides, compounds which may play a role in the plant-pathogen interaction

of Easter lily. Five steroidal glycosides, including two steroidal glycoalkaloids and three

furostanol saponins, were isolated from L. longiflorum and evaluated for fungal growth

inhibition activity against B. cinerea, using an in vitro plate assay. All of the compounds

showed fungal growth inhibition activity; however, the natural acetylation of C-6′′′ of the

terminal glucose in the steroidal alkaloid, (22R, 25R)-spirosol-5-en-3 -yl O- -L-


increased antifungal activity by inhibiting the rate of metabolism of the compound by the

B. cinerea. Acetylation of the glycoalkaloid may be a plant defense response to the

evolution of detoxifying mechanisms by the pathogen. The biotransformation of the

steroidal glycoalkaloids by B. cinerea led to the isolation and characterization of several

fungal metabolites. The fungal metabolites that were generated in a model system were

also identified in Easter lily tissues infected with the fungus by LC-MS. In addition, a

steroidal glycoalkaloid, (22,R 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-

-D-glucopyranoside, was identified as both a fungal metabolite of the steroidal

glycoalkaloids and as a natural product in L. longiflorum for the first time.

164

4.2. Introduction

Botrytis cinerea Pers. Fr. is a necrotrophic plant pathogenic fungus with a broad

host range and is the cause of grey mold disease, one of the most important post-harvest

diseases of fruits and vegetables worldwide. B. cinerea infects over 200 species of

economically important plants and is a causal organism of blossom blight of Easter lily

(Lilium longiflorum Thunb.) and other ornamental lily species (Wehlburg et al., 1975;

Raabe et al, 1981; Williamson et al., 2007). In contrast to Botrytis eliptica, the cause of

fire blight of Easter lily, B. cinerea typically does not infect healthy plant tissues and is

often found growing on stressed, wounded, or senescing tissues (Van Baarlen et al.,

2004). B. cinerea over winters as sclerotia in crop debris and penetrates young plant

tissues where it remains latent until the environmental conditions are conductive to

infection (Williamson et al., 2007). The fungus flourishes in cool temperatures and high

humidity, often during the end of the growing season in late summer and early fall

(McRae, 1998). In fact, it is recommended to treat Easter lily flower buds with fungicide

prior to cold storage as a post-harvest management strategy for the control of blossom

blight in potted Easter lily plants and cut flowers prior to shipment (McAvoy, 2010).

Host defense responses to B. cinerea have been investigated in many plants

including thale cress, Arabidopsis thaliana, and tomato, Solanum lycopersicum

(Williamson et al., 2007). In response to infection by B. cinerea, activation of host

defense pathways including the production of antifungal metabolites and pathogenesis

related proteins have been reported (Van Baarlen et al., 2004). In addition to the

165

activation of inducible defense pathways, steroidal glycosides including steroidal

saponins and steroidal glycoalkaloids, constitutively present in plant tissues, have been

shown to play a role in host defense in several plant species (Osbourn, 1996; Morrissey

and Osbourn, 1999; Osbourn, 1999; Papadopoulou et al, 1999; Osbourn et al., 2003;

Osbourn, 2003; Hughes et al., 2004).

Steroidal saponins are widely distributed secondary metabolites and have been

found in over 100 plant families (Güçlü-Üstündağ and Mazza., 2006). They are

characterized by a steroid type skeleton glycosidically linked to sugar moieties. Steroidal

glycoalkaloids are similar in structure to steroidal saponins; however, they have nitrogen

present in the steroidal aglycone. In contrast to steroidal saponins, steroidal

glycoalkaloids are only found in the Solanaceae and Liliaceae (Li et al., 2006;

Ghisalberti, 2006).

Due to the amphipathic nature of the molecules, steroidal glycosides have been

shown to disrupt cell membranes both in vitro and in vivo (Steel and Drysdale, 1988;

Roddick et al., 2001). Some studies suggest that membrane disruption may be due either

to the interaction of the aglycone with membrane bound sterols, resulting in the formation

of membrane pores (Armah et al., 1999) or the extraction of membrane bound sterols,

causing loss of lipid bilayer integrity and membrane leakage (Keukens et al., 1992;

Keukens et al., 1995). The antifungal mechanisms of steroidal glycosides remain unclear

and the exact mechanism remains to be elucidated.

Fungal plant pathogens such as Gaeumannomyces graminis and Stagonospora

avanae have the ability to enzymatically detoxify host plant saponins (Bowyer et al.,

1995; Morrissey et al., 2000). Interestingly, B. cinerea has been shown to produce

166

enzymes that can metabolize a variety of plant defense compounds from active forms to

inactive forms (Staples and Mayer et al., 1995). In tomato, B. cinerea metabolizes the

antifungal steroidal glycoalkaloid, -tomatine, to an inactive form by enzymatic cleavage

of the entire saccharide moiety, or by the cleavage of the terminal xylose by a -

xylosidase enzyme (Verhoeff and Liem, 1975; Quidde and Osbourn, 1998). In addition,

other plant pathogenic fungi such as Septoria lycopersici and Fusarium oxysporum f.sp.

lycopersici detoxify -tomatine through independent metabolic pathways (Arneson and

Durbin, 1967; Ford et al., 1977). Investigations have been conducted on the interaction of

steroidal glycosides and B. cinerea; however, to date there are no studies on the

interaction of steroidal glycosides from L. longiflorum and B. cinerea.

Easter lily is a rich source of steroidal glycosides. In the Chapter two and Chapter

three, two steroidal glycoalkaloids, (22R, 25R)-spirosol-5-en-3 -yl O- -L-

rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside (1), (22R,

25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-acetyl- -D-

glucopyranosyl-(1→4)]- -D-glucopyranoside (2), and three furostanol saponins, (25R)-

26-O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol 3-O- -L-rhamnopyranosyl-

(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside (3), (25R)-26-O-( -D-


arabinopyranosyl-(1→3)- -D-glucopyranoside (4) and (25R)-26-O-( -D-


xylopyranosyl-(1→3)- -D-glucopyranoside (5) were identified and quantified in the

various plant organs of L. longiflorum. Steroidal glycosides are known to be inhibitory to

fungal growth and may play a role in the plant-pathogen interaction. The goal of this

167

study was to (1): evaluate the biological activity of five steroidal glycosides isolated from

the bulbs of L. longiflorum on the growth of the plant pathogenic fungus B. cinerea, using

an in vitro assay and (2): to use a model system to generate fungal metabolites of the

steroidal glycosides, isolate and characterize the metabolites, and determine if the fungal

metabolites were present in plant tissue infected with B. cinerea.

168


4.3.1. Plant material.

L. longiflorum, cultivar 7-4, plants were provided from the Rutgers University lily

breeding program. Young bulbs were treated with Captan (Bayer CropScience AG,

Monheim am Rhein, Germany) fungicide prior to planting. Bulbs were planted in raised

beds containing Pro-Mix (Premier Horticulture Inc., Quakertown, PA) and were grown to

mature plants under greenhouse conditions for 9 months prior to harvest. The greenhouse

temperatures were set to provide a minimum day temperature of 24 °C and a minimum

night temperature of 18 °C. Plants were fertilized biweekly with a 100 mg L-1

solution of

NPK 15-15-15 fertilizer (J.R. Peters Inc., Allentown, PA). Each plant produced 3 – 5 new

bulbs, which were used for extraction. For the purification of steroidal glycosides 1 – 5,

each plant was harvested by hand and the bulbs were manually separated, immediately

frozen under liquid nitrogen, lyophilized on a VirTis AdVantage laboratory freeze dryer

(SP Industries inc.,Warminster, PA) and stored at -80°C until extraction. For the fungal

inoculation studies, small sections of aerial stems and adjacent leaves of healthy growing

plants were chosen. Small sections were carefully excised from intact plants

approximately 5 cm below the apical meristem.

169

4.3.2. Fungal cultures.

An isolate of Botrytis cinerea was obtained from the Plant Diagnostic Laboratory

at Rutgers Cooperative Research and Extension (New Jersey Agricultural Experiment

Station). Cultures were maintained on potato dextrose agar (PDA, 39 g L-1

deionized

water) (Thermo Fisher Scientific Inc., Fairlawn, NJ) and incubated in the dark at 25 °C.

4.3.3. Chemicals.

The following compounds were obtained commercially: Sephadex LH-20,

hydrochloric acid, sodium hydroxide, Tween-80, chloroform-d (0.03% v/v TMS),

methanol-d4 (0.03% v/v TMS), and pyridine-d5 (0.03% v/v TMS) were purchased from

Sigma-Aldrich (St. Louis, MO); and (22R, 25R)-spirosol-5-ene-3 -ol (Glycomix Ltd,

Reading, UK). All solvents (acetonitrile, chloroform, ethanol, ethyl acetate, formic acid,

n-butanol, and n-pentane) were chromatographic grade and purchased from Thermo

Fisher Scientific Inc. (Fairlawn, NJ). Potato dextrose agar and potato dextrose broth was

purchased from Thermo Fisher Scientific Inc. (Fairlawn, NJ). Water was deionized (18

MΩ cm) using a Milli-Q-water purification system (Milli-Q, Bedford, MA).

170

4.3.4. Isolation and Purification of Steroidal Glycosides 1 – 5 from Lilium longiflorum.

Closely following the procedure reported in chapter two, the following five steroidal

glycosides were isolated from lyophilized L. longiflorum bulbs. The compounds were

obtained as white amorphous powders in high purity > 98%, as determined by LC-MS

and NMR.

Compound 1, (22R, 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)- -D-

glucopyranosyl-(1→4)- -D-glucopyranoside. 1H NMR and

13C NMR were consistent

with the literature (Mimaki and Sashida, 1990).

Compound 2, (22R, 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-

acetyl- -D-glucopyranosyl-(1→4)]- -D-glucopyranoside. 1H NMR and

13C NMR were

consistent with the literature (Munafo et al., 2010).


rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside. 1H NMR

and 13

C NMR were consistent with the literature (Ori et al., 1992).


rhamnopyranosyl-(1→2)- -L-arabinopyranosyl-(1→3)- -D-glucopyranoside. 1H NMR

and 13

C NMR were consistent with the literature (Munafo et al., 2010).


rhamnopyranosyl-(1→2)- -L-xylopyranosyl-(1→3)- -D-glucopyranoside. 1H NMR and

13C NMR were consistent with the literature (Munafo et al., 2010).

171

4.3.4.1. Nuclear Magnetic Resonance Spectroscopy (NMR).

1H NMR and

13C NMR spectra were acquired on an AMX-400 spectrometer

(Bruker, Rheinstetten, Germany). For NMR analysis, all compounds were dissolved in

pyridine-d5, except for compounds 7 and 10 which were dissolved methanol-d4 and

chloroform-d, respectively. Chemical shifts were generated as δ values with reference to

tetramethylsilane (TMS).

4.3.4.2. Liquid Chromatography-Mass Spectrometry (LC-MS).

LC-MS analysis was performed using a HP 1100 series HPLC system (Agilent

Technologies Inc., Santa Clara, CA) equipped with an auto injector, quaternary pump,

column heater, and diode array detector, interfaced to a Bruker 6300 series ion-trap mass

spectrometer equipped with an electrospray ionization chamber. Chromatographic

separations were performed using a Prodigy C18 column (250mm x 4.6mm i.d.; 5.0 m

particle size) (Phenomenex, Torrance, CA). The flow rate was set to 1.0 mL min-1

and

the column temperature was set to 25 °C. The binary mobile phase composition consisted

of (A) 0.1% formic acid in deionized water and (B) 0.1% formic acid in acetonitrile.

Separations were performed using a linear gradient of 15 – 43% B over 40 min and then

to 95% B over 5 min; thereafter, elution with 95% B was performed for 10 min. The re-

equilibration time was 10 min. For instrumentation control and data acquisition, HP

ChemStation and BrukerData Analysis software was used. All mass spectra were

172

acquired in positive ion mode over a scan range of m/z 100 – 2000. Ionization parameters

included: capillary voltage, 3.5 kV; end plate offset, -500V; nebulizer pressure, 50 PSI;

drying gas flow, 10 ml min-1

; and drying gas temperature, 360 °C. Trap parameters

included: ion current control, 30000; maximum accumulation time, 200 ms; trap drive,

61.2; and averages, 12 spectra.

4.3.4.3. Partial acid hydrolysis of compound 1.

Compound 1 (1 mg) was refluxed in a reaction vial (1 mL) (Reacti-Vial, Thermo

Fisher Scientific Inc., Fairlawn, NJ) at 80 °C for 2 hours in a solution of 1N HCl in

methanol (0.5 mL). After hydrolysis and titration to pH 7 with NaOH (4N), the sample

was evaporated to dryness under reduced pressure (30 °C; 1.0 x 10-3

bar) using a

Labarota 4003 rotary evaporator (Heidolph Brinkman LLC, Elk Grove Village, IL). The

residue was dissolved in ethanol and water (7:3, v/v; 2 mL), mixed on a vortex mixer (1

min) and filtered through 0.45 m PTFE syringe filter (Thermo Fisher Scientific Inc.,

Fairlawn, NJ) prior to LC-MS analysis (Figure 4.1).

173

Figure 4.1. Total ion chromatogram (TIC) of the partial acid-catalyzed hydrolysis

products of compound 1. Compound 9 is (22,R 25R)-spirosol-5-en-3 -yl O- -D-

glucopyranosyl-(1→4)]- -D-glucopyranoside, compound 7 is (22,R 25R)-spirosol-5-en-

3 -yl O- -D-glucopyranoside, and compound 10 is (22,R 25R)-spirosol-5-en-3 -ol

(solasodine).

4.3.5. B. cinerea growth inhibition assay.

Antifungal activity was assessed by an in vitro fungal growth inhibition assay

modified from Nicol et al. (Nicol et al., 2001). Fungi were maintained on potato dextrose

agar (PDA, 39 g L-1

deionized water) and incubated in the dark at 25 °C. The cultures

were continuously maintained by transferring a 5 mm plug of mycelium cut with a cork

bore from the periphery of actively growing colonies to freshly prepared media. The

fungal growth inhibition of compounds 1 – 5 were evaluated at three concentrations (1,

10, 100 mol) in the final media. Solutions of compound 1 – 5 were prepared in ethanol

and water (7:3, v/v), filter sterilized with a 0.22 m sterile syringe filter (Thermo Fisher

0

1

2

3

7 x10

10 15 20 25 30 Time [min]

9

7

10

174

Scientific Inc., Fairlawn, NJ) and incorporated into autoclaved PDA that was allowed to

cool to 50 °C. The media (each plate; 5 mL) was then transferred to polystyrene Petri

dishes (50 mm x 12 mm) (VWR International Inc., West Chester, PA), and allowed to

solidify. The final concentration of carrier solvent was 1% of the final volume of media

for all treatments and control. Plates were inoculated with a 5 mm plug taken from the

periphery of an actively growing stock culture. Plates were incubated in the dark at 25 °C

and the radial growth of each colony was measured using an ABS Solar Digimatic

Caliper (Mitutoyo America Corporation, Aurora, IL). Treatment and control colonies

were measured when the control colonies reached approximately 80% of plate diameter.

The average control colony diameter minus the average treatment colony diameter was

used to calculate the relative growth inhibition. (% inhibition = (average control diameter

mm – treatment diameter mm)/average control diameter (mm).

4.3.6. In vitro fungal metabolism of compounds 1 and 2.

Solutions of compound 1 and 2 were separately prepared in ethanol and water

(7:3, v/v), filter sterilized with a 0.22 m syringe filter and incorporated into autoclaved

PDA that was allowed to cool to 50 °C. The media (each plate; 5 mL) was then

transferred to polystyrene Petri dishes (50 mm x 12 mm) and allowed to solidify. A total

of 4 plates were prepared for each compound. The final concentration for each compound

was 100 molar. Plates were inoculated in the center with a 5 mm plug taken from the

periphery of an actively growing stock culture. Plates were incubated in the dark at 25 °C

and harvested at 48 and 72 hours, respectively. At harvest, the total contents of 2 plates

175

(total; 10 mL) were transferred to a centrifuge tube (50 mL) (Thermo Fisher Scientific

Inc., Fairlawn, NJ) containing ethanol and water (7:3, v/v; 35 mL). Each sample was then

extracted on a wrist-action autoshaker (15 min) (Burrell Scientific, Pittsburg, PA),

sonicated in an ultrasonic water bath (15 min) (B3500A-DTH ultrasonic bath, VWR

International Inc., West Chester, PA), and centrifuged (5000 rpm for 10 min) (Sorvall

RC-3C Plus, Thermo Fisher Scientific Inc.). The supernatant was then filtered through

0.45 m PTFE syringe filter prior to LC-MS analysis

4.3.7. Scale-up fungal metabolism of compound 1.

Potato dextrose broth (PDB, 39 g L-1

deionized water) (each; 100 mL) was

prepared and transferred to an Erlenmeyer flask (250 mL) and autoclaved. Once the

broth reached room temperature, it was inoculated with B. cinerea and incubated on an

orbital platform shaker at 200 rpm for 48 hours at 25 °C. After 48 hours, compound 1 (35

mg) was dissolved in ethanol and water (7:3, v/v; 1 mL), filter sterilized with a 0.22 m

sterile syringe filter and introduced to the flask. The reaction was monitored by sampling

aliquots (each; 0.5 mL) every 24 hours for 96 hours (Figure 4.2; Figure 4.3; Figure 4.4;

Figure 4.5). Each aliquot was diluted (1:3, v/v) with ethanol and water (7:3, v/v; 1.5

mL), and filtered through 0.45 m PTFE syringe filter prior to LC-MS analysis. At the

completion of the reaction, the content of the flask was immediately frozen under liquid

nitrogen, lyophilized and stored at -80 °C until extraction.

176

Figure 4.2. Extracted ion chromatograms of m/z 885 (compound 1) from samples taken

every 24 hours over the course of 96 hours. This illustrates the decrease over time due to

its metabolism by B. cinerea.

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

OH

HO

OH

18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 Time [min]

0.0

0.2

0.4

0.6

0.8

8 x10

Intens.

24 hrs

72 hrs

48 hrs

96 hrs

177

Figure 4.3. Extracted ion chromatograms of m/z 723 (compound 6) of samples taken

every 24 hours over the course of 96 hours. This illustrates the increase of the fungal

metabolite, compound 6, derived from the metabolism of compound 1 by B. cinerea.

O

HOO

OH

HO

OH3CHO

OH OH

O

O

HN

18.0 18.5 19.0 19.5 20.0 20.5 21.0 21.5 Time [min]

0

1

2

3

4

5

6

7 x10

Intens.

24 hrs

72 hrs

48 hrs

96 hrs

178

Figure 4.4. Extracted ion chromatograms of m/z 577 (compound 7) of samples taken



O

HOO

OH

HO

OH

O

HN

19 20 21 22 23 24 25 Time [min]

0.0

0.5

1.0

1.5

2.0

2.5

8 x10

Intens.

24 hrs

72 hrs

48 hrs

96 hrs

179

Figure 4.5. Extracted ion chromatograms of m/z 414.6 (compound 10) of samples taken



HO

O

HN

30 31 32 33 34 Time [min]

0.0

0.5

1.0

1.5

2.0

7 x10

24 hrs

72 hrs

48 hrs

96 hrs

Intens.

180

4.3.7.1. Semi-preparative RP-HPLC isolation of the fungal metabolites of compound 1.

The lyophilized reaction mixture, as described above, was ground into a fine

powder with a laboratory mill (IKA Labortechnik, Staufen, Germany) and extracted with

ethanol and water (7:3, v/v; 2 x 50 mL) on an autoshaker at room temperature for 15

minutes. After centrifugation (5000 rpm for 10 minutes), the supernatant was collected

and the residue discarded. The supernatant was then evaporated under reduced pressure,

dissolved in a mixture of 0.1% formic acid in deionized water and 0.1% formic acid in

acetonitrile (75:25, v/v; 5 mL), and filtered through 0.45 m PTFE syringe filtered prior

to purification. Chromatographic separations were achieved by semipreparative RP-

HPLC performed on a Luna C18 column (250 mm x 21.2 mm i.d.; 10 m particle size)

(Phenomenex, Torrance, CA). Chromatography was performed on a Shimadzu LC-6AD

liquid chromatograph (Shimadzu Scientific Instruments Inc, Columbia, MD) using a

UV/VIS detector and a 2 mL injection loop. Mixtures of (A) 0.1% formic acid in

deionized water and (B) 0.1% formic acid in acetonitrile were used as the mobile phase.

The flow rate was set to 20 mL min-1

, the column temperature was 23 ± 2 °C and UV

detection was recorded at = 210 nm (Figure 4.6). Chromatography was performed

using a linear gradient of 5 – 30% B over 45 min and then to 90% B over 10 min;

thereafter, elution with 90% B was performed for 10 min. The re-equilibration time was

10 min.

181

Figure 4.6. RP-HPLC chromatogram (λ = 210 nm) of compounds 6, 7, and 10 isolated

from the biotransformation of compound 1 by B. cinerea.

The target compounds were collected, freed from solvent under reduced pressure and

lyophilized, yielding 6 (2 mg), 7 (5 mg), and 10 (1 mg) as white amorphous powders in

high purity > 98%, as determined by LC-MS (Figure 4.7) and NMR.

Compund 6. (22,R 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)- -D-

glucopyranoside. 1H NMR and

13C NMR were consistent with the literature (Mimaki and

Sashida, 1990).

Compund 7. (22,R 25R)-spirosol-5-en-3 -yl O- -D-glucopyranoside. 1H NMR and

13C

NMR were consistent with the literature (Kim et al, 1996).

Compound 10 (22,R 25R)-spirosol-5-en-3 -ol. 1H NMR and

13C NMR were consistent

with the literature (Bird et al., 1979).

Inte

nsity (

210)

time (min)

30 35 40 50 45

10

20

30

40

6

7

10

182

Figure 4.7. Total ion chromatograms (TIC) of compounds 6, 7, and 10 isolated

by RP-HPLC.

0

1

2

3

4

5

8 x10

10 15 20 25 30 Time [min]

10

0

2

4

6

8 x10 7

0.0

0.5

1.0

1.5

8 x10

Intens.

6

183

4.3.8. Isolation and Purification of Compound 6 from Lilium longiflorum bulbs.

4.3.8.1. Sequential Solvent Extraction of Lyophilized L. longiflorum Bulbs.

Lyophilized lily bulbs (100 g) were frozen in liquid nitrogen, ground into a fine

powder with a laboratory mill and extracted with n-pentane (3 x 100 mL) on an

autoshaker at room temperature for 30 minutes. After centrifugation (5000 rpm for 10

minutes), the organic layers were discarded and the pellet was freed from residual

solvent. The residual material was then extracted with a mixture of ethanol and water

(7:3, v/v; 2 x 150 mL) on an autoshaker for 45 minutes at room temperature. After

centrifugation (5000 rpm for 10 minutes) and vacuum filtration through a Whatman 114

filter paper (Whatman International Ltd., Maidstone, UK), the supernatant was collected

and the residue discarded. The supernatant was then evaporated under reduced pressure

and lyophilized, yielding a crude bulb extract (12.9g). The lyophilized crude bulb extract

was then dissolved in deionized water (100 mL), washed with ethyl acetate (5 x 100 mL)

and the organic phase was discarded. The aqueous phase was then extracted with n-

butanol (5 x 100 mL) and the aqueous phase was discarded. The organic phase was then

evaporated under reduced pressure (30 °C; 1.0 x 10-3

bar) and lyophilized, yielding a

crude glycoside extract (2.2g).

184

4.3.8.2. Gel Permeation Chromatography (GPC).

Crude glycoside extract (1.0 g) was dissolved in a solution of ethanol and water (7:3,

v/v; 5.0 mL), filtered with a 0.45 m PTFE syringe filter and then applied onto a standard

threaded 4.8cm x 60cm glass column (Kimble Chase Life Science and Research Products

LLC, Vinland, NJ) packed with Sephadex LH-20 (Amersham Pharmacia Biotech,

Uppsala, Sweden) that was washed and conditioned in the same solvent mixture

overnight. Chromatography was performed with isocratic ethanol and water (70:30, v/v)

at a flow rate of 3.5 mL min-1

. The first 200 mL of effluent was discarded and 25

fractions (25 mL each) were collected and analyzed by LC-MS as described above. Based

on the LC-MS analysis, GPC fractions 8 through 10 contained the highest levels of

compound 6 and were combined, evaporated under reduced pressure, and lyophilized,

yielding GPC fraction A (25mg) (Figure 4.8).

185

Figure 4.8. Isolation scheme for compound 6 purified from the bulbs of L. longiflorum.

EtOH, ethanol; EtAC, ethyl acetate; n-BuOH, n-butanol; ACN, acetonitrile.

4.3.8.3 Semipreparative Reverse-Phase High Performance Liquid Chromatography (RP-

HPLC).

Purification of compound 6 from GPC fraction A was achieved by

semipreparative RP-HPLC under the same conditions as described for above (Figure

4.9).

186

Figure 4.9. RP-HPLC chromatogram (λ = 210 nm) of compound 6 isolated from L.

longiflorum bulb n-butanol extract fractionated by gel permeation chromatography

(GPC).

Compound 6 was collected, freed from solvent under reduced pressure, and lyophilized

yielding 6 (3 mg) as a white amorphous powder in high purity > 98%, as determined by

LC-MS and NMR. 1H NMR and

13C NMR were consistent with the literature (Mimaki

and Sashida, 1990).

time (min)

20 25 30 40 35

Inte

nsity (

210)

5

6

7

8

6

187

Figure 4.10. Structures of compounds 1 – 10.

O

R5OO

OH

R4O

OH3CHO OHOH

O

O

OH

O

HO

OH

HO

OH

O

R1O

O

HN

O

HO

OH

OH

O

HOHO

OH

R1

R4 R5

H

H

H

O

HO

OH

HO

OHS1 S3

S4

S5

S6

S2

S1

O

HO

OH

R3O

OR2

S2

R2

S4

R3

S1

O

HO

O

HO

OH

O

OH3CHO OHOH

S5 S6

S2 S4 S3

S2 S4 H

S2 H

S2 S3

H

H

S2 S1H

R1 R2 R3

1

2

3

4

56

7

8

9

10

11

1213

1415

16

17

18

19

20

21

22

2324

2526

27

1'2'

4'

1''

1''''

1

2

3

4

56

7

8

9

10

1112

13

1415

16

17

18

19

20

21

22

2324

2526

27

-D-Glcp

1

2

4

5

-L-Arap -D-Xlyp

3

8

9

10

6

7

6-Ac- -D-Glcp

H

-D-Glcp

-L-Rhap

188

4.3.9. Infection of L. Longiflorum tissue and sample preparation for LC-MS analysis.

Small sections of aerial stems were excised from intact plants approximately 5

cm below the apical meristem, including several small (~3 cm) leaves. The plant tissue

was surface sterilized (10 % bleach and 0.01% tween-80, v/v) for 10 minutes, rinsed with

sterilized DI water, and transferred aseptically to a Petri dish (90 mm x 15 mm).

Treatment tissues were inoculated with two 5 mm plugs of B. cinerea and incubated at in

the dark at 25 C. Control samples were treated under the same conditions without fungal

inoculation. Once fully colonized with mycelium (7 days) the samples were frozen under

liquid nitrogen and lyophilized. The lyophilized material was ground with a mortar and

pestle and passed through a sieve (pore size; 270 mesh) (W.S. Tyler Inc., Mentor, OH).

The fine powder (0.5 g) was transferred to a centrifuge tube (15 mL), extracted with

ethanol and water (7:3, v/v; 5 mL) on an autoshaker at room temperature for 10 minutes

and sonicated in an ultrasonic water bath (10 min). After centrifugation (5000 rpm for 10

minutes), the supernatant was collected and filtered through 0.45 m PTFE syringe

filtered prior LC-MS analysis. LC-MS analysis was performed as described above.

4.3.10. Statistical Analysis.

Data was subjected to analysis of variance (ANOVA) and regression analysis

using SAS version 9.2 for Windows (SAS Institute Inc., Cary, NC).

189


4.4.1. Fungal growth inhibition assay.

Analysis of variance was performed to determine if there was a significant effect

of treatment (compounds 1 – 5), rate (1, 10, 100 mol), and the interaction between

treatment and rate. There was a significant interaction between treatment and rate (P <

0.0001), thus the main effects were ignored and a further investigation of the interaction

was performed (Table 4.1). An equation describing the relationship between the response

and the rate was generated for each treatment (Figure 4.10).

Table 4.1. ANOVA for treatment (compounds 1 – 5), rate (1, 10, 100 mol), and the

interaction between treatment and rate.

Source SS df MS F P-value

Treatment 0.01124694 4 0.00281174 3.88 0.0117

Rate 0.73396072 2 0.36698036 506.92 < 0.0001

Treatment x Rate 0.20059674 8 0.02507459 34.64 < 0.0001

Error 0.02171806 30 0.00072394

Corrected Total 0.96752246 44

190

Figure 4.11. Growth inhibition activity of compounds 1 – 5 on radial mycelia growth of

B. cinerea. Dashed lines represent steroidal glycoalkaloids and solid lines represent

furostanol saponins.

191

All five compounds were weakly inhibitory to B. cinerea. The furostanol

saponins, compounds 3 – 5, all had similar activity ranging from approximately 25 – 30%

growth inhibition as compared to control at the highest concentration tested. The steroidal

glycoalkaloid, compound 1, had similar inhibitory activity to the furostanol saponins. The

steroidal glycoalkaloid, compound 2, had the highest inhibitory activity of 49.2 % at the

highest concentration, approximately two times the activity of compound 1 (Figure

4.11). Steroidal glycoalkaloids 1 – 2 are similar in structure and only differ by the

presence of an acetyl group linked to the C-6′′′ hydroxy position of the terminal glucose

of carbohydrate moiety. The acetylation of the terminal glucose unit resulted in an

increased rate of fungal growth inhibition, as compared to compound 1. Similar to the

solanaceous glycoalkaloids, -chaconine and -tomatine, compounds 1 and 2 occur

together as a pair, share the same aglycone, only differ in the carbohydrate moiety, and

exhibit differential biological activity (Roddick et al., 2001). Friedman and MacDonald

suggested that glycoalkaloid pairs may occur as a plant defense response to the adaptive

ability of the pathogen to detoxify the plant‘s antifungal compounds (Friedman and

McDonald, 1997).

192

Figure 4.12. Growth inhibition activity of (A) compound 1 and (B) compound 2 on the

radial mycelia growth of B. cinerea. At the highest concentration, the mycelia growth

inhibitory activity was 24.9 and 49.2 % for compounds 1 and 2, respectively.

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

OH

HO

OH O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

O

HO

OH

O

1 2

A B

24.9% 49.2 %

control 1 mol 10 mol 100 mol control 1 mol 10 mol 100 mol

193

4.4.2. Metabolism of compound 1 and 2 by B. cinerea.

Based on the observation that compound 2 had a two-fold increase in fungal

growth inhibition as compared to compound 1, an investigation on the ability of B.

cinerea to cleave the sugar residues of compounds 1 and 2 was conducted (Figure 4.12).

Figure 4.13. ESI+–MS mass spectra of steroidal glycoalkaloids 1 and 2.

After 48 hours of in vitro metabolism of compound 1 by B. cinerea, only trace quantities

of compound 1 could be detected by LC-MS (Figure 4.13).

414.6 576.4 738.5

884.9 1

0

1

2

3

4

7 x10 Intens.

414.6576.4

738.5

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

OH

HO

OH

200 400 600 800 1000 1200 1400 1600 1800 m/z

200 400 600 800 1000 1200 1400 1600 1800

271.3

414.3

576.4 780.5

926.9 2

0.0 0.5

1.0

1.5

2.0 2.5

7 x10

m/z

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

O

HO

OH

414.3576.4

780.5

O

Intens.

194

Figure 4.14. Metabolism of compound 1 by B. cinerea: (A) Total ion chromatogram

(TIC) of fungal media spiked with compound 1 (time = 0) and (B) TIC of metabolites of

compound 1 (48 hours).

A metabolite of compound 1, compound 6, was observed with a based peak at m/z 722.8

(Figure 4.14). This ion was consistent with a molecule containing one less glucose

molecule then compound 1. Additionally, ion fragments at 576.4 [M– Rha+H]+ and 414.5

[M–Glu–Rha+H]+ were observed and were consistent with a disaccharide moiety

containing one glucose and one rhamnose molecule.

0.0

0.5

1.0

1.5

8 x10

10 7

10.0 12.5 15.0 17.5 20.0 22.5 25.0 27.5 30.0 Time [min]

Intens. 6

0

2

4

6

8

8 x10

1

Intens.

195

Figure 4.15. ESI+–MS mass spectra of compund 6.

Compound 6 was then isolated by semi-preparative RP-HPLC from a scale up

fermentation and subjected to further chemical and spectroscopic analysis. Based on

ESI+–MS, and comparison of

1H NMR and

13C NMR with the literature, compound 6,

was confirmed to be solasodine 3-O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranoside

(Mimaki and Sashida, 1990), previously isolated from the bulbs of Lilium brownii

(Figure 4.15).

414.5 576.4

722.8

0.0

0.5

1.0

1.5

7 x10

Intens. 6

200 400 600 800 1000 1200 1400 1600 1800 m/z

196

Figure 4.16. Molecular structure and fragmentation of compound 6.

O

HOO

OH

HO

OH3CHO

OH OH

O

O

HN

414.5

576.4

In addition, another metabolite, compound 7, was observed with a base peak at m/z

576.7 (Figure 4.16).

Figure 4.17. ESI+–MS mass spectra of compund 7.

414.5

576.7

0.0

0.5

1.0

1.5

2.0

2.5 6

x10 7

200 400 600 800 1000 1200 1400 1600 1800 m/z

Intens.

197

The mass spectrum of the metabolite was consistent with a molecule containing one less

glucose and one less rhamnose from compound 1 or one less rhamnose from compound

6. Additionally, an ion fragment at 414.5 [M–Glu+H]+ was observed and was consistent

with a monosaccharide moiety containing one glucose molecule. Compound 7 was then

isolated by semi-preparative RP-HPLC from a scale up fermentation and subjected to

further chemical and spectroscopic analysis (Figures 6 and 7). Based on comparison of

the retention time of the partial hydrolysis products of compound 1, ESI+–MS, and

comparison of 1H NMR and

13C NMR with the literature, compound 7 was confirmed to

be solasodine 3-O- -D-glucopyranoside (Bite and Rettegi, 1967; Kim et al., 1996),

previously isolated from Solanum umbelliferum (Figure 4.17).


O

HOO

OH

HO

OH

O

HN

414.5

A third metabolite, compound 10, was observed with a based peak at m/z 414.5 (Figure

4.18). The mass spectrum was consistent with a molecule containing the loss of two

glucose units and one rhamnose from compound 1, one rhamnose and one glucose from

198

compound 6, or one glucose from compound 7. Additionally, an ion fragment at m/z

271.3 was observed.

Figure 4.19. ESI+–MS mass spectra of compound 10.

Compound 10 was then isolated by semi-preparative RP-HPLC from a scale up

fermentation and subjected to further chemical and spectroscopic analysis (Figures 6 and

7). Based on comparison of the retention time with an authentic standard, ESI+–MS, and

comparison of 1H NMR and

13C NMR with the literature, compound 10 was confirmed to

be solasodine (Bird et al., 1979), a common aglycone of steroidal glycoalkaloids (Figure

4.19).

271.3

414.5

0

1

2

3

4

5

7 x10

200 400 600 800 1000 1200 1400 1600 1800 m/z

10 Intens.

199


HO

O

HN

HO

m/z 271.3

271.3

Similar to the metabolism of -tomatine by Alternaria solani, sequential cleavage of all

of the sugars of the carbohydrate moiety were observed in the model system (Schlösser,

1975). In addition to compounds 6, 7 and 10, several other fungal metabolites with

differential degrees of glycosylation and regiospecific mono- and polyhydroxylation of

the aglycone were observed.

The fungal metabolism of compound 2 by B. cinerea was markedly different than

that of compound 1, as a result of acetylation of the 6′′′ hydroxy position of the terminal

glucose unit. After 48 hours of in vitro metabolism of compound 2, most of compound 2

was still present in the media (Figure 4.20), whereas after 48 hours of metabolism of

compound 1, only trace amounts were present (Figure 4.13).

200

Figure 4.21. Metabolism of compound 2 by B. cinerea: (A) Total ion chromatogram

(TIC) of fungal media spiked with compound 2 (time = 0), (B) TIC of metabolites of

compound 2 (48 hours), and (C) TIC of metabolites of compound 2 (72 hours).

0.0

0.5

1.0

1.5

2.0

2.5

8 x10

10 15 20 25 30 Time [min]

2

7

10

8

0.0

0.5

1.0

1.5

2.0

2.5

8 x10

2

8 7

0

0.5

1.0

1.5

2.0

2.5

8 x10

2

Intens.

201

In contrast to the metabolism of compound 1, the metabolite compound 6 was not

detected; however, a new metabolite, compound 8, was observed with a based peak at

m/z 780.5 (Figure 4.21).


The mass spectrum of the metabolite was consistent a loss of rhamnose from

compound 2. Additionally, ion fragments at 576.4 [M–Glu–Ac+H]+ and 414.5 [M–2Glu–

Ac+H]+ were observed and were consistent with a disaccharide moiety containing

glucose and an acetylated glucose moiety. Based on mass spectral analysis of this

metabolite, compound 8 is likely to be (22,R 25R)-spirosol-5-en-3 -yl O- -L-

rhamnopyranosyl-(1→2)-O-[6-O-acetyl- -D-glucopyranoside; however, preparative

isolation and full characterization is needed for unequivocal confirmation (Figure 4.22).

414.5 576.4

780.5

0.0

0.5

1.0

1.5

6 x10 8

200 400 600 800 1000 1200 1400 1600 1800 m/z

Intens.

202

Figure 4.23. Proposed molecular structure and fragmentation of compound 8.

O

HOO

OH

O

OH

O

HN

O

HO

O

HO

OH

414.5576.4O

In addition, compound 7 was detected as a metabolite of compound 2 and is

consistent with the loss of one rhamnose and an acetylated glucose moiety of compound

2, or the loss of the acetylated glucose moiety of compound 8. Compound 10, was also

observed and is consist with the metabolite of compound 1. The mass spectrum was

consistent with a molecule containing the loss of one rhamnose, one glucose and an

acetylated glucose from compound 2, the loss of one glucose and an acetylated glucose

from compound 8, or one glucose from compound 7.

Due to the fact that only a small portion of compound 2 was metabolized after 48

hours of incubation, the metabolism experiment was continued for an additional 24

hours. After 72 hours of metabolism of compound 2 by B. cinerea, compound 2 was still

present in the media (Figure 8) as compared to the metabolism of compound 1 which

only trace amount were present after 48 hours (Figure 4). The decreased metabolism rate

of compound 2 may play a role in the increased fungal growth inhibition of compound 2

as compared to compound 1. Interestingly, after 72 hours, compound 10 increased and

203

small amounts of compound 9 and compound 1 were detected (Figure 8). Compound 9

had a base peak m/z 738.8 (Figure 4.23).


This ion was consistent with the de-acetylation of compound 8. Ion fragments at

576.4 [M–Glu+H]+ and 414.5 [M–2Glu+H]

+ were observed and were consistent with a

disaccharide moiety containing a two glucose moieties. Additionally, Compound 9 had

the same retention time and mass spectrum as the product of the partial acid hydrolysis of

compound 1 (Figure 4.24).

414.5 576.4

738.8

0.00

0.25

0.50

0.75

1.00

1.25 6 x10 9

200 400 600 800 1000 1200 1400 1600 1800 m/z

Intens.

204

Figure 4.25. (A)Total ion chromatogram (TIC) of the metabolites of compound 2 (72

hours), (B) extracted ion chromatogram (EIC) for compound 9 (m/z 738.8) from the

metabolite mixture derived from compound 2, and (C) TIC of the partial acid-catalyzed

hydrolysis products of compound 1.

0

1

2

3

7 x10

10 15 20 25 30 Time [min]

9

7

10

0.0

0.2

0.4

0.6

0.8

1.0

6 x10

9

0.0

0.5

1.0

1.5

2.0

2.5

8 x10

2

7

10

8

9

Intens.

205

Accordingly, compound 9 is likely to be (22,R 25R)-spirosol-5-en-3 -yl O- -D-

glucopyranosyl-(1→4)]- -D-glucopyranoside; however, preparative isolation and full

characterization is needed for unequivocal confirmation (Figure 4.25). The presence of

small amounts of compound 1 and compound 9 after 72 hours of incubation suggests

acetylase activity; however, due to the presence of a greater abundance of compound 8,

the cleavage of the rhamnose moiety at the C-2′ position of the inner glucose is favored

over de-acetylation of the acetyl moiety of the C-6′′′ position of the terminal glucose

under these conditions.

Figure 4.26. Proposed molecular structure and fragmentation of compound 9

414.5576.4

O

HOO

OH

O

OH

O

HN

O

HO

OH

HO

OH

Based on these data, the metabolism of compound 1 occurs by the sequential

removal of the sugars of the trisaccharide moiety with compounds 6, 7 and 10 as

intermediates (pathway A; Figure 4.27). In parallel, hydroxylation of the aglycone

occurs. Regiospecific microbial hydroxylation of diosgenin and solasodine is well known

and is utilized for production of pharmaceutical steroids (Sato and Hayakawa, 1963a;

Sato and Hayakawa, 1963b). In tomato, B. cinerea has been shown to metabolize the

steroidal glycoalkaloid, -tomatine, by both cleavage of the entire carbohydrate moiety

206

and by the cleavage of the terminal xylose (Verhoeff and Liem, 1975; Quidde et al.,

1998). Sequential sugar cleavage in compound 1 is similar to the metabolism of -

tomatine by Alternaria solani, where all four sugars of the tetrasaccharide moiety are

sequentially cleaved (Schlösser, 1975). In the case of compound 2, acetylation of the

terminal glucose moiety inhibits cleavage from the inner glucose and metabolism

proceeds through the cleavage the rhamnose at the C-2′ position of the inner glucose. The

major metabolic pathway proceeds sequentially with compounds 8, 7, and 10 as

intermediates (pathway B; Figure 4.26). Alternatively, evidence of acetylase activity

was observed, and two minor metabolic pathways of compound 2 are proposed. One

minor metabolic pathway may proceed with de-acetylation of compound 2 with

compounds 8, 9, 7, and 10 as intermediates (pathway B1; Figure 4.26) and a second

minor metabolic pathway may proceed with compounds 1, 6, 7, and 10 as intermediates

(pathway B2; Figure 4.26).

207

Figure 4.27. Proposed partial metabolic pathways for compounds 1 and 2 (thick arrows):

(A) Major metabolic pathway for compound 1. (B) Major metabolic pathway for

compound 2. (B1 and B2) Minor metabolic pathways for compound 2. De-Ac, de-

acetylation; R-OH, mono/polyhydroxylation of aglycone.

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

O

HO

OH

O

O

HOO

OH

O

OH3CHO

OH OH

O

O

HN

O

HO

OH

HO

OH

O

HOO

OH

O

OH

O

HN

O

HO

O

HO

OH

O

O

HOO

OH

HO

OH3CHO

OH OH

O

O

HN

O

HOO

OH

O

OH

O

HN

O

HO

OH

HO

OHO

HOO

OH

HO

OH

O

HN

HO

O

HN

1

2

6

7

8

9

10

B1 De-Ac

De-Ac

A B

B2

R-OH

R-OH

R-OH

R-OH

R-OH

208

4.4.3. In planta identification of compounds 6 – 10 by LC-MS.

Based on the in vitro fungal growth inhibition studies and the characterization of the

fungal metabolites of compounds 1 and 2, the objective of this part of the study was to

determine if B. cinerea has the ability to metabolize compounds 1 and 2, in planta, into

the fungal metabolites that were identified in the model system. In order to investigate

this question, plant tissue that was infected with B. cinerea was compared to a control

tissue by LC-MS analysis. All fungal metabolites that were characterized in the model

system were detected in the infected plant tissue (Figure 4.27; Figure 4.28; Figure 4.29;

Figure 4.30; Figure 4.31). None of the metabolites were detected in the control tissue

with the exception of compound 6. Although the infected tissue had elevated levels of

compound 6 as compared to the control, interestingly, compound 6 was also present in

the control sample that was not infected with B. cinerea (Figure 4.31).

209

Figure 4.28. (A) Extracted ion chromatograms (EIC) for compound 8 (m/z 780.5) of

control plant tissue, and (B) plant tissue infected with B. cinerea.

0.0

0.5

1.0

1.5

7 x10

8

10 15 20 25 30 Time [min]

B

0

2

4

6

7 x10 A

210



0.0

0.5

1.0

8 x10

10 15 20 25 30 Time [min]

9 B

0.0

0.5

1.0

1.5

2.0

7 x10 A

211



0.0

0.5

1.0

1.5

8 x10 7

10 15 20 25 30 Time [min]

B

0.0

0.5

1.0

1.5

2.0

2.5

8 x10 A

212



0

1

2

3

4

5

8 x10

10 15 20 25 30 Time [min]

10

B

0

2

4

6

7 x10 A

213



0

1

2

3

8 x10

10 15 20 25 30 Time [min]

6

B

0

1

2

3

4

8 x10

6

A

214

4.4.4. Isolation and identification of compound 6 from L. Longiflorum bulbs.

In order to confirm the presence of compound 6 as a natural product in L.

Longiflorum, compound 6 was isolated and purified from L. Longiflorum bulbs. Briefly,

lyophilized lily bulbs were washed with n-pentane and extracted with ethanol and water.

After the removal of solvent, the extract was dissolved in deionized water, washed with

ethyl acetate and extracted with n-butanol yielding a crude steroidal glycoside extract.

The crude glycoside extract was fractionated by gel permeation chromatography and

repeated semi-preparative RP-HPLC to yield compound 6 (Figure 4.32). Based on 1H

NMR and 13

C NMR, compound 6 was confirmed as (22,R 25R)-spirosol-5-en-3 -yl O-

-L-rhamnopyranosyl-(1→2)- -D-glucopyranoside, previously isolated from L. brownii

(Mimaki and Sashida, 1990). These data confirms that compound 6 is not only a fungal

metabolite of compounds 1 and 2, but it is also constitutively present in L. longiflorum.

Figure 4.33. Total ion chromatogram (TIC) of compound 6 isolated by RP-HPLC from L.

longiflorum bulbs.

10 15 20 25 30 Time [min]

0.0

0.5

1.0

1.5

8 x10

6

Intens.

215

4.5. Conclusion

In this chapter, two steroidal glycoalkaloids and three furostanol saponins,

isolated from L. longiflorum, were evaluated for fungal growth inhibition of the plant

pathogenic fungus, B. cinerea. All compounds showed weak fungal growth inhibition

activity. In addition, five fungal metabolites of the glycoalkaloids 1 and 2, were

characterized from a model system and were observed in living plant tissue infected with

the fungus. Furthermore, a structure-function relationship for the fungal growth inhibition

for compounds 1 and 2 was established based on the acteylation of the terminal glucose

moiety. On the basis of these results, B. cinerea can metabolize compounds 1 and 2 by

the sequential removal of the sugars of the trisaccharide moiety. Additionally, these data

suggests that a decreased rate of metabolism of compound 2 may play a role it the

increased fungal growth inhibition activity. Moreover, compound 6 was determined to be

both a fungal metabolite of compounds 1 and 2 and a natural product constitutively

present in L. longiflorum. This is the first report of compound 6 from L. longiflorum. This

study can be used as a model of characterizing fungal metabolites of plant derived natural

products and a means for the generation of new natural products with novel biological

activities. In addition, the antifungal activity of the compounds can be pursued further

with pathogenic organisms not active in L. longiflorum.

216

Summary and Concluding Remarks

This research project was designed to: (1) Isolate and characterize new steroidal

glycosides from the bulbs of L. longiflorum, (2) quantify their contents in all of the

organs of L. longiflorum, and (3) perform studies on the antifungal activity and fungal

metabolism of the compounds. The results of this study led to the discovery of new

steroidal glycosides, a better understanding of their distribution within the different plant

organs, and provided insight into structure-function relationships and fungal metabolism

of the compounds.

Based on novel isolation methodologies and a combination of extensive

spectroscopic and chemical analyses including 1D and 2D NMR, IR, HRESI–TOFMS,

ESI-MS, GC-MS, entantioselective GC-FID, chromatographic data, chemical analysis,

and chemical transformations, several novel steroidal glycosides were isolated from L.

longiflorum and structures elucidated. L. longiflorum contains two types of steroidal

glycosides: steroidal glycoalkaloids and steroidal saponins. A new acetylated steroidal

glycoalkaloid and two new furostanol saponins, along with three known steroidal

glycosides, were isolated from the bulbs of L. longiflorum. The new steroidal

glycoalkaloid was identified as (22R, 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-

(1→2)-[6-O-acetyl- -D-glucopyranosyl-(1→4)]- -D-glucopyranoside. The new

furostanol saponins were identified as (25R)-26-O-( -D-glucopyranosyl)-furost-5-en-

3 22 -triol 3-O- -L-rhamnopyranosyl-(1→2)- -L-arabinopyranosyl-(1→3)- -D-

glucopyranoside and (25R)-26-O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol 3-O-

-L-rhamnopyranosyl-(1→2)- -L-xylopyranosyl-(1→3)- -D-glucopyranoside.

Additionally, two known steroidal glycoalkaloids, (22R, 25R)-spirosol-5-en-3 -yl O- -

217

L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside and (22,R

25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranoside, and a

known furostanol saponin, (25R)-26-O-( -D-glucopyranosyl)-furost-5-en-3 22 -triol

3-O- -L-rhamnopyranosyl-(1→2)- -D-glucopyranosyl-(1→4)- -D-glucopyranoside,

were isolated from L. longiflorum for the first time.

In order to investigate the natural distribution of the newly identified compounds

within the plant, a LC-MS/MS method performed in multiple reaction monitoring

(MRM) mode was developed for the simultaneous quantitative analysis of the five new

steroidal glycosides in the different organs of L. longiflorum. The highest concentrations

of total steroidal glycosides were detected in flower buds, lower stems, and leaves. The

steroidal glycoalkaloids were detected in higher concentrations as compared to the

furostanol saponins in all of the plant organs except for the fibrous and fleshy roots. The

proportions of steroidal glycoalkaloids to furostanol saponins were higher in the plant

organs exposed to light and decreased in proportion from the aboveground organs to the

underground organs. The highest concentrations of the steroidal glycoalkaloids were

detected in flower buds, leaves, and bulbs. Both steroidal glycoalkaloids had a similar

pattern of distribution in the various plant organs; however, the acetylated derivative

occurred at lower levels. The furostanol saponins were detected in the highest

concentrations in the lower stems, fleshy roots, and flower buds. In the bulbs, the

steroidal glycosides were not distributed uniformly throughout the bulb scale tissue, but

accumulated in the basal plate, bulb scale epidermal cells, and vascular bundles. This

work has led to a better understanding of the natural distribution of these compounds

within the different plant organs and plant tissues of L. longiflorum.

218

To gain insight into the plant-pathogen interaction, purified steroidal glycosides

were evaluated for fungal growth inhibition activity against the plant pathogenic fungus,

Botrytis cinerea, using an in vitro plate assay. All of the compounds showed weak fungal

growth inhibition activity; however, the natural acetylation of C-6′′′ of the terminal

glucose in the steroidal alkaloid, (22R, 25R)-spirosol-5-en-3 -yl O- -L-


increased the antifungal activity by inhibiting the rate of metabolism of the compound by

the B. cinerea. Acetylation of the glycoalkaloid may be a plant defense response to the

evolution of detoxifying mechanisms by the pathogen. A model system was developed to

generate fungal metabolites of the steroidal glycoalkaloids which led to the identification

of several new fungal metabolites. The fungal metabolites characterized from the model

system were subsequently identified by LC-MS to naturally occur in Easter lily tissues

infected with the fungus.

The extraction and purification procedures reported in this work may be used for

the production of sufficient quantities of pure compounds for biological investigations.

These new compounds from L. longiflorum can be used for studies on the biological role

of steroidal glycosides in plant development and plant-pathogen interactions, as well as

for studies in food and human health. Furthermore, the quantitative analysis of steroidal

glycosides in the different organs of L. longiflorum is the first step to developing insight

into the biological role these compounds play in plant metabolism, plant development,

and plant-pathogen interactions. An understanding of the distribution of steroidal

glycosides in the different organs of L. longiflorum may aid in the development of

optimized extraction and purification methodologies for food, health, and industrial

219

applications. Moreover, the results from the fungal metabolism work can be used as a

model for characterizing fungal metabolites of plant derived defense compounds, gaining

insight into plant-pathogen interactions, and a means for the generation of new natural

products with novel biological activities. The structural modification of plant defense

compounds may be an adaptive response to plant pathogens and the acetylation of the

glycoalkaloid, (22R, 25R)-spirosol-5-en-3 -yl O- -L-rhamnopyranosyl-(1→2)-[6-O-

acetyl- -D-glucopyranosyl-(1→4)]- -D-glucopyranoside, may be a plant defense

response to the evolution of detoxifying mechanisms by the pathogen. A plant defense

strategy aimed at inhibiting the pathogen‘s ability to detoxify plant defense compounds

may be an alternative strategy in plant defense. The characterization of detoxification

pathways utilized by plant pathogenic fungi helps to provide a fundamental

understanding of the plant-pathogen interaction and suggests strategies to chemically

modify these anti-fungal compounds to prevent pathogen degradation.

.

220

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CURRICULUM VITA

John Peter Munafo, Jr

Education

2011 Ph. D. Plant Biology, Department of Plant Biology and Pathology,

Rutgers - The State University of New Jersey

1998 B. S. Biology, The Richard Stockton College of New Jersey

Professional Experience

2007–Present Flavor Research Scientist, Mars Chocolate North America LLC

2006–2007 Flavor Chemistry Technician, Masterfoods USA

2005–2006 Natural Products Chemistry Technician, Masterfoods USA (Contractor)

1999–2005 Manager Member, Medicinal Natural Products LLC

Publications

Munafo, J.; Ramanthan, A.; Jimenez, L.; Gianfagna, T. Isolation and structural

determination of steroidal glycosides from the bulbs of Easter Lily (Lilium longiflorum

Thunb.). J. Agric. Food Chem. 2010, 58, 8806–8813.

Munafo, J.; Gianfagna, T. Quantitative Analysis of Steroidal Glycosides in Different

Organs of Easter Lily (Lilium longiflorum Thunb.) by LC-MS/MS. J. Agric. Food Chem.

2010, 59, 995–1004.

Munafo, J; Gianfagna, T. Antifungal Activity and Fungal Metabolism of Steroidal

Glycosides of Easter Lily (Lilium longiflorum) by the Plant Pathogenic Fungus, Botrytis

cinerea. J. Agric. Food Chem. - In Press

Documents

©2011 John Peter Munafo, Jr ALL RIGHTS RESERVED