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dissertation entitled

PHYTOCEUTICALS FROM HEMEROCALLIS FLOWERS
AND ROOTS WITH ANTIOXIDANT, ANTICANCER,
MOSQUITOCIDAL, AND SCHISTOSOME
INHIBITORY ACTIVITIES

presented by

 

Robert Henry Cichewicz

has been accepted towards fulfillment
of the requirements for

Ph . D . degree in Horticulture

W

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31/ 3/ a 2

MS U is an Affirmative Action/Equal Opportunity Institution 0-12771

 

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Michigan State
University

 

 

 

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DATE DUE I DATE DUE DATE DUE

 

DEC 3 o zoqiEs

 

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6/01 cJClRC/DateDuepes-sz

PHYTOCEUTICALS FROM HEMEROCALLIS FLOWERS AND ROOTS WITH
ANTIOXIDANT, ANTICANCER, MOSQUITOCIDAL, AND SCHISTOSOME

INHIBITORY ACTIVITIES

By

Robert Henry Cichewicz

A DISSERTATION
Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of
DOCTOR OF PHILOSOPHY

Department of Horticulture

2002

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ABSTRACT

PHYTOCEUTICALS FROM HEMEROCALLIS FLOWERS AND ROOTS WITH
ANTIOXIDANT, ANTICANCER, MOSQUITOCIDAL, AND SCHISTOSOME
INHIBITORY ACTIVITIES
By

Robert Henry Cichewicz

Daylilies (Hemerocallis spp., Hemerocallidaceae) are widely consumed in
eastern Asia as both a traditional food and medicine. In this study, Hememcallis
flowers and roots were examined in order to identify bioactive constituents that
may exhibit antioxidant, anticancer, mosquitocidal, and schistosome inhibitory
properties. An investigation of Hemerocallis cv. Stella de Oro flowers led to the
isolation of nine kaempferol, quercetin, and isorhamnetin 3-O-glycosides (1-9),
phenethyl B-D-glucopyranoside (10), orcinol B-D-glucopyranoside (11), phloretin
2'-O-B-D-g|ucopyranoside (12), phloretin 2'—O-B-D-xylopyranosyl-(1—>6)—B-D-
glucopyranoside (13), a new napthalene-glycoside, stelladerol (14), and an
amino acid (longitubanine A) (15). An examination of Hemerocallis fulva
‘Kwanzo’ Kaempfer roots led to the isolation of seven new anthraquinones,
kwanzoquinones A (16), B (17), C (19), D (20), E (21), F (22), and G (24), two
known anthraquinones, 2-hydroxychrysophanol (18) and rhein (23), one new
naphthalene glycoside, 5-hydroxydianellin (26), one known naphthalene
glycoside, dianellin (25), one known flavone, 6-methylluteolin (27), and a-

tocopherol.

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Several new compounds such as kwanzoquinones A (16), B (17), C (19),
E (21), and kwanzoquinone A and B monoacetates (16a and 17a, respectively)
exhibited promising cancer cell growth inhibitory activity. In addition, the known
compounds 2-hydroxychry50phanol (18) and rhein (23) inhibited cancer cell
growth. Three compounds, kwanzoquinone D (20), stelladerol (14), and 5—
hydroxydianellin (26), demonstrated remarkable antioxidant activity by inhibiting
lipid oxidation more than 90% in an in vitro assay system. Three compounds, 2-
hydroxychrysophanol (18) and kwanzoquinones C (19) and E (21), exhibited
mosquitocidal properties against fourth instar Ades aegyptii larvae. Two
compounds, 2-hydroxychrysophanol (18) and kwanzoquinone E (21), were
discovered as novel agents for the prevention and treatment of schistosomiasis.
These compounds were found to inhibit the motility and induce mortality in
Schistosoma mansoni cercariae and adults. The bioactive constituents from
daylilies are being further investigated in order to determine their modes of action

and for development as new phytoceuticals.

 

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ACKNOWLEDGMENTS

Partial funding of this project was provided by the MSU Agricultural
Experiment Station and the Center for Plant Products and Technologies. The
MSU Mass Spectrometry Facility is partially supported by a grant from the NIH
(DRR-00484). Purchase of the NMR instruments used in this study was made
possible by grants from the NIH (1-S10-RR04750) and NSF (CHE-88-OO77O and
CHE-92-13241). Travel funding for the presentation of this work was provided by
the MSU Graduate Studies Program and Dr. S. K. Ries.

I would like to thank Dr. L. S. Barnes, Mr. J. C. Halinar, and Mr. D. Walters
for providing the daylilies used in this study. I would like to acknowledge Dr. J. H.
McKerrow and Mr. K.-C. Lim for performing the schistosome inhibitory studies. I
am grateful for assistance from Dr. E. Walker for providing the Aedes aegyptii
eggs and for post-mortem observations of the larvae. I would also like to thank
Mr. K. Johnson and Dr. L. Le for technical assistance conducting selected NMR
experiments. In addition, I would like to acknowledge Dr. B. Borhan and Mr. B.
Travis for assistance obtaining IR and optical rotation data. Furthermore, I would
like to thank past and present members of the Bioactive Natural Products and
Phytoceuticals Laboratory for their kind assistance. I am especially indebted to
Dr. L. J. Clifford for her unremitting support. Finally, I would like to acknowledge
my major professor and committee chair, Dr. M. G. Nair, and the other committee
members, Dr. L. D. Bourquin, Dr. R. E. Schutzki, and Dr. G. M. Strasburg, for

their time and guidance in the preparation of this document.

 

 

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TABLE OF CONTENTS

LIST OF TABLES .............................................................................................. vii
LIST OF FIGURES .......................................................................................... viii
KEY TO ABBREVIATIONS ................................................................................ xi
INTRODUCTION ............................................................................................... 1
CHAPTER ONE
LITERATURE REVIEW ..................................................................................... 4
Introduction ............................................................................................. 4
Botany of Hemerocallis ........................................................................... 4
Chemical Constituents of Hemerocallis ................................................... 6
Biological Activity of Hemerocallis ......................................................... 26
CHAPTER TWO

ISOLATION AND CHARACTERIZATION OF
STELLADEROL, A NEW NAPHTHALENE
GLYCOSIDE AND OTHER GLYCOSIDES

FROM EDIBLE DAYLILY (HEMEROCALLIS) FLOWERS ............................... 29
Abstract ................................................................................................. 29
Introduction ........................................................................................... 30
Methods and Materials .......................................................................... 31

General Experimental Procedures .............................................. 31

Plant Material ............................................................................. 31

Extraction and Isolation of

Compounds 1, 3, 5-7, and 9. ...................................................... 32

Extraction and Isolation of

Compounds 2, 4, 8, and 10-15 ................................................... 33

Stelladerol (14) ........................................................................... 36
Results and Discussion ......................................................................... 37
Conclusions ........................................................................................... 43

CHAPTER THREE

KWANZOQUINONES A-G AND OTHER CONSTITUENTS

OF HEMEROCALLIS FUL VA ‘KWANZO’ ROOTS .......................................... 44
Abstract ................................................................................................. 44
Introduction ........................................................................................... 45
Methods and Materials .......................................................................... 46

General Experimental Procedures .............................................. 46
Plant Material ............................................................................. 47
Extraction and Isolation of Compounds 16-28 ............................ 47

 

 

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Kwanzoquinones A and B (16 and 17) ....................................... 52

Acetylation of Compounds 16 and 17 ......................................... 52
Kwanzoquinone A and B
Monoacetates (16a and 17a) ...................................................... 53
2-Hydroxychrysophanol (18) ...................................................... 53
Kwanzoquinone C (19) ............................................................... 56
Kwanzoquinone D (20) ............................................................... 56
Kwanzoquinone E (21) ............................................................... 57
Kwanzoquinone F (22) ............................................................... 57
Kwanzoquinone G (24) ............................................................... 58
Dianellin (25) .............................................................................. 58
5-Hydroxydianellin (26) ............................................................... 59
6-Methylluteolin (27) ................................................................... 59
Hydrolysis of Compounds 19, 20, 22, 25, and 26 ....................... 60
Results and Discussion ......................................................................... 61
Conclusions ........................................................................................... 76
CHAPTER FOUR

BIOLOGICAL ACTIVITIES OF COMPOUNDS
ISOLATED FROM HEMEROCALLIS CV
STELLA DE ORO FLOWERS AND

HEMEROCALLIS FULVA ‘KWANZO’ ROOTS ................................................ 78
Abstract ................................................................................................. 78
Introduction ........................................................................................... 80
Methods and Materials .......................................................................... 80

Cancer Cell Growth Inhibition Assay .......................................... 80
Antioxidant Assay ....................................................................... 81
Cyclooxygenase Inhibition Assay ............................................... 82
Mosquito Larvicidal Assay .......................................................... 83
Nematicidal Assay ...................................................................... 83
Schistosoma mansoni Cercaricidal Assay .................................. 84
Schistosoma mansoni Schistosomulacidal Assay ...................... 85
Schistosoma mansoni Adult Schistosomicidal Assay ................. 85
Topoisomerase Inhibition Assay ................................................. 86
Results and Disscussion ....................................................................... 87
Overview of Results .................................................................... 87
Anticancer Activity ...................................................................... 87
Antioxidant Activity ..................................................................... 92
Mosquitocidal Activity ................................................................. 95
Schistosome Inhibitory Activity ................................................... 95

CHAPTER FIVE

SUMMARY AND CONCLUSIONS ................................................................. 102

REFERENCES .............................................................................................. 106

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LIST OF TABLES

Table 1.1 Chemical constituents reported from
Hemerocallis spp. ........................................................................

Table 1.2. Conditions treated and reputed beneficial
effects of Hemerocallis spp. .........................................................

Table 2.1. Percent yield of 15 compounds isolated

from methanol and aqueous methanol extracts of

edible Hemerocallis cv. Stella de Oro flowers and

literature sources containing comparative spectroscopic data .....

Table 3.1. NMR spectral data for kwanzoquinones A (16)
and B (17) in CDCI3 .....................................................................

Table 3.2. 13C NMR assignments for compounds 18-22 and 24

Table 3.3. Yield of 12 compounds isolated from
Hemerocallis fulva ‘Kwanzo’ roots. ..............................................

Table 4.1. Summary of the results of bioassays
performed on compounds obtained from Hemerocallis cv. Stella
de Oro flowers and H. fulva ‘Kwanzo’ roots .................................

Table 4.2. Growth inhibitory effects of anthraquinones
isolated from Hemerocallis fulva ‘Kwanzo’ roots against four
human cancer cell lines ...............................................................

vii

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LIST OF FIGURES

Figure 1.1. Some unique amino acids found in
Hemerocallis spp. ............................................................................................ 19

Figure 1.2. Fulvanines A-E found in the aerial
portion of H. fulva ............................................................................................. 19

Figure 1.3. Anthocyanins found in the flowers of

Hemerocallis spp. ............................................................................................ 21
Figure 1.4. Phenolic compounds found in Hemerocallis spp. ......................... 21
Figure 1.5. Anthraquinones found in Hemerocallis spp. ................................. 22

Figure 1.6. Carotenoids found in the flowers of
Hemerocallis spp. ............................................................................................ 24

Figure 1.7. Structures of hemerosides A and B obtained
from the aerial portion of H. fulva. .................................................................... 25

Figure 1.8. A unique 2,5-dimethoxytetrahydrofuran,
fulvanol, from the aerial portion of H. fulva ....................................................... 25

Figure 2.1. Structures of kaempferol, quercetin, and
isorhamnetin 3-O—glycosides (compounds 1-9) isolated
from Hemerocallis cv. Stella de Oro flowers. ................................................... 39

Figure 2.2. Structures for compounds 10-15 isolated from
Hemerocallis cv. Stella de Oro flowers. ........................................................... 40

Figure 2.3. Selected HMBC (A) and difference NOE (B)
correlations used to determine the structure of stelladerol (14). ...................... 42

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Figure 3.1. Structures for compounds 16-27 isolated
from Hemerocallis fulva ‘Kwanzo’ roots. .......................................................... 63

Figure 3.2. Difference NOE ( —+ ) and long-range COSY ( —)
correlations used to establish the structures of
kwanzoquinones A (16) and B (17) .................................................................. 66

Figure 3.3. Selected HMBC correlations used to determine
the structure of kwanzoquinone D (20). ........................................................... 70

Figure 3.4. Selected HMBC correlations used to determine
the structure of kwanzoquinone E (21). ........................................................... 72

Figure 3.5. Selected HMBC correlations used to determine
the structure of 5-hydroxydianellin (26). ........................................................... 75

Figure 4.1. Inhibition of LUV phospholipid oxidation by

synthetic antioxidants (panel A) and compounds 1-11

(panel A) and compounds 12-21 and 23-26 (panel B).

Compounds were tested in triplicate at 10 uM (except the

mixtures of 16-17 and 16a-17a at 10 ug/mL). Results are

expressed as the mean percent inhibition i one standard deviation ................ 93

Figure 4.2. Dose-response effect of 2-hydroxychrysophanol

(18), kwanzoquinone C (19), and kwanzoquinone E (21) on

fourth instar A. aegyptii larvae mortality. Results are

expressed as the percent mortality i one standard deviation of

the larvae following 24 h of incubation with test compounds at three

different concentration (pg/mL). Experiments were performed

with replicates (n=5) of test tubes containing 10-15 larvae. ............................. 96

Figure 4.3. Schistosome life-cycle .................................................................. 98

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Figure 4.4. Dose-response effect of 2-hydroxychrysophanol

(18) and kwanzoquinone E (21) on S. mansoni cercariae

mobility. Motility was accessed based on the movement

and swimming behavior of the invasive aquatic larval

stage 10 min after the addition of test compound. Data

are expressed as the mean i one standard deviation of the

percent of immobilized cercariae (n=10). ....................................................... 101

Ac20
BHA
BHT
BuOH
CHCI3
CH20l2
CH3CN
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DQF-COSY
D20
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FABMS
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HMBC
HMQC
HOAc
HPLC
HREIMS
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KEY TO ABBREVIATIONS

Acetic anhydride

Butylated hydroxyanisole

Butylated hydroxytoluene

Butanol

Chloroform

Dichloromethane

Acetonitrile

Correlation spectroscopy

Cultivar

Distortionless enhancement by polarization transfer
Dimethyl sulfoxide

Double-Quantum Filtered Correlation Spectroscopy
Deuterium oxide

Electron impact ionization mass spectrometry

Ethanol

Ethyl acetate

Fast atom bombardment mass spectrometry

Fourier transform

Galactose

Glucose

Hydrochloric acid

Formic acid

Heteronuclear multiple bond coherence

Heteronuclear correlation through multiple quantum coherence
Acetic Acid

High performance liquid chromatography

High resolution electron impact ionization mass spectrometry
High resolution fast atom bombardment mass spectrometry
Water

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MeOH
mp
MPLC
MS
NMR
NOE
NOESY
ODS
PTLC
Si
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TBHQ
TFA
TLC
UV
le

Sulfuric acid

Infrared

Methanol

Melting point

Medium pressure liquid chromatography
Mass spectrometry

Nuclear magnetic resonance

Nuclear Overhauser effect

Nuclear Overhauser spectroscopy
Octadecyl silica

Preparative thin layer chromatography
Silica

Species

tert-Butylhydroquinone

Trifluoroacetic acid

Thin layer chromatography

Ultraviolet

Xylose

xii

INTRODUCTION

In the last century, perennial gardens across the United States have been
transformed by the addition of the now ubiquitous daylily (Hemerocallis spp.).
Generally regarded by gardeners as relatively vigorous, low-maintenance, and
pest-free plants, daylilies add an appreciable degree of color and beauty to the
garden landscape with their brilliant floral hues and prominent cascading foliage.
However, elsewhere in the world, daylilies are valued for much more than just
their ornamental qualities. For millennia, daylilies have been cultivated in their
native land of Asia where they are still widely regarded as an important source of
food and medicine. Despite a long and rich history of utilization by humans,
science still possesses a paltry understanding of the pharmacological potential
and phytochemical constituents of daylilies.

One of the earliest references to daylilies can be found in an ancient
Chinese materia medica penned for the Emperor Huang Ti nearly 5000 years
ago (Schabell, 1990). Further references to the medicinal uses of Hemerocallis
spp. can be found throughout the pages of recorded Chinese and Japanese
history in which daylilies are referred to by a variety of colloquial names. One of
the most prevalent of the terms ascribed to daylilies is wangyoucao (Chinese) or
wasure-gusa (Japanese), which translates as ‘forget sorrow plant’ (Carr, 1997).
As these names literally imply, daylilies were widely regarded for their reputed

antidepressant properties. In addition, Hemerocallis spp. have been extensively

 

flip .

used throughout Asia to treat a variety of other ailments including anemia, fever,
insomnia, and schistosomiasis.

Daylilies first appeared in Europe during the late sixteenth century where
they were cultivated solely for ornamental purposes (Schabell, 1990). These first
Hemerocallis spp. were rather unremarkable in terms of their orange and yellow
ephemeral flowers, and for years daylilies lingered in the shadows of garden
recesses as inconspicuous perennials. It was not until the early twentieth
century that plant breeders in Europe and the United States (Schabell, 1990)
took an interest in daylilies and embarked upon intensive breeding programs that
lead to the extraordinary variety and beauty in floral form and color for which
daylilies are now highly regarded. Today, several national and international
societies such as the American Hemerocallis Society and the International
European Daylily Society have been established by daylily enthusiast in order to
promote, propagate, and educate the public about the more than 40,000 named
daylily cultivars that now exist.

Throughout humankind’s existence, plants have served as an important
source of bioactive compounds. Today, researchers continue to exploit the
chemical diversity of the botanical world as a source of novel compounds that
possess interesting biological activities. Based on the extensive use of
Hemerocallis spp. as an important phytomedicinal agent in Asian cultures as well
as their many reported pharmacological properties, it is conceivable that daylilies
possess a number of new biologically active constituents. Therefore, the

objective of this study was to isolate and elucidate the structures of bioactive

 

 

 

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constituents from Hemerocallis spp. that may have phytoceutical applications.
Crude extracts and their respective isolates were subjected to a panel of
bioassays in order to evaluate their antioxidant, anticancer, cyclooxygenase
inhibitory, mosquitocidal, nematocidal, schistosome inhibitory, and
topoisomerase inhibitory activities.

This dissertation is composed of a series of chapters detailing the results
of this research. Chapter 1 is a literature review in which the botany, chemical
constituents, traditional uses, and pharmacological properties of daylilies are
outlined. In Chapter 2, the results of the isolation and structure elucidation study
performed on daylily (Hemerocallis cv. Stella de Oro) flowers are presented. An
accounting of the isolation and structure elucidation of compounds obtained from
the roots of Hemerocallis fulva ‘Kwanzo’ Kaempfer is detailed in Chapter 3. All
of the compounds obtained from the flowers and roots of Hemerocallis spp. were
investigated in order to evaluate their antioxidant, anticancer, cyclooxygenase
inhibitory, mosquitocidal, nematocidal, schistosome inhibitory, and
topoisomerase inhibitory activities. The results of these studies and the methods
used for these experiments are presented in Chapter 4. Chapters 2-4 provide
data that are derived from published and submitted peer reviewed journal articles
and a patent application, and are arranged here as manuscripts each with an
abstract, introduction, materials and methods, and results and discussion

sections.

 

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CHAPTER ONE

LITERATURE REVIEW

Introduction

In Europe and North America, daylilies are regarded as common garden
perennials that are grown for their intriguingly shaped and richly colored flowers.
However, in Asia daylilies are utilized as both a food item and medicinal agent.
The botany, chemical constituents, traditional uses, and pharmacological

properties of daylilies are outlined in this chapter.

Botany of Hemerocallis

Daylilies are herbaceous, clump-forming, spreading, perennial monocots
whose familial affiliation remains in question, although evidence now suggests
that daylilies should be placed in their own family, the Hemerocallidaceae. The
generic name for daylilies, Hemerocallis, is derived from the Greek words for
‘beauty’ and ‘day’ in reference to the fact that daylily flowers bloom and
subsequently senesce over the period of a single day.

Daylilies are indigenous to Asia; however, they can now be found growing
wild throughout portions of Europe and North America as a result of having
escaped from cultivation. Debate remains regarding the number of species that
comprise the genus Hemerocallis with estimates ranging from approximately 12-

30 or more (Grenfell, 1998). Some of the reported species of daylilies include

 

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Hemerocallis altissima Stout, Hemerocallis aurantiaca Baker, Hemerocallis
citn'na Baroni, Hemerocallis disticha Donn, Hemerocallis esculenta Koidzumi,
Hemerocallis fulva L., Hemerocallis Iilioasphodelus L. (syn. Hemerocallis flava L.
and Hemerocallis Iilio-ashpodelus L.), Hemerocallis Iongituba Miq., Hemerocallis
minor Miller, and Hemerocallis thunbergii Barr ex Baker. Intensive breeding
programs have led to the development of thousands of cultivars with some
estimates indicating the existence of over 40,000 named cultivars at the present
time (Grenfell, 1998). The following description of daylily characteristics has
been adopted from Jones and Luchsinger (1986), Zomlefer (1994), and from
personal observations.

Hemerocallis spp. are typically observed bearing rich green fans of
alternate, linear leaves that are lanceolatus in character. Numerous, tall, arching
simple leaves arise from a subsurface crown bearing a pattern of linear venation
and appearing to fold along the midrib. The leaves typically dieback in autumn;
however, semi-evergreen varieties may continue to bear leaves year-around.

The rhizomatous roots of the daylilies display a range of morphologies,
although they can generally be characterized as having a tapered appearance
with numerous fleshy tuberous or spindle-shaped swellings. The root thickness
may range from nearly fibrous to thick and cylindrical. A variety of colors are also
observed for the interior of the roots ranging from white to reddish in nature.

Hemerocallis flowers are characterized as bisexual and actinomorphic
bearing six tepals composed of three petals and three sepals. The funnel-form

lily-like flowers are cymose and borne on a leafless scape. Stamens possess

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elongated filaments with linear anthers opening via a lengthwise slit. The pistil is
composed of three united carpels and three locules bearing numerous ovules.
The daylily fruit is a capsule that contains a number of small, iridescent, black

seeds.

Chemical Constituents of Hemerocallis

Phytochemical investigations have revealed the presence of a diverse
array of chemical constituents in Hemerocallis spp. (Yang and Li, 2002). A
summary of compounds previously reported from Hemerocallis is provided in
Table 1.1. The known chemical constituents of Hemerocallis can be divided into
four general groups based on their biosynthetic origins. These groups include
the amino acids and their derivatives, fatty acids and related aliphatic
compounds, phenolic constituents, and terpenoid and steroid compounds (Table
1.1).

Many common amino acids have been identified in Hemerocallis spp.
(Takemoto and Kusano, 1966) (Table 1.1). However, some uncommon amino
acids isolated from daylilies include pinnatanine (Grove et al., 1973; Yoshikawa
et al., 1994) and oxypinnatanine (Grove et al., 1973; Kruger et al., 1976; Inoue et
al., 1990) from the leaves, flowers, stems, and seeds of H. fulva and the roots of
H. Iongituba (Figure 1.1). In addition, Iongitubanines A and B (Yoshikawa et al.,
1994) have been found in the roots of H. Iongituba (Figure 1.1). A series of

unique amino acid derivatives, fulvanines A-E (Figure 1.2), have been isolated

 

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18

OH NH2

H
H , N
lfiCZC—Czfi COOH
0
OH
pinnatanine
H OH NH2
O “N
‘ COOH
— 0
CH3

longitubanine A

OH NH2

COOH
o
CHz—OH

oxypinnatanine

CH3

longitubanine 8

Figure 1.1. Some unique amino acids found in Hemerocallis spp.

OH
O 0 CH3 0 CH3
0&- : H0513 0&0?
a H O a H O , H O
CH CH

fulvanine A

OH

O
“’0 ~—
,H O

ibCHg

fulvanine D

fulvanine B

'bCHa

fulvanine C

O
H O
Hofi?
O

fulvanine E

Figure 1.2. Fulvanines A-E found in the aerial portion of H. fulva.

19

 

In

from the aerial portion of H. fulva. It has been proposed that these 2,5-
dihydrofuryl-y-lactams are metabolites of oxypinnatanine (Inoue et al. 1990).

Gas chromatography has been used to analyze various daylily tissues
providing evidence for the presence of numerous long-chain fatty acids, alcohols,
aldehydes, and alkenes (lsono et al., 1976; Sarg et al., 1990; Wang et al., 1994)
(Table 1.1). Most of these compounds are common, unbranched Iipophilic
constituents such as capric, decanoic, Iauric, linoleic, myristic, and palmitic acids.
Other constituents include branched species such as 3,3-dimethyl butanoic acid,
3,7-dimethyI-1-octanol, 2-methyl-6-ethyl octane, and 2-propyl-1-heptanol (Wang
etaL,1994)

A variety of phenolic pigments have been identified in daylilies (Table 1.1).
For example, four anthocyanins, cyanidin 3-glucoside, cyanidin 3-rutinoside,
delphinidin 3-glucoside, and delphinidin 3-rutinoside (Asen and Arisumi, 1968;
Yoshitama et al., 1980; Griesbach and Batdorf, 1995) (Figure 1.3), have been
identified as constituents in the flowers of H. fulva and other daylily cultivars.
Other phenolic compounds include a unique isoflavone, hemerocallone, and a
substituted naphthalene, mi-hemerocallin (Figure 1.4), have also been isolated
from the roots of H. minor (Xiu et al., 1982). Another compound, a naphthalene
dimer known as stypandrol (Figure 1.4), is a potent neurotoxin that has been
identified in the roots of several Hemerocallis spp. (Xiu et al., 1982; Wang et al.,
1989). Early investigations of stypandrol from daylilies resulted in an incorrect
structural assignment for this compound and as a result, it was identified as a

novel structure and given the generic name hemerocallin (Wang et al., 1989).

20

 

HO
HO

OH

cyanidin 3-glucoside: R1=H, R2=H

cyanidin 3-rutinoside: R1=H, R2=a-L-rhamnose
delphinidin 3-glucoside: R1=OH, R2=H

delphinidin 3-rutinoside: R1=OH, R2=a-L-rhamnose

Figure 1.3. Anthocyanins found in the flowers of Hemerocallis spp.

OH

 

mi-hemerocallin

 

stypandrol

Figure 1.4. Phenolic compounds found in Hemerocallis spp.

21

OH 0 0R1

 

0
R1 R2 R3
chrysophanol H H CH3
obtusfolin CH3 OH CH3
2-methoxy-obtusfolin CH3 OCH3 CH3
aloe-emodin H H CHZOH
3-carbomethoxy 18 H H OCOCH3
dihydroanthraquinone
3-methoxy 1,8- H H OCH3
dihydroanthraquinone
rhein H H COOH
hemerocal CH3 OH CHZOH

 

 

 

Figure 1.5. Anthraquinones found in Hemerocallis spp.

22

Several anthraquinones have been identified in the roots of Hemerocallis
(Table 1.1) (Figure 1.5). These include many known compounds such as
chrysophanol, obtusfolin, 2-methoxy-obtusfolin, aloe-emodin, 3-carbomethoxy
1,8-dihydroxyanthraquinone, 3-methoxy 1,8-dihydroxy anthraquinone, and rhein
(Yang and Li, 2002). A new anthraquinone, hemerocal, was also isolated from
the roots of H. citrina and identified as 3-hydroxymethyl 1-methoxy 2,8-
hydroxyanthraquinone (He et al. 1982).

Many other compounds have been identified in the leaves, root, and
flowers of daylilies including a number of terpenoids and steroids (Table 1.1).
For example, several carotenoid pigments (Figure 1.6) have been identified in
Hemerocallis flowers including derivatives of B-carotene, B-cryptoxanthin, lutein,
lycopene, and zeaxanthin (Valadon and Chapman, 1984; Griesbach and Batdorf,
1995; Tai and Chen, 2000). In addition, two new steroidal saponins,
hemerosides A and B, were obtained from the aerial portion of H. fulva (Konishi
et al., 2001) (Figure 1.7).

A unique 2,5-dimethoxytetrahydrofuran named fulvanol (Figure 1.8) was
isolated from the aerial portion of H. fulva (Konishi et al., 1996). It was proposed
that this compound, a 4-methoxy methyl-a-L-apioside, might be related to other

branched aldofuranose compounds that play important regulatory roles in plants.

23

\\\\\\\\\

B-carotene

 

lycopene

‘\\\\\\\\
HO

zeaxanthin

Figure 1.6. Carotenoids found in the flowers of Hemerocallis spp.

24

   

CH3

OH
OH HO O O
HO
OH 0 HO 0 0
HO 0
HM OH
OH
hemeros:de B

Figure 1.7. Structures of hemerosides A and 8 obtained from the aerial portion

of H. fulva.

H3C0fi7,OCH3
HO 3 "

- 'OH
HO/

fulvanol
Figure 1.8. A unique 2,5-dimethoxytetrahydrofuran, fulvanol, from the aerial

portion of H. fulva.

25

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Biological Activity of Hemerocallis

Plants are widely used across Asia as traditional medicines. One group of
medicinal plants that are encountered throughout eastern Asia is daylilies.
Daylilies have been reportedly used for treating a host of diseases including
depression, inflammation, insomnia, and schistosomiasis (Table 1.2). A limited
number of tests have been carried out to examine the effects of Hemerocallis
extracts on biological systems. For example, it had been reported that daylily
flowers were capable of alleviating insomnia (Uezu, 1998). Based on these
anecdotal claims, Uezu (1998) assessed the effect that freeze-dried H. fulva
flowers had on sleep behavior in mice. It was determined that mice fed a diet
containing daylily flowers exhibited a significant increase in the duration of slow
wave and paradoxical sleep as compared to control animals. In another study
conducted by Hsieh et al. (1996), the effects of chloroform, ethyl acetate, n-
butanol, and aqueous extracts of Hemerocallis flava L. roots were examined for
their impact on motor activity in rats. These researchers found that the aqueous
daylily root extract significantly inhibited the motor activity of the test animals.
Upon further examination, it was determined that this extract significantly
decreased levels of norepinepherine in the cortex as well as reduced the
concentrations of dopamine and serotonin in the brain stem tissues of the
experimental rats.

Hemerocallis spp. have been used throughout Asia for the treatment of
schistosomiasis (Shiao et al., 1962a; Wang et al., 1989). Studies have shown

that a preparation containing H. thunbergii roots exhibited a

26

 

 

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27

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schistosomistatic effect in mice; however, toxic side-effects of this treatment
regime were noted including a degeneration of spinal and optic nerve tissues and
liver damage. Other researchers had previously noted the same toxicity in a
variety of other animals and had gone further to provide compelling evidence
implicating that the polyaromatic compound stypandrol was responsible for these
effects (Wang et al., 1989; Yunping and Kangnan, 1989; Huaitao et al., 1987).

A number of other studies have presented information suggesting that
Hemerocallis spp. possess a variety of other biological activities. For example,
Sarg and colleagues (1990) as well as Roia and Smith (1977) reported that
daylily flowers and roots exhibited modest antimicrobial properties. It has also
been found that daylilies have diuretic properties (Xui et al., 1982). Others found
that Hemerocallis was able to inhibit fibroblast proliferation (He, 1994) and to
induce cancer cells to undergo differentiation (Hata et al., 1998). Furthermore, it
was recently reported by Hsieh and colleagues (1996) that daylilies possessed
antimalarial properties.

Despite a long and rich history of utilization by humans, science still
possesses a paltry understanding of the pharmacological potential and
phytochemical constituents of daylilies. Therefore, this research focused on the
isolation, structure elucidation, and biological testing of compounds from daylily
roots and flowers. The following chapters provide details of the results of these
studies and provide a foundation upon which the development of new daylily-

based phytoceutical entities can be based.

28

CHAPTER TWO

ISOLATION AND CHARACTERIZATION OF STELLADEROL, A NEW
NAPHTHALENE GLYCOSIDE AND OTHER GLYCOSIDES FROM EDIBLE
DAYLILY (HEMEROCALLIS) FLOWERS

Abstract

Daylily (Hemerocallis spp.) flowers are utilized as an important ingredient in
traditional Asian cuisine and are also valued for their reputed medicinal effects.
Studies of the bioactive (antioxidant) methanol and aqueous methanol extracts of
lyophilized Hemerocallis cv. Stella de Oro flowers, lead to the isolation of
kaempferol, quercetin, and isorhamnetin 3-O-glycosides (1-9), phenethyl B-D-
glucopyranoside (10), orcinol B-D—glucopyranoside (11), phloretin 2'-O—B-D-
glucopyranoside (1 2), phloretin 2’-O—B-D-xylopyranosyl-(1—>6)-8-D-
glucopyranoside (13), a new napthalene-glycoside, stelladerol (14), and an

amino acid (longitubanine A) (15).

29

Intro:

easfia
tenil
1997)
posse
Bofli

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SI'lOWl
1998)
bveh
Phytc
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1968
Urifor

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IeSee
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Introduction

Daylilies (Hemerocallis spp., Hemerocallidaceae) have been harvested in
eastern Asia for thousands of years where they have been utilized as both a food
item (Tai and Chen, 2000) and medicinal agent (Tiejun and Tao, 1997; Uezu,
1997) for the treatment of a host of diseases. Daylilies have been reported to
possess antidepressant properties, reduce inflammation, and promote digestion.
Both fresh and dried daylily flowers are widely consumed as an important
component in traditional eastern Asian cuisine. Pharmacological studies have
shown that daylilies can facilitate neurological changes in sleeping mice (Uezu,
1998) and impact motor activity in rats as a result of alteration to the normal
levels of several central nervous system neurotransmitters (Hsieh et al., 1996).
Phytochemical investigations of Hemerocallis spp. have identified an assortment
of chemical constituents including carotenoids (Tai and Chen, 2000), fulvanine
lactams (Inoue et al., 1990; Inoue et al., 1994), anthocyanins (Asen and Arisumi,
1968; Griesbach and Batdorf, 1995), and anthraquinones (He et al., 1982).
Unfortunately, very little is known regarding the chemical composition of edible

daylily flowers.

This investigation was undertaken in order to examine the bioactive
antioxidant chemical constituents of edible daylily flowers. Specifically, this
research focuses on the bioactive phenolic glycosides since these compounds
are known to have a significant impact on the status of human health and

disease prevention. The isolation and structure elucidation of 14 phenolic

30

glycosides and one amino acid from lyophilized Hemerocallis cv. Stella de Oro

flowers are reported in this chapter.

Materials and Methods

General Experimental Procedures. 1H NMR spectra were recorded at 300,
500, and 600 MHz on Varian (Palo Alto, CA) INOVA (for 300 and 600 MHz) or
VRX (for 500 MHz) instruments. 13C NMR spectra were obtained at 75 and 125
MHz on Varian INOVA and VRX instruments, respectively. All spectra were
recorded in DMSO-d5. Standard pulse sequences were employed for all NMR
experiments. FAB mass spectra were acquired at the Michigan State University
Mass Spectrometry Facility using a JEOL HX-110 double-focusing mass
spectrometer (Peabody, MA) operating in the positive ion mode. The UV spectra
were recorded in MeOH using a Shimadzu UV-260 recording spectrophotometer
(Kyoto, Japan). Sephadex LH-20 was purchased from Sigma-Aldrich (St. Louis,
MO). Si gel PTLC plates (20 x 20 cm; 250, 500, and 1000 pm thick) were
obtained from Analtech, Inc. (Newark, DE). Preparative HPLC was performed on
a Japan Analytical Industry Co. model LC-20 recycling preparative HPLC with
tandem JAIGEL-C13 columns (10 pm, 20 mm x 250 mm). All solvents and
chemicals were purchased from Sigma-Aldrich (St. Louis, MO) and were of ACS

analytical grade.

Plant Material. Approximately 12,000 Hemerocallis cv. Stella de Oro

(Hemerocallidaceae) flowers (24.6 kg) were hand-harvested from Walters

31

Gank
Theft

kg of

pook
Dortir
ekne

afiOr

Unde

and

Gardens, Inc. (Zeeland, MI) on September 3 and 10, 1999 and frozen at -20 °C.
The frozen flowers were lyophilized and ground in a Waring blender, yielding 2.8

kg of fine yellow powder that was stored at —20 °C until extracted.

Extraction and Bioassay Guided Isolation of Compounds 1, 3, 5-7, and 9.
Powdered daylily flowers (1.8 kg) were successively extracted with hexane (5 x 6
L) (27 g), EtOAc (6 x 6 L) (20 g), and MeOH (8 x 8 L) (5849). Four 146-g
portions of the bioactive MeOH extract, were each applied to XAD-16 resin and
eluted with H20 (2 L) followed by MeOH (1.5 L). The total MeOH eluate (14.5 g)
was divided into eight 1.8-g fractions and further fractionated by C18 MPLC under
isocratic conditions with CH3CN-H20 (3:2). The bioactive constituents from each
MPLC column were eluted as a single, dark, UV-absorbing (7t 366 nm) band and
pooled (12 g). This material was dissolved in EtOH (3 x 300 mL) and the soluble
portion (9 g) was again applied to a column of XAD-16 resin and sequentially
eluted with H20 (2 L) followed by 30 (2 L), 60 (2.5 L), and 100 % (2 L) MeOH

affording 2.9, 2.7, 1.8, and 1.2 9 fractions, respectively.

The 60% MeOH eluate from XAD-16 (1.8 g) was subjected to C18 MPLC
under a 50-100% MeOH-H20 gradient. Eleven-milliliter fractions were collected
and pooled based on their TLC (CHClg-EtOAc saturated with HzO-MeOH-
HCOOH, 1:8:2:0.1) profiles. Fractions A-C, composed of subfractions 20-25, 29-
37, and 50-64, respectively, were determined to be bioactive and subjected to
further purification. PTLC of fraction A (44 mg) with CHZClz-MeOH-toluene

(22:5:1) yielded one fraction (35 mg) that was further purified by C18 preparative

32

HPLC under a 40-60% MeOH-H20 (with 0.1% TFA) gradient to give compound 9
(beige amorphous solid; 8.3 mg). Using C13 preparative HPLC under a 40-60%
MeOH-H20 (with 0.1% TFA) gradient, fractions B (68 mg) and C (45 mg)
provided compounds 6 (yellow powder; 43.1 mg) and 5 (yellow powder; 28.0

mg), respectively.

The 100% MeOH eluate (1.2 g) from XAD-16 was subjected to C18 MPLC
under a 40-60% MeOH-H20 gradient. Eleven-milliliter fractions were collected
and pooled based on their TLC (CHClg-EtOAc saturated with water-MeOH-
HCOOH, 1:8:2:0.1) profiles. Bioactive fractions D and E, composed of
subfractions 41-70 and 71-100, respectively, were subjected to further
purification. PTLC of fraction D (235 mg) with CHZCI2-MeOH-toluene (130:1522)
provided fractions D1 (10 mg) and DZ (60 mg). Further purification of fractions
D1 and D2 by C13 preparative HPLC under a 40-60% MeOH-H20 (with 0.1%
TFA) gradient afforded compounds 3 (yellow-brown amorphous solid; 3.0 mg)
and 7 (yellow amorphous solid; 5.2 mg), respectively. Fraction E (36 mg) was
also subjected to PTLC with CHzClz-MeOH-toluene (130:1522) yielding fraction
E1 (25 mg) that was subjected to C13 preparative HPLC under a 40-60% MeOH-

HzO (with 0.1% TFA) gradient yielding compound 1 (yellow amorphous solid; 2.0

mg).

Extraction and Isolation of Compounds 2,4, 8, and 10-15. A 1.0 kg portion of
the lyophilized flowers was exhaustively extracted with 1:1 MeOH-H20 (6 x 5 L)
and the extract reduced in vacuo yielding 390 g of gummy amber extract. The

extract was divided into three, 130 9 portions and 500 mL water was added to

33

each. Each portion was partitioned with hexane (3 x 200 mL) and then
chloroform (3 x 250 mL). The resultant aqueous extracts were combined,

concentrated in vacuo, and applied to a XAD-16 column. The column was eluted

with water (2 L) followed by 20% MeOH (2 L) and 100% MeOH (2.5 L).

The 20% MeOH eluate (11 g) was subjected to C13 MPLC under a 10-40%
CH3CN-H20 gradient and 200 mL fractions were collected affording fractions F
and G. Fraction F (320 mg) was dissolved in 15 mL of warm MeOH and left on
the bench-top for 14 days. Upon standing, fraction F yielded 176 mg of a
powdery off-white precipitate. The precipitate was analyzed by HPLC (MeOH-
H20, 3:7) and determined to be composed of an unresolved mixture of several
compounds. The mother liquor (144 mg) was subjected to repeated isocratic
preparative HPLC (MeOH-H20, 3:7) to give compound 15 (White powder; (34.0

mg).

Fraction G (400 mg) was applied to a Sephadex LH-20 column and eluted
with MeOH and 15-mL fractions were collected. Fractions 9-11 were pooled
based on their TLC (n-BuOH-HOAc-CHClg-HZO, 5:1:124, upper phase) profiles
providing a 220-mg fraction that exhibited a strong UV absorption at it 254 nm.
This fraction was further purified by PTLC with n-BUOH-HOAC-CHCI3-H20
(5:1:1:4, upper phase) and gradient preparative HPLC under 5-30% CH3CN

affording compound 11 (clear, glass-like amorphous solid; 13.9 mg).

The 100% MeOH eluate from XAD-16 (20 g) was repeatedly purified by

C13 MPLC under a 20-100% MeOH-H20 gradient giving fractions H and I.

34

Fraction H (600 mg) was applied to Sephadex LH-20 and eluted with 70% MeOH
giving 15 mL fractions that were pooled based on their TLC (n-BuOH-HOAc—
CHCl3-H20, 5:1:1 :4, upper phase) profiles affording fractions H1-H3. Fraction H1
(230 mg) was subjected to PTLC with CHzClz-MeOH-toluene-HCOOH
(15:6:0.2:0.2) to yield fractions H1A-H1C. Fractions H1A (20 mg) and H1C (17
mg) were further purified by PTLC with CHClg-EtOAc-MeOH-HCOOH
(3:7:1.5:0.1) (yields 13 and 5 mg, respectively) and isocratic C13 preparative
HPLC (10% CH3CN) to give compounds 10 (clear glass-like amorphous solid; 9.0
mg) and 13 (clear glass-like amorphous solid; 4.0 mg), respectively. Fraction
H1B (15.0 mg) was purified by isocratic C13 preparative HPLC (10% CH3CN),

yielding compound 14 (yellow amorphous solid; 12.0 mg).

PTLC of fraction H2 (130 mg) with CH2Clz-MeOH-toluene-HCOOH
(15:6:0.2:0.2) afforded a major dark, UV-absorbing band (it 366 nm) (120 mg)
that was applied to Sephadex LH-20 (MeOH) to give fractions H2A (85 mg) and
H28 (17 mg). Fractions H2A and H23 were both further purified by gradient
preparative HPLC under 40-60% MeOH with 0.1% TFA (yields were 10 and 5
mg, respectively) followed by additional gradient preparative HPLC under 10-
30% CH3CN affording compounds 4 (yellow-brown amorphous solid; 4.2 mg) and

2 (yellow amorphous solid; 2.5 mg), respectively.

Fraction H3 (100 mg) was subjected to further purification by gradient
preparative HPLC under 40-60% MeOH with 0.1% TFA (yield was 15 mg)
followed by gradient preparative HPLC under 10-30% CH3CN yielded compound

8 (yellow-brown amorphous solid; 2.3 mg).

35

Fraction l (1 g) was purified by repeated column chromatOgraphy on
Sephadex LH-20 eluted with 70 and 100% MeOH, respectively. This provided a
120 mg fraction that was further purified by PTLC with CHCI3-EtOAc-MeOH-
HCOOH (3:7:1.5:0.1) (yield 20 mg) and gradient C15 preparative HPLC (5-35%

CH3CN) to give compound 12 (clear glass-like amorphous solid; 13.0 mg).

Stelladerol (14) (1-(1,5,8-trihydroxy-3-methyl-napthalen-2-yl)-ethanone-8-O—
B-D-xylopyranosyl-(1—>6)—B-D-glucopyranoside): yellow amorphous solid; UV
(MeOH) Xmax (log a) 223 (4.97), 313 (4.15), 345 (4.18) nm; 1H NMR (600 MHz,
DMSO-d5) 6.. 9.84 (1H, s, -OH, exchange with D20), 9.63 (1H, s, -OH, exchange
with 020), 7.41 (1H, s, J=1.5, H-4), 7.27 (1H, d, J=8.3, H-7), 6.76 (1H, d, J=8.3,
H-6), 4.85 (1H, d, J=7.5, H-1'), 4.23 (1H, d, J=7.5, H-1”), 4.02 (1H, d, J=10.5, H-
6'), 3.69 (1H, dd, J=5.3, 11.3, H-5"), 3.58 (1H, m, H-6'), 3.31 (2H, m, H-2', H-5’),
3.29 (1H, m, H-4"), 3.18 (1H, t, J=9.0, H-4'), 3.11 (2H, m, H-3', H-3”), 3.02 (2H,
m, H-2", H-5"), 2.51 (3H, s, -COCH3), 2.25 (3H, d, J=1.5, -CH3); 13C NMR (75
MHz, DMSO-d5) (Sc 204.9 (s, C=O), 150.2 (s, C-1), 148.2 (s, C-5), 146.8 (s, C-8),
131.3 (s, C-3), 126.2 (s, C-10), 125.5 (s, C-2), 114.0 (s, C-9), 113.9 (d, C-4),
112.1 (d, C-7), 109.0 (d, C-6), 104.2 (d, C-1"), 103.4 (d, C-1’), 76.5 (d, C-3', C-
3"), 76.2 (d, C-5'), 73.4 (d, C-2', C-2"), 70.0 (d, C—4'), 69.6 (d, C-4"), 68.6 (t, C-
6'), 65.6 (t, C-5"), 32.0 (q, -COCH3), 19.4 (q, -CH3); FABMS m/z 549 [M+Na]*,
527 [M+H]*, 395 [M-Xyl+2H]+, 233 [M-Xyl-Glc+2H]"; HRFABMS m/z 527.1756

[M'I'Hr (calcd fOI' 0241131013, 521.1765)

36

Results and Discussion

Methanol and aqueous methanol extracts of edible Hemerocallis cv. Stella
de Oro flowers were subjected to a series of chromatographic procedures,
including C13 MPLC and preparative HPLC, silica gel PTLC, and Sephadex LH-
20 column chromatography, affording 15 compounds (Table 2.1). The structures
of these compounds, including nine flavonol-3-O—glycosides (1-9) (Figure 2.1),
phenethyl B-D-glucopyranoside (10), orcinol B-D-glucopyranoside (11), two
dihydrochalcone-glycosides, phloretin 2'-O-8-D-glucopyranoside and phloretin 2'-
O-B-D-xylopyranosyI-(1—>6)-B-D-glucopyranoside (12 and 13, respectively), one
new naphthalene-glycoside, stelladerol (14), and one amino acid, longitubanine
A (15) (Figure 2.2) were established based on uv, NMR (1H, 130, DEPT,
difference NOE, DQF-COSY, HMQC, and HMBC), and MS experiments and by
comparisons with literature data (Table 2.1). All of these compounds are
reported here for the first time as components of edible daylily flowers. The 3J
coupling constants of the anomeric protons were used to determine the absolute
a-(L-arabinose and L-rhamnose) or B-(D-galactose, D-glucose, and D-xylose)
configuration of the common, naturally-occurring sugar residues found in each of
the glycosides.

Examination of the 1H and 13C NMR spectra of compound 14 indicated
that it was composed of a highly substituted naphthalene moiety conjugated with
a disaccharide. The aglycone spins in the 1H NMR spectrum were represented

by three doublets at a. 7.41 (1H, J=1.5), 7.27 (1H, J=8.3), and 6.76 (1H, J=8.3).

37

Table 2.1 .

Yield of 15 compounds isolated from methanol and aqueous

methanol extracts of edible Hemerocallis cv. Stella de Oro flowers and literature

sources containing comparative spectroscopic data

 

 

yield (mg/kg
compound dry material) reference

1 kaempferol 3-O-a-L-arabinopyranoside 1.1 Vasange et al., 1997

2 quercetin 3-O-B-D-xylopyranoside 2.5 Dick et al., 1987

3 kaempferol 3-O-8-D-glucopyranoside 1.7 Markham et al., 1982

4 quercetin 3-O-B-D-glucopyranoside 4.2 Markham et al., 1982

5 kaempferol 3-O-a-L-rhamnopyranosyl- 15.6 Markham et al., 1982
(1 —>6)-B-D-glucopyranOSIde

6 quercetin 3-O-a-L-rhamnopyranosyl- 23.9 Markham et al., 1982
(1 —>6)-B-D-glucopyran05lde

7 quercetin 3-O-a-L-rhamnopyranosyl- 2 9 Agrawal and Bansal,
(1 —>6)-B-D-galactopyranoside ' 1989
quercetin 3-O—a-L-rhamnopyranosyl- S' .

lewek et al., 1984,

8 (1—>6)-[a-L-rhamnopyranosyl-(1-+2)]-B- 2.3 Webby and Boase, 1999
D-glucopyranOSIde
isorhamnetin 3-O—a-L-rhamnopyranosyl-

9 (1—>6)-[or-L-rhamnopyranosyl-(1—>2)]-B- 4.6 Masterova et al., 1991
D-glucopyranoside

10 phenethyl B-D-glucopyranoside 9.0 Kitajima et al., 1998

. . Chung et al., 1999;

11 orClnol B-D-glucopyranOSIde 13.9 Kuster et al., 1996

12 phloretin 2'-O-B-D-glucopyranoside 13.0 Lu and Foo, 1997

13 phloretin 2'-O-B-D.-xylopyranosyl-(1—>6)- 4.0 Lu and Foo, 1997
B-D-glucopyranOSlde
stelladerol (1 -(1 ,5,8-trihydroxy-3-methyl-

14 napthalen-Z-yl)-ethanone-8-O-B-D- 12 0 new
xylopyranosyl-(1-+6)-B-D- ' compound
glucopyranoside)

15 longitubanine A 15.0 Yoshikawa et al., 1994

 

38

 

R1 R2

1 H a-L-arabinopyranoside

2 OH B-D-xylopyranoside

3 H B-D-glucopyranoside

4 OH B-D-glucopyranoside

5 H a-L-rhamnosyl-(1—>6)-B-D-glucopyranoside
6 OH a-L-rhamnosyI-(1—>6)-B-D-glucopyranoside
7 OH ct-L-rhamnosyl-(1—>6)-B-D-galactopyranoside
8 OH a-L-rhamnopyranosyl-(1—>6)-[a-L—rhamnosy|-

(1 —>2)]-B-D-glucopyranoside
OMe a-L-rhamnopyranosyI-(1—+6)-[or-L-rhamnosyl-
(1 —>2)]-B-D-glucopyranoside

@

Figure 2.1. Structures of kaempferol, quercetin, and isorhamnetin 3-O-

glycosides (compounds 1-9) isolated from Hemerocallis cv. Stella de Oro flowers.

39

 

 

O OH
OH
R
12 B-D-glucopyranoside
13 B-D-xylopyranosyl-(1 —>6)-
B-D-glucopyranoside
OH
OH OH
,
O
dww
1..
14
H3C H OH NH2
2’ N 5 1 OH
/ 3
O O O
5.
15

Figure 2.2. Structures for compounds 10-15 isolated from Hemerocallis cv.

Stella de Oro flowers.

40

Analysis of the DQF-COSY spectrum confirmed the correlation between the
ortho coupled protons at 6.. 7.27 and 6.76, while the proton at (34 7.41 couple
weakly with the methyl doublet at (2.. 2.25. The multiplicities of the aglycon spins
were determined by DEPT demonstrating that the naphthalene nucleus was
composed of three methine carbons (60 113.9, 112.1, and 109.0) and seven
quaternary spins (69 150.2, 148.2, 146.8, 131.3, 126.2, 125.5, and 114.0). In
addition, two methyls (63 32.0 and 19.4) and one carbonyl (63 204.9) were
observed. Based on these data, HMQC and HMBC experiments were used to
establish the structure of the aglycon as shown in Figure 2.3.

Additional methylene and methine spins were observed in compound 14
between 69 65.6 and 104.2 that were assigned to xylopyranose and
glucopyranose residues. A 1—>6 linkage was confirmed between the two sugar
moieties based on 3J HMBC correlations between the H-6' protons ((5.. 3.58 and
4.02) and C-1" (53 104.2) (Figure 3). Further confirmation of the structure for
compound 14 was obtained as a result of 1D difference NOE experiments in
which reciprocal NOE enhancements were observed between H-7 and H-6, H-7
and H-1’,—CH3 (6H 2.25) and H-4, as well as —CH3 (5,, 2.25) and —COCH3 (cit
2.51) (Figure 2.3). Therefore, the structure of the new naphthalene glycoside 14
was established as that illustrated in Figure 2.2. Compound 14 has been given

the trivial name stelladerol in recognition of its biogenic source.

41

 

HO

Figure 2.3. Selected HMBC (A) and difference NOE (B) correlations used to

determine the structure of stelladerol (14).

42

Conclusions

Daylily flowers have been used extensively throughout eastern Asia as an
important traditional food item and medicinal agent. Yet despite this rich history
of use, very little was known regarding the chemical composition of the flowers.
In this study of Hemerocallis cv. Stella de Oro flowers, a number of compounds
were reported here for the first time as constituents of daylily flowers. These
compounds include kaempferol, quercetin, and isorhamnetin 3-O—glycosides (1-
9), phenethyl B-D—glucopyranoside (10), orcinol B-D-glucopyranoside (11),
phloretin 2’-O-B-D-glucopyranoside (12), phloretin 2'-O-l3-D-xylopyranosyl-(1—>6)-
B-D—glucopyranoside (13), a new napthalene-glycoside, stelladerol (14), and an
amino acid (longitubanine A) (15). The biological of activities of these

compounds have been investigated and are reported in Chapter Four.

43

CHAPTER THREE

KWANZOQUINONES A-G AND OTHER CONSTITUENTS OF

HEMEROCALLIS FULVA ‘KWANZO’ ROOTS

Abstract

Daylilies (Hemerocallis spp.) have been used in Asia for the treatment of
schistosomiasis; however, the active principles have not been fully characterized.
In this study of Hemerocallis fulva ‘Kwanzo’ Kaempfer roots, several compounds
were isolated including seven new anthraquinones, kwanzoquinones A (16), B
(17), C (19), D (20), E (21), F (22), and G (24), two known anthraquinones, 2-
hydroxychrysophanol (18) and rhein (23), one new naphthalene glycoside, 5-
hydroxydianellin (26), one known naphthalene glycoside, dianellin (25), one
known flavone, 6-methylluteolin (27), and' a-tocopherol. The structures of the

compounds were elucidated by spectroscopic and chemical methods.

44

Introduction

Daylily roots (Hemerocallis spp., Hemerocallidaceae) have been used in
Asia to treat schistosomiasis (Shiao et al., 1962a; Shiao et al., 1962b). However,
this method of treatment has fallen into disfavor due to a host of toxic side effects
and deaths associated with the administration of Hemerocallis root extracts to
humans (Wang et al., 1989). Previous efforts to identify the active constituent
responsible for the therapeutic properties of Hemerocallis roots led to the
isolation of a neurotoxic binaphthalenetetrol known as stypandrol (Wang and
Yang, 1993) which had been shown to cause paralysis, blindness and death in
mammals (Main et al., 1981; Colegate et al., 1985). In another report (Chen et
al., 1962), researchers obtained a yellow powdery isolate to which was ascribed
both the biological activity against schistosomes, as well as the toxic side effects
associated with the use of Hemerocallis roots; however, its structure was never
identified. While other studies have described additional compounds found in
daylilies, none of these efforts have addressed the need to fully characterize the
bioactive schistosome inhibitory chemical constituents from Hemerocallis roots.

In this study of the roots of Hemerocallis fulva ‘Kwanzo’ Kaempfer roots, a
series of seven new and two known anthraquinones, one new and one known
naphthalene glycosides, and one flavone were obtained. The isolation and

structure elucidation of these compounds is reported in this chapter.

45

Materials and Methods

General Experimental Procedures. 1H NMR spectra were recorded at 500 and
600 MHz on Varian (Palo Alto, CA) VRX (500 MHz) and INOVA (600 MHz)
instruments, respectively. 13C NMR spectra were obtained at 125 MHz on a
Varian VRX instrument. NMR spectra of compounds 16 and 17 were obtained in
CDCI3 while all other spectra were recorded in DMSO-d5 (Cambridge Isotope
Laboratories, Inc., Andover, MA). Standard pulse sequences were employed for
all 1D (1H, 130, DEPT, selective ‘H decoupling, and difference NOE) and 20
(DQF-COSY, long-range COSY, NOESY, HMQC, and HMBC) NMR
experiments. Mass spectra were acquired at the Michigan State University Mass
Spectrometry Facility using a JOEL AX-505H double-focusing mass
spectrometer operating at 70 eV for EIMS analysis and a JEOL HX-110 double-
focusing mass spectrometer (Peabody, MA) operating in the positive ion mode
for FABMS experiments. The UV spectra were recorded in EtOH using a
Shimadzu UV-260 recording spectrophotometer (Kyoto, Japan). IR spectra were
obtained on a Mattson Galaxy Series F,T|R 3000 using WinFlRST software
(Thermo Nicolet, Madison, WI). Optical rotations were measured with a Perkin-
Elmer Polarimeter 341 (Shelton, CT). Melting points were determined using a
Thomas Model 40 Hot Stage (Philadelphia, PA). Sephadex LH-20 was
purchased from Sigma-Aldrich (St. Louis, MO). Si gel (particle size 40-63 pm)
was obtained from Fischer Scientific (Pittsburgh, PA). Amberlite XAD-16 resin
was purchased from Supelco (Bellefonte, PA). LC-SORB SP-A-ODS gel

(particle size 25-40 pm) was obtained from Dychrom (Santa Clara, CA). Si gel

46

PTLC plates (20 x 20 cm; 250, 500, and 1000 pm thick) were acquired from
Analtech, Inc. (Newark, DE). Preparative HPLC was performed on a Japan
Analytical Industry Co. model LC-20 recycling preparative HPLC with a JAIGEL-
C18 column (10 um, 20 mm x 250 mm). Standards (a-tocopherol, D-
glucopyranose and L-rhamnopyranose ) were purchased from Sigma-Aldrich (St.
Louis, MO). All other solvents and chemicals were purchased from Spectrum
Laboratory Products, Inc. (New Brunswick, NJ) and were of ACS analytical

grade.

Plant Material. Hemerocallis fulva ‘Kwanzo’ plants were purchased from Dr.
Linda Sue Barnes, Perennial Patch (Wade, North Carolina) in August 1999. The
plants were grown on the Michigan State University campus before being

harvested in April 2001. The leaves were removed and the roots and crowns of
124 plants (10 kg) were washed and frozen at —4 °C. The frozen roots were
lyophilized and ground in a Waring blender, yielding 2.2 kg of fine, light-brown

powder.

Extraction and Isolation of Compounds 16-28. The lyophilized, powdered
roots (2.0 kg) were sequentially extracted with 3 x 8 L portions of hexane, EtOAc,
and MeOH yielding 25, 23, and 130 g of extracts, respectively. The hexane
extract was redissolved in 500 mL of hexane and partitioned with 3 x 500 mL
portions of MeOH. The MeOH fractions were pooled, yielding 15 g of extract that

was applied to Si gel VLC and eluted with 4 L hexane, 3 L hexane-acetone (9:1),

47

and 3 L hexane-acetone (3:2). The hexane eluate (8.5 g) was subjected to Si gel
MPLC under gradient conditions with 100% hexane to 100% acetone, and a total
of 30 fractions, each 200 mL, were collected. All fractions were analyzed by TLC
and pooled according to similarities in their profiles, yielding fractions A1 -A4.

The hexane-acetone (9:1) eluate from the Si gel VLC (4.5 g) was
subjected to Si gel MPLC under gradient conditions with 100% hexane to
hexane-acetone (1:1) providing 900 mL fractions B1-B4. Fraction B2 (1.5 g) was
rechromatographed by Si gel MPLC under gradient conditions with 100% hexane
to 100% EtOAc and a total of 18 fractions, 'each with a volume of 200 mL, were
collected and pooled based on TLC profiles giving fractions C1-C4. Fractions A3
(19), A4 (1g), C2 (300 mg), and C3 (300mg) were pooled based on further
examination by TLC and applied to Si gel MPLC. Elution was carried-out under
gradient conditions with 100% hexane to 100% CHCI3 to CHClg-ethanol (1 :1) and
18 mL fractions, D1-D90, were collected. ' Fractions D1-D10 were pooled (500
mg) and further subjected to Si gel MPLC under gradient conditions with 100%
hexane to hexane-acetone (97:3) and 15 mL fractions E1-E4O were collected.
Fractions E6-E20 (200 mg) were composed of primarily one major component
and thus were pooled and subjected to sequential Si gel PTLC with hexane-
EtOAc (10:1) (72 mg), hexane- diethyl ether (6:1) (51 mg), and benzene-CHCI3
(20:1), yielding 30 mg of a-tocopherol as a clear oil that exhibited spectral
characteristics matching those reported in the literature (Baker and Myers, 1991),

and was found identical in all respects to an authentic standard.

48

Fractions D12-D45 (300 mg) were combined, applied to Si gel PTLC
plates, and developed twice in benzene-CHCI3 (10:1). A bright yellow band (44
mg) was obtained and following extraction from the Si gel, it was dissolved in a
minimal volume of CHCIa, and hexane was added drop-wise until a slight degree
of turbidity was noted. The solution was stored at —20 °C, yielding an
inseparable 1:1 mixture (based on 1H NMR) of compounds 16 and 17 as fine
yellow needles (12 mg). Compounds 16 and 17, and their monoacetates 16a
and 17a, were subjected to a variety of chromatographic techniques including
further Si gel TLC and MPLC, as well as, ODS MPLC and ODS preparative
HPLC, but failed to separate them as single entities.

The MeOH extract of the roots was dissolved in 800 mL MeOH-H20 (3:1)
and left at 4 °C until a precipitate formed. The mixture was centrifuged (16,000 x
g, 15 min, 4 °C) and the supernatant decanted to give 30 g of extract. The
extract was applied to a column of XAD-16 resin and eluted with 10 L H20, 6 L
25% aqueous MeOH, and 8 L 100% MeOH. The MeOH eluate (18 g) was
dissolved in 500 mL H20 and partitioned with CHCI3 (3 x 300 mL). The CHCI3
fractions were pooled and evaporated under reduced pressure, yielding 2 g of
extract that was applied to ODS MPLC, eluted with 50-100% MeOH, and 16 mL
fractions F1-F166 were collected. Fractions F116-F125 were pooled giving 100
mg of residue that was dissolved in MeOH-acetone (3:1) and stored at -20 °C,
yielding 7 mg of compound 23 as a yellow powder. Compound 23 was identified
as rhein based on comparisons of its physical and spectral data to those reported

in the literature (Danielsen and Aksnes, 1992).

49

The aqueous phase (16 g), from partitioning with CHCI3, was dissolved in
50 mL of MeOH and 450 mL of acetone was slowly added while stirring, and the
mixture was left at 4 °C. The supernatant (14 g) was applied to ODS MPLC and
eluted with 45-100% MeOH under gradient conditions, yielding 750-mL fractions
G1-G6. Fraction G3 (1 g) was again applied to ODS MPLC and eluted with
CHgCN-MeOH-H2O-TFA (25:25:50:0.1 to 30:30:40:0.1) under gradient conditions
yielding fractions H1-H6. Fraction H5 (170 mg) was applied to Sephadex LH-20
with MeOH. The major component eluted as a yellow band (25 mg) and was
further purified by ODS preparative HPLC with CHacN-MeOH-H2O-TFA
(5022023001), yielding 16 mg of compound 24 as a yellow powder.

Fraction G1 (10 g) was applied to ODS MPLC with 10-50% CH3CN under
gradient conditions and 550 mL fractions (l1-l7) were collected. Fraction IS (410
mg) was chromatographed on Sephadex LH-20 with MeOH, yielding 80 mg of
yellow amorphous solid. This material was further purified by successive Si gel
PTLC chromatography with EtOAc-CHCI3-MeOH-H2O-HCOOH
(65:25:10:0.8:0.1) (75 mg) followed by CHClg-MeOH- H2O (8:221) (70 mg). Final
purification by ODS preparative HPLC with 60% MeOH gave 61 mg of compound
25 as a clear-yellow, glass-like solid.

Fraction l4 (1.5 g) was applied to Sephadex LH-20 and eluted with MeOH
giving 150-mL fractions J1-J6. Fractions J3-J6 (400 mg), I7 (300 mg), and H2-
H4 (700 mg) were pooled and subjected to ODS MPLC with CH3CN-MeOH-H2O-
TFA (20220260201-40:40:2020.1) under gradient conditions and 16-mL fractions

K1-K105 were collected. Fractions K22-K38 (430 mg) were combined and

50

chromatographed on Sephadex LH-20 with MeOH, giving fractions L1-L2.
Fraction L1 (300 mg) was applied to Si gel PTLC and developed twice with
CHCl3-MeOH-H2O (822:0.1) giving a single band that was further purified by ODS
preparative HPLC with 60% MeOH to yield 31 mg of compound 26 as a clear,
glass-like solid.

Fraction L2 (130 mg) was applied to Sephadex LH-20 and eluted with
MeOH to give 80 mg of a yellow amorphous solid. This material was dissolved in
MeOH and placed at —20 °C yielding 62 mg of precipitate. The precipitate was
chromatographed twice by ODS preparative HPLC with CH3CN-MeOH-H2O-TFA
(40:15:45:0.1) to give 30 mg of yellow amorphous solid. Further purification of it
was achieved by using 60-100% MeOH as the solvent under gradient conditions,
yielding a single fraction. It was reduced in vaccuo and kept at —20 °C yielding 1
mg of compound 22 as a yellow powder.

Fractions K50-K55 were combined (98 mg), subjected to Sephadex LH-20
chromatography using MeOH as the eluant, and 125-mL fractions (M1-M5) were
collected. Fraction M5 (40 mg) was dissolved in MeOH and left at room
temperature, whereupon 25 mg of compound 19 was obtained as fine, yellow
needles.

Fractions K56-K62 were pooled (130 mg), applied to Sephadex LH-20 and
eluted with MeOH to yield fractions N1-N3. Fraction N1 (50 mg) was subjected
to further Sephadex LH-20 chromatography with MeOH, giving a fraction (35 mg)
that was chromatographed again on ODS preparative HPLC using CH3CN-

MeOH-H2O-TFA (50:20:30:0.1). A single fraction was collected, reduced in

51

vaccuo, and placed at —20 °C, yielding 6 mg of compound 20 as golden-yellow
needles. Fraction N2 (7 mg) was further purified by ODS preparative HPLC
using CH3CN-MeOH-H2O-TFA (50:20:30:0.1) as the mobile phase to yield 1 mg
of compound 27 as a yellow glass-like solid.

Fractions K63-K77 were pooled and subjected to Sephadex LH-20
chromatography using MeOH as the mobile phase, and 100 mL fractions (01-
OS) were collected. Fraction 03 (30 mg) was applied to ODS preparative HPLC
using CH3CN-MeOH-H2O-TFA (50:20:30:0.1) as the mobile phase and yielded
an amorphous yellow solid (6 mg). This material was further purified by ODS
preparative HPLC under the same conditions, and the resultant fraction was
reduced in vaccuo and placed at -20 0C to yield 4 mg of compound 21 as fine
yellow needles.

Fractions K94-K100 were reduced in vaccuo to dryness, yielding 13 mg of
an orange amorphous solid. This material was dissolved in a minimal volume of

MeOH and left at —20 °C providing 7 mg of compound 18 as orange needles.

Kwanzoquinones A and B (16 and 17). Yellow needles; 165-167 °C; UV Amax

(EtOH) 212, 262, 287, 403 nm; IR (KBr) 17...... 3438, 1700, 1696, 1691, 1685,
1670, 1652, 1630, 1595, 1559 cm"; 1H NMR 13c NMR data, see Table 3.1;

HRFABMS m/Z 295.0971 [Mt-H]+ (calcd for C13H1504, 295.0970).

Acetylation of Compounds 16 and 17. A portion (4 mg) of the 1:1 mixture of

compounds 16 and 17 was dissolved in 1 mL of pyridine and 1 mL of A020 was

52

added ,and the solution was stirred at room temperature for 16 h. Deionized H2O
was added to the reaction mixture, and it was subsequently partitioned with
CHC13 (x 3). The CHCl3 fractions were reduced in vaccuo and applied to a silica
gel PTLC plate. The plate was repeatedly developed (x 3) in hexane-
diethylether-CHCI3 (5:1:0.1) giving two UV active bands. Band 1 (Rf = 0.4) (0.7
mg) was found to be identical to kwanzoquinones A and B (1 and 2) while band 2
(Rf = 0.2) (3.6 mg) was crystallized from MeOH to give yellow needles that were
an inseparable mixture of kwanzoquinone A and B monoacetates (16a and 17a,

respectively).

Kwanzoquinone A and B Monoacetates (16a and 17a). Yellow needles; IR
(KBr) 17...... 1773, 1706, 1675, 1592, 1457, 1438, 1368, 1328, 1251, 1187 cm"; 1H
NMR (CDCI3) (5.. 8.10 (4H, m, H-5’s and H-8’s), 8.02 and 7.99 (2H, s, H-4’s), 7.55
(2H, m, H-6 and H-7 for compounds 17a and 16a, respectively), 2.49 (12H, brs,
H-12’s and -OCOCH3's), 2.45 (6H, s, H-14’s), 2.41 (6H, s, H-13’s); HRFABMS at

m/z 337.1068 [M+H]+ (calcd for C2oH1705, 337.1076).

2-Hydroxychrysophanol (18). Orange needles; mp 239-240 °C; UV Amax
(EtOH) (1098) 208 (4.19), 235 (4.05), 258 (4.11), 426 (3.73) nm; IR (KBr) vmax
3408, 1653, 1620, 1560, 1473, 1456, 1434, 1310, 1271, 1190 1023 cm"; 1H
NMR (DMSO-d6) (St 12.04 (1H, brs, 1-OH), 11.90 (1H, s, 8-OH), 10.34 (1H, brs,

2-OH), 7.76 (1H, dd, 8.0, 7.5, H-6), 7.66 (1H, dd, 7.5, 1.0, H-5), 7.55 (1H, s, H—4),

7.31 (1H, dd, 8.0, 1.0, H-7), 2.26 (1H, s, 3-CH3); 13C NMR, see Table 3.2; EIMS

53

Table 3.1. NMR spectral data for kwanzoquinones A (16) and B (17) in CDCI2,a

 

 

 

 

16 17
position 3.. (J in Hz) " 80¢ HMBC” 5H (J in Hz) b 30‘ HMBC”
1 - 159.6 (s) 1-OH - 159.6 (s) 1-OH
e 1-OH, H- e 1-OH, H-13,
2 - 114.4 (s) - 114.5 (s)
13, H4 H-4
3 - 144.7'(s) H-4, H-13 - 144.9'(s) H-4, H-13
4 7.61 (s) 121.5 (d) H-13 7.61 (s) 121.5 (d) H-13
4a - 133.1 (s) H-4 - 133.1 (5) H4
8.04 (s) 127.8 (d) H-14 8.13 (d, 7.5) 127.7 (d) H-6
- 146.2 (s) H-8, H-14 7.58 (d, 7.5) 135.6 (d) H-8, H-14
7.58 (d, 7.5) 135.1 (d) H-5, H-14 - 145.6 (s) H-5, H-14
8.15 (d, 7.5) 127.1 (d) H-7 8.05 (s) 127.2 (d) H-14
8a - 133.4 (s) H-8 - 131.2 (s) H-8
9 188.1 (s) H-8 - 188.5 (s) H-8
9a 135.79(s) 1-OH, H-4 135.89(s) 1-OH, H-4
10 182.3 (s) H4, H-5 - 181.9 (s) H-4, H-5
10a 130.9 (s) H-5 - 133.0 (s) H-5
11 203.0 (s) H-12 - 203.0 (s) H-12
12 2.59 (s) 31.9 (q) - 2.59 (s) 31.9 (q) -
13 2.37 (s) 20.2 (q) H-4 2.37 (s) 20.2 (q) H4
14 2.51 (s) 21.9” (q) H-5, H-7 2.51 (s) 22.0" (q) H-6, H-8
1-OH 12.95 (s) - - 12.90 (s) - -

 

8All spectra were recorded using 12 mg of a 1:1 mixture of compounds 16 and 17
dissolved in 1 mL of CDCI3 with a 5 mm probe at 25 °C.
”Recorded at 500 MHz.
0Recorded at 125 MHz. Multiplicities were determined by DEPT experiment.

”HMBC data were recorded using a JCH = 8 Hz and are expressed as protons

exhibiting 2'3qu couplings to the carbons as indicated.

”Assignments bearing the same letter may be interchanged.

54

Table 3.2. 13C NMR assignments for compounds 18-22 and 24"

 

 

position 18 19 20 21 22 24
1 149.4 s 153.9 s 153.9 3 149.1 s 153.4 s 158.6 s
2 150.2 3 147.7 s 147.7 s 148.4 s 146.0 s 131.2 s
3 132.3 s 141.4 s 141.5 s 136.7 s 145.3 s 140.4 3
4 122.8d 121.5d 121.4d 119.0d 117.9d 112.0d
43 123.1 s 128.0 s 128.0 s 123.3 s 128.2 3 136.2 s
5 119.0d 119.0d 119.0d 119.1d 119.1d 118.1d
6 137.3 d 137.1 d 137.1 d 137.4 d 137.2 d 135.9 d
7 123.7 d 124.1 d 124.0 d 123.7 d 124.0 d 124.2 d
8 161.23 161.23 161.23 161.33 161.23 161.33
8a 115.93 115.93 115.93 116.03 116.13 116.73
9 192.23 191.53 191.43 192.33 191.73 189.23
93 114.33 115.23 115.23 114.63 115.73 122.43
10 180.1 s 180.8 s 180.6 3 180.2 s 180.8 s 181.8 3
10a 133.7 s 133.2 s 133.1 s 133.8 s 133.3 s 132.3 3
11 16.4 q 17.2 q 17.2 q 57.8 t 58.1 t 19.5 q
12 - - - - - 167.8 s
1' - 102.9 d 102.8 d - 102.7 d -
2’ - 74.2 d 74.0 d - 74.1 d -
3’ - 76.3 d 76.0 d - 76.2 d -
4’ - 69.7 d 69.7 d - 69.7 d -
5' - 77.3 d 73.8 d - 77.2 d -
6' - 60.8 t 63.7 t - 60.7 t -
1" - - 166.4 s - - -
2” - - 41.1 t - - -
3" - - 167.4 s - - -

 

aData recorded in DMSO-d5 at 125 MHz at 25 °C. Multiplicities were determined

by DEPT experiment and confirmed by. analysis of HMQC spectra.

55

m/z 270 [M]+ (100), 253 (2), 242 (8), 213 (4), 196 (3), 185 (2), 168 (5), 139 (11);
HREIMS m/z 270.0532 [M]+ (calcd for C15H1005, 270.0528) (for literature values

refer to Li and McLaughlin, 1989; Midiwo and Arot, 1993).

Kwanzoquinone C (19). Fine yellow needles; mp 233-234 °C; [(11200 -46° (c
0.031, EtOH); UV Amax (EtOH) (loge) 206 (4.20), 227 (4.23), 260 (4.17), 429
(3.78) nm; IR (KBr) vmax 3433, 1671, 1624, 1559, 1473, 1382, 1373, 1293, 1263,
1067 cm"; 1H NMR (DMSO-d5) 5.. 12.04 (1H, brs, 8-OH), 12.00 (1H, s, 1-OH),
7.79 (1H, dd, J = 7.5, 8.0 Hz, H-6), 7.70 (1H, dd, J = 1.0, 7.5 Hz, H-5), 7.61 (1H,
3, H-4), 7.36 (1H, dd, J = 1.0, 8.0 Hz), 5.07 (1H, d, J = 7.5 Hz, H-1'), 3.60 (1H,
ddd, J = 2.0, 5.5, 12.0 Hz, H-6a'), 3.42 (1H, ddd, J = 6.0, 11.5, 11.5 Hz, H-6b'),
3.31 (1H, m, H-2'), 3.25 (1H, m, H-3'), 3.16 (1H, m, H-4'), 3.13 (1H, m, H-5’),
2.42 (3H, s, H-11); 13C NMR data, see Table 3.2; HRFABMS m/z 433.1139

[M+H]+ (calcd for C21H21Om, 433.1135).

Kwanzoquinone D (20). Golden-yellow needles; mp 174-175 °C; [0429.) —313° (c
0.008, EtOH); uv 2...... (EtOH) (logo) 205 (4.28), 227 (4.35), 260 (4.31), 290 sh
(3.91), 430 (3.96) nm; IR (KBr) cm... 3430, 1734, 1717, 1699, 1670, 1653, 1559,
1457, 1268, 1066 cm"; 1H NMR (DMSO-d5) 5.. 12.57 (1H, brs, 1-OH), 11.96 (1H,
s, 8—OH), 7.77 (1H, dd, J = 7.5, 8.0 Hz, H-6), 7.67 (1H, dd, J = 1.0, 7.5 Hz, H-5),
7.57 (1H, s, H4), 7.33 (1H, dd, J = 1.0, 8.0 Hz), 5.06 (1H, d, J = 7.5 Hz, H-1'),
4.27 (1H, dd, J = 2.5, 11.9 Hz, H-6a'), 4.12 (1H, dd, J = 6.5, 11.9 Hz, H-6b'), 3.38

(1H, m, H-5'), 3.33 (1H, m, H-2'), 3.28 (1H, m, H-3'), 3.23 (2H, s, H-2"), 3.21 (1H,

56

m, H-4'), 2.37 (3H, s, H-11); 130 NMR data, see Table 3.2; HRFABMS m/z

519.1139 [M-l-H]+ (calcd for Cz4H23O13 519.1151).

Kwanzoquinone E (21). Fine yellow needles; mp 196-197°C; UV kmax (EtOH)
(logs) 209 (4.32), 235 (4.10), 258 (4.27), 354 (3.72), 426 (3.76) nm; IR (KBr) v”...
3469, 1652, 1619, 1559, 1473, 1458, 1382, 1321, 1273, 1092 cm"; 1H NMR
(DMSO-d5) a. 12.06 (1H, brs, 1-OH), 11.92 (1H, s, 8-OH), 10.47 (1H, brs, 2-OH),
7.87 (1H, d, J = 0.5 Hz, H-4), 7.78 (1H, dd, J = 7.8, 7.8 Hz, H-6), 7.70 (1H, dd, J
= 0.5, 7.8 Hz, H-5), 7.33 (1H, dd, J = 0.5, 7.8 Hz, H-7), 5.40 (1H, brs, 11-OH),
4.59 (2H, s, H-11); 13c NMR data, see Table 3.2; EIMS m/z 286 [Mr (62), 268
(89), 240 (56), 212 (100), 184 (50), 155 (14), 128 (19), 120 (19); HREIMS m/z

286.0479 [M]+ (calcd for C15H1005, 286.0477).

Kwanzoquinone F (22). Yellow powder; mp 204-206°C; [61290 —38° (c 0.01,
EtOH); UV 2...... (EtOH) (loge) 228 (4.04), 259 (4.03), 291 (3.57), 432 (3.68) nm;
IR (KBr) um... 3450, 1698, 1684, 1652, 1635, 1559, 1540, 1457, 1262, 1027 cm";
1H NMR (0.75 mL DMSO-d5/2 drops D20) 3. 7.88 (1H, s, H-4), 7.79 (1H, dd, J =
7.5, 8.0 Hz, H-6), 7.71 (1H, dd, J = 1.0, 7.5 Hz, H-5), 7.36 (1H, dd, J = 1.0, 8.0
Hz, H-7), 5.07 (1H, d, J = 7.5, H-1'), 4.37 (1H, d, J = 16.0 Hz, H-11a), 4.65 (1H,
d, J = 16.0 Hz, H-11b), 3.60 (1H, d, J = 3.0, 12.5 Hz, H-6a'), 3.40 (1H, dd, J =
5.5, 12.0 Hz, H-6b'), 3.26 (1H, m, H-2'), 3.25 (1H, m, H-3'), 3.15 (1H, m, H-4'),
3.12 (1H, m, H-5'); 13C NMR data, see Table 3.2; HRFABMS m/z 433.1132

[M+H]+ (calcd for C21H21010, 433.1135).

57

Kwanzoquinone G (24). Yellow powder; mp 235-236°C; kmax (EtOH) (I098) 219
(4.25), 283 (4.19), 413 (3.63) nm; IR (KBr) cm... 3420, 1717, 1700, 1670, 1634,
1577, 1365, 1320, 1261, 1223 cm"; 1H NMR (DMSO-d5) 3, 12.82 (1H, s, 8-OH),
12.81 (2H, brs, 1-OH and 12-OH), 7.67 (1H, dd, J = 8.1, 8.1 Hz, H-6), 7.57 (1H,
dd, J = 1.2, 8.1 Hz, H-5), 7.56 (1 H, s, H4), 7.28 (1H, dd, J = 1.5, 8.1, H-7), 2.67
(3H, s, H-11); 13C NMR data, see Table 3.2; HRFABMS m/z 299.0547 [M+H]*

(calcd for CranOe, 299.0556).

Dianellin (25). White needles; mp 156-157 °C; [61290 —137° (c 0.01, EtOH); UV
1...... (EtOH) (logs) 225 (4.75), 301 (3.80), 334 (3.78) nm; IR (KBr) vma, 3416,
2923, 1651, 1633, 1579, 1467, 1443,1356, 1270, 1067 cm"; 1H NMR (DMSO-
d6) 54 9.53 (1H, brs, 1-OH), 7.47 (1H, dd, J = 1.0, 8.0 Hz, H-5), 7.40 (1H, dd, J =
8.0, 8.0 Hz, H-6), 7.30 (1H, dd, J = 1.0, 8.0 Hz, H-7), 7.21 (1H, s, H4), 5.04 (1H,
d, J = 7.5 Hz, H-1'), 4.62 (1H, d, J = 1.5 Hz, H-1"), 3.93 (1H, dd, J =15, 11.0 Hz,
H-6a'), 3.68 (1H, m, H-2"), 3.59 (1H, m, H-5'), 3.50 (2H, m, H-6b’ and H-3"), 3.49
(1H, m, H-5"), 3.39 (1H, m, H-2'), 3.36 (1H, m, H-3’), 3.20 (1H, m, H-4"), 3.18
(1H, m, H-4'), 2.52 (3H, s, H-12), 2.25 (3H, s, H-13), 1.12 (3H, d, J =6 Hz, H-6");
13c NMR (DMSO-d5) ac 204.4 (s, 041), 154.2 (s, C-8), 150.2 (s, 04), 135.7 (s,
010), 132.8 (s, 03), 127.3 (d, C-6), 125.2 (s, 0.2), 122.3 (d, 05), 119.4 (d, c-
4), 113.2 (s, 69), 110.7 (d, 07), 102.6 (d, 0.1), 100.7 (d, c-1~), 76.2 (d, 03),

76.0 (d, C-5'), 73.3 (d, C-2'), 71.9 (d, C-4"), 70.7 (d, C-3"), 70.4 (d, C-2"), 70.1

58

(d, C-4'), 68.4 (d, c-5"), 66.6 (t, C-6’), 31.9 (q, C-12), 19.0 (q. 013), 17.7 (q, o-

6"); HRFABMS m/z 525.1970 [M+H]+ (calcd for C25H33012, 525.1972).

5-Hydroxydianellin (26). Yellow amorphous solid; mp 152-153 °C; [or]290 —212°
(c 0.01, EtOH); uv Am... (EtOH) (logs) 224 (4.92), 318 (4.13), 346 (4.15) nm; IR
(KBr) vmax 3420, 1698, 1684, 1653, 1635, 1559, 1457, 1364, 1257, 1059 cm"; 1H
NMR (DMSO-d5) 5.. 9.71 (2H, brs, 1-OH and 5-OH), 7.43 (1H, s, H-4), 7.16 (1H,
d, J=8.0, H-7), 6.76 (1H, d, J=8.0, H-6), 4.87 (1H, d, J=7.5, H-1'), 4.61 (1H, m, H-
1"), 3.92 (1H, m, H-6a'), 3.69 (1H, brs, H-2"), 3.52 (1H, m, H-6b'), 3.51 (1H, m,
H-5'), 3.50 (1H, m, H-3"), 3.48 (1H, H-5"), 3.34 (2H, m, H-2' and H-3'), 3.21 (1H,
m, H—4"),3.18(1H, m, H-4'), 2.51 (3H, s, H-12), 2.26 (3H, s, H-13), 1.14 (3H, d, J
= 6 Hz, H-6");13C NMR (DMSO-d5) 5,; 204.7 (s, 011), 150.2 (s, 04), 148.3 (s,
05), 146.7 (s, C-8), 131.3 (s, 03), 126.4 (s, 010), 125.6 (s, 02), 114.2 (s, c-
9), 113.8 (d, 04), 111.9 (d, 07), 108.6 (d, C-6), 103.5 (d, c-1'), 100.7 (d, c-1~),
76.3 (d, 03), 75.9 (d, 0.5), 73.3 (d, 072'), 72.0 (d, C-4"), 70.8 (d, c-3"), 70.5 (d,
02'), 70.0 (d, 04'), 68.4 (d, 05'), 66.6 (t, C-6’), 31.9 (q, 012), 19.3 (q, 013),

17.7 (q, C-6"); HRFABMS m/z 541.1910 [M+H]+ (calcd for C25H33013, 541.1921).

6-Methylluteolin (27). Yellow glass-like solid; UV and IR data were identical to
literature values (Milovanovic et al., 1996); 1H NMR (DMSO-d5) (3.. 10.92 (1H, s,
5-OH), 9.71 (1H, s, 7-OH), 9.55 (1H, s, 4'-OH), 9.23 (1H, s, 3’-OH), 7.40 (1H, d, J

= 2.0 HZ, H-2'), 7.16 (1H, dd, J = 2.0, 8.5 Hz, H-6'), 6.80 (1H, d, J = 8.5 Hz, H-5'),

59

6.47 (1H, s, H-3), 6.32 (1H, s, H-8), 1.92 (3H, s, -CH3); 130 NMR (DMSO-d6) (30
180.1 (s, C4), 165.0 (s, 05), 164.2 (s, 09), 154.5 (s, 07), 147.4 (s, 04'),
145.6 (s, 03), 145.3 (s, 02), 123.9 (d, C-6'), 123.5 (s, 04), 117.5 (d, 02),
115.8 (d, C-5'), 109.8 (d, 03), 105.8 (s, C-6), 102.8 (s, 010), 90.2 (d, C-8), 7.5

((1. -CH3); HRFABMS m/z 301.0709 [M-l-H]+ (calcd for C15H1305, 301.0712).

Hydrolysis of Compounds 19, 20, 22, 25, and 26. Approximately 0.5 mg of
compounds 19, 20, 22, 25, and 26 were each combined with 2.5 mL of 15%
aqueous HCI and left at 50 0C for about 6 h with constant stirring. The mixtures
were each neutralized with the drop-wise addition of 5% aqueous NaOH and
partitioned with EtOAc. The EtOAc fractions were reduced in vaccuo, the
resultant residues were spotted on an analytical silica gel TLC plate along with
compounds 18 and 21, and the plate was developed in toluene-EtOAc-HOAc
(4:2:0.1). After development, the plate was dried, lightly sprayed with 10%
aqueous H2SO4, and charred with a heat-gun. The hydrolysate from compounds
19 and 20 exhibited a bright pink spot (Rf = 0.8) that was identical to that
observed for 2-hydroxychrysophanol (18). Similarly, the hydrolysate of
compound 22 yielded a pink spot (Rr = 0.4) that was identical to kwanzoquinone
E (21). The aqueous portions from compounds 19, 20, 22, 25, and 26 were also
reduced in vaccuo and the residues spotted on analytical silica gel TLC plates
along with D-glucopyranose and L-rhamnopyranose. The plate was developed in
n-ButOH-HOAc-H2O (321:1), air dried, sprayed with 10% aqueous H2804, and

charred with a heat-gun. The hydrolysate from compounds 19, 20, 22, 25, and

60

26 each exhibited a black spot (Rf = 0.6) that was identical with that observed for
D-glucopyranose. In addition, compounds 25 and 26 also exhibited a second

dark greenish-black spot (Rf = 0.7) that matched L-rhamnopyranose.

Results and Discussion

The roots of H. fulva ‘Kwanzo’ were successively extracted with hexane,
EtOAc, and MeOH. The hexane and MeOH extracts were selected for further
study and subsequently subjected to a combination of chromatographic
procedures including Si gel MPLC and PTLC, ODS MPLC and preparative
HPLC, and crystallization. This work led to the isolation of seven new
anthraquinones, kwanzoquinones A-G (16, 17, 19-22, 24), and a new
naphthalene glycoside (26). The structures and complete 1H and 13C NMR
spectral assignments for these new compounds, as well as those for compounds
18, 25, and 27, were defined based on extensive 1D and 2D NMR studies and
are reported here for the first time and are presented in Figure 3.1. The yield of
the compounds obtained from the H. fulva ‘Kwanzo’ roots is presented in Table
3.3.

The hexane extract was subjected to a series of chromatographic
procedures, leading to the isolation of 12 mg of fine, yellow needles following
crystallization from CHClg-hexane. Initial inspection of the‘H and 13C NMR
spectra of this product indicated a doubling of most proton and carbon signals
that suggested it was perhaps a large dimeric compound composed of more than

31 unique carbon nuclei. However, positive FABMS indicated a major signal at

61

Table 3.3. Yield of 12 compounds isolated from Hemerocallis

fulva ‘Kwanzo’ roots

 

 

yield (mg/kg

compound dry material)
16 and 17 kwanzoquinones A and B 9.5
18 2-hydroxychrysophanol 1 1.1
19 kwanzoquinone C 18.0
20 kwanzoquinone D 5.7
21 kwanzoquinone E 3.8
22 kwanzoquinone F 0.9
23 rhein 4.7
24 kwanzoquinone G 8.2
25 dianellin 15.5
26 5-hydroxydianellin 30.5
27 6-methylluteolin 0.5

 

62

 

16 R1=H, R2=CH3, R3=H
163 R1=AC, R2=CH3, R3=H
17 R1=H, R2=H, R3=CH3
17a R1=AC, R2=H, R3=CH3

GHQ

21 R=H
22 R=B-D-glucopyranoside

C)?

OHO

000°

18 R=HO

19 R=B-D-glucopyranoside

20 R=malonyl-(1—>6)-
B-D—glucopyranoside

OHO

 

Figure 3.1. Structures for compounds 16-27 isolated from Hemerocallis fulva

‘Kwanzo’ roots.

63

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pre
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ins

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17

9V

m/z 295 [M+H]+ that suggested the product was a mixture of two structurally
related isomers each with a formula of C18H14O4. This was supported by the
presence of a significant fragment ion at m/z 273 [M+H-H2O]*. Further evidence
was also provided by HMBC experiment that showed two sets of contours
representing the 2‘3JCH connectivities for two compounds, each composed of 18
carbon and 14 proton spins. Extensive efforts to separate these two compounds
by Si gel MPLC and TLC, ODS MPLC and preparative HPLC, and crystallization
with a variety of solvent systems proved unsuccessful. Further attempts were
made to separate the acetylated products (16a and 17a) from one another, but
this method also failed. Therefore, the structure elucidation and full 1H and 13C
NMR assignments of compounds 16 and 17 (Table 3.1) were performed on the
inseparable 1:1 mixture of these two constitutional isomers.

Compounds 16 and 17 were determined to each be composed of
substituted 1-hydroxyanthraquinone moieties. Evidence for this came from a
combination of HRFABMS with m/z 295.0971 [M+H]+ (calc. 295.0970) and
spectroscopic studies. The IR spectrum of compounds 16 and 17 exhibited a
number of diagnostic absorption bands at 3438 (broad, O-H stretch), 1670 (C=O
stretch, non-chelated), and 1633 cm'1 (C=O stretch, chelated). The UV spectrum
showed a Amax at 403 nm that suggested the presence of a single penlhydroxyl
functionality (Schripsema et al. 1999). This was supported by the 1H NMR
spectrum that revealed two sharp singlets at ($4 12.90 and 12.95 for compounds
17 and 16, respectively, that were both exchanged upon addition of D2O. Further

evidence for the presence of a single hydroxyl functionality in compounds 16 and

64

17 came from their acetylation products 16a and 17a. Both of these acetyl
derivatives exhibited the same molecular ion with HRFABMS at m/z 337.1068
[M+H]+ (calcd for C20H1705, 337.1076) confirming the addition of a single acetate
to 16 and 17. The 1H NMR spectrum of 16a and 17a no longer displayed
downfield peaks between 54 12 and 13 while the 13C NMR spectrum exhibited
new signals at 60 19.6 (-OCOQH3) and 169.0 (-OQOCH3).

1H NMR and DEPT experiments revealed the presence of two aromatic
(60 20.2 q x 2, 21.9 q, and 22.0 q) and one acetyl (63 31.9 q x 2) methyl groups
in both compounds 16 and 17. Data from the HMBC experiment (Table 3.2)
provided evidence for the assignment of these functionalities for compounds 16
and 17. Further support in favor of this conclusion was obtained from long-range
COSY and difference NOE experiments (Fig. 3.2). Both compounds 16 and 17
exhibited reciprocal NOE correlations upon irradiation of the methyl protons of C-
12 (both (3.. 2.59) and 1-OH’s ((54 12.95 and 12.90, respectively). In addition,
NOE enhancements and long-range COSY correlations were noted between the
methyl protons of C-13 (both 84 2.37) and the H-4 aromatic singlet (both 64. 7.61).
Together, these data confirmed the proposed ring B assignments for compounds
16 and 17.

Compound 16 exhibited reciprocal NOE enhancements and COSY
correlations Fig. 3.2) amongst H-7 (3. 7.58 d, J=7.5 Hz) and H-8 (6H 8.15 d,
J=7.5 Hz), as well as between the methyl protons of C-14 ((34 2.51 s) and protons
at positions H-7 and H-5 (6H 8.04 s). This evidence confirmed that the aromatic

methyl C-14 (6H 21.9) was attached at position 6 on ring A of compound 16.

65

   

Figure 3.2. Difference NOE ( —> ) and long-range COSY ( — ) correlations used

to establish the structures of kwanzoquinones A (16) and B (17).

66

Compound 17 differed from compound 16 by displaying reciprocal NOE
enhancements and long-range COSY correlations between the methyl protons of
C-14 ((5.4 2.51 s) and protons H-6 (5,; 7.58 d, J=7.5 Hz) and H-8 (3.. 8.05 3).
Similar NOE and COSY correlations were noted between H-6 and H-5 (5.. 8.13 d,
J=7.5 Hz). Therefore, the assignment of the aromatic methyl C-14 (62 22.0) was
confirmed at position 7 on ring A of compound 17. Compounds 16 and 17 have
been named kwanzoquinones A and B, respectively, in recognition of their
biogenic source.

The MeOH extract was subjected to repeated ODS and Sephadex LH-20
gel column chromatography yielding compounds 18-27. Following purification,
compound 18 was obtained from MeOH as orange needles. HREIMS (m/z
270.0532 [M]+ (calcd for C15H1005, 270.0528)) and spectral evidence (IR, UV, 1D
and 2D NMR) confirmed that compound 18 (1,2,8-trihydroxy 3-
methylanthraquinone) had been previously isolated from Myrsine afn'cana L.
(Myrsinaceae) and was given the trivial name 2-hydroxychrysophanol (Li and
McLaughlin, 1989). Previous studies had only given partial 1H and no 13C NMR
assignments for this compound; therefore, we undertook a thorough NMR
investigation of 18 in order to confirm its proposed structure. This is the first
report of compound 18 from daylilies and its complete 13C NMR spectral data are
presented in Table 3.3.

Compound 19 was obtained as yellow needles and exhibited many
spectral characteristics similar to 18. The IR spectrum of 19 revealed absorption

bands at 3455 (broad, O-H stretch), 1671 (C=O stretch, non-chelated), and 1624

67

cm'1 (C=O stretch, chelated). The UV spectrum presented a 4mm, at 429 nm that
was accordant with the presence of two pert-hydroxyl functionalities (Schripsema
et al., 1999; Brauers et al., 2000; Li et al., 2000). In addition, the 1H NMR
spectrum showed two downfield peaks (cit 12.00 and 12.04) that were
exchanged with D2O. Together this evidence supported the presence of a 1,8-
dihydroxyanthraquinone moiety for compound 19.

FABMS gave m/z 433 [M+H]+ that represented a molecular composition of
C21H2101o. 1H NMR provided important evidence for the substitution pattern of
rings B and A in compound 19. Three protons representing an ABC spin system
at a. 7.70 (dd, J = 1.0, 7.5 Hz), 7.79 (dd, J = 7.5, 8.0 Hz), and 7.36 (dd, J = 1.0,
8.0 Hz) were assigned to C-5, C-6, and C-7, respectively, on ring A of compound
19. 13C NMR and DEPT experiments (Table 3.2) provided further evidence for
the identity of the substituents attached to ring B of compound 19 with one
methine (60 121.5), one C-linked ((52 141.4) methyl (69 17.2), and two quaternary
carbon (60 147.7 and 153.9) linked with a hetero-atom. These carbons were
assigned positions in ring B of compound 19 based on their respective chemical
shifts and the results from HMBC and HMQC experiments. Five additional
methine (50 69.7, 74.2, 76.3, 77.3, and 102.9) and one methylene (60 60.8) spins
were observed that exhibited chemical shift values that coincided with those for a
glucopyranose moiety. Further evidence of the presence of a glucopyranose
moiety in compound 19 was obtained by comparative TLC of the hydrolysate with

authentic D-glucopyranose. The glucopyranose was assigned a B-configuration

based on the coupling of H-1' (6); 5.07, d, J = 7.5 Hz). The complete structure of

68

compound 19 was confirmed by HMBC experiment. Compound 19 is a new
anthraquinone glycoside and has been given the name kwanzoquinone C.

The molecular formula of compound 20 was determined to be C24H22013
based on FABMS analysis that exhibited m/z 519 [M+H]+. The spectral data of
20 were very similar to those obtained for compound 19. The most significant
difference was observed in the 1H and 13C NMR (Table 3.2) spectra with the
addition of three new carbon signals at 60 41.1, 166.4, and 167.4 and a new
proton resonance at 64 3.23 integrating for two hydrogens. These chemical shifts
were characteristic of those expected for a malonyl moiety. The linkage of the
malonyl group in compound 20 was established as malonyl-(146)430-
glucopyranoside based on the observed downfield shift of C-6' to 53 63.7 verses
that observed for compound 19 (A = +2.9 ppm). This was verified by HMBC
analysis (Figure 3.3) which exhibited weak 4JCH correlations from H-6a' ((3.. 4.12)
and H-6b’ (6H 4.27) to C-1" (62 41.1) and H-2" (3. 3.23) to C-6' (5c 63.7). This
confirmed that compound 20 was a new anthraquinone malonyl-glucoside named
kwanzoquinone D.

EIMS analysis of compound 21 gave a molecular ion of m/z 286 [M]+
indicating a molecular formula of C15H1006. The UV (xlmax at 426 nm) and IR
(absorption bands at 3469 (broad, O-H stretch), 1667 (C=O stretch, non-
chelated), and 1620 cm’1 (C=O stretch, chelated)) spectra suggested a 1,8-
dihydroxyanthraquinone chromaphore for compound 21. The 1H NMR spectrum
provided evidence for four exchangeable protons at 5 12.06, 11.92, 10.47, and

5.40 representing three aromatic and one aliphatic hydroxyl functionalities. An

69

 

Figure 3.3. Selected HMBC correlations used to determine the structure of

kwanzoquinone D (20).

70

ABC spin system was observed with protons at 34 7.70 (dd, J = 0.5, 7.8 Hz), 7.78
(overlapping dd, J = 7.8, 7.8 Hz), and 7.33 (dd, J = 0.5, 7.8 Hz) which occupied
contiguous positions attached to C-5, C-6, and C-7, respectively, on ring A of
compound 21.

1H and ‘30 (Table 3.2) NMR and DEPT experiments of compound 21 gave
evidence that ring B possessed quaternary carbons with ortho—hydroxyl
functionalities (6 149.1 s and 148.4 s), a hydroxy-methylene moiety (64 4.59 s,
2H and 2% 57.8 t) attached to a quaternary carbon (53 136.7), and a methine (5c
119.0). An HMBC experiment was used to make full assignments of these
proton and carbon spins as shown for compound 21 (Figure 3.4). Compound 21
was identified as a new anthraquinone and has been named kwanzoquinone E.

Compound 22 exhibited spectral data similar to 21 with the addition of five
methine (60 69.7, 74.1, 76.2, 77.2, and 102.7) and one methylene (50 60.7) spins
that coincided with those for a glucopyranose moiety. The addition of a
glucopyranose moiety in compound 22 was confirmed by HRFABMS which gave
a molecular ion of m/z 449.1082 [M+H]+ (calcd 449.1084 for C21H21O11) that
represented a molecular formula of C21H20011. The glucopyranose moiety was
determined to be O-linked at position 11 due to the downfield shift of this carbon
signal to 60 58.1 (A = +0.4) and the change in the splitting pattern of the attached
protons. While the enantiotopic C-11 protons of compound 21 ((5.1 4.59, 2H)
appeared as a singlet, the diastereotopic C-11 protons of compound 22 (5.. 4.65 ,
1H and 4.73, 1H) were each a doublet (J = 16.0 Hz) in achiral solvent (0.75 mL

DMSO-d5 with 2 drops of D2O). Further evidence in support of the composition

71

 

 

Figure 3.4. Selected HMBC correlations used to determine the structure of

kwanzoquinone E (21).

72

71
I

 

of compound 22 was obtained from acid hydrolysis that yielded kwanzoquinone
E and D—glucopyranose based on co-TLC with authentic sugar samples. The
assignments of all proton and carbon (Table 3.2) spins in compound 22 were
confirmed by HMBC experiment. Compound 22 is a new conjugated
anthraquinone glucoside and has been given the name kwanzoquinone F.

Compound 23 was obtained as an amorphous yellow powder and its
spectral data were found to match those reported for rhein, a known
anthraquinone (Danielsen and Aksnes, 1992). Compound 24 exhibited spectral
data similar to 23 with the main differences in the 1H NMR spectrum being the
loss of an aromatic doublet (ca 64 8.15, 1H, J = 1.5 Hz) and the concomitant loss
of splitting in the proton signal at 6.. 7.59 (s, 1H). This indicated that position 2 in
ring B of compound 24 was substituted. These observations coincided with the
appearance of an aromatic methyl (64 2.67 s, 3H and 62 19.5 q) and the
downfield shift of C-2 in compound 23 from 6; 124.2 to 131.2 (A = +7.0 ppm) in
compound 24 (Table 3.2). HMBC experiment was able to confirm that this
methyl was a substituent of C-2 based on the long-range coupling of the C-11
methyl protons to C-2 (6c 131.2) and C-3 (60 140.4). Compound 24 is a new
anthraquinone and has been named kwanzoquinone G.

FABMS of compound 25 provided a molecular ion of 525 [M+H]+ and the
13C NMR spectrum exhibited ten 3p2 carbon signals between 110 and 155 ppm
along with 12 additional 31p3 carbon signals that were characteristic of a rutinose
moiety. In light of the presence of three additional carbon signals that

represented an aromatic methyl (6; 19.0) and an acetyl moiety (60 31.9 and

73

-. if!

 

II'L'QK - Z n

204.4), it was determined that compound 25 was a substituted naphthalene
diglycoside. Acid hydrolysis of compound 25 yielded D-glucopyranose and L-
rhamnopyranose based on co-TLC with authentic sugar samples. HMQC and
HMBC experiments established the aglycone portion of compound 25 as 2-
acetyI-3-methyl-1,8-dihydroxynaphthalene, dianellidin. Further scrutiny of the
HMBC data provided for the assignment of an 8-O-linkage to the rutinoside
moiety based on a correlation from H—1' (64 5.04, d, J = 7.5 Hz) to C-8 (60 154.2
3). According to these data, compound 25 was identified as dianellin, previously
isolated from Diane/la spp. (Liliaceae)‘(Batterham et al., 1961). This is the first
account of compound 25 from daylilies and the first report detailing its 1H and 13C
NMR spectral data.

The 1H, 13C and DEPT NMR data of compound 26 were very similar to
those observed for 25. The loss of one aromatic methine spin in compound 25
was replaced by a quaternary carbon (6c 148.3) that was linked to a hetero-atom.
The FABMS analysis of compound 26 yielded a molecular ion at m/z 541 [M+H]+
indicating a molecular formula of C25H32O13. A comparison of the 1H and 13C
NMR spectral data for the aglycone portion of compound 26, with the reported
values for the naphthalene glycoside stelladerol (Cichewicz and Nair, 2002),
demonstrated that both possessed the same aglycone moiety. However, these
compounds differed with respect to their glycosidic moiety. Acid hydrolysis of the
new naphthalene glycoside revealed the presence of D-glucopyranose and L-
rhamnopyranose moieties in compound 26. HMBC correlation data for

compound 26 (Figure 3.5) showed that it possessed an 8-O-[3-D-

74

 

OH OH OH

   

Figure 3.5. Selected HMBC correlations used to determine the structure of 5-

hydroxydianellin (26).

75

77477471

 

rhamnopyranosyl-(1—>6)-B-D-glucopyranoside moiety. Significant HMBC
correlations that were used to deduce these connectivities included those
observed for H-1' (64 4.87) to C-8 (6; 146.7), and H-6a’ (64 3.92) and H-6b' (64
3.52) to C-1" (6c 100.7), as well as, from H-1" (6.. 4.61) to C-6' (6c 66.6). Based
on these data, compound 26, 5-hydroxydianellin (1-(1,5,8-trihydroxy-3-methyl-
napthalen-2-yl)—ethanone-8-O-B-D-rhamnopyranosyl-(1—+6)—B-D-
glucopyranoside), was identified as a new naphthalene glycoside.

Compound 27 was obtained as a yellow, clear, glass-like solid and
identified as 5,7,3,4-tetrahydrow-6-methylflavone (6-methylluteolin) that was
previously reported from Salvia nemorosa L. (Lamiaceae) (Milovanovic et al.,
1996). The structure of compound 27 was confirmed based on detailed 10 and
2D NMR studies and by comparisons of its UV and IR spectral data with those
reported. This is the first report of compound 27 from daylilies and the first

detailed account of its 1H and 13C NMR spectral properties.

Conclusions

Daylily roots have been used extensively throughout eastern Asia as a traditional
treatment for schistosomiasis. However, the bioactive constituents have never
been fully characterized. In this study of Hemerocallis fulva ‘Kwanzo’ Kaempfer
roots, several compounds were isolated including seven new anthraquinones,
kwanzoquinones A (16), B (17), C (19), D (20), E (21), F (22), and G (24), two
known anthraquinones, 2-hydroxychrysophanol (18) and rhein (23), one new

naphthalene glycoside, 5-hydroxydianellin (26), one known naphthalene

76

glycoside, dianellin (25), one known flavone, 6-methylluteolin (27), and 0t-
tocopherol. The biological of activities of these compounds have been

investigated and are reported in Chapter Four.

77

CHAPTER FOUR

BIOLOGICAL ACTIVITIES OF COMPOUNDS ISOLATED FROM
HEMEROCALLIS CV STELLA DE ORO FLOWERS AND HEMEROCALLIS

FUL VA ‘KWANZO’ ROOTS

Abstract

Daylilies (Hemerocallis spp.) have been used in eastern Asia for the treatment of
a variety of medical conditions. Isolation studies conducted with Hemerocallis
cv. Stella de Oro flowers and H. fulva ‘Kwanzo’ roots yielded a variety of
compounds that have been evaluated for their biological activities. These
compounds were assayed for anticancer, antioxidant, cyclooxygenase inhibitory,
mosquitocidal, nematocidal, schistosome inhibitory, and topoisomerase inhibitory
effects. The new anthraquinones, kwanzoquinones A (16), B (17), C (19), E (21),
and kwanzoquinone A and B monoacetate analogues (16a and 17a,
respectively), inhibited the proliferation of human cancer cells in vitro. In addition
to these new anthraquinones, the known compounds 2-hydroxychrysophanol
(18) and rhein (23) inhibited cancer cell growth. Three compounds,
kwanzoquinone D (20), stelladerol (14), and 5-hydroxydianellin (26),
demonstrated remarkable antioxidant activity by inhibiting lipid oxidation by more
than 90% in an in vitro assay system. Two compounds, 2-hydroxychrysophanol
(18) and kwanzoquinone E (21), were discovered as novel agents for the

prevention and treatment of schistosomiasis. These compounds were found to

78

 

 

innit

I

adul,

 

mod

four

dem

inhit

 

inhibit the motility and induce mortality of Schistosoma mansoni cercariae and
adults. Kwanzoquinones C and E and 2-hydroxychrysophanol exhibited
moderate mosquitocidal properties. These compounds induced mortality of
fourth instar Aedes aegyptii larvae within 24 h. None of the compounds
demonstrated nematocidal, cyclooxygenase inhibitory, or topoisomerase

inhibitory activities.

79

 

 

Introduction

Hemerocallis spp. have been used extensively throughout eastern Asia as
a traditional food item and medicinal agent for treating a variety of conditions
(Table 1.2). A small number of pharmacological studies performed using crude
Hemerocallis extracts have indicated the presence of bioactive components in
daylilies; however, the chemical constituents responsible for these effects have
not been conclusively determined. Phytochemical investigations of daylilies have
revealed the presence of a limited number of chemical constituents in the flowers
and roots of these plants, but the biological activities of these components have
not been determined. Additional work is needed to further evaluate the

bioactivities of compounds from Hemerocallis spp.

Materials and Methods

Cancer Cell Growth Inhibition Assay. Compounds 1-21 and 23-26 were
tested for their activity against breast, central nervous system (CNS), colon, and
lung human tumor cell lines. The breast (MCF-7), CNS (SF-268), and lung (NCI-
H460) cultures were purchased from the National Cancer Institute (Bethesda,
MD) while the colon culture (HCT-116) was purchased from the American Type
Culture Collection (Rockville, MD). All cell lines were maintained in a humidified
chamber at 37 °C with 5% CO2 in RPMI-1640 medium supplemented with 10%
fetal bovine serum, penicillin (1 unit/100mL), and streptomycin (1 ug/100mL). All
cell lines were sub-cultured according to their individual growth profiles in order

to ensure exponential growth throughout the experiments. Breast (10,000

80

 

 

cells/well), CNS (15,000 cells/well), colon (7,500 cells/well), and lung (7,500
cells/well) cancer cells were aliquoted (100 pL) into 96-well plates and allowed to
grow for 24 h before the addition of test compounds to the media. Compounds
were dissolved in DMSO and diluted in sterile media as necessary to obtain the
appropriate concentration. The test compounds were added to the sample wells
in 100-pL aliquots so that the final concentration of DMSO did not exceed 0.25%.
Test compounds, standards, and DMSO control were incubated for 48 h, after
which the assay was terminated via the addition of cold trichloroacetic acid. The

plates were incubated for one hour at 4 °C, washed with deionized water (x 5),

and air-dried. A 100-uL aliquot of 0.4% sulforhodamine B stain in 1% acetic acid
was added to each well and the plates were incubated for 30 min at room
temperature. Following incubation, the wells were rinsed with 1% acetic acid (x
5) and the bound stain was dissolved in 100 pL of 10 mM Trizma base. The
plates were shaken for five minutes on a gyrorotary shaker after which the
absorbance of each well was recorded with an automated microplate reader
(model EL800, Bio-Tek Instruments, Inc., Winooski, VT) at 515 nm. Three
independent experiments were performed in triplicate using at least five drug
concentrations inclusive of the 50% growth inhibitory concentration. Results are
expressed as the concentration of compound required to inhibit cellular growth
50% (Gl5o) i SE Further details regarding these procedures have been reported

(Boyd and Paull, 1995; Skehan et al. 1990).

81

Antii
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Antioxidant Assay. Compounds 1-21 and 23-26 were tested in vitro for their
ability to inhibit the oxidation of large unilamellar vesicles (LUVs). The vesicles
were prepared by combining the phospholipid 1-stearoyI-2-IinoleoyI-sn-glycero-3-
phosphocholine (Avanti Polar Lipids, lnc., Alabaster, AL) with the fluorescent
probe 3-[p-(6-phenyl)-1,3,5-hexantrienyljphenyI-propionic acid (Molecular Probe,
Inc., Eugene, OR) (in a molar ration of 350:1 (lipid:probe). A buffer maintained in
Chelex resin and composed of 0.15 M NaCl, 0.01 M MOPS (pH 7.0), and 0.1 mM
EDTA (all purchased from Sigma-Aldrich, St. Louis, MO) was used to suspend
the lipid-probe mixture and this was exposed to ten freeze-thaw cycles in a dry
ice-ethanol bath. The resultant material was passed through a 100 nm
polycarbonate filter 29 times to give the LUVs.

Experiments were conducted by combining LUVs, 100 mM NaCl, and 50
mM Tris-HEPES (pH 7.0) and test compound in DMSO (final concentration of 10
pM test compound in 2 mL). Oxidation of the lipid-probe substrate was initiated
by the addition of 20 uL of a 0.5 mM FeCl2 solution. Data represent the relative
fluorescence intensity of the probe-lipid-test compound mixture as compared to a
probe-lipid control. All compounds were tested in triplicate and results are
reported as the mean i one standard deviation after fifteen minutes of
incubation. The antioxidant standards tert-butylhydroquinone (TBHQ), butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT) and vitamin E (or-
tocopherol), were purchased from Sigma-Aldrich (St. Louis, MO). Full
experimental details have been previously reported (Arora et al., 1997; Wang et

al., 1999).

82

Cyclooxygenase Inhibition Assay. Compounds 1-21 and 23-26 were tested
for their ability to inhibit cyclooxygenase I and II enzymes in vitro. These
experiments were performed and components obtained from sources as
previously described (Wang et al., 1999). Briefly, cyclooxygenase enzyme
preparations were incubated at 37 °C with arachidonic acid and test compound
(100 (M) was delivered in DMSO. The initial rates of oxygen consumption were

recorded and compared to controls. All compounds were tested in triplicate.

Mosquito Larvicidal Assay. Compounds 16-21 and 23-26 were tested for their
activity against fourth instar larvae of Aedes aegyptii (Rockefeller strain) as
previously described (Roth et al., 1998) with minor modifications. Larvae were
reared from eggs in deionized water at 27 °C with the addition of bovine liver
extract (25 mg/L). Approximately 10-15 fourth instar larvae were placed in test
tubes in 990 uL of deionized water. The test compounds were dissolved in
DMSO and 10 )uL of each sample were added to the larvae so as to give a final
concentration of 50, 25, or 12.5 ug/mL of test compound. The larvae were
observed at 15 and 30 min and 1, 2, 4, 12, 24, and 36 h for changes in motility
and mortality. Compounds were tested in replicate (n=5) with DMSO controls.

No mortality was observed among the control larvae up to 36 h.

83

Nematicidal Assay. Compounds 1-21 and 23-26 were tested for activity against
two nematode strains, Caenorhabditis elegans and Panagrellus redivivus, as
previously described (Nair et al., 1989) with minor modifications. Caenorhabditis
elegans was maintained in a mixed culture with Escherichia coli in a medium
containing NaCl (3 g), peptone (1.25 g), agar (8.5 g), KH2PO4 (1.5 g), K2HPO4
(0.3 g), CaCl2 (0.1 g), M9804 (0.1 g), cholesterol (2.5 mg), and deionized water
(500 mL). Panagrellus redivivus was grown in a medium containing peptone (2.5
g), glucose (5 g), molasses (10 g), agar (9 g), and deionized water (500 mL).
Tests were performed by placing 10-15 nematodes in 48 uL of media into each
well of a 96-well plate. Two microliters of test compound dissolved in DMSO
were added to each test well such that the well contained a final concentration of
25 pg/mL of test compound in a volume of 50 pL. The nematodes were
observed at 15 and 30 min and 1, 2, 4, 12, 24, and 48 h for changes in motility

and mortality. Tests were performed in triplicate with DMSO controls.

Schistosoma mansoni Cercaricidal Inhibition Assay. Compounds 16-26
were examined for their effects on S. mansoni (Puerto Rican strain) cercariae
motility and mortaility. Cercariae were obtained from infected Biompha/aria
glabrata snails by light induction. Details regarding the methods used for the
maintenance of both S. mansoni and B. glabrata cultures have been previously
reported (Salter et al., 2000). A total of 50-100 cercariae in 100-uL distilled H2O
were collected and placed in 96-well vinyl assay plates (Costar, Acton, MA).

Stock solutions of compounds 1-11, including 1a and 2a, were prepared by

84

dissolving 1 mg of test compound in 100 pL of DMSO and 19.9 mL of distilled
H2O. This stock solution was further diluted as needed and 100 uL aliquots were
added to each well. Cercariae motility (i.e. tail movement and swimming
behavior) was observed under a dissecting microscope. Viability of the cercariae
was determined by removing the test compounds after ten hours and replacing it
with fresh water. Recovery from exposure to the test compounds was assessed

after 24 h.

Schistosoma mansoni Schistosomulicidal Inhibition Assay. Compounds 16-
26 were tested for their effects on S. mansoni schistosomula motility and
mortality. Schistosomula were prepared from S. mansoni cercariae by shearing
the tails and incubating the organisms for 2 days in RPMI-1640 media with fetal
bovine serum plus antibiotics in flat-bottomed 96-well culture plates. Test
compounds were added to the media as described for the cercariae assay and
the schistosomula were observed for changes in movement, feeding, and

viability.

Schistosoma mansoni Adult Schistosomicidal Assay. Compounds 16-26
were tested for their ability to alter the motility and mortaility of S. mansoni adults.
Adult worms were perfused from Syrian Golden hamsters as described (Davies
et al., 2001). Twenty male and female adult worms were cultured in 24 well
Falcon plates at 37 °C in one milliliter of RPMI-1640 media supplemented with 2

g/L glucose, 0.3 g/L L-glutamate, and 2 g/L N8HCO3, 15% fetal bovine serum

85

(heat inactivated), antibiotics, and 15 uL of hamster red blood cells (washed with
RPMl). Five-microliter aliquots of test compounds in DMSO or DMSO control
were added to each well. The movement, feeding, and viability of the adult
worms were monitored for 24 h. The media was removed and replaced with
fresh media to which the test compounds were added again and the adult worms
observed for another 24 h. Finally, the media was again removed and replaced
with fresh media without test compounds and the recovery of the adult worms

was monitored for another 24 h.

Topoisomerase Inhibition Assay. Compounds 1-21 and 23-26 were tested for
their ability to inhibit the activity of topoisomerase I and II enzymes in vitro as
previously described (Roth et al., 1998). Three mutant strains of Saccharomyces
cerevisae were provided by Dr. John Nitiss (St. Jude Children’s Hospital,
Memphis, TN) that possess recombinant forms of topoisomerase I and II
enzymes. The S. cerevisae strain JN394.-1 lacks the topoisomerase I enzyme
and is resistant to the topoisomerase inhibitor camptothecan. The S. cerevisae
strain JN394(-2-5 possesses a mutated topoisomerase II enzyme and is resistant
to etoposide and other topoisomerase II inhibitors, but is sensitive to
topoisomerase I inhibitors. Another strain of S. cerevisea, JN394, contains
recombinant forms of both topoisomerase I and II and is sensitive to both
topoisomerase I and II inhibitors. Organisms were grown in a liquid culture
medium composed of peptone (10.0 g), yeast extract (5.0 9), glucose (10.0 g),

and deionized water (500 mL) at 25 °C. The organisms were lawned onto the

86

surface of an agar plate. Compounds were dissolved in DMSO and applied to
the surface of the cultured plates such that 2 uL contained 25 pg of test
compound. The plates containing the test compounds and DMSO controls were
incubated at 30 °C for 72 h and were observed every 24 h for signs of growth

inhibition. All experiments were performed in triplicate.

Results and Disscussion

Overview of Results. The compounds obtained from the flowers and roots of
Hemerocallis were tested for a range of biological activities against a panel of
bioassays. These tests included an examination of the anticancer, antioxidant,
cyclooxygenase inhibitory, mosquitocidal, nematocidal, schistosome inhibitory,
and topoisomerase inhibitory effects of the daylily-derived compounds. A
summary of the results of these studies is presented in Table 4.1. Several
compounds exhibited promising anticancer, antioxidant, mosquitocidal, and
schistosome inhibitory effects. Details of these results are noted below. None of
the compounds demonstrated, nematocidal, cyclooxygenase inhibitory, or

topoisomerase inhibitory activities.

Anticancer Activity. It was previously reported that crude Hemerocallis extracts
inhibited fibroblast proliferation (He, 1994) and induced cancer cells to undergo
differentiation (Hata et al., 1998); however, the active constituents were never
identified. In these studies, several compounds were obtained from H. fulva

roots that exhibited growth inhibitory effects against human breast, CNS, colon,

87

 

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89

and lung cancer cell lines. The Glso concentrations of these compounds are
presented in Table 4.2. The mixtures of compounds 16 and 17, as well as, 16a
and 17a exhibited strong growth inhibitory effects against breast cancer cells with
Glso values of 2.6 i 0.6 and 1.8 i 0.2 mg/mL, respectively. In contrast, the Glso
concentration of these compounds was approximately three to six times higher
against the other three cell lines (Table 4.2).

Compounds 18, 19, and 21 exhibited consistent activity against all four
cancer cell lines (Table 4.2). The in vitro cytotoxicity of compound 18 against
three tumor cell lines including lung (A-549, E050 3.1 ug/mL), nasal (KBMRI,
EDso 5.7 pg/mL), and colon (HT-29, EDso 2.8 jug/mL) had been previously
described (Li and McLaughlin, 1989). The results obtained here for compound
18 are consistent with the previously reported findings. Compounds 19 and 21
are both new compounds that represent 2-O-glucopyranose conjugated and 3-
hydroxymethyl derivatives, respectively, of compound 18. Both of these
compounds were found to possess cancer cell growth inhibitory properties
against all cell lines tested at concentrations similar to that of compound 18.
Compound 23 exhibited moderate activity against all four cancer cell lines.

In light of the growth inhibitory activity exhibited by these compounds
against a number of cancer cell lines and the current cancer chemotherapeutic
application of other anthraquinone derivatives, compounds 16-19, 21, and 23
warrant further investigation to determine their mode of action and potential

clinical applications.

90

Table

Heme

COT

16a

ad
3V3

Table 4.2.

Growth

inhibitory effects of anthraquinones

isolated from

Hemerocallis fulva ‘Kwanzo’ roots against four human cancer cell lines

 

cell line (G150, pg/mL i SE)

 

 

compound MCF-7 SF-268 HCT-116 NCl-H460
(breast) (CNS) (colon) (lung)

16 and 17 2.63.0.6 14.7:25 13.5i0.9 10.3:12
16a and 17a 1.8 i 0.2 5.3 i 0.8 10.5 i 0.7 8.5 i 0.6
18 6.5 i1.2 2.4 i1.8 6.3 i 0.8 6.3 i 0.8

19 6.7 i 0.4 6.1 2.1.0 7.4 i- 0.6 3.8 i- 0.3

21 2.8 i 0.3 3.8 i 0.7 5.0 i 0.3 7.3 i 0.7

23 17.2i0.8 16.3:18 21.1 :18 15.4:17
adriamycina 1.7 i 0.2 1.9 i 0.7 2.1 i 0.6 1.7 i 0.4

 

aValues for adriamycin are expressed in (M.

91

Antioxidant Activity. Compounds 1-15, 18-21, and 23—26 were evaluated for
their potential antioxidant activity at 10 pM, while the mixtures of compounds 16
and 17 and 16a and 17a were tested at 10 ug/mL. The results of these
experiments are presented in Figure 4.1. Under these experimental conditions,
stelladerol (14) and 5-hydroxydianellin (26) exhibited strong antioxidant activity
(94.6 i 1.4 and 99.6 i 2.0% inhibition, respectively) that was more pronounced
than that of the commercial synthetic antioxidants TBHQ, BHA, and BHT (81.8 i
1.2, 80.0 _+. 1.0, and 86.4 i 1.3%, respectively) and vitamin E (15.7 i 0.6%). Both
compounds 14 and 26 share a common aglycon portion composed of 2-acetyl-
1,5-dihydroxy-3-methyl naphthalene moieties. This structural feature of
compounds 14 and 26 allows for them to function as antioxdants via the
formation of oxidized resonance stabilized quinone radicals and stable naptho-
quinone products. It is interesting to note that compound 25 is structurally similar
to compounds 14 and 26, but lacks a 5-hydroxy moiety and did not exhibit any
pronounced antioxidant effects.

A number of the anthraquinones demonstrated moderate antioxidant
activity. These include compounds 18, 19, 21, and 23 that exhibited 49.9 i 4.1,
42.0 i 4.8, 28.6 i 3.1, and 25.8 i 2.0% inhibition of oxidation, respectively. The
most striking activity was noted for compound 20, an anthraquinone 2-O-malonyl-
(1—>6)-B-D-glucopyranoside, which inhibited oxidation by 99.9 i 2.0%. This
represents an approximate 58% increase in activity compared to compound 19,
which lacks a 6'-malonyl moiety. The reason for the pronounced difference in

activity between these two compounds remains unclear.

92

100

% Inhibition
M (II N
0 (ll 0 0|
BHA _
I
TBHQ _

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I.” I" N n V m o N Q a, O P
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P
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r: 'U
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F (B
CO
t-

Figure 4.1. Inhibition of LUV phospholipid oxidation by synthetic antioxidants
(panel A) and compounds 1-11 (panel A) and compounds 12—21 and 23-26
(panel B). Compounds were tested in triplicate at 10 pM (except the mixtures of

16-17 and 16a-17a at 10 jig/mL). Results are expressed as the mean percent

inhibition : one standard deviation.

93

Several of the flavonol 3-O—glycosides in which quercetin represented the
aglycon moiety, such as compounds, 2, 4, and 6, exhibited more modest
antioxidant effects with 28.2 i 1.5, 28.6 i 0.8, and 31 i 2.3% inhibition,
respectively. In comparison, the flavonol 3-O-glycosides that possessed a
kaempferol or isorhamnetin aglycon moiety generally exhibited lower antioxidant
inhibitory effects at the same concentration. Our results are in agreement with
previously published studies demonstrating that substitutions to the B-ring of
flavonoids, such as the hydroxyl substituents at C-3', 4’ in quercetin, make this
flavonol a more effective antioxidant than kaempferol (C-4' hydroxyl) or
isorhamnetin (C-3’ methoxy and C-4' hydroxyl) due to their comparatively
hindered ability to chelate metal ions (Arora et al., 1998; Rice-Evans, 1999).

Phenolic compounds are highly regarded for their important dietary roles
as chemopreventive agents (Bravo, 1998). The noted beneficial effects of these
bioactive compounds are mitigated in part by means of their antioxidant effects
as free radical scavengers or metal ion chelators (Arora et al., 1998; Rice-Evans
et al., 1997; Gordon and Roedig-Penman, 1999). Today, in vivo oxidative events
are widely recognized as factors affecting the onset and progression of various
diseases such as cancer, arteriosclerosis, and neural degenerative disorders
(Bland, 1995). In light of the complex array of phenolic compounds observed in
daylily flowers, in addition to the host of antioxidant carotenoids present in these
tissues, it can be conjectured that the dietary consumption of Hemerocallis

flowers may convey a variety of beneficial chemopreventive effects to humans.

94

 

 

Mosquitocidal Activity. Compounds 16-26, including 16a and 17a, were tested
their activity against fourth instar A. aegyptii larvae. Compounds 18, 19, and 21
exhibited mosquitocidal activity at 50 pg/mL (Figure 4.2). Within 15-30 min of
exposure to compounds 18, 19, and 21, the mosquito larvae began to exhibit a
darkening of the gastric caeca and Malpighian tubules. After approximately 1 h,
the midgut region and anus were also darkened. However, after 12-24 h the
darkening of the midgut was greatly reduced in size and intensity. The
significance of these observations remains unclear. The active compounds were
further tested at 25 and 12.5 pg/mL (Figure 4.3). At 25 mg/mL, compound 18
(71.5 i 5.6% mortality) exhibited the greatest activity, followed by compounds 21
and 19 (42.3 i 6.1and 24.1 i 7.2% mortality, respectively). Post-mortem
dissection of the larvae treated with compounds 18, 19, and 21 revealed an
enlargement of the still darkened gastric caeca and a general dissolution of the

alimentary canal.

Schistosome Inhibitory Activity. Schistosomiasis is a disease caused by
parasitic digenetic trematodes of the genus Schistosoma. The World Health
Organization estimates that Schistosoma species currently infect 200 million
people, while another 600 million are at risk (Chitsulo et al., 2000). A large
number of schistosomes are known; however, only five appear to be primarily
responsible for human infections. These include Schistosoma haematobium,

Schistosoma intercalatum, Schistosoma japonicum, Schistosoma mansoni, and

95

 

 

 

 

 

 

 

 

 

100 a
concentration
/mL)
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3‘ I50.0
E I25.0
5° 50 - 1312.5
°\, 'Ai‘ :1:-
‘+
25 ‘ *
0 _ I i
18 19
Compound

Figure 4.2. Dose-response effect of 2-hydroxychrysophanol (18),
kwanzoquinone C (19), and kwanzoquinone E (21) on fourth instar A. aegyptii
larvae mortality. Results are expressed as the percent mortality i one standard
deviation of the larvae following 24 h of incubation with test compounds at three
different concentrations (pg/mL). Experiments were performed with replicates

(n=5) of test tubes containing 10-15 larvae.

96

Schistosoma mekongi (Elliot, 1996; Schafer and Hale, 2001). Schistosomes
pass through a complex life-cycle (Figure 4.3) in which free-swimming cercariae
emerge from their intermediate freshwater snail hosts and infect humans by
attaching to the skin via mucus secretions. Cercariae then penetrate the skin by
releasing proteolytic enzymes (McKerrow and Salter, 2002). Concurrently, the
cercariae shed their tails and transform into schistosomula that enter the venous
vascular system where they are carried to the heart and lungs, before reaching
the systemic circulation. Ultimately, the schistosomula arrive at the liver where
they grow into sexually mature adults. Male and female adults form copulating
pairs in the portal venous system. Later, they migrate to the mesenteric or vesical
veins depending on the specific species of schistosome, and begin laying eggs
for a period of typically 3 to 5 years. The eggs evoke a host immune response
that results in the formation of granulomas leading to fibrosis and the sequelae of
clinical manifestations (Bica et al., 2000; Elliot, 1996; Morris and Knauer, 1997;
Schafer and Hale, 2001). These clinical manifestations of schistosomiasis may
include bloody diarrhea and hematuria, portal and pulmonary hypertension,
hepatosplenomegaly, and death. There are limited options available for the
chemotherapeutic treatment for Schistosoma infections with the drug-of—choice
being the pyrazionoisoquinoline, praziquantel (Elliot, 1996). Unfortunately, the
long-term, worldwide application of the drug coupled with the recent discovery of
praziquantel-tolerant schistosomes has generated concern over the development

of drug-resistant Schistosoma strains (Cioli, 1998, 2000; William et al., 2001).

97

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98

With few other options available for combating schistosomiasis, there is an
urgent need to develop new methodologies for the treatment and prevention of
Schistosoma infections (Cioli, 1998, 2000).

Daylily roots (Hemerocallis spp., Hemerocallidaceae) have been used in
Asia to treat schistosomiasis (Shiao et al., 1962a; Shiao et al., 1962b). However,
this method of treatment has been disfavored due to a host of toxic side effects
and deaths associated with the administration of Hemerocallis root extracts to
humans (Wang et al., 1989). Previous efforts to identify the active constituents
responsible for the therapeutic properties of Hemerocallis roots led to the
isolation of a neurotoxic binaphthalenetetrol known as stypandrol (Wang and
Yang, 1993) which had been shown to cause paralysis, blindness and death in
mammals (Main et al., 1981; Colegate et al., 1985). In another report (Chen et
al., 1962), researchers obtained a yellow powdery isolate to which was ascribed
both the biological activity against schistosomes, as well as the toxic side effects
associated with the use of Hemerocallis roots; however, its structure was never
identified. While other studies have described additional compounds found in
daylilies, none of these efforts have addressed the need to fully characterize the
bioactive anti-schistosome chemical constituents from Hemerocallis roots.

Compounds 16-26, including 16a and 17a, were tested in vitro for their
activity against multiple life-stages (cercariae, schistosomula, adult) of the human
pathogenic trematode S. mansoni. At a concentration of 25 pg/mL, compounds
18 (2-hydroxychrysophanol) and 21 (kwanzoquinone E) exhibited significant

activity by completely immobilizing all cercariae within 15 s and 14 min,

99

 

re

Ci

[1

m

O)

A}

respectively. The dose-response effect of these compounds is shown in Figure
4.4. The potency of compound 18 was not diminished even when diluted to a
concentration of 3.1 pg/mL. After 30 min of exposure to test compounds, the test
solution was removed and replaced with fresh medium. Cercariae treated with
compound 18 exhibited 80% mortality after 24 h while those exposed to
compound 21 were all dead. None of the other compounds isolated from H. fulva
roots, including the glycosides of compounds 18 and 21, compounds 19 and 22,
respectively, exhibited any activity at 25 pg/mL. The adult worms were also
immobilized within 16 h by compounds 18 and 21 at 50 pg/mL. Following
removal of the compounds, 35 and 55% of the adults exposed to compounds 18
and 21, respectively, were dead. In contrast to the effects on the cercariae and
adults, the intermediate schistosomula stage was refractory to all compounds at
25 pg/mL. Based on these promising results, compounds 18 and 21 are being
investigated further in order to determine their mode of action and for potential
development as topical anti-cercarial agents for the prevention of Schistosoma

infection.

100

 

 

 

 

 

 

 

 

 

 

 

 

 

I 2-hydroxychrysophanol (18)

100 ~
1:
o
.5
.3 8O .
(E: D kwanzoquinone E (21)
.§ 50 -
o
.2
L
0
2 40 .
o
0
‘E
g 20 -
0
IL

0 I V I 7 ’
control 1.6 3.1 6.3 12.5 25.0

Concentration (pg/mL)

Figure 4.4. Dose-response effect of 2-hydroxychrysophanol (18) and
kwanzoquinone E (21) on S. mansoni cercariae mobility. Motility was accessed
based on the movement and swimming behavior of the invasive aquatic larval
stage 10 min after the addition of test compound. Data are expressed as the

mean i one standard deviation of the percent of immobilized cercariae (n=10).

101

CHAPTER FIVE

SUMMARY AND CONCLUSIONS

Daylilies (Hemerocallis spp.) have been used for thousands of years in
eastern Asia as an important food item and medicinal agent for the treatment of a
host of diseases. Yet, despite a long and rich history of use, very little was
known about the bioactive components that are present in this plant. In Chapter
One, a summary of all known research conducted prior to these studies
regarding the chemistry and pharmacological activity of Hemerocallis spp. was
presented. Based on this review, it was determined that daylilies should be
investigated as a source of new bioactive compounds for the treatment of a
variety of diseases.

The flowers of Hemerocallis cv. Stella de Oro were subjected to a series
of extraction and isolation procedures that led to the procurement of fifteen
compounds that were tested for biological activity. The compounds obtained
from the flowers are presented in Chapter Two. These compounds included
kaempferol, quercetin, and isorhamnetin 3-O-glycosides (1-9), phenethyl B-D-
glucopyranoside (10), orcinol B-D-glucopyranoside (11), phloretin 2'-O-[3-D-
glucopyranoside (12), phloretin 2'-O-B-D-xylopyranosyl-(1—->6)-B-D-
glucopyranoside (13), a new napthalene-glycoside, stelladerol (14), and an
amino acid (longitubanine A) (15). The structures of these compounds were

determined based on thorough spectral and physical analyses including UV, MS,

102

and NMR experiments. This is the first report of these compounds as
components of edible daylily flowers.

It had been widely reported that daylily roots were used throughout Asia
as a traditional treatment for schistosomiasis. However, the active components
responsible for the root’s therapeutic properties had never been fully
characterized. The results of an isolation study performed on H. fulva ‘Kwanzo’
roots are presented in Chapter Three. As a consequence of this work, a number
of compounds were discovered in daylily roots including seven new
anthraquinones, kwanzoquinones A (16), B (17), C (19), D (20), E (21), F (22),
and G (24), two known anthraquinones, 2-hydroxychrysophanol (18) and rhein
(23), one new naphthalene glycoside, 5-hydroxydianellin (26), one known
naphthalene glycoside, dianellin (25), one known flavone, 6-methylluteolin (27),
and a-tocopherol. The structures of the compounds were elucidated by a
combination of spectroscopic and chemical methods. All of these compounds
are reported here for the first time as components of the daylily roots.

The compounds obtained from the flowers and roots of daylilies were
subjected to a variety of bioassays in order to determine their potential as new
anticancer, antioxidant, cyclooxygenase inhibitory, mosquitocidal, nematocidal,
schistosome inhibitory, and topoisomerase inhibitory agents. The results of
these studies are presented in Chapter Four. Based on this work, several
compounds reported in Chapters Two and Three exhibited promising cancer
cell growth inhibitory, antioxidant, mosquitocidal, and schistosome inhibitory

activities. Kwanzoquinones A (16), B (17), C (19), E (21), and kwanzoquinone A

103

and B monoacetates (16a and 17a, respectively) and the known anthraquinones,
2-hydroxychrysophanol (18) and rhein (23), exhibited cancer cell growth
inhibitory properties against a panel of four human tumor cell lines. Additional
studies are currently underway in order to further characterize their mode of
action. Three new compounds exhibited strong antioxidant properties in this
investigation. These compounds were stelladerol (14), kwanzoquinone D (20),
and 5-hydroxydianellin (26). All three compounds inhibited lipid oxidation by
more than 90%. Three compounds exhibited mosquitocidal properties.
Compounds 18, 19, and 21 were found to induce mortality in fourth instar A.
aegyptii larvae. Two compounds were discovered to inhibit schistosome activity.
2-Hydroxychrysophanol (18) and kwanzoquinone E (21) were found to inhibit the
motility and induce mortality in S. mansoni cercariae and adults. Both of these
compounds are being further investigated in order to determine their mode of
action and for development as potential agents for prevention and treatment of
schistosomiasis.

Together, these studies have significantly expanded the available body of
information regarding the chemical composition and phytoceutical potential of
Hemerocallis flowers and roots. Both the flowers and roots are widely consumed
in eastern Asia as a medicinal agent and component of traditional cuisine. A
variety of new and known compounds were reported here for the first time as
components of the edible flowers and roots of Hemerocallis. Several of these
compounds can serve as models for the development of new anticancer,

antioxidant, mosquitocidal, and schistosome inhibitory agents. The schistosome

104

inhibitory properties of compounds 18 and 21 are particularly intriguing. At this
time, more that 200 million people worldwide are infected by Schistosoma spp.
Recently, signs of schistosomes resistant to praziquantel, have emerged
underscoring the urgent need for the creation of new agents for the treatment of
schistosomiasis. It is hoped that compounds 18 and 21 will provide new avenues

for the development of a novel, safe, and effective treatment for schistosomiasis.

105

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