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A SURVEY OF INSECTICIDAL ACTIVITY FROM
DELPHINI UM X CUL T ORUM CV. MAGIC F OUNTAINS FLOWERS

By

Jennifer Elizabeth Christina Miles

A THESIS

Submitted to
Michigan State University
in partial fulfillment of the requirements
for the degree of

MASTER OF SCIENCE

Department of Horticulture

1998

ABSTRACT
A SURVEY OF INSECTICIDAL ACTIVITY FROM
DELPHINI UM X CUL TORUM CV. MAGIC FOUNTAINS FLOWERS
Jennifer Elizabelt}: Christina Miles

The genus Delphinium (Ranunculaceae) is well known for the production of many
structurally complex diterpenoid alkaloids. Numerous publications are available on the
discovery, classification and biological activity of these alkaloids. This research
highlights compounds isolated by bioassay-directed fractionation of the crude hexane and
ethyl acetate extracts of Delphinium X cultorum flowers. The bioassays performed
include insecticidal assays using 4th instar mosquito larvae (Aedes aegyptii L.), neonate
gypsy moth (Lymantria dispar L.) and corn earworm (Helicoverpa zea Boddie)
caterpillars, and adult Colorado Potato Beetles (Leptinotarsa decemlineata Say), and
Japanese Beetles (Popilliajaponica Newman); nematocidal assays using Caenorhabditis
elegans Dought and Panagrellus redivivus Goody; antimicrobial assays using gram
negative and gram positive bacteria, and various plant pathogenic fungi; and
topoisomerase enzyme inhibitory assays using mutant Saccharomyces cerevisiae strains.

Bioassay-directed fractionation of the hexane extract from D. X cultorum flowers
led to the isolation of six volatile compounds that were characterized as
ethylmethylbenzene, 1-isopentyl-2,4,5-tn'methylbenzene, 2-penten-2-ylbenzoic acid, two
isomers of 3-butlidene-3H-isobenzofuran-1-one, and a novel compound (2-hex-3-ene-2-
one)-phenylmethylketone from GC/MS studies. Bioassay-directed fractionation of the
ethyl acetate extract from these flowers led to the isolation of 4-hydroxybenzoic acid and

bis-(4-hydroxyphenyl)methanol. Their structures were confirmed by 1H and 13C NMR

experiments.

To Grandma Polcik

iii

ACKNOWLEDGEMENTS

I wish to express my appreciation to my long-time advisor, Professor Muralee Nair, who
helped to direct me into the challenging and rewarding field of natural products
chemistry. I give many thanks to the members of my commitee, Professor John Kelly,
one of the first to help shape my interest in horticulture and offer encouragement, and
Professor David Smitley for his expertise in entomology. My deepest gratitude goes out
to Dr. Russel Rameswak, Dr. Amitabh Chandra, and Dr. James Nitao for offering so
much advice, encouragement, patience and friendship. I also want to thank my lab-mates:
Yu Chen Chang, Andrew Erikson, Holly Homer, Mark Kelm, Rafik Momin, Geoffery
Roth, Haibo Wang, and Di Zhang who always offered a sympathetic ear and lots of fun to
the workday. I am very grateful to my sister, Meredith Phares for helping me dig through
old dusty volumes of Chemical Abstracts during the writing of my literature review, and
for weighing many caterpillars. Also, I wish to express my appreciation to Dr. Lowell
Ewart for providing me with information on Delphiniums, Drs. Alexander Raikel and
Alan Hayes for hundreds of A. egyptii larvae, Drs. Long Le and Kermit Johnson for their
technical support with the NMR, Dr. Beverly Chamberlain for producing my GCMS data,
Dr. Cliff Beninger for use of his lyophilizer and greenhouse space, Bodger Seed
Company for supplying me with all of the seeds for my research on D. X cultorum,
Walter's Gardens for the D. bellamosum plants, NC State Insectory for corn earworm and

tobacco earworm eggs, Dr. Dan Herms for supplying gypsy moth eggs, and Dr. Robert

iv

Hemer for his support. To my mother, Kay Phares, brother, Nicholas Phares and
grandparents, John and Doris Phares and Marjorie Polcik, mother-in-law Rose Miles my
love and thanks for their words of encouragement. Lastly, to my husband, Christopher,
my love for sharing my triumphs, holding me up during my pitfalls, surviving through

my fits, and understanding. I would not have made it without the help from these people.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................... ix
LIST OF FIGURES .................................................................................. x
LIST OF SCHEMES ................................................................................ xii
LIST OF ABBREVIATIONS ..................................................................... xiii
LIST OF APPENDICES ........................................................................... xvi
CHAPTER I - Introduction .......................................................................... 1
History and Classification .................................................................. 1
Traditional uses .............................................................................. 4
Background and Project Objectives ....................................................... 4
CHAPTER II - Horticultural Aspects of D. X cultorum and D. bellamosum ................ 7
CHAPTER III - Literature Review ............................................................... 11
Chemistry of Delphinium ................................................................. 11

Acetogenins ......................................................................... 12

Terpenoids .......................................................................... 26

vi

Nitrogen Containing Compounds ............................................... 28

Biologically Active Compounds Reported from Delphinium ........................ 43
Toxicity and Pharmacological Studies .......................................... 43
Alellopathic Studies ............................................................... 57
Insecticidal Activity ............................................................... 6O
Antimicrobial Activity ............................................................ 67

CHAPTER IV - Chemical Evaluation of the Hexane Extract from D. X cultorum

Flowers ........................................................................... 70
Abstract ...................................................................................... 70
Introduction .................................................................................. 71
Experimental ................................................................................ 71
Results and Discussion .................................................................... 84

CHAPTER V - Chemical Evaluation of the Ethyl Acetate Extract from D. X cultorum

Flowers ............................................................................. 99
Abstract ....................................................................................... 99
Introduction ................................................................................ 1 00
Experimental .............................................................................. 104
Results and Discussion .................................................................. 108

vii

CHAPTER VI - Summary and Conclusions ................................................... 114

APPENDICES ..................................................................................... 1 17
Appendix I ................................................................................. 1 17
Appendix II ................................................................................ 123

LITERATURE CITED ............................................................................ 126

viii

LIST OF TABLES

Table 3.1. Alkaloids isolated from D. X cultorum and D. bellamosum ..................... 41
Table 4.1. Average weights of H. zea Boddie larvae after 6 days ........................... 86
Table 4.2. Average weights of L. dispar L. larvae after 6 days .............................. 87
Table 4.3. Average weights of H. zea Boddie larvae after 6 days ........................... 88
Table A.l. Mosquitocidal assay of MPLC fractions ........................................... 121

Table A.2. Average weight of corn earworm (H. zea Boddie) after 6 days when tested

against MPLC fractions ............................................................ 122
Table A.3. YMG agar media recipe ............................................................ 123
Table A.4. Emmons agar media recipe ......................................................... 123
Table A.5. YPDA agar media recipe ............................................................ 123
Table A.6. PDA agar media recipe ............................................................. 124
Table A.7. NG media recipe ..................................................................... 124
Table A.8. Phosphate buffer recipe ............................................................. 125
Table A.9. Basal media recipe ................................................................... 125

ix

LIST OF FIGURES

Figure 3.1 Fatty acids reported from Delphinium species ..................................... 14
Figure 3.2. F lavonoids isolated from Delphinium species .................................... 17
Figure 3.3. Anthocyanins isolated from Delphinium species .................................. 20
Figure 3.4. Another anthocyanin isolated from Delphinium species ........................ 21
Figure 3.5. Phenolic compounds isolated from Delphinium species ......................... 24
Figure 3.6. Sterols isolated from Delphinium species ......................................... 25
Figure 3.7. Diterpenoid alkaloids and their proposed biosynthetic precursors ............. 31
Figure 3.8. Delphinium alkaloids studied to determine their biosynthesis .................. 33
Figure 3.9. Gibberellin and delcosine ............................................................ 35
Figure 3.10. Norditerpenoid alkaloid skeletons ................................................. 36
Figure 3.11. Diterpenoid alkaloids ................................................................ 38
Figure 3.12. Bisditerpenoid alkaloid skeleton .................................................. 39
Figure 3.13. Alkaloids toxic to cattle ............................................................ 47
Figure 3.14. Alkaloids from D. cashmirianum which produce arrhythimia ................ 51
Figure 3.15. Alkaloids with pharmacological activity ......................................... 52
Figure 3.16. Curarifonn alkaloids ................................................................ 53
Figure 3.17. Alkaloids studied for binding activity with nAChR ............................ 56
Figure 3.18. Alkaloids with pharmalogical activity ............................................ 58
Figure 3.19. Alkaloids studied for allelopathic activity ....................................... 59

Figure 3.20. Stomach and contact poisons on insects from delphinium species ........... 61

Figure 3.21. Insecticidal and mammalian alkaloid toxins .................................... 65
Figure 3.22. Antifungal alkaloids from D. denudatum ........................................ 68
Figure 4.1. Ethyl-methylbenzene and MS fragmentation pattern ............................ 89

Figure 4.2. 1-Isopentyl-2,4,5-trimethylbenzene and its MS fragmentation pattern ........ 90

Figure 4.3. 2-(Hex-3-ene-2~one)-phenylmethylketone and its MS fragmentation
pattern .................................................................................. 92

Figure 4.4. 2-(Hex-3-ene-2-one)-pheny1 methylketone ........................................ 93

Figure 4.5. 3-Butylidene-3H-isobenzofi1ran—1-one and its MS fragmentation pattern ..... 94

Figure 4.6. 2-Pentylbenzoic acid and its MS fragmentation pattern ......................... 95
Figure 4.7. B-sitosterol ............................................................................. 97
Figure 5.1. Average weights of H. zea larvae after 6 days .................................. 109
Figure 5.2. D—mannitol (1) and p-hydroxybenzoic acid (2) .................................. 111
Figure 5.3. Bis-(4-hydroxyphenyl)-methanol ................................................. 112

xi

LIST OF SCHEMES

Scheme 4.1. Accelerated solvent extraction procedure ........................................ 74

xii

CC14
CI-IC13
CNS

CV.

DMSO
DNA
eV

EI

F TIS
GC
GC/MS
GLC
HPLC

IR

LIST OF ABBREVIATIONS

OOOOOOOOOOOOOOOOOOOOO

ooooooooooooooooooooo

ooooooooooooooooooooo

ooooooooooooooooooooo

ooooooooooooooooooooo

OOOOOOOOOOOOOOOOOOOOO

ooooooooooooooooooooo

carbon nuclear magnetic resonance
proton nuclear magnetic resonance
analysis of variance

broad singlet

carbon tetrachloride

chloroform

central nervous system

cultivar

doublet

dimethylsulfoxide

dioxyribonucleic acid

electron volts

electron impact

Fourier transform infrared spectroscopy
gas chromatography

gas chromatography/mass spectroscopy
gas liquid chromatography

high performance liquid chromatography
infrared

xiii

LDroo
LDSO

LSD

MPLC

MSAL

nAChR
NIRS

NMR

SAR

spp.
TLC

ooooooooooooooooooooo

ooooooooooooooooooooo

ooooooooooooooooooooo

ooooooooooooooooooooo

OOOOOOOOOOOOOOOOOOOOO

lethal dose (100%)

lethal dose (50%)

least significant difference

multipet

meta

molar

methanol

megahertz

methyllycacontine

medium pressure liquid chromatography
N-methylsuccinyl)-anthranoyllycoctonine
mass to charge ratio

acetylcholine receptor subtypes

near infrared reflectance spectroscopy
nuclear magnetic resonance

para

ribonucleic acid

retention time

singlet

structure activity relationship

species

thin layer chromatography

xiv

ultra violet

vacutun liquid chromatography

XV

LIST OF APPENDICES

Appendix 1. Survey of D. bellamosum flower and stem, and D. X cultroum stem and leaf

extracts for Biological Activity ................................................... 117

Appendix II. Recipes for Media Used in Bioassays ........................................... 123

xvi

CHAPTER I

Introduction

History and Classification

Greek tradition tells a tale of the hero Ajax related to the origin of the delphinium.
In this legend, the mother of the great warrior Achilles expresses a wish that her dead
son's armor be presented to the most-worthy warrior in the Achaean host. Of the two
claimants, Ajax and Odysseus, Odysseus was the most celebrated and was given the
honor. But Ajax, upon hearing the decision, was embittered and took his own life in
protest. From that blood-soaked earth, grew a delphinium plant with the Greek letters Al
(the first letters of Ajax's name) upon the petals. This gave miraculous support to Ajax's
claim and the injustice done to him (Dwyer and Rattray, 1986).

Delphiniums were named by the ancient Greeks due to the flower's apparent
resemblance to the dolphin, the Greek word for dolphin being ‘delphis.’ In 1640, John
Parkinson, apothecary to Charles I, cultivated a famous garden in London, and in writing
about his plants in Paradisi in Sale mentioned D. elatiusflore pleno diversiforum
colorum (Bishop, 1949; Phillips, 1933). This was the first documentation of cultivated
delphiniums in England. In Tudor England, akin to the delphinium larkspur was so
named because the nectary resembled the spur of a lark’s claw.

Delphiniums grew to be a very popular and sought-after garden plant. Their tall
stature and often true-blue color was not shared by any other plant in cultivation.
Increased interest in breeding delphiniums during the mid-nineteenth century greatly
added to the number and quality of available varieties. Work on delphinium breeding into
the twentieth century by Blackmore and Langdon afforded gardeners the named varieties

of the large delphiniums grown in the present day (Dodeswell, 1997).

Delphinium is a member of the natural order Ranunculaceae. Perennial
delphiniums often are referred to as ‘true delphiniums’. Related to it is the larkspur (D.
ajacis L., D. consolida L., etc. ), also a member of the delphinium genus, sometimes
called the “annual delphinium” (Puttock, 1959). However, both delphinium and larkspur
species contain annual and perennial types, delphinium and larkspur belong to the
crowfoot family. This family includes species of anemone, hepatica, buttercup, aconite,
and clematis. Some of these species, in addition to delphinium, are notorious for
producing poisonous compounds (Kuder, 1947).

Larkspurs are sometimes called wild or naturally occurring delphiniums. Two of
the important species of larkspur, D. consolida L. and D. ajacis L., are collectively called
the consolida group (Kuder, 1947). Consolida-type larkspurs have since been classified
into their own genus. D. consolida L. and Consolida is now used interchangeably
depending upon the source. These species were introduced to the United States as garden
varieties, but have since escaped cultivation and have been naturalized as prairie
wildflowers.

The perennial larkspurs are subdivided into two categories, the low larkspurs and
the tall larkspurs. Perennial larkspurs colonize stable, closed habitats, and form dense
colonies. Vegetative propagation plays the largest role in reproduction because self-
fertilization occurs in only 0-1% (Blanche, 1990). Annuals called “plains larkspur”
colonize unstable, open habitats. They have a higher germination rate, and do not
multiply by vegetative means (Blanche, 1990). Low larkspur, such as D. nelsonii and D.
andersonii A. Gray, are smaller plants, and inhabit dry low-lying areas. Tall larkspur,
such as D. barbeyi, D. occidentale, and D. glaucum S. Wats., are larger plants growing on
high mountainous areas, or areas with abundant soil moisture (James and Johnson, 1976).
Plains larkspur, such as D. geyeri Greene, grows on high windswept short grass plains

(Ralphs et al., 1989). All of these larkspurs are toxic.

The cultivated varieties of delphinium are known to horticulturists as “larkspurs”
referring to the annual delphinium, and “true delphinium” referring to the perennial type
(Kuder, 1947). It is impossible to unmistakably classify delphiniums according to their
botanical and ancestral characteristics due to the high level of cross-breeding of these
species. The modern garden delphiniums are the result of the hybridization of many
species. A brief outline of classification of cultivated delphiniums was published by
Robert Kuder (1947).

Cultivated Delphiniums
I. The Annual Larkspurs
A. The Ajacis type
1. Tall Rocket Larkspurs
2. Dwarf Rocket Larkspurs
3. Hyacinth-flowered Larkspurs
B. The Consolida type
1. Double Stock-flowered larkspurs
2. Emperor or Double Giant Imperial
Larkspurs
II. The Perennial Delphiniums
A. The Elatum type
1. Candle Larkspurs
2. Bee Larkspurs
B. The Cheilanthum type
1. Garland Larkspurs
2. Belladonna Larkspurs
C. The Nudicaule or Ruysii type
1. Red Larkspurs

D. elatum L. and D. belladonna Hort. are the parents of all the common garden
varieties (Puttock, 1959). D. elatum L. originates from the Swiss Alps and can be found
in the upper slopes of the mountains of West Virginia and across Europe from the
Pyrennes to Siberia (Genders, 1963). The species name is from the Latin ‘elatus’ meaning
tall. The origin of D. belladonna Hort. is unknown. Those delphinium species
resembling D. elatum L. are known as the elatum type (Bishop, 1949). The genus
delphinium is distributed almost exclusively in the temperate and cold regions of the
Northern Hemisphere, with some from the high mountains of southeast Africa (De la

Fuente and Reina, 1990).

Traditional Uses

The delphinium genus has long been known as a group of poisonous plants.
Italians called these plants “Little Spur” or “King’s Flower” (Phillips, 1933). They were
known to the French as “Knight’s Spur” or “King’s Comfrey". These names refer to the
use of this plant to rid oneself of body lice. Sap from larkspur was suggested as a remedy
for colic, dropsy, and spasmodic asthma (Cronin and Neilson, 1990). Delphinium
denudatum Wall. is known as nirbisi, nirvisha, judivar in the following languages; Hindi,
Sanskrit, and Punjab, respectively. The root of this plant was used by Indians of southeast
Asia to relieve toothaches, as a stimulant (Jain and Defilipps, 1991), alliterative, and tonic
(Atta-ur-Rahman et al., 1997). Forking larkspur (Delphinium consolida L.) seeds were
used in India for their insecticidal properties (Chopra et al., 1969). In ancient times,
crude alkaloidal extracts from delphinium plants were used as a poisonous dip for
arrowheads. D. caeruleum Watt. has been used in traditional Chinese medicine to treat
traumatic injuries.

D. elatum L. was classified by Carolus Linneaus in 1753 (Missouri Botanical
Garden, 1997 ). Its seeds were used in Indian medicine as a verrnifuge and for their
insecticidal, anthelmintic, diuretic, aperient, anti-itch, and emetic properties (Chopra et
al., 1956; Beckstrom et al., 1994) The activity, or poisonous nature, of these plants is said

to be related to the concentrations of different types of alkaloids.

Background and Project Objectives

Historically, plants have been used for sustenance, pest control, as medicine, and
for recreational, and ceremonial purposes. Today, a renewed interest in plants and natural
products may be is developing in the light of new knowledge as an alternative to
synthetic products, testing the efficacy of the time honored traditional uses. Additionally,
it is important to note that natural products are the templates from which these synthetic

products created. For these reasons, it is easy to understand the importance of natural

products research. Pure natural products are of interest to those who want a return to the
natural, while synthetic derivatives of these natural compounds can be powerfirl new
alternatives for combating disease and infestation problems in agriculture as well as
medicine.

Natural products are defined as organic compounds from natural sources, which
are unique to one or a small group of related organisms. Natural products are secondary
metabolites and are touted as non-essential to the originating organism i.e. plant, animal,
or microorganism (Mann, 1987). Natural products from plants are perceived by many as
less harmful than their synthetic counterparts. While this idea does not always hold to be
true, natural plant products already exist in the environment, and therefore may be broken
down, or controlled more readily than synthetic compounds. Natural products may also
have more specific modes of action due to the fact that these plants have evolved along
with the pests and pathogens that we are attempting to control. The goal of this project
was to isolate, purify, and identify the compounds found in D. X cultorum and D.
bellamosum that have biologically active properties.

The idea for this project was initiated in 1994 when a high school student from
Three Rivers, Michigan, Carrie Scott, wrote to Dr. Lowell Ewart about the Magic
Fountain delphiniums that were growing in her garden at home. Carrie reported her
observation on plants she believed to be D. X cultorum cv. Magic Fountains. Carrie
noticed that Japanese beetles were feeding on the flowers of these plants, slowly dying,
and falling to the ground around these plants.

If accurate, Scott’s observation seemed to reflect the findings of some of the
toxicology studies done on other delphinium species. Therefore, it was my hypothesis
that Delphinium X cultorum. cv. Magic Fountain contained compounds that have the
potential to control Japanese beetles, along with other non-alkaloidal compounds with

significant biological activity.

This thesis is comprised of a chapter describing the horticultural aspects of
delphinium growth and production, a comprehensive literature review of non-alkaloidal
compounds from delphinium species, a chapter on the chemical evaluation of the hexane
extract of D. X cultorum flowers, and finally a chapter on the chemical evaluation of the
ethyl acetate extract from D. X cultorum flowers. Appendix I describes preliminary
investigation of D. bellamosum flower and stem extracts, in addition to an evaluation of

D. X cultorum L. stem extracts.

CHAPTER II
Horticultural aspects of D. X cultorum and D. bellamosum

The Magic Fountains Series is classified as D. X cultorum. It is of hybrid origin
but directly related to D. elatum L. All elatum-type delphiniums are of hybrid origin.
Bailey (1976) describes D. X cultorum Voss (elatum-type) as a garden plant. D. elatum
L. is a perennial with a knotty crown. Stems are spicatem and can be as tall as 6 feet. The
leaves are large, palmate with 5-7 segments that are parted near the base. Also, the leaves
can appear palmate with three upper segments parted. Ultimately, the leaf segments can
be over 1/ ” wide. Racemes are dense and erect. Flowers are up to 1-inch long. The
sepals are blue, blunt, glabourous, and 1/2-inch long. The petals are dark shades of dull
purple, the two lower petals can be yellow and bearded. The seeds are wrinkled, not scaly
(Bailey, 1976).

Elatum-type delphiniums have spire-like flowers which can reach up to six feet
tall. The botanical term for this type of flower head is raceme. The flowers are usually
blue, white, lavender, purple or pink in color. A mature plant may be anywhere from 1 to
6 feet in height depending on the cultivar. The delphinium cultivar Magic Fountains
ranges in height between 1.5 and 3 feet (Genders, 1963). Bee delphiniums such as the
Magic Fountains series are so named because early growers of delphinitun thought that
the off-colored corolla resembled a pollinating bumble bee (Genders, 1963). The "flower"

being the calyx.

The elatum-type delphiniums originated in the Pyrennees mountains, and
eastwards in Siberia (Wyman, 1977). In the wild, D. elatum L. inhabits the clearings,
woodland margins, and river valleys in thin birch and aspen forests of Siberia (Everett,
1981)

D. elatum L. is a natural tetraploid. The sterility of these plants was overcome by
Dr. Legro (Marshall Cavendish, 1970). He crossed species at the diploid level, which
produced tetraploids. The tetraploids were then crossed with hybrid delphiniums.
However, elatum-types have four sets of eight chromosomes and therefore, seed
development is difficult.

Cheilanthum-type delphiniums, or garland delphiniums have racemes with more
sparse florets. The blue varieties are closer to true blue than those of D. elatum L.
Delphinium bellamosum has an average height of 1.5 to 2 feet. They often are referred to
as garland delphinium. and appear as 2-3-foot, erect perennial plants. The flower stalks
are usually branched. The most popular species in cultivation is D. formosum Boiss. &
Huet. The horticultural forms are offered as “belladonna” or “bellamosa” (Taylor, 1961 ).
Similarly, garland delphiniums originated in Siberia or eastern Asia (Wyman 1977; Wise
1990). Hortus Third describes D. bellamosum as equal to D. X Belladonna Hort D.
bellamosum is similar to D. elatum L. but lacking a pronounced central raceme. The
sepals are rich blue with a spur over l-inch long.

Both types of delphiniums provide excellent cut flowers. Delphiniums bloom
during the late spring and summer and also in autumn if the flowers are removed before
the seed heads are formed. The autumn flowers are smaller than those produced in the

spring. Delphiniums grown in a greenhouse may flower more than once if the plants are

cutback after blooming. The second and third crop of flowers will be smaller than the
first.

Most delphiniums grow as perennial plants in Hardiness Zones 3-7. To plant
delphiniums outdoors in beds, the plants should be placed 12 to 24 inches apart, with the
crown 1 to 2 inches below the soil surface. The plants require full sun, and a well-drained
soil that has been enriched with compost or manure (Time Life Virtual Garden, 1997).
Delphiniums lose vigor after 2 to 3 years and should be replaced with new seedlings. If
the plants are grown in a greenhouse, supplemental lighting is recommended.
Delphiniums do not have a photoperiodic requirement for flowering. However, the more
light they receive over time, the larger the flower head and more ofien they will flower.

Delphinium seeds should be sown in a disease-free sowing mix. The seeds should
be covered lightly with sowing mix and kept watered enough so that the media surface is
kept from drying out. The germination container should be maintained in a warm place
which receives light and has good air circulation. It is of the utmost importance not to
overwater delphinium seeds or plants. Seedlings are especially susceptible to damping off
caused by root rot pathogens such as Phytophthera, Rhizoctom'a, or Pythiwn species
(Dowdeswell, 1997).

Chilling the seeds for at least one week at approximately 4°C increases the
number of seeds that germinate. Germination temperatures between 18 to 24°C are ideal.
The temperature should not exceed 26°C. For growing, air and media temperature should
be in the range of 15-25 ° C (Dowdeswell, 1997). Delphiniums thrive with a soil pH at
6.0 to 6.5. Delphiniums can be propagated vegetatively by division in spring or fall or by

cuttings. Seeds are the more favored means of production.

The diseases to which delphinium are susceptible are as follows: bacterial spot,
damping off, mosaic virus, root rot, powdery mildew, black spot, crown rot (Wise, 1990;
Genders, 1963). The common insect pests of delphinium in the field or garden are wood-
lice, wirewonn, Tortrix moth caterpillar, leafminer, cutworrn, cockchafers, capsid, thrip,
slugs, and leather jacket (daddy long legs larvae) (Genders, 1963). Cyclamen mite and
two spotted spider mite are voracious pests in the greenhouse. The damage from mite
infestation causes “the blacks,” a condition in which the new growth turns black and
becomes malformed. This irreversible condition appears mainly at the new growing
points.

Carpenter and Boucher’s (1992) research provided growers with temperature
requirements for storage and germination of D. X cultorum seed (elatum-type). The most
rapid and uniform germination rate was achieved at 20°C. Holding the seeds at 20°C for 3
weeks reduced the time to germination by 50%. A study on the effect of nitrogen level on
the vegetative growth and flower production of D. grandiflorum L. was reported by
Hassan and Khattab (1987). They used nitrogen levels of 0 ,0.5, 1, 1.5, 2, 2.5, and 3 g per
plant. The highest leaf number, tallest stem length, and heaviest stem and dry leaf matter
were induced at the level of 1.5 g of nitrogen per plant (7.5 g of ammonium sulfate). The
authors stated that perhaps these data can be extrapolated to D. elatum L. production
(Hassan and Khattab, 1987).

Information on growing delphiniums commercially can be accessed on the World
Wide Web (www) at hgpzl/wwwdelphiniumconz and on delphinium diseases can be

obtained on the www at http://msue.msu.edu/msue/imp/mod03/01700333.html.

10

CHAPTER III

Literature Review

Chemistry of Delphinium

Many classes of compounds have been identified from the Delphinium genus. The
compounds of interest are generally referred to as secondary metabolites. Secondary
metabolites have enormously diverse chemistry and are presumably non-essential to plant
growth and development. Their principal function is for ecological interactions
(Gershenzon, 1996). This section is organized into specific classes of compounds with
some attention being paid to their ecological significance. This review is a compilation of
information on compounds isolated from many delphinium species. It shows the breadth
of work that has been done on this genus. The alkaloids from this genus have been the
primary focus of most of the phytochemical studies, and for this reason only those
alkaloids isolated from D. elatum L. are discussed. It is important to note that no two
plant species have the same profiles of secondary metabolites.

In addition to providing protection to the plant, secondary compounds are often
toxic to the plant itself. Therefore, these compounds are either compartmentalized in the
plant, or stored in inactive forms. A common way for plants to store secondary
metabolites is to add sugar moieties, proteins, or to form salts for the production
relatively inert compounds (Bemays and Chapman, 1994).

The initial compounds that an insect, pathogen, herbivore would contact with are

the low-molecular-weight, volatile compounds. This class includes a wide variety of

11

CHAPTER III

Literature Review

Chemistry of Delphinium

Many classes of compounds have been identified from the Delphinium genus. The
compounds of interest are generally referred to as secondary metabolites. Secondary
metabolites have enormously diverse chemistry and are presumably non-essential to plant
growth and development. Their principal function is for ecological interactions
(Gershenzon, 1996). This section is organized into specific classes of compounds with
some attention being paid to their ecological significance. This review is a compilation of
information on compounds isolated from many delphinium species. It shows the breadth
of work that has been done on this genus. The alkaloids from this genus have been the
primary focus of most of the phytochemical studies, and for this reason only those
alkaloids isolated from D. elatum L. are discussed. It is important to note that no two
plant species have the same profiles of secondary metabolites.

In addition to providing protection to the plant, secondary compounds are often
toxic to the plant itself. Therefore, these compounds are either compartmentalized in the
plant, or stored in inactive forms. A common way for plants to store secondary
metabolites is to add sugar moieties, proteins, or to form salts for the production
relatively inert compounds (Bemays and Chapman, 1994).

The initial compounds that an insect, pathogen, herbivore would contact with are

the low-molecular-weight, volatile compounds. This class includes a wide variety of

11

short-chain alcohols, aldehydes, ketones, esters, aromatic phenols, lactones,
monoterpenes, and sesquiterpenes. The characteristic odors of some plants often are
produced by a group of closely related volatile compounds (Bemays and Chapman,
1994). Volatile compounds produced by many plants are rich sources of secondary
metabolites. To date no research on the volatile constituents of delphinium has been
reported.

In addition, the epicuticular waxes that accumulate on leaf surfaces are another
source for secondary metabolites. The waxy layer or cuticle is made up of complex
mixtures of lipophilic compounds such as fatty acids, esters, hydrocarbons, triglycerides,
phospholipids, glycolipids, and steroids (Bemays and Chapman, 1994). The cuticular

waxes are the next level of plant defense against insects, pathogens, or herbivores.

Acetogenins

The compounds that comprise the cuticle are biochemically related and are
classified as polyketide compounds. They are collectively referred to as acetogenins.
These compounds are formed from condensation of acetate molecules (Ikan, 1991). The
most common polyketide compounds are lipids. Phenolic compounds fall under the
classification of acetogenins, also. Phenolics are characterized by at least one aromatic
ring bearing one or more hydroxyl substituents. Phenolic compounds are abundant in
plant tissues. There are several important classes of phenolic compounds such as
flavonoids, coumarins, phenyl propanoids, tannins which are polymers of phenols, and
phenolic acids and their glycosides. Phenolics are derived fiom the shikimate or malonate

biosynthetic pathways (Taiz and Zeiger, 1991).

12

Lipids. Lipids can be classified broadly into three groups: fats, oils, and waxes. Fats are
mixtures of glycerides. Glycerides can be saponified into glycerol backbone molecules,
and the salts of the fatty acids. Generally, the fatty acids are from 12 to 34 carbon units in
length, and usually contain an even number of carbon atoms. The bulk of the unsaturated
fatty acids, including the non-conjugated acids, occur in the cis form. Waxes are the
esters of fatty acids and monohydric alcohols, containing anywhere from 24 to 36 carbon
atoms (Ikan, 1991). They make up the protective cuticle that prevents water loss and
inhibits pathogen attack (Taiz and Zeigler, 1991). Fats and oils are storage forms of
reduced carbon found in seeds, which are converted into carbohydrates during
germination. Figure 3.1 shows some examples of fatty acids isolated from delphinium
species.

Oil extracted with ether from D. hybridum Hort. seed was prepared to form a
mixture of methyl esters. The mixture was reported to contain 9,10-dihydroxypalmitic,
palmitic, tetrahydroxystearic, dihydroxystearic, stearic, tetrahydroxyeicosanioc, 11,12-
dihydroxyeicosanoic, and ll-eicosenoic N-hydroxyamide acids (Chisholm and Hopkins,
1956). This was the first report of C20 free fatty acids from the seed oils, i.e.
tetrahydroxyeicosanioc and 11-eicosenoic acids, from this plant family.

An unsaturated hydrocarbon, C32H53, b.p. 200°/0.1 mm of Hg, n34 p 1.4800 with
no rotation, and a monohydric unsaturated alcohol, C16H3OO, b.p 156-160° /0.1 mm of
HG, n34 1) 1.4646 with no rotation were isolated from the petroleum ether extract of the

roots of D. denudatum Wall. The compounds were purified by adsorption analysis on

13

Capric Acid

Laurie Acid

Myn'stic Acid

Palmiu'c Acid

Stearic Acid

Oleic Acid

Linolenic Acid

/\CH=CHACH=CHACH=CH'\/\/\/\£-OH

1 I-Eioosanoic acid

0
/\/\/\/\CH=CH/\/\/W\él:-OH

Figure 3.1. Fatty acids reported from Delphinium species.

14

alumina followed by distillation. Purity was checked by thin layer chromatography (TLC)
on silica (Qureshi and Ahsan, 1965).

The fatty acid composition of D. denudatum Wall. was quantified by separation
on TLC and gas liquid chromatography (GLC). The fatty acids identified and quantified
by weight were capric (2.3 %), lauric (1.6%), myristic (3.8%), palmitic (16%),
palrnitoleic (2.5%), stearic (1.3%), oleic (23%), linoleic (30.2%), and linolenic acids
(19.3%) (Asif et al., 1981). The seed oil of D. staphisagria L. was analyzed for its
chemical constituents. Palmitic and steric acids were present in the highest amounts.
Fatty acids from C6 to C25 were detected by GC analysis (Costa de Pasquale etal., 1985).

Icosenoic acid was isolated from the seed oil of C. regalis S. F. Gray by Dabi et
al. (1986). After methylation and analysis of GC/MS, NMR, and IR spectra, it was
determined that it was a 20:1 fatty acid. Using two eluent systems, its bromophenacyl
ester was identified as 20:1 (cis -11) by comparison of HPLC retention times of 20:1 (cis-
5) as well as several other larger and smaller bromophenacyl esters. The seeds of C.
regalis S. F. Gray contain 30% of oil, which is comprised of 25% of 20:1 fatty acid. The
main fatty acid was 18:1 (Dabi et al., 1986).

Octacosanoic acid and N-acetylanthranilic acid were isolated from the roots of D.
iliense. These compounds, along with some alkaloids, were identified by chemical and
spectral means such as UV, IR, 1H-NMR, and l3C-NMR (Dong et al., 1991).

A study by Hasan and Osman (1993) suggests that D. ajacis L. should be
considered for use as an agronomic crop due to its seed oil components. It was found to
contain linoleic acid (<40%) but no linolenic acid. Oils with this composition belong to

the class of non-drying oils. The oil component of these seeds was reported to be 32 %.

15

The methyl ester composition of the oils was reported for 16:0, 18:0, 18:1, 18:2, 18:3 and
20:1 as being 6.5, 1.0, 46.5, 18.9, and 27.1 %, respectively. D. ajacis L. seeds yielded
high oleic content (46.5%) seed oil, as well as, eicos-cis-l l-enoic acid (27.1%) (Hasan

and Osman, 1993).

F lavonoids. Flavonoids are phenolic compounds. They consist of two aromatic rings, A
and B, linked by a three-carbon chain or ring C. They are often present in plant tissue as
glycosides. Ring A originates fiom the malonate pathway whereas the B ring is
synthesized from the shikimic acid pathway via phenylalanine (Taiz and Zeigler, 1991).
A number of physiological activities in humans and animals have been linked to
flavonoids. Figure 3.2 shows some representative flavonoids isolated from delphinium
species.

D. zalil Biernert ex. Boiss. is an Indian dye plant known for the production of
“gandhaki”. The dye was attributed to two coloring substances, one of which was very
soluble in alcohol (quercetin) and another which was only sparingly soluble [Quercetin
monomethyl ether (iso-rhamnetin)] (Perkin et al., 1898).

Several Ranunculaceae plants were investigated to determine the types of flavonol
glycosides they contained. Glucose usually was not found in the seven position of the
flavonol glycosides. However, rhamnose, and to a lesser extent xylose, were found to be
the sugar moiety at the C-7 position. The flavonol glycosides of Ranunculaceae are used
as chemotaxonomic tools for this family (Egger and Keil, 1965).

Different parts of D. consolida L. were monitored for their flavonoid content

during growth and development. The typical flavonoid, isorhamnetin, was absent

16

 

Figure 3.2. F lavonoids isolated from Delphinium species.

17

throughout the study. For this reason, it was recommended that this plant be classified
into a new genus, Consolida (Mel’nichuk et al., 1971).

Leaves of D. flexiosum and D. elisabethae yielded quercetin, isoquercetin, and 7--
L-rhamnofuranoside (Arazashvili et al., 1973). Arazashvili et al. (1974a) analyzed
flavonoids from the leaves of four species of delphinium. All four contained rutin. In
addition, leaves of D. freym'i, D. smalhausenii, D. tamarae contained quercetin,
hyperoside, and kaempferol and astragalin, respectively. Similarly, D. dzawachischwilii
flowers contained kaempferol-rutinosides.

Strzelecka (1976) reported four flavonoid compounds from D. grandiflorum L. In
three of the flavonoid glycosides, the aglycone was kampferol. The carbohydrate
constituents were rhamnose and glucose, rhamnose and arabinose, and arabinose and
glucose, respectively. In the fourth glucoside, the aglycone was quercetin. The flavonoids
quercetin, rutin, astragalin, and kaempferol-3-rutinoside were isolated fi'om the aerial
parts of D. peregrinum (Mericli et al., 1991b).

The flavonoids quercetin (3’ ”-benzoyl-2"-glucosyl)-3-glucoside-7-rhamnoside
and partially characterized quercetin (xylosyl, benzoyl)-3-glucoside, and
quercetin(benzoyl, g1ucosyl)-3-glucosides were isolated from D. carolinianum Walter
during a systematic study of the genus. Several known compounds such as galactosides,
glucosides , rharnnosides and xylosides of quercetin and kaempferol were isolated, also

(Wamock et al., 1983).

18

Anthocyanins. Anthocyanins are the most widespread group of pigmented flavonoids,
followed by carotenoids. They occur as glycosides having a sugar moiety at the three-
position. These secondary products apparently function as attractants of animals and
insects for pollination and seed dispersal. The color of an anthocyanin is influenced by
the number and position of the hydroxyl and methoxy substituents on the B ring, the
presence of chelating metals, the presence of flavone or flavonol c0pigments and the pH
of the cell vacuole in which these compound are stored. The anthocyanin, delphinin, was
discovered in D. consolida L. by Willstater and Mieg (1915) for the first time. Figures
3.3 and 3.4 show some anthocyanins isolated from delphinium species.

Anthocyanins were of primary interest as dyes before more easily obtained
synthetic dyes were available. Lushchevs’kaya (1937) studied D. consolida L., the red
delphinium, as a dye-yielding plant. Galfayan (193 8) extracted the pigment from D.
orientale flower petals. It was tested for its use an indicator for alkalimetry and
acidirnetry. The pigment was pink in color in acid solution, blue in neutral solution, and
green in alkaline solution. The pH interval for the blue color is 4.2 to 6.3. Ringer (1950,
1951) measured the absorption of dyes in solution from several plants including D.
consolida L.

Later, delphinidin diglycoside was isolated from D. ajacis L. (Krishnarnoorthy et
al., 1962). In 1975, Asen and colleagues re-examined the pigment, delphinidin, from
fiom blue larkspur cv. Dark Blue Supreme and reported the structure as delphinidin 3-di-
(p-hydroxybenzoyl)-g1ucosy1glucoside. Reddish-purple flowers of this cultivar showed
distinctive in vivo absorption bands which were found to be related to pH (Asen et al.,

1975). The pH of epidermal peels from young, moderate, reddish-purple larkspur flowers

19

 

Cyanodelphin

Figure 3.3. Anthocyanins isolated from Delphinium species.

20

 

Viodelphin

Figure 3.4. Another anthocyanin isolated from Delphinium species.

21

was 5.5. As the flowers aged, the pH increased to 6.6 and the color changed to light
purplish blue to light blue.

Many older cells contained strong blue crystals of anthocyanin. The anthocyanin
and flavonol glycosides in this species are delphinidin, 3-di (p-hydroxybenzoyl)
glucosylglucoside,kaempferol 3-galactoside-7-rhamnoside (robinin), kaempferol 3-
rutinoside, kaempferol 7-rhamnoside, and kaempferol 3-(caffeylgalactosylxyloside)-7-
rhamnoside. Also, absorbance of buffered solutions containing these compounds were
measured. Delphinidin 3-di (p-hydroxybenzoyl) glucosylglucoside at pH 5.6 was reddish-
purple in color and was not stable. Addition of kaempferol glycoside c0pigments resulted
in a stable complex, a slight bathochromic shift, and sharpening of the visible absorption
maxima. The absorbance maxirna of senescent light purplish-blue cells could not be
matched by the absorption curves of a 10'2 M solution of delphinidin 3-di (p-hydroxy-
benzoyl)glucosylglucoside in a pH range of 6.6 - 7.2 (Asen et al., 1975).

An anthocyanin from D. hybridum Hort. cv. Black Night, Viodelphin, was isolated
and characterized by Kondo et al. (1990). It is a delphinidin derivative containing two
molecules of p-hydroxybenzoic acid (Kondo et al., 1990). Kondo et al. (1991) reported
the structure of cyanodelphin, a tetra-p-hydroxybenzoated anthocyanin from D. hybridum

Hort. cv. Blue Springs.
Coumarins and other phenolics. Coumarins generally are found throughout all plant

parts. Some coumarins are thought to protect against herbivory, while others are thought

to be responsible for attracting pollinators. Coumarins are synthesized from phenyl-

22

alanine from the shikimic acid biosynthetic pathway. Figure 3.5 shows a range of
phenolic compounds isolated from delphinium species.

Delphoside, an isocoumarin glucoside, was isolated from the leaves of an
unspecified species of delphinium (Arazashvili et al., 1974b). Lomadze et al. (1976a)
used polargraphic methods to determine coumarins in D. flexuosum and D. speciosum
Bieb. Delphoside and flexidine were detected before, during and after flowering in D.
flexuosom. These were detected in D. speciosum Bieb. as well, but concentration varied
with geographic location. The level of rutin in the leaves of D. speciosum Bieb. was
found to be 0.58-2.5 (Lomadze et al., 1976b).

Cis-p-coumaric acid, a-amyrin (Figure 3.6), and a-amyrin acetate were isolated
from aerial parts of D. peregrinum (Mericli et al., 1991b). The aerial parts of D.
venulosum Boiss yielded five aromatic compounds including one new compound, 2,5,6
trihydroxypiperonylic acid methyl ester. The other four were reported as cis and trans p-
coumaric acids m and p—hydroxybenzoic acids, and protocatechuic methyl ester (Mericli
et al., 1991a).

The ethanolic extract from D. formosum Bioss.& Huet. afforded a new
benzoxepine derivative oxforrnasine (1,1',7-trimethyl-2-oxo-3,8-dihydroxy-6-methoxy-
benztetrahydrooxepine) (Mericli et al., 1996). The roots of D. denudatum Wall. yielded

3-hydroxy-2-methyl-4H-pyran-4-one (Attar-ur-Rahman et al., 1997).

23

”0 \ CH.

O
”OCH, HO CH=CH—C-OH
o O O
OH
H p-oomnaric acid
H
Delphoside
OH
OH 0 CH3
“*3 - :1
O o O OH
”.00 0,, o
1,1',7-trirmthyl-2-oxo-3,8-dihydroxy~ m-hydroxybmzoic acid
6-n'ethoxy-bermeu'ahydooxpine
O 0
6i“ ”0 I)
l l
O HO OH
CH. 4mg
8
3-hydmxy-2-rmd1yl-4H—pyranA—one 2,5,6-nihyioxypipumylic acid "ethyl ester

Figure 3.5. Phenolic compounds isolated from Delphinium species.

24

W W
H
M ”m“

Emmi

. at?”

 

Grolesterol

Avenastaol

24 Whip/lent: cholstanol 24 with: cholestanol

Figure 3.6. Sterols isolated from Delphinium species.

25

Carbohydrates. Carbohydrates are one of the most abundant groups of plant constituents.
For this reason, they generally are not considered secondary metabolites. Carbohydrates
play important multiple rolls in all life forms. They are the energy stores in plant tissue,
and the structural framework of DNA, RNA, and cell walls. Most of the reports on
carbohydrates from delphinium are the glycosides of true secondary metabolites such as
flavonoids, anthocyanins, coumarins, etc. However, one paper reported the isolation of
D-mannitol (Figure 5.1) from D. flexiosum, D. elisabethae, D. tamarae, and D.

dzawachischvili (Dekanosidze et al., 1974).

Terpenoids

Terpenoids form the largest and most diverse class of compounds found in plants.
They range from constituents of essential oils such as monoterpenes and sesquiterpenes
to nonvolatile triterpenes, sterols, and carotenoids. They are generally lipophilic
compounds. Terpenes often serve the plant as defensive compounds against herbivores.
They are derived from acetyl-CoA via the mevalonic acid pathway. Steroids are widely
distributed in nature. Most steroids are hydroxylated at C-3, and other sites. Sitosterol and
stigmasterol are the most abundant plant sterols (Ikan, 1991). A number of plant steriods

have pharmacological activity on humans and animals.

Sterols and triterpenes. It is not surprising that B-sitosterol, being one of the ubiquitous

sterols, has been reported from many delphinium species. (Qureshi, 1965; Mericli et al.,

1991a). In addition to B-sitosterol, many other sterols and triterpenes have been isolated

26

from plants of this genus. Figure 3.6 shows some representative sterols isolated from
delphinium species.

Trace amounts of cholesterol and AS-avenasterol were detected in D. denudatum
Wall. roots along with characteristic higher plant fatty acids and the ubiquitous sterols,
i.e. B-sitosterol, stigmasterol, etc. (Asif et al., 1981).

Sterols, 4-methylsterols, 4,4-dimethylsterols, and triterpenes from whole
nonsterile D. ajacis L., and from sterile cultures of callus tissues of germinated seedlings
were analyzed by Waller and Mangiafico (1978). Among the 26 sterols and triterpenoids
identified, tin-stigmasterol was identified in both tissue types, whereas, 24-
ethylidenecholestanol was tentatively identified in callus tissue and 24-
methylenecholestanol only in non-sterile tissue. Several other differences also were
observed. Qualitatively the contents of sterile and non-sterile tissue was similar,
suggesting that these sterols fit into a metabolic role in which enzymatic reactions
proceed from cycloartenol through 4,4 dimethylsterols and 4-methylsterols to sterols. The
difference in quantity may be due to lack of organogenetic controls in the rapidly
proliferating tissue grown aseptically, in comparison with the slow-growing nonsterile
plants (Waller and Mangiafico, 1978).

Later, whole plants and sterile callus tissue of D. ajacis L.were studied for
production and the metabolic relationship of sterols. These studies began as an attempt to
produce diterpenoid alkaloids in tissue-culture specimens. However, these cultures
produced large amounts of sterols rather than alkaloids. The major sterols in the whole-

plant tissues were B-sitosterol, carnpesterol, and stigmasterol. The major sterols in the

callus tissue were stigmasterol, 24-ethylidenelophenol, and Azz-stigmasterol. Five of the

27

sterols reported from callus tissue were not present in the whole plant. A partial
metabolic pathway was proposed for sterol production in D. ajacis L. by combining the
data from this study and from others (Waller et al., 1981).

Sterols, such as cholesterol, campesterol, stigmasterol, A7-campesterol, B-sitosterol, A5-
avenasterol, and A7-avenasterol, were isolated from the fatty seed oil of D. staphisagria

L.(Costa de Pasquale, 1985).

Nitrogen-Containing Compounds

Diterpenoid and Norditerpenoid Alkaloids. Alkaloids form one of the largest classes of
plant secondary metabolites. Alkaloids occur in concentrations of more than 0.01% dry
weight, and are found in at least 15% of vascular plants (Bemays et al., 1994 ). The
concentrations of N-containing compounds are usually highest in young tissue. As the
plants mature, the concentrations of these compounds slowly decline.

The classic definition of a “true” alkaloid is a compound that has a heterocyclic
ring containing a basic nitrogen, is related to amino acids, has a complex molecular
structure, exhibits significant, often toxic, pharmacological activity and is produced by
members of the plant kingdom (Pelletier et al., 1983). A modern and more appropriate
definition for an alkaloidal compound was proposed by S. W. Pelletier in 1983, as a
cyclic organic compound containing nitrogen in a negative oxidation state, which is of
limited distribution among living organisms. This description encompasses a larger
number of pharrnacologically active, nitrogen-containing compounds, including the

diterpenoid alkaloids, previously referred to as psuedoalkaloids (Pelletier et al., 1983).

28

Diterpenoid alkaloids are the most extensively investigated compounds from
delphiniums due to their extreme toxicity. A summary of the procedure for isolating
delphinium alkaloids is published in Chapter 12 of "Toxicants of Plant Origin," (Olsen
and Manners, 1989). At least 150 diterpenoid alkaloids have been identified in
delphinium (Olsen, 1990). In addition to Delphinium (Ranunculaceae), these types of
alkaloids have been investigated in Aconitum (Ranunculaceae), Garrya (Comaceae), and
Inula (Compositae) species (Waterman, 1993). As of 1990, 40 % of the diterpenoid
alkaloids isolated from larkspur are exclusive to this family (Blanche, 1990).
Furthermore, 30% of the lycoctonine-type, 50 % of the hetisine-subtype and 50 % of the
aconitine-type are exclusive to the annual delphinium (Blanche, 1990). The classification

of diterpenoid and related alkaloids follows:

I. Diterpenoid alkaloids
A. Atisine-type
B. Veatchine-type
C. Delnudine-type
II. Norditerpenoid alkaloids
A. Aconitine-type
1 . Aconitum-type
2. Pyrodelphine-type
3. Heteratisine-type

B. Lycoctonine-type
III. Bisditerpenoid alkaloids
Diterpenoid alkaloids are distributed throughout all the parts of delphinium plants.
Alkaloids accmnulate in the vacuoles and periplasmic space in plants. The alkaloidal
content of most delphinituns is up to about two percent (Kuder, 1947).
Diterpenoid alkaloids are divided into two categories, a hexacyclic C-19 skeleton

(norditerpenoid), and those based on 020 skeletons (diterpenoid). Pelletier (1980a)

29

suggested that these two types are derived from tetracylic or pentacyclic diterpenes in
which the nitrogen atom of methylamine, ethylarnine, or B-aminoethanol is linked to the
C-17 and C-19 in the C-19 skeleton (e.g. lycoctonine) and to 019 and C-20 in the C-20
skeleton (e. g. atisine). These linkages form a substituted piperidine ring.

Diterpenoid alkaloids are referred to as isoprenoid alkaloids. Unlike the classical
alkaloids, biogenetically related to amino acids, diterpenoid alkaloids are thought to be
formed from the precursor, acetyl CoA, in the mevalonic acid pathway. Specifically,
diterpenoid alkaloids of the lycoctonine , heteratisine, veatchine, and atisine types are
thought to be formed from the precursors primaiadiene and phyllocladine type
intermediates with the incorporation of nitrogen (Figure 3.7.) (Gross et a1, 1985;
Figuernido et al. 1995).

Early attempts to test whether or not specific delphinium alkaloids were of
terpenoid origin was demonstrated by the incorporation radiolabled mevalonic acid into
the alkaloid structure. Herbert and Kirby (1963) fed D. elatum L. plants with DL- [214C]
mevalonic acid through the cut ends of leaf stalks. The plant was found to contain a non-
saponifiable extract which exhibited 1.3% incorporation of radioactivity. The radioactive
component was shown to be B-sitosterol. Delpheline isolated from the same plant
showed no significant radioactivity. This experiment was conducted during the rapid
growth phase of the plant, also known to be the time during the delphinium life cycle to
produce the highest level of alkaloids. The authors suggested that delpheline lacks the
incorporation of mevalonic acid, because the precursor is converted into the non-basic

plant terpenoid before it reaches alkaloid synthesis (Herbert and Kirby, 1963).

30

   

19’ 18
Atisine Skeleton Heteratisine

   

Lycoctonine Skeleton Veatchine Skeleton

Primaradiene Phyllocladene

Figure 3.7. Diterpenoid alkaloids and their proposed biosynthetic precursors.

31

A later communication from Benn and May (1964) reported that browniine and
lycoctonine became radioactive in twelve specimens after being supplied with
mevalonate-23°C dibenzylethylenediamine by stem injection (Figure 3.8). Sodium
acetate - 1-‘4C, -2-”C were incorporated to a lesser degree. This experiment was carried
out twice as long as the previous experiment, and radioactive incorporation was low.
However, these results were consistent with those expected for normal terpene
biosynthesis. Benn and May (1964) suggested that the unsuccessful incorporation of
radio-labelled mevalonate in delphiline by Herbert and Kirby occurred because they used
detached leaves. The primary site for alkaloid biosynthesis is in the roots. Furthermore,
there may have been significant competition for incorporation by non-alkaloidial
terpenes.

A study by Ovezov (1965) showed that l"C02 was incorporated into elatine. The
incorporation of radiolabelled compounds was greater in the roots, supporting
Benn and May’s (1964) previous idea. Ovezov (1965) suggested that diterpenoid
alkaloids were synthesized from several precursors, and the rate of synthesis was
different for each alkaloid observed. A later report supported to the claim that diterpenoid
alkaloids are synthesized at different rates. Acetate- 2-‘4C was incorporated mainly into
elatinic acid and to a lesser degree in to elatidine and other alkaloids. The low inclusion
of acetate- 2-‘4C into elatidine was explained by the competition for incorporation of the
radiolabelled substance into non-alkaloidal terpenes (Ovezov, 1966). Methanol-”C was
fed to roots of D. elatum L. plants, and this produced a lower level of alkaloids in the

plant. However, the 14C was incorporated into the alkaloids, elatine and

32

 

Delpheline

 

Lycoctonine

Figure 3.8. Delphinium alkaloids studied to determine their biosynthesis.

33

methyllycaconitine from elatinic acid. Methanol-”C was not shown to be a direct
precursor of the methoxy groups in alkaloids (Ovezov, 1967).

The structure of diterpenoid alkaloids have been compared to that of the
gibberellins (Figure 3.9). Diterpenoid alkaloids and gibberellins may share
similar early biosynthetic pathways (Waller et al., 1969). The C-19 or
norditerpenoid alkaloids have been subdivided into two groups,the aconitine-type and
the lycoctonine-type (Figure 3.10). These two groups share some common elements.
Both possess a hexacyclic skeletal system comprised of one 7-membered, three 6—
membered, and two 5-membered rings. Both contain a tertiary nitrogen substituted with
an ethyl or methyl group (Keith et al.,l970). The C-1 and 08 are attached to an oxygen-
containing moiety which can be hydroxyl, methoxy, or ester functionalities. The C-14
has an ot-oxygen functional group, C-16 a methoxy group in the B-configuration. The
C-18 usually possesses either a methoxy or an N-substituted ester of anthranilic acid
(Keith et al., 1970).

Lycoctonine-type alkaloids are medially toxic, methoxy] monoesters of the
alkaloidal amino alcohol bearing benzoic acid or anthranilic acid ester substituents. In
general, O7 is always oxygenated, and these types of alkaloids are widely distributed in
nature (Stern, 1954). Aconitines are acutely toxic with a lethal dose on the order of 2-5
mg for human. They are heavily substituted with methoxy groups, two of the alcoholic
hydroxyl groups being esterified, one by acetic acid and the other by an aromatic acid
(e.g. benzoic acid or a p-methoxy- or 3,4-dimethoxy derivative) (Stern, 1954). Usually,
O7 is not oxygenated when compared to the lycoctonine-type alkaloids. Those

lycoctonine-type alkaloids with N-(methylsuccinyl)anthranilic acid at the C-18 position

34

 

 

Figure 3.9. Gibberellin and delcosine.

35

 

Aconitine-type Skeleton

 

Lycoctonine-type Skeleton

Figure 3.10. Norditerpenoid alkaloid skeletons.

36

have been reported to be the most toxic of this type. The LDso values were
between 2.7 and 10 mg/kg in mice. Structure-activity relationship studies show that toxic
norditerpenoid alkaloids must contain a tertiary nitrogen and an anthranilic acid ester
(Manners et al., 1995).

Aconitines can be subdivided into three groups based on their different skeletal
arrangements, such as (1) Aconitum-type in which the O7 is either not substituted or is
substituted with anything other than oxygen, (2) Pyrodelphinine-type in which there is a
double bond between C-8 and C-15, and (3) Heteratisine-type in which there is a lactone
moiety present (Pelletier et al., 1980a). The chemistry of C-1 9 diterpenoid alkaloids was
reviewed by Pelletier and coworkers (1980a).

The C-20 diterpenoid alkaloids can be divided into three groups, the atisine-type,
veatchine-type, and the delnudine-type (Figure 3.11) (Pelletier and Mody, 1981).
Atisines are simple non-toxic amino alcohols which are not extensively oxygenated and
which contain at most one methoxy moiety (Stern, 1954). The three different C-20
skeletons isolated differ in attachment of the C-15 and C-16 bridge at either 01 1, C-12,
or C-13. The chemistry of the C-20 diterpenoids alkaloids is reviewed by Pelletier
(1980a).

The largest and most complicated diterpenoid alkaloids are the bis-diterpenoid
alkaloids isolated from D. staphisagria L. (Figure 3.12) (Pelletier et al., 1980a).
Bisditerpenoids consist of two C-20 diterpenoid alkaloids attached at the C-16 position

(Pelletier and Mody, 1981).

37

 

Veatchine-type Skeleton

\
\

h
l
I
| \

    

 

Delnudine

Figure 3.11. Diterpenoid alkaloids.

38

 

Figure 3.12. Bisditerpenoid alkaloidal skeleton.

39

Alkaloids are by-products of plant metabolism, i.e. secondary metabolites. It has been
conjectured that alkaloids are reserve materials for protein synthesis, supporting the
earlier definition of an alkaloid. A more popular and modern view is that alkaloids are
defense compounds and serve to protect the plants from pathogen infection, insect
infestation, or mammalian herbivores. Countless alkaloids have been found in numerous
species of delphinium. The alkaloids reported from D. elatum L. are listed in Table 3-1.
The only work reported on D. belladonna Hort, a close relative of D. bellamosum
was a study of the quantitative estimation of the alkaloids in the different organs of this
plant (Hashem et al., 1974a). Total alkaloid content was measured in roots, stems,
leaves, flowers, fruits, and seeds before flowering, during flowering, and during fruiting.
The highest alkaloid content was in the leaves before and during flowering, and in the
fruits during fruiting. The highest overall level was in the seeds during fruiting. Ajacine
was identified, and its levels were monitored in this study as well. Aj acine's highest level
was found in the seeds of this plant. The trend for the alkaloid level in this plant during
growth and development was the alkaloid percentage increased in stems, leaves, and
roots during the flowering stage and decreased in the fruiting stage (Hashem et al.,
1974a). This plant was reported to have insecticidal properties due to the alkaloid
content. Phytochemical screening was carried out on the different organs of this plant.
Alkaloids were detected in the root, stem, leaf, flower, pericarp and seeds. These
alkaloids were identified by IR spectral studies. The fingerprint regions for ajacine,
delpharnine, and ajaconidine were identified by comparison to previously reported

literature values (Hashem et al., 1974b).

4o

Table 3.1 Alkaloids Isolated from D. elatum L. and D. bellamosum

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

 

Alkaloid Species“ Plant part ** References

andersonidine e s Wada et al., l992;Atta-ur-Rahman et
al., 1995

ajacine b s Hashem et al., 1974b; Pelletier and
Page, 1978

ajaconine e s Pelletier et al., 1990;Yunusov, 1993

ajacinoidine b s Hashem et al., 1974b; Pelletier and
Page,1978

blacknidine e w Park et al., 1995

blacknine e w Park et al., 1995

delatine e s Goodson, 1943; Stern, 1954

delatisine e 5 Ross et al., 1991; Atta-ur-Rahman et
al., 1995

delcorine e s Wada et al., l992;Atta-ur-Rahman et
al., 1995

delcosine b s Hashem et al, 1974b; Pelletier and

(delphamine) Page, 1978

delectinine e w, s Vaospv, 1993; Park et al.,
1995;Yunusov, 1991

delelatine e w, s Park et al., 1995; Pelletier et al., 1989;
Yunusov, 1991

deltaline e w, s Rabinovich, 1954; Kuzovkov, 1956;
Feofilaktov, 1954

delphelatine e w Feofilaktov, 1954

delpheline e w, s Goodson, 1943; Cookson, 1954;
Herbert et al., 1963; Pelletier et al.,
1989; Bando et al., 1989; Park et al.,
1995; Stern, 1954; Olsen and Manners,
1989

eladine e s Pelletier et al., 1989; Wada et al.,
1992; Yunusov, 1991 ; Attar-ur-
Rahman, 1995

elanine e s Pelletier et al., 1990; Yunusov, 1993

elasine e s Pelletier et al., 1990; Yunusov, 1993

elatine e w, s Rabinovich, 1952, 1954; Dozortseva,
1956; Kuzovkov, 1956; Ovezov,
1966,1967; Pelletier et al., 1989; Ross
et al., 1991; Olsen and Manners, 1989

elatidine e w Ovezov, 1966

eldelidine e w Rabinovich, 1952; Strzelecka, 1968

(deltarnine)

 

41

Table 3.1 cont. Alkaloids Isolated from D. elatum L. and D. bellamosum

 

Alkaloid Species“

Plant Part* *

References

 

eldeline (deltaline) e

W

Rabinovich, 1952,] 954; Kuzovkov et
al., 1956; Strzelecka, 1968

 

 

 

 

 

 

 

 

 

 

 

 

 

 

isodelpheline e s Pelletier et al., 1989; Wada et al.,
1992; Yunusov, 1991; Attar-ur-
Rahman, 1995

lycoctonine e s Pelletier et al., 1989; Yunusov, 1991

methyllcaconitine e w, s, r Goodson, 1943; Strzelecka, 1966,
1967,1968; Ovezov, 1967; Pelletier et
al., 1989; Stern, 1954

monoacetyl- b s Hashem et al., 1974b; Pelletier and

delcosine Page, 1978

nudicaulidine e w Dela Fuente, 1990; Yunusov, 1993

14-deacetyl- e w, s Vaospv, 1993; Park et al., 1995;

nudicauline Pelletier et al., 1989; Yunusov, 1991

nudicauline e w Vaospv, 1993; Pelletier et al., 1989;
Yunusov, 1991

pacidine e s Wada et al., 1992; Att-ur-Rahman et
al., 1995

pacifiline e s Wada et al., 1992; Att-ur-Rahman et
al., 1995

pacifidine e s Wada et al., 1992; Att-ur-Rahman et
al., 1995

paciliLne e Bando et al. ,1989

pacifinine e Wada et al., 1992; Att-ur-Rahman et
al., 1995

pacinine e s Pelletier, et al., 1990; Bando etal.,
1989; Yunusov, 1993

yunnadelphinine e s Wada et al., 1992; Atta-ur-Rahman et

al., 1995

 

 

*e= D. elatum L., b= D. bellamosum
**s=seeds, w= whole plant, r=roots

42

Biologically Active Compounds Reported From Delphinium

Many secondary metabolites are known poisons for insects, mammals, and
microorganisms. Some types of secondary metabolites protect the plants from abiotic
factors or competitive interactions against other plants. These poisonous attributes can be
exploited for use as drugs, pesticides, herbicides, antibiotics, antifungals, etc. All of the
biological activities reported from delphinium species are due to norditerpenoid and
diterpenoid alkaloids produced by these plants. The review chapter titled "The
Toxicology and Pharmacology of Diterpenoid Alkaloids" by Benn and Jacyno (1983) in
Volume four of Alkaloids Chemical and Biological Perspectives reviews the activities of

diterpenoid alkaloids from Delphinium, Aconitum, and Garrya species.

Toxicity and Pharmacological Studies. Delphiniums produce cardiac poisons possessing
a depressant action (Martin, 1953). Delphinium species contain many complex and
biologically active diterpenoid alkaloids. At least 150 diterpenoid alkaloids have been
identified in delphinium (Olsen, 1990). Several investigators found that alkaloid
concentration varies as a function of environmental conditions, and stage of plant growth.
The apex or reproductive raceme retains higher concentration of alkaloids than other
parts (Ralphs et al., 1988). Of the wild range plants, tall larkspurs have the highest
concentration of alkaloids during their vegetative growth stages, whereas low larkspurs
display a constant level of alkaloids (Manners et al., 1993).

Delphiniums grow wild in pastures, are consumed by stock, and are the leading
cause of cattle loss in the western rangelands. As expected, the toxicity of delphinium

plants on cattle is attributed to their complex diterpenoid alkaloids. Toxicity decreases as

43

the plants mature, but increases again during seed development. The most highly
substituted alkaloids produce the highest toxicity. Autopsies of animals killed by larkspur
showed inflammation of the stomach, small intestine, and the lining of the windpipe. The
blood vessels on the surface of the body and the kidneys were extremely congested
(Marsh et al., 1918). The alkaloids in these plants produce central vagal stimulation with
slowing of the heart rate. This is followed by a sudden increase in the rate and force of
the heartbeat due to a central sympathetic stimulation. There is a direct increase of
excitability of heart muscle which in fatal cases leads to death from ventricular
fibrillation (Martin, 1953). This condition is referred to as “Delphinosis”. The common
names of some delphiniums reflect this effect (e.g. poison weed and staggerweed)
(Kuder, 1947).

Alkaloid concentration is inversely correlated with palatability in some plants.
Alkaloids are considered to be bitter due to their basic nature. However, cattle readily
graze on delphiniums under conditions of low availability of fodder. Rabbits, guinea pigs,
cats, and rats were used in many experiments to outline the parameters of larkspur
toxicity, determine the symptoms, and identify the types of compounds responsible for
this phenomena (Atianasova-Shopova, 1969). Suchtaneous injection of delphinium
ethanolic extracts at different levels allowed researchers to determine at what time during
the growing season certain species were most toxic, and which species were most toxic
overall (Crawford, 1907).

To better coordinate cattle grazing with areas having high populations of wild
larkspurs, Olsen (1977b) reported the toxicity of three tall larkspurs, D. barbeyi, D.

gIaucescens, and D. occidentale. Olsen (1977a) developed a rat bioassay that would

estimate the toxicity of D. barbeyi. This assay was designed so that the toxicity of the
injected solution was expressed in terms of the equivalent amount of dried plant material.
Median lethal dose (LD 50) was determined by rats injected subcutaneously with ethanol
extracts dissolved in saline solution from the aerial delphinium parts prior to flowering.
This stage of grth produced median lethal doses ranging form 5.0 to 6.7 mg/g of body
weight. The initial effects were central nervous system depression and an increased
respiratory rate. Loss of motor function followed in the cervical muscles and then the
thoracic limbs. Periods of apparent excitement and sudden exaggerated muscle activity
occurred, also. Finally after which the rats became paralyzed and died. Death from
larkspur poisoning occurred from 1.5 to 18 h after injection. From this study, it was
determined that larkspur’s toxicity was due to total alkaloid concentration. D. barbeyi
was about four times more toxic than D. glaucescens, and approximately 10 times more
toxic than D. occidentale (Olsen, 1977b). D. barbeyi has been classified as one of the
most dangerous species related to forage poisoning in cattle (Keeler, 1975).

Clark et al. (1987) published a study designed to determine the total alkaloid
concentration in larkspur and lupine with Near Infrared Reflectance Spectroscopy
(N IRS). Total alkaloid concentration is used as a preliminary indicator of plant toxicity.
NIRS was the first reliable method for monitoring total alkaloid levels when ranchers
were trying to determine safe or dangerous ranging periods.

The most recent and perhaps most reliable method of quantitative analysis of
norditepenoid alkaloids in larkspur, Fourier Transform Infrared Spectroscopy (FTIS),
was reported by Gardener and coworkers (1997). Plant samples with known

concentrations of alkaloids determined by high-pressure liquid chromatography (HPLC),

45

and gravimetric methods were used to construct a calibration curve. The measurements of
toxic and total alkaloid concentrations were shown for D. barbeyi, D. occidentale, and D.
glaucesens. Olsen (1978) compared the toxicity of larkspur on sheep and cow by
determining LD50 for both types of animals. The results showed that sheep tolerate about
four times more larkspur than cattle.

Ingestion of wild delphiniums by cattle can produce teratogenic effects, as well as
death (Keeler, 1969). Larkspur toxicity is usually acute, resulting in death 3 to 8 h after
an animal ingests a lethal dose. Death generally occurs from paralysis followed by
respiratory failure. The clinical signs of larkspur poisoning in cattle are as follows:
restlessness, stiff gate, stradle-stance followed by collapse, nausea, and abdominal pain
(Ralphs et al., 1989). Other signs have been described as loss of muscle control,
salivation, trembling, rapid and weak respiration and heart action, bloating, and
ultimately respiratory paralysis (Keeler, 1975).

Aiyar and colleagues (1979) collected leaves of D. brownii Rybd. and tested
extracts from this material, attempting to determine which of the chemical constituents
were responsible for cattle deaths due to poisoning. The primary symptom of D. brownii
Rybd. poisoning is paralysis. Pharmacological research on some diterpenoid alkaloids
suggests that a primary site of action of these toxins is the neuromuscular junction.
Purification of the alkaloid fraction from the methanol extract yielded three major
constituents, MLA, browniine and browniine acetate (Figure 3.13). The depressant
effects of these alkaloids was tested on the responses of the electrically-induced muscle
twitch of the isolated rat phrenic nerve-diaphragm preparation and an electrically

stimulated longitudinal muscle strip of guinea pig ileum. The individual alkaloids did not

46

 

Browniine

Figure 3.13. Alkaloids toxic to cattle.

47

exhibit activity when tested on the ileum preparation. However, the crude tertiary
alkaloid fraction showed significant nicotinic blocking action in the phrenic nerve-
diaphragm prepartion. MLA was the most effective, and most of the activity on the
nerve—diaphargm test was attributed to this compound. A dose response curve comparing
MLA to reference compounds such as, aconitine hydrochloride and (+)-tubocurarine
further illustrated this conclusion.

Preliminary assays using sciatic nerve-sartorius muscle preparation further
supported the conclusion that MLA produces competitive blockage at nicotinic receptors,
as well as effecting sodium channels (Aiyar et al., 1979). The blockage by MLA was at
least partially reversed by eserine. The alkaloid could slowly be washed out on
equilibration of the paralyzed tissue with fresh physiological solution. Sciatic nerve-
sartorius preparations from frog (Rana pipiens) were stimulated electrically and treated
with MLA (10'7 M). This concentration produced a 50 % inhibition of post-synaptically
recorded action potentials within 20 sec. A concentration of 10‘8 M the 50 % inhibition
was obtained after 50 sec (Aiyar et al., 1979).

A follow-up study to the in vitro mammalian assays was done in vivo on calves to
see if the site of action was the same. Calves injected with MLA showed signs of its
having a neuromuscular site of action as expected from the previous assays (Nation et al.,
1982). Physostigmine was tested and it proved to be an effective antidote when MLA
injections were at lethal levels. This further supports that MLA has a neuromuscular site
of action (Nation et al., 1982).

A study of the relationship of relative total alkaloid concentration and toxicity of

D. occidentale Wats., tall duncecap larkspur, on mouse bioassay over the entire growing

48

season showed that mouse bioassay is better correlated to cattle toxicity rather than to
total alkaloid content (Olsen, 1983). Saline solutions of the alkaloid mixtures were
administered by subcutaneous injection between the scapula (Olsen, 1983). Later, D.
barbeyi, D. occidentale, and D. glaucescens were evaluated for total alkaloid
concentration. The variation in concentration of individual alkaloids across the three
species was analyzed by GC. Of all the different alkaloids isolated, MLA was shown to
be the most toxic using, the mouse bioassay developed earlier by Olsen in 1983
(Manners, 1991). The authors stated that MLA was only one of the toxic alkaloids
present in these plants.

In order to find a suitable laboratory animal model to test larkspur toxicity,
researchers tested the toxicity of larkspur extracts on mice, hamsters, rats and sheep. The
extracts were given orally or by subcutaneous injection (Olsen et al., 1991). By
comparing LDsos and susceptibility to oral dosage versus subcutaneous injection, the
authors concluded that, of the animals tested, mice would be the best choice for testing
toxicity of larkspur. They had high susceptibility, rapid response time, and a small dose
requirement (Olsen et al., 1991).

The arrhythmogenic and heart rate effects of several diterpenoidal alkaloids from
D. cashmirianum Royle have been measured on isolated guinea atria. Aconitine
produced a dose-related increase in heart rate previous to the arrhythmias. Arrhythmias
were produced at concentration of 3 x 10'5 M. Only lappaconitine showed an
arrhythmogenic effect at 1 x 104M, without a prior increase in heart rate. An in vivo
study with rabbits showed that lappaconitine produced a decrease in heart rate, followed

by arrhythimias. N-deacetyllappaconitine produced arrhythimias in conjunction with a

49

decrease in blood pressure. Lycaconitine produced a small increase in blood pressure
while the alkaloid cashmiradelphine produced a small decrease. However, these
responses were not dose- related. Aconitine caused an increase in heart rate followed by
arrhythimias and decreasing blood pressure, which was reversible at 0.005 mg/kg. These
activities were limited to compounds having an anthranoyl or N-substituted anthranoyl
group in position 4 (Figure 3.14) (Shamma et al., 1979).

Tulyaganov and coworkers (1976) published the pharmacology of some aconite
alkaloids. Lycoctonine, delcorine, and eldeline showed a myorelaxant, antidepolarizing
effect on nerve muscle transmission, low toxicity, did not produced cardiac arrhythmias,
did not stimulate different nerve receptors, and did not affect the central nervous system
(CNS). Delphinine administered at (0.01 mg/kg) I.p. showed significant deleterious
effects on liver function in adult male rats (Figure 3.15) (El-Maraghy, 1995). Elatine
from D. elatum L. , condelphine from D. confitsum, and MLA and delsemine form D.
dictyocarpum are referred to as curariform alkaloids (Figure 3.16).

The effects of J adwar or D. denudatum Wall. were investigated by testing on rats.
The rats were treated with CC14 (3.0 mg/kg) which caused a severe depletion of glycogen
and adrenal ascorbic acid. It caused an increase in levels of serum-free fatty acids,
bilirubin, glutamic-oxaloacetic transaminase, glutamic-pyruvic transaminase, cholesterol,
triglycerides, and blood urea. Oral administration of the aqueous extract (1 g/ kg on three
consecutive days) of D. denudatum Wall, prior to CC14 treatment antagonized the effect
of CC14 by significantly elevating the levels of liver glycogen and adrenal ascorbic acid,
and depletion of the levels of the other metabolites, thus showing a protective effect on

the liver against CCl4-induced hepatoxicity (Khan et al., 1981).

50

 

Aconitine

 

Lycoctonine

/ OMe
“OMe

 

 

Cashmiradelphine

 

Me’fjp

Lappaconitine

Figure 3.14. Alkaloids from D. cashmirianum Royle. which produce arrhythimia.

51

 

Delcorine

 

 

Delphinine

_———‘

_—-——'

    

Me OAc

Deltaline

Figure 3.15. Alkaloids with pharmacological activity.

52

  
 

Elatine

 

Delcorine

Figure 3.16. Curariform alkaloid.

53

Studies by Alkondon and colleagues (1992) showed that MLA is a highly potent,
yet reversible inhibitor of the 125I- or bungarotoxin binding site in adult rat hippocampal
membranes. It is comparable to or more effective than other a-toxins from snakes. For
this reason the authors suggested it be considered for use in the characterization of
neuronal nicotinic receptors.

Now, MLA is used as a ligand for distinguishing neuronal nicotinic acetylcholine
receptor subtypes (nAChR). These receptors are the focus for pharmaceuticals, because
of their involvement in Alzheimer’s Disease (Jacyno, 1996). MLA has high affinity for
a7 type nAChRs. Lycaconitine differs by the absence of methyl substituent from the
succinimide ring. The pharmacology or toxicology of the two have been virtually
unstudied. An LDso (mouse, iv) of ca 15 mg/kg of lyaconitine is less toxic than MLA
(LDso mouse iv 3 mg/kg). MLA was compared to lyaconitine in rat brain preparations.
Lyaconitine is half as potent as MLA in the inhibition of binding of [3H]-(-)cytisine in
its affinity for a402 nAChR subtypes. Lyaconintine displayed higher activity in the
inhibition of binding of [1251]-or-bugarotoxin (ol7 nAChR subtype) but less than MLA.
MLA is a potent and specific antagonist of the aBGT sensitive nAChR of cultures
hippocampal neurons in rat. Its action is reversible (Jacyno, 1996). In addition,
Wonnacott and colleagues (1993) showed that MLA is able to discriminate among
nicotinic acetylcholine receptor subclasses. Yum and colleagues (1996) demonstrated
that MLA was effective in dinstinguishing between certain subtypes of nicontinic
receptors in chicks.

A study by Hardick and Co-workers (1996) compared the binding activities of

nudicauline, elatine, and MLA as well many other norditerpenoid alkaloids (Figure

54

3.17). From this study these workers established further support for the necessity of the
methylsuccinimido ring for potency of these type of alkaloids on binding with the
mammalian neuronal nAChR.

The tertiary nitrogen on MLA was reported to have a conformation suitable to fit
the nicotinic pharmacophore, similar to that of the quantemary nitrogen present in
acetylcholine. Semi-synthetic drugs with quantemary nitrogen moieties such as
delphinine methochloride and delphonine methochloride were shown to have greater
reactivity, further supporting the evidence of the alkaloid nitrogen as the binding site for
interaction with the nicotinic receptor site (Manners et al., 1995).

MLA is known as delatine, delsemidine, and mellictine (Coates et al., 1994). It
finds continued use in Russian medicine as a muscle relaxant for surgery. MLA was
detected in D. grandiflorum L., D. crassifolium, and D. triste. All three of these species
have been shown to produce curare-like effects, D. crassifolium having the most activity
(Mats, 1972). MLA is referred to as the most potent, non-proteinaceuos, neuronal
nicotinic acetylcholine receptor antagonist found to date (Hardick et al., 1994). By
regioselective anthranoylation of demethylated aconitine, a novel analogue of MLA was
prepared. This novel semi-synthetic alkaloid was synthesized for use in the study of the
roles of the anthranilate and succinimide moieties in the selective biochemical
pharmacology of MLA at protein receptors (Hardick et al., 1994). An evaluation of toxic
N- (methylsuccinyl)-anthranoyllycoctonine (MSAL) norditerpenoid alkaloids determined
that the presence of the methylsuccinimide group and probably the ester carbonyl and the
lycoctonine nitrogen are primary factors in establishing toxicity of the norditerpenoid

alkaloids in the mammalian system (Manners et al., 1994).

55

OMe

   

Nudicauline

Elatine

 

Methyllycaconitine

Figure 3.17. Alkaloids studied for binding activity with nAChR.

56

A few of the alkaloids from D. elatum L.have been studied for their
pharmacological activity. Elatine has curare-like action on the central nervous system
(Chopra et al., 1969) (Figure 3.17). Deltaline (eldeline) has been isolated from D. elatum
L. among other delphinium species. It is an example of 10-hydroxylated diterpenoid
alkaloids which have been reported as relatively uncommon (Figure 3.18). It showed
weak effects on smooth muscle using rabbits and guinea pigs. Another alkaloid isolated
from D. elatum L. , delcorine, was shown to depress blood pressure and respiratory rate in
cats and dogs (Figure 3.18). It showed inhibiton of contractions in smooth muscle tissue
in rat and rabbit, and produced contractions in smooth muscle tissue of guinea pig. Also,

it proved to be a ganglionic blocking agent (Benn and Jacyno, 1983).

Alellopathic Studies. Alkaloids isolated from D. ajacis L., delcosine, ajaconine, and an
unknown designated as LBA-III, were tested on pea plants to observe their affect on plant
growth (Figure 3.19) (Waller et al., 1969). Segments of 4-day, old pea cambium were
attached with tubing containing a lanolin +1/20 M sucrose solution. Auxin was applied to
these segments either above or below the lanolin, sucrose, alkaloid solution. Kinetin was
added to the auxin in some treatments, but had no effect. Evaluation of the treatments
was made by weighing the top 10 mm of the segments and comparing them to the control
weights, or by preparing microscopic sections from below the tip and projecting them
onto a planimetcr to measure the surfaces of xylem and phloem (Waller et al., 1969).
Delcosine and LBL-III produced inhibition to both xylem and phloem. Ajaconine showed
no significant effect on grth (Waller et al., 1969). It was proposed that delcosine and

LBL-III may compete with the gibberellic acids for enzyme active sites or that they might

57

  
 

Deltaline

Elatine

 

Delcorine

 

Condelphine

Figure 3.18. Alkaloids with pharmacological activity.

58

 

Aj aconine

 

Delcosine

Figure 3.19. Alkaloids studied for allelopathic activity.

59

be a feedback control of gibberellic acids in some manner (Waller et al., 1969).

Insecticidal Activity. Some of the earliest work on the biological activity of the
delphinium species was reported by W. M. Davidson (1929). The oils from D. consolida
L. and Stavesacre (D. staphisagria L.) were prepared as soap emulsions. The D.
consolida L. emulsion was effective against Tetranychus telarius L., Paratetranychus
pilosus C.& F., and a variety of aphid species (Aphis rumicis, Myzus persicae, M. rosea,
M. cerasi, A. pomi DeGeer). The D. staphisagria L. emulsion was found to give effective
control (>95%) of red spider mites (T. telarius), a number of aphid species (A. rumicis,
M. persicae. M. rosea, M. cerasi, A. pomi DeGeer), and whitefly (A. vaporariorum). The
alkaloids present in this emulsion, delcosine, delsoline, and delphinine were identified as
both stomach and contact poisons on some insect species (Figure 3.20). Delphinine-HO]
tested up to 0.16% concentration was inefficient against the green peach aphid (M.
persicae), mealy bugs, red spider mites, Heliothripsfemoralis and the fall webworrn
(Hyphentria cunea). However, at a concentration of 0.085% Delphinine-HCI killed onion
thrips (Thrips tobaci) and cabbage worm. Delcosine (0.63 %) was effective against A.
rumicis, M. persicae. T. tabaci, H. cunea, P. rapae. Delsoline (0.042%) was effective
against A. rumicis, M. persicae. A 0.085% solution of this alkaloid with soap was
effective against T. tabaci (Davidson, 1929).

Plants used in traditional medicine were tested by Soviet scientists in 1945.
Among them six species of delphinium including, D. elatum L. were tested. D. elatum L.

was the most effective against a variety of insect pests (Petrischeva, 1945). The

60

OCH

 

OCH,

Delsoline

 

Delphinine

Figure 3.20. Stomach and contact poisons on insects from Delphinium species.

61

delphinium plants showed to possess potent insecticidal activity even after 2-3 years of
freezer storage. Later, Gatty-Kostyal and Stawowczyk (1956) compared
the quality, quantity, and content of active substance in above- and below-ground plant
parts of D. elatum L. with D. oxysepalum Borb. & Pax. The insecticidal activity of D.
consolida L. and D. orientale was proposed to be due to their alkaloids (Avramova et al.,
1964). D. ajacis L. a.k.a C. ambigua L. is known to posses insecticidal and growth
inhibitory activities (Pelletier et al., 1980b). D. crassifolium, a Russian folk-remedy plant
was among the plants collected during the Transbaikal medical expedition of 1936. This
plant was used as a fly repellent in old Russia (Efros, 1946). Another delphinium species
found to have insecticidal activity was D. delavayi. Whole plants were extracted with a
variety of organic solvents and tested on several species of insects (Lee et al., 1943).

Extracts from five species of delphinium were tested for their effectiveness
against bedbugs (C imex lectularius) and lice (Pediculus humanus L.). The extracts were
tested as sprays, dips, and droplets applied directly to the insects by a micropipet. The
Delphinium alkaloids were active on contact and produced paralysis. The activity was
attributed to the alkaloids. However, it was not considered that the alkaloids are general
contact insecticides, but rather stomach posions. D. elatum L. was one of the most potent
of the species tested. The treatment was lethal to the lice, but the bedbugs were able to
recover from the treatment (Busvine, 1946).

Seeds of D. hybrid Hort. cv. Pacfic Giant, King Arthur were investigated for
insecticidal properties. Chloroform extracts of crushed seeds were tested against
arrnyworrn (Spodoptera eridania), bean aphid (A. fabae), mite (T. urticae), spotted

cucumber beetle larvae (Diabrotica undecimpunctat howardr), malaria mosquito

62

(Anopheles quadrimaculatus) , Empoasca abrupts, Heliothis virescens, and the common
housefly Musca domestica. In all cases, the extract produced either mortality or feeding
inhibition (Jennings et al., 1986).

The same extract was tested for nicotinic activity against insect cholinergic
receptors. The inhibition of [3H]-propionyl-a-bungarotoxin binding to M. domestica head
homogenate. or- bungarotoxin is an antagonist of nicotinic acetylcholine receptors in
insects. The seed extract displayed potent inhibition of [3H]-propionyl-ot-bungarotoxin
(Jennings et al., 1986). The compound responsible for this activity was MLA showing the
highest level of inhibition. In fact, the level of cholinergic activity of this alkaloid at the
insect nicotinic receptor is much more potent than that reported for the rat muscle
receptor. Lycoctonine inhibited [3H]-propionyl-a-bungarotoxin binding to the insect
cholinergic receptor at higher concentration than MLA. Lycoctonine is an analogue of
MLA lacking the aromatic ester function. However, the investigators surmised that T.
urticae and A. quadrimaculatus were killed by saponins in the extract, rather than by the
alkaloids. The authors proposed that MLA may have evolved in Ranunculaceae species
as a highly specific ligand for the insect nicotinic receptor and acts as a
antifeedant/insecticide through its action on this target site in the insect (Jennings et al.,
1986)

The cholinergic activity of this alkaloid at the insect nicotinic receptor is much
more potent that that reported for the rat muscle receptor, where the EDso was determined
to be 2.3 x 10'6 M. Aconitine, which is more potent on the rat muscle nicotinic receptor,
was found to be less active. The rank in order of potency for inhibition of ot-bugarotoxin

binding in the insect preparation is MLA>lycoctonine>aconitine and aconitine>MLA>

63

lycoctonine in the rat phemic nerve-diaphragm prepartion (Figure 3.21) (Jennings,
1986)

The inhibition reported for MLA in the fly head nicotinic receptor assay is the
most potent ever reported for non-proteinaceous toxin at this site in insects (Jennings,
1986). Later review of this work by the authors brought them too the conclusion that the
previous experiments on the vertebrate preparations were performed at levels to high to
affect the housefly receptor. They concluded that the acetylcholine receptors of insects
and vertebrates have significant differences, and they should be studied further (Jennings
et al., 1987). They proposed that MLA may be useful for studying these differences.
Knowledge of these differences could aid in the development of more effective new
pesticides.

Seeds of garden hybrid delphinium closely related to the cultivar Pacific Giant
and the species D. elatum L. have been used as a source for homogenous MLA for
structure activity relationship (SAR) studies. Investigators were able to confirm the
absolute stereochemistry of MLA as S (Coates et al., 1994). The biological activity of
this pure compound was tested by ligand-binding assays on rat membranes. The nACHR
subtype was identified by [1251]-ot-bungarotoxin labelling. The pure MLA showed high
affinity for inhibiting [1251]-a-bungarotoxin-binding. These values were similar to those
found with the assay testing the citrate salt of MLA. This study was the beginning of the
modeling of the nicotinic pharrnacophore to determine SAR of MLA (Coates et al.,
1994)

Later, 17 Gig-diterpenoid alkaloids isolated from Delphinium spp. were tested for

inhibition of radiolabelled otBGTx binding to rat and housefly neural tissue

64

 

Methyllycaconitine

 

 

Aconitine

Figure 3.21. Insecticidal and mammalian alkaloid toxins.

65

(Kukel, 1994). This series of alkaloids exhibited a range of inhibitory potencies (ICso)
from 42 pM in the house fly head prepartion to in excess of 100 aM in the rat brain
preparation. The most potent alkaloids in the series tested, all possessed the C13
succinimide moiety functionalities of MLA, confirming that the succinimide group
contributes to the potent activity seen with these alkaloids (Jennings, 1986). These results
are consistent with previous assays by Jennings (1986).

D. staphisagria L. has been used since ancient times to kill and prevent
infestation of body parasites. It goes by many common names such as Lousewort,
Stavesacre, Lice-Grains, and Stephans Grains. Seeds from this plant were mixed into
oatmeal bait to kill rats, and were sprinkled around the bases of fruit trees to ward off ants
(Smith et al., 1981). The insecticidal nature of this plant was thought to be related to its
alkaloids, as are the activities shown by many Delphinium species. Trexler (1960)
patented the insect repellents or deterrents containing Staphisagria (U .S. 2,939,816).

An alkaloid insecticide from the extract of D. consolida L. was active against
head lice and nits (Strzelecka et al., 1957). In 1966, Strzelecka showed that the benzene
and chloroform (CHCI3) extracts containing total alkaloid of C. regalis L. produced
100% mortality of lice and nits in 2 h. Extracts without alkaloids showed no activity. The
alkaloid delsoline and the alkaloid mixture produced 100% mortality at a 1:2000 dilution

ratio after 1 h.

66

Antimicrobial Activity. The alkaloids from D. flexuosum were shown to have bactericidal
effect in a 1:10,000 dilution when tested on gram positive rather than gram negative
bacteria (Zolotnitskaya et al.,1962). A bacteriostatic effect was observed on
Staphylococcus aureus at 12104-1 07 dilution and Escherichia coli at l:10"-105
(Zolotnitskaya et al., 1962).

Siddiqui and co-workers (1990) demonstrated that D. denudatum Wall. had
antimicrobial and immuno-modulating properties. Organic extracts were tested against 30
gram-negative and 10 gram-positive bacterial strains. These extracts showed activity
against gram negative bacteria such as Pseudomonas, Yersinia, and Plesionmonas strains.
Gram-positive bacteria, Staphylococcus aurues and Bacillus suvtilus, were inhibited by
these extracts. Ethanolic extracts from the roots of D. denudatum Wall. exhibited
antifungal activity against Stachybotrys atra, Trichophyton longifusus, C urvularia lunata,
Drechslera rostrata, Epidermophyton flocosum, Microsporum canis, Nigrospora oryzae,
Ganoderma applanatum, Allescheria boydii, E. floccosum Dutarium rotatum, Pleutortus
ostreatus, and Dutarium rotatum (Atta-ur-Rahman, 1997). 8-acetylheterophyllisine and
vilrnorrianone displayed antifirngal activity against A. boydii, E. floccosum, and A. niger.
Panicutine exhibited antifungal activity against A. boydii, S. atra, P. ostreatus, N. oryzae,

and D. rotatum (Atta-ur-Rahman, 1997)(Figure 3.22).

67

 

-- Panicutine

 

Villmorrianone

Figure 3.22. Antiflmgal alkaloids from D. denudatum Wall.

68

It is clear from this review published on the phytochemistry of the Delphinium
genus, that the bulk of research has focused on the norditerpenoid and diterpenoid
alkaloids produced from these plants. The biological activity associated with delphiniruns
is for the most part due to these alkaloidal compounds. The following chapters report new
non-alkaloidal compounds isolated from the hexane and ethyl acetate extracts from D. X
Cultorum flowers using bioassay-directed fractionation and purification procedures.

There are no phytochemical reports available for D. X C ultorum, a hybrid of D. elatum L.

69

CHAPTER IV

Chemical Evaluation of the Hexane extract from D. X cultorum Flowers

Abstract

Delphinium species are known for producing complex norditerpenoid and
diterpenoid alkaloids, which show a wide range of biological activities. We have isolated
six compounds from the mosquitocidal fraction resulting from the hexane extract of D. X
cultorum flowers. It is our hypothesis that this biological activity is due to one or more of
these non-alkaloidal compounds. Although compounds ethylmethylbenzene (1), 1-
Isopentyl-2,4,5-trimethylbenzene (2), and 2-Penten-2-ylbenzoic acid (6) were reported
earlier, they have not been isolated fiom plant tissue, until now. This is the first report of
3-Buty1idene-3H-isobenzofuran-1-one (4) and an isomer (5) thereof being isolated from
any delphinium species. All of these compounds and a novel derivative of acetophenone,
2-(hex-3-ene-2-one)-phenylmethylketone (3) were characterized from their GC-EIMS
spectral data. In addition, B-sitosterol (7) was isolated and characterized by spectral

methods.

70

Introduction

The temperate plant, Delphinium elatum L. (Ranunculacae) native to East Asia, is
known for its diterpenoid and norditerpenoid alkaloid constituents. Many traditional uses
of delphinium species are related to their insecticidal activity (Efros, 1946; Smith et al.,
1981; Chopra et al., 1969). More recently, investigators linked this insecticidal activity to
the diterpenoid and norditerpenoid alkaloids produced by these plants (Coates et al.,
1994; Jennings et al., 1986; 1987; Kukel, 1994). In addition to insecticidal activity, these
alkaloids possess allelopathic activity (Waller et al., 1969), antimicrobial activity against
gram-negative and gram-positive bacteria, and a variety of fungi (Atta-ur-Rahman, 1997;
Siddiqui et al., 1990 Zolotnitskaya et al., 1962). Also, some of these compounds are
being considered for pharmacuetical applications (Jacyno, 1996). In this chapter, we
report that D. X cultorum, a hybrid species from D. elatum L. possesses insecticidal
compounds that are not related to these alkaloidal components. Volatile components
characterized by GC-EIMS are reported from a mosquitocidal fraction isolated from the

hexane extract of D. X cultorum flowers.

Experimental
Plant Material
D. X cultorum seeds cv. Magic Fountians Dark Blue/ White Bee were donated by Bodger
Seed, Ltd. South El Monte, CA. The seeds were germinated in high porosity, peat-based
growing mix in styrofoarn trays (4”x 6” x 2”) and grown until the first leaf appeared. The
seedlings then were planted in pots (4”x 4” x 6”) containing high porosity, peat-based

growing mix and grown to the five-leaf stage. At that point, the plants were transplanted

71

into large clay pots containing high porosity, peat-based growing mix and were grown
until maturity. The plants were grown year round in the Pesticide Research Center
Greenhouses at Michigan State University.

Plant parts were harvested when 75-85% of the florets were fully expanded on
each raceme. The florets and leaves were removed from each stem and the stem was cut
into two to 3.5 cm sections. The separate plant parts were then packed into reclosable
plastic bags and stored at -20°C. Frozen plant parts were weighed on a Mettler BD601
balance (Mettler-Toledo, Inc., Hightstown, NJ). The separated plant material was spread
evenly onto three metal lyophilizer trays (29x 31.5 x 3.4 cm) and lyophilized using a bulk
tray lyophilizer (FTS Systems, Inc., Stone Ridge, NY) at 5°C for 48h. The dried plant
material was macerated into a fine powder in a commercial Waring blender, packed into
reclosable plastic bags and stored at -20°C until analysis. The water content of the fresh

D. X cultorum flowers was approximately 80%.

General Experimental

The components of the hexane extract of D. X cultorum flowers were analyzed by
thin layer chromatography (TLC). Concentrated solutions of the crude extracts were
prepared in CHC13. The solutions then were applied to silica plates (20 x 5 cm, 250 pm
with inorganic binder and UV 254) with 5 pL capillary pipettes. The spots were allowed
to dry. The TLC plates were placed into solvent chambers equilibrated with 15 mL of the

following solvent systems: CHC13/ MeOH (4:1), (10:1), and (1:1), and hexane/ acetone

72

(4:1). TLC plates (250 u) and preparative TLC plates GF (20 x 20 cm2, 1000 u) were
purchased from Analtech Inc. (Newark, Delaware).

GC-EIMS was performed using a 30 m DB5 column (0.32 x 0.25 mm) on a
JEOL-JMS-AXSOSH mass spectrometer. The column temperature was set at 50 °C and
programmed to increase to 190°C at 10 °C /min. The ionization energy was 70 eV. The
solvent was acetone and the carrier gas was helium. Mass spectral data were obtained at
the Michigan State University Mass Spectrometry Facility which is supported, in part, by
a grant (DRR-00480) from the Biotechnology Research Technology Program, National
Center for the Research Resources, National Institutes of Health. NMR spectra were
recorded on a Varian VXR 300 MHz spectrometer (V arian, Palo Alto, CA) at room
temperature at the Max T. Rogers NMR facility, Chemistry Department, Michigan State

University.

Extraction

Accelerated Solvent Extraction. An ASE 200 Accelerated Solvent Extractor
(Dionex Corp., Sunnyvale, CA) was used to extract the plant material. The dry,
macerated plant material was mixed with diatomaceous earth to yield a 10:1 mixture by
weight. This mixture was hand-packed tightly into 33 mL cells which contained a
cellulose filter in the bottom. The extraction was done under 1000 psi and 60°C. The
extraction solvent (10 mL per cycle) was collected in 60 mL vials. The plant material was

extracted sequentially with hexane and MeOH (Aldrich, Milwaukee, WI) (Scheme 4-1.).

73

 

D.elatum flowers/Celite (10:1)

 

 

1. Extracted with hexanes. 50 °C. 1000 p.s.i., X 3.
2. 10 min. static cycle X 3.
3. Extract collected separately for each cycle.

L L

Residue I I Crude Extract I

 

 

 

 

l Evaporated in vacuo at 45°C.

 

 

Crude Hexane Extract I

 

1. Extracted with MeOH, 50°C. 1000 p.s.i., X 3.
2. 10 min. static cycle X 3.
3. Extract collected separately from each cycle.

 

 

V
Residue I

 

 

l I Crude Extract I

 

 

l Evaporated in vacuo at 45°C.

 

Discarded I

 

Crude MeOH Extract

 

 

 

Scheme 4-1. Accelerated solvent extraction procedure.

74

Large-scale extraction of D. X cultorum flowers. A large glass column was used
for the extraction of D. X cultorum flowers to obtain a sufficient quantity of crude hexane
extract. Lyophilized and macerated flowers (409 g) were packed in the column that
contained a cotton plug at the bottom. The plant material was extracted sequentially with
hexane (3.5 L x 3) and ethyl acetate (2.0 L x 3). The plant material was soaked in solvent
for at least 72 h, with exchange of fresh solvent every 12 h. Solvent was removed in
vacuo at 40°C on a rotary evaporator yielding crude hexane extract (8.68 g) and ethyl
acetate extract (3.61 g). The dried extract was stored at -20°C until bioassay and

purification. The residual plant material was discarded.

Insecticidal Assays

Corn Earworm Assay. Com earworm eggs (Helicoverpa zea Boddie) and dry
corn earworm diet was purchased from North Carolina State Insectory, Department of
Entomology, North Carolina State University, Raleigh, North Carolina. The eggs were
hatched in an incubator at 27°C. The dry diet was dispensed into scintillation vials (940
mg) for each treatment. Crude extracts or purified compounds were dissolved in DMSO
to give a concentration of 1250 pg'25 uL", unless otherwise stated. Twenty-five uL of
these stock solutions were mixed thoroughly with the portions of dry diet. A 25 pL of
DMSO was used as a control. Agar solution (1.4%) was mixed and autoclaved for 5 min
at 15 psi and 125°C to melt the agar. This solution was held in a water bath at 45°C, and
added to the dry diet until the total diet weighed 5 g. The final concentration of test

extracts and compounds was 250 ppm. The wet diet was mixed thoroughly, and 3-4 drops

75

of diet were dispensed into 3.5 mL polystyrene vials. The freshly poured portions of diet
were allowed to cool and dry for at least 1 h. After drying, one neonate larvae was placed
in each vial, and the vials were capped. The treatment and control vials were held in a
growth chamber at a photoperiod of 16 h day and 8 h night with day temperature at 28°C
and night temperature at 24°C. Each treatment had 15 replicates. The treatments were
arranged in a completely randomized design. The larvae were weighed (mg) on a Mettler
AE163 analytical balance (Mettler-Toledo, Inc., Hightstown, NJ) after six days.
Statistical analysis was conducted using AN OVA and the F test. Least Significant
Difference (LSD) was calculated for those treatments with significant F values, and used
to compare treatment mean weights with controls.

Gypsy Moth Caterpillar Assay. Gypsy moth eggs (Lymantria dispar L.) were
obtained from the Insect Production Unit of the Canadian Forest Service, Sault Ste.
Marie, Canada. The dry diet was mixed in the Bioactive Natural Products Laboratory,
Department of Horticulture, Michigan State University. The recipe was as follows: wheat
germ (36 g), casein (7.5 g), Wesson’s salt mix (2.4 g), sorbic acid (0.6 g), methylparaben
(p-hydroxy-benzoic acid methyl ester) (0.3 g), Hoffrnan-Larouche #26862 vitamin
mixture (Hoffrnan-Larouche, Inc., Nutley, NJ) (3.0 g). The eggs were hatched in an
incubator at 27°C. The dry diet was dispensed into scintillation vials (845 mg) for each
treatment. Crude extracts or purified compounds were dissolved in DMSO to give a
concentration of 1250 pg ‘25 uL“, unless otherwise stated. Twenty-five uL of these stock
solutions were mixed thoroughly with the aliquots of dry diet. A 25 uL aliquot of DMSO

was used as a control. Agar solution (1.4 %) was mixed and autoclaved for 5 min at 15

76

psi and 125°C to melt the agar. This solution was held in a water bath at 45°C, and added
to the dry diet until the total diet weighed 5 g. The final concentration of test extracts and
compounds was 250 ppm. The wet diet was mixed thoroughly, and 3-4 drops of diet were
dispensed into 3.5 mL polystyrene vials. The freshly poured portions of diet were allowed
to cool and dry for at least 1 h. After drying, one neonate larvae was placed in each vial,
and the vials were capped. The treatment and control vials were held in a growth chamber
at a photoperiod of 16 h day and 8 h night with day temperature at 28°C and night
temperature at 24°C. Each treatment had fifteen replicates. The treatments were arranged
in a completely randomized design. The larvae were weighed (mg) on an analytical
balance after six days. Statistical analysis was conducted using AN OVA and the F test.
LSD was calculated for those treatments with significant F values, and used to compare
treatment mean weights with controls.

Mosquito Assay. First instar mosquito larvae (A edes aegypti L.) were provided by
Drs. Alexander Raikel and Alan Hays, Department of Entomology, Michigan State
University. The larvae were raised in 500 mL beakers containing 150-250 mL of reverse
osmosis water in an incubator at 26°C, for three days. Ten to 12 fourth instar larvae were
placed in 980 uL of reverse osmosis H20 in 12 x 75 mm borosilicate glass test tubes.
Crude extracts or purified compounds were dissolved in DMSO, unless otherwise stated,
to give a concentration of 250 pg '20 uL', unless otherwise stated. A 20 uL aliquot of
DMSO was used as a control. Treatments and controls were covered and left at room
temperature. There were three replicates per treatment. The number of dead larvae were

recorded at 2 h intervals, up to and including 24 h. Statistical analysis was conducted

77

using AN OVA and the F test. LSD was calculated and used to compare treatment means
with controls.

Japanese Beetle Assay. Adult Japanese beetle (Papilliajaponica Newman) were
collected for 8 h on sunny, calm days at the Michigan State University Tennis Facility
with a hanging floral lure/ pheromone trap provided by Dr. Dave Smitley, Department of
Entomology, Michigan State University. The beetles were collected 1-2 days prior to the
assay. They were kept in plastic containers (IL) with perforated lids. The beetles were fed
leaves from a climbing rose tree or a grapevine. Test solutions for the micro-application,
direct dip, and leaf assays were prepared at 250 and 100 pg 'mL" concentrations of crude
delphinitun flower extracts in acetone. The micro-application assay was performed by
treating the insects, held upside down, with l pL of the test solution on the abdomen. The
droplet was allowed to dry. Twenty beetles per treatment were placed into 150 x 15 mm
polystyrene disposable petri plates containing two pieces of filter paper. Each treatment
and the control was replicated three times. The beetles were fed with either grape or rose
leaves depending on what they were fed when captured. The petri plates were kept in a
growth chamber at 25°C and a photoperiod of 16 h day, 8 h night. The leaves and filter
paper were changed daily. Mortality was recorded first at 2 h followed by 24 h intervals
for 96 h.

For the direct dip assay, five beetles were placed in a covered strainer. The
strainer was dipped into the solution for 5 s. The beetles were removed from the strainer
and allowed to dry. The strainer was rinsed with acetone between treatments. Twenty

beetles per treatment were placed into 150 x 15 mm polystyrene disposable petri plates

78

containing two pieces of filter paper. Each treatment and the control were replicated three
times. The beetles were fed either grape or rose leaves, depending upon what they were
fed when captured. The petri plates were kept in a growth chamber at 25°C and a
photoperiod of 16 h day, 8 h night. The leaves and filter paper were changed daily.
Mortality was recorded first at 2 h followed by 24 h intervals for 96 h.

For the leaf dip assay, grape leaves were dipped into the treatment solutions and
allowed to dry. The leaves were fitted with small glass vials containing reverse osmosis
water and stoppered with cotton. One leaf was added to each 150 x 15 mm polystyrene
disposable petri dish containing two pieces of filter paper and 5 beetles. Each treatment
and the control was replicated three times. After 24 h the remaining treated foliage was
removed and replaced with fresh untreated foliage. The petri plates were kept in 3 grth
chamber at 25°C and a photoperiod of 16 h day, 8 h night. The leaves and filter paper
were changed daily. Mortality was recorded first at 2 h followed by 24 h intervals for 96
h.

A no-choice feeding assay was performed on 20 beetles placed into two 150 x 15
mm polystyrene disposable petri plates containing two pieces of filter paper. One large
mature leaf and one small new leaf and flowers from D. X cultorum flowers were fitted
with small glass vials containing cotton, fitted with cotton plugs and placed into the
dishes. One dish contained leaves, the other flowers. The petri plates were kept in a
growth chamber at 25°C and a photoperiod of 16 h day, 8 h night. Mortality was recorded
first at 2 h followed by 24 h intervals for 96 h.

Colorado Potato Beetle Assay. Adult Colorado potato beetles (Leptinotarsa

decemlineata Say) were collected from Michigan State University Entomology Research

79

Station, courtesy of Dr. Ed Grafius. The potato beetles were fed potato leaves from plants
grown in the Pesticide Research Center Greenhouses at Michigan State University. Test
solutions for the micro-application, direct dip, leaf assays were prepared at 250 and 100
rrg'mL'l concentrations of crude delphinium flower extracts in acetone. The micro-
application, leaf dip, and direct dip assays were performed exactly like that of the

Japanese beetle assay, except potato leaves were used instead of grape or rose leaves.

Nematocidal Assay

This assay was conducted on two nematode cultures, Caenorhabditis elegans
Dought and Panagrellus redivivus Goody kept in culture at the Bioactive Natural
Products Laboratory, Michigan State University. C. elegans Dought was kept on NG agar
media in disposable petri plates wet with 24 mL of physiological saline solution. C.
elegans Dought was grown with a strain of Escherichia coli on which it fed. P. redivivus
Goody was kept in axenie, liquid Basal-Heme media (5 mL) in scintillation vials. The
cultures were stored at room temperature and subcultured every four weeks. Appendix 11
lists the recipes for NO and Basal-Heme media.

The nematodes were added to 1 mL of physiological saline solution in a
scintillation vial. This solution was diluted until the nematodes numbered 15-20 in a 48
uL aliquot. Forty-eight pL of dilute nematode solution was delivered to each of three
wells per treatment in a polystyrene 96 well plate. Crude extracts or purified compounds
were dissolved in DMSO, to give a concentration of 12.5 ug'2 uL', unless otherwise

stated. Two pL of the test solution or DMSO alone, as a control was added to each well

80

bringing the final concentration to 250 ppm. The plate was covered, sealed with parafilm,
and kept in a humid chamber. The number of dead nematodes was recorded every two h

up to and including 24 h.

Anti-microbial and Topoisomerase Inhibitory Assays

Micro-organisms (Aspergillus parasiticus, Candida albicans, Escherichia coli,
Staphylococcus epidermidis, Streptococcus aureus, F usarium moniliform, F usarium
oxysporum, Rhizoctonia solanii, Saccharomyces cerevisiae strain 394, S. cerevisiae strain
394 H, and S. cerevisiae strain 394,“) were stored at -80°C in 1.2 mL Nalgene cryogenic
vials containing a 70 % glycerol solution. The cryogenic culture was thawed and 50 uL
of the suspension was spread onto a 100 x 15 mm disposable polystyrene petri plate
containing an agar media specifically for that organism. (Appendix 11 lists the recipes
used for each micro-organism.) The transfer was performed in a laminar air-flow hood
(Contamination Control, Inc. Lansdale, PN) to maintain sterile conditions. The cultures
were grown in an Equatherm incubator (Curtin Matheson Scientific, Inc., Kennesaw, GA)
at 275°C until the organism covered the plate, approximately 24 h for bacteria and yeast,
and 48 to 72 h for fungi. Two to 3 mL of physiological saline solution (8.5 g/L) was used
to dislodge the colonies or hyphal mat from the petri plate. This solution was placed into
a 16 mm OD. x 125 mm long, 19 mL, disposable, borosilicate glass test tube and diluted
to 10 mL with saline solution. These cultures were stored at 4°C until the assay.

The culture suspensions were mixed on a Vortex Genie mixer (Fisher Scientific,

Norcross, GA) until all cells or hyphae were suspended uniformly. Fifty uL of the

81

suspension was spread onto a petri plate containing the designated agar media in a
laminar air-flow hood to maintain sterile conditions. The plates were left in the hood to
dry for 30 min. With a permanent marker, the plate was divided into 3 segments by
drawing lines on the outer-underside of the plate. Crude extracts or purified compounds
were dissolved in DMSO, to give a concentration of 250 ug'20 uL", unless otherwise
stated. These spots were allowed to dry for 30 min. After drying, the petri plates were
sealed with parafilm and placed in an incubator at 27.5°C. The zone of inhibition was

measured in cm after 24, 48, and 72 h.

Purification of Hexane Extract

A preliminary separation of the crude hexane extract from D. X cultorum was
performed by solvent-solvent partition. A solution of the crude hexane extract (4 g) in
hexane (200 mL) was extracted in a 1L separatory funnel with MeOH: H20 (95:5) (300
mL). The hexane layer was removed, and the aqueous MeOH layer was extracted twice
with hexane (200 mL). The combined hexane extracts were evaporated in vacuo at 45°C,
yielding a yellow-green hexane-soluble fraction (3.8 mg). Similarly, the aqueous MeOH
(95:5) extract afforded a green-waxy fiaction (283 mg). This green-waxy fraction (283
mg) was purified firrther using preparative TLC on silica plates developed with CHC13.
This yielded fractions A-E with weights of 3.4, 4.6, 9.9, 3.4, and 177.4 mg, respectively.
The bands were removed after viewing under UV light at 254 and 366 nm, and then
eluted with MeOH. Fraction B was dissolved in acetone (2 mg ' mL") and analyzed by

GC-EIMS. Fifteen peaks were obtained in the Total Ion Current (TIC) chromatograrn.

82

Six compounds with retention times of 3.00, 6.98, 13.1 1, 13.22, 13.75, 13.90 min and
relative abundances of 5, 9, 100, 15, 33, %, respectively were identified from the TIC
chromatogram.

Compound 1: m/z values (% rel. int.): 55(6), 91(12.5), 105(100), 120 (Mi, 28.75).
These m/z values are identical to the reported values (National Institutes of Standards and
Technology, 1998) for the isomers of ethylmethylbenzene.

Compound 2: m/z values (% rel. int.): 51(3.75), 77 (6.25), 105 (26.25), 133 (100),
190(M‘, 10.63). This fragmentation pattern matches that reported by Kingston et al.
(1988) for 1-isopentyl-2,4,5-trimethylbenzene.

Compound 3: m/z values (% rel. int): 55 (16.25), 65 (2.5), 77 (3.75), 83 (100), 91
(12.5), 97 (7.5), 105 (8.75), 119 (67.5), 132 (21.25), 145 (2.5), 173 (2.5), 201 (18.75),
216 (M+, 47.5).

Compound 4: m/z values (% rel. int): 55 (3.75), 77 (11.25), 89 (2.5), 103 (18.75),
131 (27.5), 146 (30), 159 (100), 188 (M+, 28.75). This fragmentation pattern matches that
of the published data for 3-Butylidene-3H-isobenzofuran-l-one (Uozumi et al., 1989;
Ogawa et al., 1995; Watanabe et al., 1993).

Compound 5: m/z values (% rel. int): 50 (5), 77(7.5), 103 (16.25), 131(20),
146(36.25), 159(100), 188 (M*, 26.25). This fragmentation pattern matches that of the
published data for 3-Butylidene-3H-isobenzofuran-1-one (U ozumi et al., 1989; Ogawa et
al., 1995; Watanabe et al., 1993).

Compound 6: m/z values (% rel. int.): 55 (27.5), 63 (3.75), 91 (12.5), 106 (32.5),
120 (11.25), 134 (21.25), 148 (78.75), 161 (100), 190 (M”, 72.5). This fiagmentation

pattern was identical to that of the published MS data by Li ct al. (1993).

83

Fraction C obtained from the purification of the aqueous methanol extract was
recrystallized from MeOH to yield compound 7 (6.2 mg). Compound 7 was characterized
by 1H and 13C NMR analysis.

Compound 7: 1H NMR (CDCl,): 60.68(3H, s, 18-H X 3), 0.81(6H, d, 27-H X 3,
26-H X 3), 0.92(3H, m, 21-H X 3), 1.0(3H, bs, 19-H X 3), 1.12(6H, m), 1.24(3H, m),
1.51(10H, m), 1.82(9H, m), 1.97(2H, m), 2.25(2H, m) 3.54 (1H, m, 6-H X 1,), 4.76(1H,
m, 3-H X l), 5.35(1H, m, 5-H X 1). 13C NMR (CDCl,): 511.86(C-18), 11.98(C-29),
18.77(C-27), 19.02 (C—21), 19.40(C-19), 19.82 (C-26), 21.08(C-11), 23.05 (C-28), 24.29
(C-15), 26.04 (C-4), 28.25(C-16), 29.13(C-23), 29.70 (025), 31.60(C-2), 31.90 (C-7,C-
8), 33.93 (C-22), 36.15(C-20), 36.51(C-10), 37.23(C-1), 39.76 (C-l2), 42.23 (C-13),
45.82 (C-24), 50.12 (C-9), 56.03 (017), 56.75 (C-l4), 71.68(C-3), 121.72 (06),
140.73(C-5). The 'H and '3C NMR chemical shift values are in agreement with the
published values for B-sitosterol (Slomp and MacKeller, 1962; Akihisa et al., 1986; Bisht

et al., 1978; Shoolery and Rogers, 1958; Holland et al., 1978).

Results and Discussion
Flowers of D. X cultorum were extracted sequentially with hexane and MeOH.
Preliminary bioassays were performed on these extracts for insecticidal, mosquitocidal,
antimicrobial, nematocidal, and topoisomerase enzyme inhibitory activities. The assays
revealed that the hexane extract exhibited potent mosquitocidal activity at 10 ppm
concentration; 100 % mortality was obtained in 2 h. The MeOH extract exhibited 100 %

mortality in 2 h at 250 ppm. Both extracts showed antifeedant activity on corn earworm

84

(Table 4.1) and gypsy moth caterpillars (Table 4.2). Neither of the extracts showed any
insecticidal activity against the Colorado potato or Japanese beetles. Similarly, these
extracts did not show antimicrobial, nematocidal or topoisomerase enzyme inhibitory
activities.

Bioassay-directed fractionation of the hexane extract was conducted using
mosquito larvae and corn earworm neonates to monitor biological activity. The hexane-
and MeOH:HZO(95:5)-soluble fractions were tested for biological activity against
mosquito larvae. Both exhibited LDloo in 2 h at 100 ppm. In addition, these fractions were
tested against corn earworm (Table 4.3). Both showed significant antifeedant activity,
however the MeOHzH20(95:5)-soluble fraction was the most active.

The mosquitocidal fraction B from the purification of the MeOHszO(95:5)-
soluble fraction was analyzed by GC-EIMS. The TIC profile indicated 15 peaks; six of
the major peaks were characterized and assigned structures based on their fragmentation
patterns. Peak 1, at 3.00 min., was assigned to have a structure identical to compound 1,
an isomer of ethylmethylbenzene as shown in Figure 4.1. The base peak in its EIMS
spectrum at m/z 105 was due to a homolytic cleavage of the methyl group. The peak at
m/z 91 is due to homolytic cleavage of the ethyl group. However, it is not clear whether
this compound is the o, m, or p isomer of ethylmethylbenzene.

The peak at 6.98 min in the TIC chromatogram of the mosquitocidal fraction is
due to compound 2, 1-Isopentyl-2,4,5-trimethyl benzene shown in Figure 4.2. The base
peak in its EIMS spectrum at m/z 133 is due to the homolytic cleavage of the isopentyl

moiety.

85

Table 4.1. Average weights of H. zea Boddie larvae after 6 days.
Treatments were tested at 250 ppm. "' p5 0.01 (compared to control).

 

Treatment Average Weight (mg)
Control 15.60
Hexane Extract 0.85“
Methanol Extract 8.57*

 

86

Table 4.2. Average weights of L. dispar L. larvae after 6 days.
Treatments were tested at 250 ppm. ‘p 5 0.01 (compared to control).

 

Treatment Average Weight (mg)
Control 18.05
Hexane Extract 8.04*
Methanol Extract 8.75*

 

87

Table 4.3. Average weights of H. zea Boddie larvae after 6 days.
Treatments were tested at 100 ppm. *p 5 0.01 (compared to control).

 

Treatment Average Weight (mg)
Control 37.22
Hexane Soluble Fraction 29.97*
Methanol:Water (95:5) Fraction 0.83*

 

88

CH3

m/z 91 (12.5)

+ +.

_l 7

j

m/z 120 (28.75)

m/z 105 (100)

Figure 4.1. Ethylmethylbenzene and MS fragmentation pattern.

89

+. +

7 1

CH2

m/z 190 (11) mlz 133 (100)

Figure 4.2. 1-Isopentyl-2,4,5-trimethylbenzene and its MS fiagmentation pattern.

90

Compound 3, as indicated by the TIC peak at 13.11 min., was characterized from
its EIMS fragmentation pattern as 2-(Hex-3-ene-2-one)-phenylmethylketone. Figure 4.3
depicts a plausible fragmentation pattern for this compound. The peak at m/z 83 was
resulting from a heterolytic cleavage at C-ll (Figure 4.4). The peak at m/z 201 was due
to the methyl group loss at C-l3. Similarly, the peak at m/z 173 was formed by the
acetate group loss at the C-1. The peak at m/z 119 with a relative abundance of 67.5%
was due to the heterolytic cleavage of the hex-3-ene-2-one side chain at C-2. A higher
abundance of this peak compared to the loss of an acetate at m/z 173 (2.5) was indicative
of a stable radicle. Similarly, the peak at m/z 105 was assigned to a homolytic cleavage of
the fragment ion m/z 173 at C-11. The ion at m/z 132 was resulting from a hydrogen
rearrangement followed by the cleavage at G9.

The TIC profile gave peaks at 13.22 and 13.75 min. in the chromatogram that
were assigned to compounds 4 and 5, respectively. However, the MS analysis revealed
that both 4 and 5 showed identical fiagrnentation profiles. Indicating that both of these
compounds are isomers of 3-Butylidene-3H-isobenzofuran-l-one (Figure 4.5). The base
peak at m/z 169 was the result of a homolytic cleavage of the ethyl group in the
butylidene side chain. The fragment ion at m/z 146 was due to a y-hydrogen
rearrangement followed by a homolytic cleavage of the propylene group in the butylidene
side chain. The GC-MS fragmentation pattern was insufficient to determine the structures
of 4 and 5.

Compound 6 was identified fiom the TTC at. 13.90 min. as 2-pent-2-ylbenzoie

acid (Figure 4.6). The base peak at m/z 161 was due to homolytic cleavage of an ethyl

91

.
of

O
m/z 145 (3)

+

(jar?

m/z 105 (9)

07 +
CH/\)\

2

m/z 83 (100)

 

cm
{in +

0 Ti
m/z 132 (21) ©\(

 

O
m/z 119 (67.5)
——. +.
— O
\ +
07
O \ W
_J
Compound 3
m/z 216 (48) m/z 173 (2.5)
‘ +
07
O
m/z 201 (19)

Figure 4.3. 2-(Hex-3-ene-2-one)-phenylmethylketone and its MS fragmentation pattern.

92

 

Figure 4.4. 2-(Hex-3~ene-2-one)-phenylmethylketone.

93

/
o
o
,/ mlz 188 (29) \
/
F
+. +
CH2_I CHZ—I
/
o o
o o
mlz 146 (so) mlz 159 (100)

Figure 4.5. 3-Butylidene-3H-isobenzofi1ran-l-one and its MS fragmentation pattern.

94

 

 

\
OH
O
/ mlz 190 (72.5)
/ \
F
+
H
\ 2 \ CHz—l
OH OH
0 O
mlz 161 (100) mlz 148 (79)

Figure 4.6. 2-Pentylbenzoic acid and its MS fragmentation pattern.

95

+

group from the pentyl side chain in compound 6. The peak at m/z 148 was resulting from
a y-hydrogen rearrangement and loss of a propylene group.

The MeOHzH20 (95:5)-soluble fraction afforded a mosquitocidal fraction with an
LDloo on mosquito larvae in 2 h at 1 ppm, and compound 7. Compound 7 (Figure 4.7)
was characterized from its 'H and 13C NMR spectral data. The spectral data indicate a
chemical formula of C29HSOO for compound 7. There was an exchangeable proton at
51.82 that was assigned to the 3-OH, and was embedded in a multiplet with 4-CH2
protons. The 5 values at 3.54 and 5.35 were assigned to the allylic protons at C-6 and C-
5, respectively. Both the 1H and 13C NMR spectra of this compound revealed that it is
identical to the NMR data of B-sitosterol.

A very low yield of the mosquitocidal fraction containing compounds 1-6 did not
allow further purification of it to yield pure compounds in order to test them for
individual

insecticidal activity. Further investigation of these compounds is necessary to determine
which of them is responsible for the insecticidal activity. In addition,
ethylmethylbenzene, 1-Isopentyl-2,4,5-trimethylbenzene, and 5-Penten-2-ylbenzoic acid
have never been reported from natural sources. 3-Butylidene-3H-Isobenzofirran-1-one
and its isomers are commonly found in essential oils of Angelica acutilboa (Takano et al.,
1990), A. glauca Edgew (Dai and Qui, 1996), A. tenuissima (Kim et al., 1989), Apium
graveolens var. graveolens (Ferh, 1979), A. graveolens var. rapaceum (Gijbels et al.,

1985), Levisticum oflicinale (Gijbels et al., 1980), Ligusticum actiloba var. sugiyamae

96

 

Figure 4.7. B-sitosterol.

97

(Yamagishi etal., 1974), L. acuminatum (Huang and Pu, 1989), L. acutilobum (Kariyone
et al., 1937), L. chuanxiong Hort. (Yu, 1983; Kimura et al., 1989), L. porteri (Delgado et
al., 1992), L. sinense Oliv cv. Chaxiong (Luo et al., 1996; Xi et al., 1987), L. wallichii
Franch (Ko et al., 1977; K0 et al., 1980), Opopanax chironium (Gijbels et al., 1983). This
is the first report of ethylmethylbenzene, 1-Isopentyl-2,4,5-trimethylbenzene, 3-
Butylidene-3H-Isobenzofirran-1-one, and 5-Penten-2-ylbenzoic acid from Delphinium
species. Also, 2-(Hex-3-ene-2-one)-phenylmethylketone is isolated as a natural product

for the first time.

98

CHAPTER V

Chemical Evaluation of the Ethyl Acetate Extract from D. X cultorum Flowers

Abstract

Little chemistry has been reported for Delphinium species, other than diterpenoid
or norditerpenoid alkaloids. In this chapter, we report the isolation of compounds 1—3
from the ethyl acetate extract of D. X cultorum flowers. The known compounds, D-
mannitol (1) and 4-hydroxybenzoic acid (2), have been isolated previously from other
Delphinium species. This is the first report of the isolation of compound 3, bis-(4-
hydroxyphenyl)methanol, from a natural source. These compounds were characterized
from their 1H- and '3 C NMR spectral data. Compounds 1-3 were not active when tested
against mosquito larvae (A edes aegyptii L.) and corn earworm neonates (Helicoverpa zea

Boddie).

99

Introduction

This Siberian native species, D. elatum L., is known predominately for its
norditerpenoid and diterpenoid constituents. However, some interesting phenolic
compounds have been isolated previously from species other than D. elatum L. Mericli,
A. et al.(1 991) isolated 2,5,6-trihydroxypiperonylic acid methyl ester from the aerial parts
of D. venulosum Boiss in addition to cis and trans p-coumaric acids m and p-
hydroxybenzoic acids, and protocatechuic acid methyl ester. D. formosum Bioss afforded
a new benzoxepine derivative oxforrnasine (l,1',7-trimethyl-2-oxo-3,8-dihydroxy-6-
methoxy-benztetrahydrooxepine) (Mericli et. a1, 1996). 3-hydroxy-2-methyl-4H-pyran-4-
one was isolated from D. denudatum Wall. (Attar-ur-Rahman et. a1, 1997). No biological
activity was reported for any of these compounds.

In this chapter and for the first time, we report insecticidal activity of the crude
ethyl acetate extract from D. X cultorum flowers, purified using bioassay directed
fractionation. With this procedure, we isolated two of the major constituents, which are
phenolic compounds, present in a corn earworm antifeedant fraction. When tested, none
of these compounds 1-3 showed activity in our other bioassays. There are no reports of

compounds 1-3 being isolated from D. X cultorum flowers, until now.

Experimental
Plant Material
D. X cultorum. seeds cv. Magic F ountians Dark Blue/ White Bee were donated by

Bodger Seed, Ltd. South El Monte, CA. The seeds were germinated in high porosity,

100

peat-based growing mix in styrofoarn trays (4”x 6” x 2”) and grown until the first leaf
appeared. The seedlings then were planted in pots (4”x 4” x 6”) containing high porosity,
peat-based growing mix and grown to the five-leaf stage. At that point, the plants were
transplanted into clay pots (1 gal.) containing high porosity, peat-based growing mix and
were grown until maturity. The plants were grown year round in the Pesticide Research
Center Greenhouses at Michigan State University.

Plant parts were harvested when 75-85% of the florets were fully expanded on
each raceme. The florets and leaves were removed from each stem and the stem was cut
into two to 3.5 cm sections. The separate plant parts were then packed into reclosable
plastic bags and stored at -20°C. Frozen plant parts were weighed on a Mettler BD601
balance (Mettler-Toledo, Inc., Hightstown, NJ). The separated plant material was spread
evenly onto three metal lyophilizer trays (29x 31.5 x 3.4 cm) and lyophilized using a bulk
tray lyophilizer (F TS Systems, Inc., Stone Ridge, NY) at 5°C for 48h. The dried plant
material was macerated into a fine powder in a commercial Waring blender, packed into
reclosable plastic bags and stored at -20°C until analysis. The water content of the fresh

D. X cultorum flowers was approximately 80%.

General Experimental
The components of the crude ethyl acetate extract were analyzed initially by thin layer
chromatography (TLC). A concentrated solution of the crude extract was prepared in

CHC13. The solution was applied to silica plates (20 x 5 cm, 250 pm with inorganic

binder and UV 254) with a 5 11L capillary pipette. The spot was allowed to dry. The TLC

101

plate was placed into a solvent chamber equilibrated with 15 mL of CHCl3zMeOH. TLC
plates were purchased from Analtech Inc. (Newark, Delaware, USA).

NMR spectra were recorded on a Varian VXR 300 MHz spectrometer (Varian,
Palo Alto, CA) at room temperature in the Max T. Rogers NMR facility at Michigan

State University.

Extraction

Extraction of D. X cultorum flowers. A large glass column was used for the
extraction of D. X cultorum flowers to obtain a sufficient quantity of crude hexane
extract. Lyophilized and macerated flowers (409 g) were packed in the column that
contained a cotton plug at the bottom. The plant material was extracted sequentially with
hexane (3.5 L x 3) and ethyl acetate (2.0 L x 3). The plant material was soaked in solvent
for at least 72 h, with exchange of fresh solvent every 12 h. Solvent was removed in
vacuo at 40°C on a rotary evaporator yielding crude hexane extract (8.68 g) and ethyl
acetate extract (3.61 g). The dried extract stored at -20°C until bioassay and purification.

The residual plant material was discarded.

Insecticidal Assays

Corn Earworm Assay. Corn earworm eggs (Helicoverpa zea Boddie) and dry
corn earworm diet was purchased from North Carolina State Insectory, Department of
Entomology, North Carolina State University, Raleigh, North Carolina. The eggs were
batched in an incubator at 27°C. The dry diet was dispensed into scintillation vials (940

mg) for each treatment. Crude extracts or purified compounds were dissolved in DMSO

102

to give a concentration of 1250 pg '25 pL". Twenty-five pL of these stock solutions
were mixed thoroughly with the portions of dry diet. A 25 pL of DMSO was used as a
control. Agar solution (1.4%) was mixed and autoclaved for 5 min at 15 psi and 125°C to
melt the agar. This solution was held in a water bath at 45°C, and added to the dry diet
until the total diet weighed 5 g. The final concentration of test extracts and compounds
was 250 ppm. The wet diet was mixed thoroughly, and 34 drops of diet were dispensed
into 3.5 mL polystyrene vials. The freshly poured portions of diet were allowed to cool
and dry for at least 1 h. After drying, one neonate larvae was placed in each vial, and the
vials were capped. The treatment and control vials were held in a growth chamber at a
photoperiod of 16 h day and 8 h night with day temperature at 28°C and night
temperature at 24°C. Each treatment had 15 replicates. The treatments were arranged in a
completely randomized design. The larvae were weighed (mg) on a Mettler AE163
analytical balance (Mettler-Toledo, Inc., Hightstown, NJ) after six days. Statistical
analysis was conducted using AN OVA and the F test. Least Significant Difference (LSD)
was calculated for those treatments with significant F values, and used to compare
treatment mean weights with controls.

Mosquito Assay. First instar mosquito larvae (A. aegyptii L.) were provided by
Drs. Alexander Raikel and Alan Hays, Department of Entomology, Michigan State
University. The larvae were raised in 500 mL beakers containing 150-250 mL of reverse
osmosis water in an incubator at 26°C, for three days. Ten to 12 fourth instar larvae were
placed in 980 pL of reverse osmosis H20 in 12 x 75 mm borosilicate glass test tubes.
Crude extracts or purified compounds were dissolved in DMSO, unless otherwise stated,

to give a concentration of 250 pg '20 pL', unless otherwise stated. A 20 pL aliquot of

103

DMSO was used as a control. Treatments and controls were covered and left at room
temperature. There were three replicates per treatment. The number of dead larvae were
recorded at 2 h intervals, up to and including 24 h. Statistical analysis was conducted
using ANOVA and the F test. LSD was calculated and used to compare treatment means

with controls.

Preparative High Performance Liquid Chromatography

Active fractions were subjected to preliminary separation on a recycling
preparative HPLC Model LC-20 (Japan Analytical Industry, Dichrom, Santa Clara, CA)
with a 2-Jaigel-GS310-F columns (id. 20 x 300mm) connected in series. The mobile
phase, MeOHzH20 (90:10), was used under isocratic conditions at a flow rate of 5 mL '
min”. The samples were filtered through a 0.22 p filter (Millipore Corp, Bedford, MA),
prior to injection. A variable-wavelength UV detector S-310A Model II (Soma Optics,
LTD., Dichrom, Santa Clara, CA) was used to collect data at 210 nm. Fractions were
purified with two Jaigel S-343-15 ODS (15p) columns (id. 20 x 250 mm) connected in
series. The mobile phase, MeOHzH20 (70:30), was used under isocratic conditions at a
flow rate of 3 mL ' min". All fractions were dried under vacuum on a Buchii RE111

Rotovapor (Brinkman, Westbury, NY) at 45°C.

Purification of Ethyl Acetate Extract
A preliminary separation of the crude ethyl acetate extract from D. X cultorum
was performed by solvent-solvent partition. Crude ethyl acetate extract (1.09 g) was

dissolved in hexane (200 mL), and MeOH: H20 (90:10) (300 mL) was added to this

104

solution. The mixture was poured into a 1L separatory funnel and shaken well. The
hexane layer was removed, and the MeOHzH20 layer was extracted twice with hexane
(200 mL X2). The hexane extracts were combined and evaporated in vacuo at 45°C to
afford fraction I (395.2 mg). Similarly, removal of solvent in vacuo at 45°C gave a
MeOH:H20 (90: 10) soluble fraction II (703 mg).

Fraction II (2 g), accumulated from several solvent-solvent partitions, was
dissolved in MeOH:H20 (90:10) at a concentration of 45 mg/mL. This solution was
fractionated further on a sep-pak C13 cartridge (Millipore Corp., Bedford, MA) using
MeOH: H20 (90:10) as the eluting solvent. Four fractions (1.25 mL each) were collected
and removal of solvent afforded 1012.1, 174.1, 78.5, and 47.9 mg, respectively, for
fraction A-D. Following this, the sep-pak cartridge was eluted with 5 ml of MeOH. The
1 combined MeOH yielded fraction B (399.2 mg). Finally, the sep-pak cartridge was
stripped with CHC13 (2 mL) and the CHC13 evaporated to dryness to give fraction F
(156.1 mg).

Fractions A-D were combined based on their biological activity against corn
earworm (H. zea Boddie) to give fraction G (1.2 g). It was then dissolved in MeOHzH20
(90: 10) at a concentration of 205 mg/mL and purified by preparative HPLC on Jaigel
GS310-F columns. This yielded six fractions A-F weighing 21.8, 323.6, 621.5, 10.2, 0.6,
123.0 mg at retention times of 0-25, 25-28, 28-35, 35-38, 38-46, 46-110 min.,
respectively. Fraction C was recrystallized from MeOH and yielded compound 1 (10.1
mg). Compound 1 was characterized by ‘H and '3 C NMR experiments.

Compound l: 1H NMR (d6-DMS0): 8 3.34 (4H, dd, J=6 Hz,1-H X 2, 6-H X 2),

3.44 (2H,dt, J=3 Hz, 2-H, 5-H), 3.55 (2H, dd, J=10.5 Hz, 3-H, 4-H), 4.13 (2H, d, J=2.4

105

Hz, l-OH, 6-0H), 4.33 (2H, m, 2-0H, 5-0H), 4.41 (2H, d, J=2.4 Hz, 3-OH, 4-0H). l3C
NMR ((CD3)2SO): 6 63.92 (C-1, C-6), 69.78 (C-2, C-5), 71.43 (C-3, C-5). These values
were analogous to those reported in Breitrnaier et al. (1987) for D-mannitol.

Fraction F (K. 46-110) dissolved in MeOHzH20 (70:30) at a concentration of 40
mg/mL and purified using the Jaigel S-343-15 ODS column. Three injections yielded six
fractions I-Vl weighing 4.6, 23.9, 9.6, 49.7, 4.2, 1.7 mg. These were collected at retention
times 22-34, 34-37, 37-42, 42-47, 47-50, 50-90 min., respectively. Fraction II and VI
were purified further on the same column using MeOHzH20 (60:40) as the mobile phase
at a flow rate of 3 mL/ min. Compound 2 (13.9 mg) was obtained from the HPLC
purification of fraction II (R.. = 52 min). Compound 3 (25.6 mg) was obtained from the
HPLC purification of fraction VI (R.. = 57min). Compounds 2 and 3 were characterized
by 1H- and 13C NMR experiments.

Compound 2: 1H NMR (dt-DMSO): 8 6.85 (2H, d, J= 7.8 Hz, 3-H, 5-H), 7.85
(2H, d, J=7.8 Hz, 2-H, 6-H), 11.2 (1H, bs, 4-OH). 13C NMR (db-DMSO): 8 110.97 (c.2,
cs, 05, C-6), 127.50 (04), 157.23 (07). Both 1H and 13c NMR chemical shift values
were identical to the published spectral data for 4-hydroxybenzoic acid (The Aldrich
Library of 13c and 'H FT NMR Spectra, 1993).

Compound 3: 1H NMR (d6-DMSO): 8 3.72 (1H, s, 1-H), 6.67 (4H, d, J=8.7 Hz, 2-
H, 2’-H, 6-H, 6’-H), 7.74 (4H, d, J=8.4 Hz, 3-H, 3’-H, 5-H, 5’-H). 13C NMR (d6-DMS0):
5 55.64 (C-7), 114.17 (C-2, C-2’, C-3, C-3’, C-5, C-5’, C-6, 06’), 131.27 (C-l, C-l’),

159.03 (C4, 04’).

106

Methylation of Compound 3.

Diazomethane was prepared by dissolving 0.5 N-nitroso-N-methylurea in 10%
KOH solution (50 mL) with ether (50 mL) over an ice bath. The mixture was stirred
gently for a few minutes and poured into a 250 mL separatory funnel. The KOH solution
was removed and the diazomethane solution was then washed with 25 mL of ice cold
water. The diazomethane solution was poured into a brown bottle containing KOH pellets
and stored at -20°C. The diazomethane solution and a small amount of MeOH was added
to a sample of compound 3 (8 mg). The sample was capped, and allowed to sit in the
hood for 2 h. The ether was evaporated with N2 gas, and removed the MeOH in vacuo at
30°C.

Compound 4: 1H NMR (Ck-DMSO)! 8 3.72 (1H, s, 1-H), 3.84(6H, s), 6.67 (4H, d,

J=8.7 Hz, 2-H, 2’-H, 6-H, 6’-H), 7.74 (4H, d, J=8.4 Hz, 3-H, 3’-H, 5-H, S’-H).

Acetylation of Compound 4.
To compound 4 a solution of pyridine (2 mL) and acetic anhydride (200 pL) was
added. The clear solution was allowed to sit for two days in the dark. The solvent solution

was removed in vacuo at 50°C.
Compound 5: ‘H NMR (dé-DMSO): 8 2.35 (3H, s), 3.72 (1H, s, 1-H), 3.84(6H, s)
6.67 (4H, d, J=8.7 Hz, 2-H, 2’-H, 6-H, 6’-H), 7.74 (4H, d, J=8.4 Hz, 3-H, 3’-H, S-H, 5’-

H).

107

Results and Discussion

D. X cultorum flowers were extracted sequentially with hexane and ethyl acetate.
A preliminary bioassay performed on the ethyl acetate extract revealed that the average
weights of the treated caterpillars (H. zea Boddie) were significantly smaller than the
controls when tested at 250 ppm (p _<_ 0.01). After partitioning, the aqueous MeOH-
soluble fraction possessed a slightly higher level of activity than the hexane soluble
fraction on H. zea neonates when tested at 250 ppm (p 5 0.01). Therefore, the aqueous
MeOH-soluble fraction was chosen for further bioassay-directed purification.

Separation of the aqueous MeOH-soluble fraction was performed on a Ctg-sep-
pak cartridge to remove as much of the non-polar compounds fi'om this fraction as
possible. The fractions obtained from this separation (A-F) were tested against corn
earworm (H. zea Boddie) to determine their activity (Figure 5.1). All of the fractions
exhibited high activity. However, the MeOH- and CHCl3-eluted fractions showed slightly
less activity and were not studied further.

Compounds 2 and 3 did not show activity against either corn earworm neonates or
mosquito larvae when tested at 250 ppm concentration. Compound 1 was isolated from a
non-active fraction in crystalline form and was not expected to posses biological activity
against these organisms, and therefore was not tested.

The 1H NMR spectral data for compound 1 showed some interesting features. The
three peaks at 8 4.13, 4.33, and 4.41, integrated for 2 H each and exchanged with D20,
indicate a polyhydroxylated molecule. The peak at 8 3.34 integrated for 4 H was assigned
to an aliphatic CH2. The doublets at 8 3.55 indicated a CH adjacent to another CH

moiety, both bearing OH groups. The triplets 8 3.44 indicated a CH adjacent to a CH2

108

 

 

 

I Control

Ill Fractlon A
I Fractlon B
E Fractlon C
Fractlon D
a Fractlon E
l Fractlon F

 

 

 

 

 

 

Treatments

 

 

 

Figure 5.1. Average weights of H. zea Boddie larvae alter six days. Treatments were
tested at 250 ppm. p _< 0.01.

109

moiety, both bearing OH groups. The carbon data suggested the presence of three sets of
carbons appearing at 8 39.2, 69.78, and 71.43. This implied that compound 1 was
symmetrical. The NMR spectral data for Compounds 1 (Figure 5.2) were in agreement
with those values reported in the literature for D-mannitol (Brietrnaier, 1987).

Some features of the lH NMR spectral data for compound 2 (Figure 5.2) were the
peaks at 8 6.85 and 7.85, both integrating for 2 H with a coupling constant of 7.8 Hz.
This is typical of an AB system indicating para-substituents on an aromatic ring. The
peak at 81 1.2, with a downfield chemical shift value, was indicative of a desheilded
proton. Peaks in this region of a 1H spectrum were indicative of a phenolic or an acidic
proton. Both 1H and 13C NMR spectral data revealed that 2 is identical to the spectral data
of p-hydroxybenzoic acid.

Compound 3 (Figure 5.2) is reported as a synthetic product (Elliger, 1985).
However, its spectral data is not available in the literature. This is the first report of the
spectral characterization of this compound. The 1H NMR of pure compound 3 indicated
an AB system as evidenced by the doublets at 8 6.85 and 7.85 with a J value of 7.8 Hz.
Both of these peaks integrated for four protons each. This suggested symmetry in the
molecule that contains two aromatic rings. The singlet peak at 8 3.72 integrating for l-H
was assigned to a CH group. The fact that this peak was a singlet further supported that 3
is symmetrical.

Features of the ’3 C NMR spectral data that should be noted are the chemical shifts
of the three carbons bearing hydroxyl moieties at 8 55.6 for O7, and 8 159.03 for C-4

and C-4’. The downfield chemical shifts clearly indicated oxygen functionalities present

110

 

Ho————H
HO--——-H
H————0H
H———0H
Ctr—0H
on
coon

Figure 5.2. D-mannitol (1) and p-hydroxybenzoic acid (2).

111

Compound 3 R1 = R2 = H
Compound 4 R. = Me, R2 = H

Compound 5 R1 = Me, R2 = OCMe

Figure 5.3. Bis-(4-hydroxyphenyl)-methanol and its analogues.

112

at these positions. Furthermore, the peak at 8 114.17 representing eight identical carbon
atoms indicated that this molecule is symmetrical.

It was evident from the 1H-NMR of 3 that it contained a total of 3-0H groups, one
each at 4 and 4’ positions. The third hydroxy group was assigned to C-7. To confirm the
presence of the phenolic hydroxyls, 3 was methylated using CH2N2. The lH-NMR of the
methylated product, 4, gave a sharp singlet for 6 H and confirmed that the 4 and 4'
hydroxy moieties were methylated. Also, the 1H-NMR of 4 gave a singlet at 8 3.91
indicating the presence of the third hydroxyl group. In order to confirm the third hydroxy
group in 3, the dimethyl product, 4, was acetylated using acetic anhydride in pyridine.

The lH-NMR analysis of the acetylated product, 5, gave a singlet integrating for 3
H, confirming the presence of a hydroxy group at C-7. Therefore, compound 3 was
assigned the structure bis-(4-hydroxyphenyl)-methanol from the 1H and '3 C NMR
spectra of the parent compound and its derivatives.

This is the first report of D-mannitol (1), 4-hydroxybenzoic acid (2) and bis-(4-
hydroxyphenyl)methanol (3) from D. X cultorum flowers. Compound 2, 4-
hydroxybenzoic acid was isolated from D. venulosum Boiss (Mericli et. al, 1991). D-
mannitol (1) was isolated from D. flexiosum, D. elisabethae, D. tamarae, and D.
dzawachischvili (Dekanosidze et. a1, 1974). However, bis-(4-hydroxyphenyl)-methanol
(3) has not been reported from any natural source, until now. While the ethyl acetate
extract yielded new chemistry from this plant, further investigation is warranted to
identify those compounds responsible for its biological activity against corn earworm

neonates.

113

CHAPTER VI

Summary and Conclusions

Delphinium X cultorum cv. Magic Fountains flowers were investigated for
insecticidal, nematocidal, antimicrobial, and topoisomerase enzyme inhibitory assays.
Lyophilized and macerated D. X cultorum flowers were sequentially extracted with
hexane, ethyl acetate, and MeOH. The crude extracts were assayed at 250 ppm
concentrations against 4“1 instar mosquito larvae (A. aegyptii L. ), corn earworm (H. zea
Boddie) and gypsy moth (L. dispar L.) neonate larvae, Japanese beetle (P. japonica
Newman), Colorado potato beetle (L. declimenta Say), nematodes (C. elegans Dought)
and (P. redivivus Goody), microbial pathogens (A. parasiticus, C. albicans, E. coli, S.
epidermidis, S. aureus, F. moniliform, F. oxysporum, R. solanii), and a topoisomerase
enzyme inhibitory assay using S. cerevisiae strain 394, S. cerevisiae strain 394 Li, S.
cerevisiae strain 394 t2-5. Based on the results of these initial bioassays, the hexane and
ethyl acetate extracts were chosen for further purification.

The hexane extract showed mosquitocidal activity against fourth-instar A. aegyptii
larvae. A 50 ppm concentration gave 100% mortality in 2h. Also, it showed antifeedant
activity on corn earworm (H. zea Boddie) and gypsy moth (L. dispar L.) neonate larvae at
250 ppm concentrations. Similarly, the ethyl acetate extract showed antifeedant activity

against corn earworm neonate larvae (H. zea Boddie) at 250 ppm concentration.

114

The hexane extract from D. X cultorum flowers was separated using solvent-
solvent partitioning and purified by preparative TLC on silica gel plates. It yielded a
mosquitocidal fraction showing 100% mortality in 2 h at 1 ppm concentration. From this
fraction, 6 volatile, non-alkaloidal compounds were characterized using GC-EIMS
experiments. The compounds characterized were ethyl-methyl benzene, l- Isopentyl-
2,4,5-trimethylbenzenc, and 2-Penten-2-ylbenzoic acid, 2 isomers of 3-Butylidene-3H-
isobenzofuran-l-one and a novel derivative of acetophenone, 2-(Hex-3-ene-2-
one)phenylmethylketone. However, 2-(Hex-3 -ene-2-one)- phenylmethylketone was the
most abundant constituent of this fiaction. Due to the low yield of these compounds, we
were unable to determine which of these compounds was responsible for the
mosquitocidal activity observed. In addition, B-sitosterol was isolated from the hexane
extract was confirmed from its 1H and '3 C NMR spectral data.

In addition to the mosquitocidal fiaction, initial bioassays with corn earworm (H.
zea Boddie) showed that the hexane extract possessed antifeedant activity. Further,
investigation of the hexane extract is necessary to determine which of the reported
compounds is producing mosquitocidal activity, as well as separation of the active
fiaction to yield pure compounds with antifeedant activity.

The ethyl acetate extract was separated by solvent-solvent partitioning and
purification using a C13 sep-pak cartridge. This extract yielded a fraction, which
displayed corn earworm antifeedant activity at 250 ppm concentration. The active
fraction was purified using a preparative HPLC with size exclusion and reverse phase C13
columns. Two compounds were isolated from this fraction, 4-hydroxybenzoic acid and

bis-(4-hydroxypheny1)-methanol. Neither of these compounds displayed corn earworm

115

antifeedant or mosquitocidal activity when tested at 250 ppm concentration. In addition,
D-mannitol was also isolated from this extract.

This is the first report of the isolation of these compounds from D. X cultorum. D-
mannitol, 4-hydroxy benzoic acid, and B-sitosterol have been isolated from other
delphinium species such as D. felxiosum, D. elisabethae, D. tamarae, and D.
dzawachischilli; D. venulsoum; and D. ajacis and D. staphisagria, respectively. 3-
Butylidene-3H-isobenzofuran-l-one is a common constituent of celery, lovage and
angelica, but has never been isolated from delphinium. The compounds ethyl-methyl
benzene, l- Isopentyl-2, 4,5-trimethylbenzene, and 2-Pentenylbenzoic acid, and bis- (4-
hydroxyphenyl) methanol have been reported as synthetic products and have not been
isolated from plant tissues until now. Finally, 2-(Hex-3-ene-2-one)-phenyl methyl ketone
is a novel compound and has not been reported until now.

As stated previously, delphiniums are well known for their diterpenoid and
norditerpenoid alkaloidal constituents. The phytochemical research reported from
delphiniums reflects this fact. To the present time, delphinium research is still focusing
on the isolation, identification, and biological activities associated with these alkaloids. A
select few researchers have published new or novel chemistry from delphinium species,
other than alkaloids. The results presented in this thesis show that delphinium should be
considered as a potential source for novel compounds with biological activities that are

not related to alkaloids.

116

APPENDICES

Appendix I

SURVEY OF D. BELLAMOSUM FLOWER AND STEM, AND D. X CUL TORUM
STEM AND LEAF EXTRACTS FOR BIOLOGICAL ACTIVITY

This appendix briefly describes the extraction, biological screening and
preliminary fractionation of crude extracts from flowers, stems, and leaves of D. X
cultorum cv. Magic Fountains Dark Blue/ White Bee, Dark Blue Dark Bee, and White/
Dark Bee. These cultivars were grown as described in chapter IV. D. bellamosum plants
were donated by Walters Gardens, Michigan. The dormant plants were planted in Bacto
mix in clay pots and grown to maturity in the Pesticide Research Center Greenhouses at
Michigan State University. The plant material was lyophilized and processed in the same
manner described in chapter IV. The bioassays referred to in this section are described in
chapter IV. The procedure for extraction of the plant material is outlined in Scheme 4.1.

The hexane and MeOH extracts of D. bellamosum flowers and stems were tested
against mosquito larvae (A. aegyptii) and showed no activity. These extracts were
investigated primarily for potential mosquitocidal compounds.

The hexane extract from D. X cultorum stem showed antifeedant activity against
corn earworm (H. zea Boddie) and gypsy moth (L. dispar L.) neonates at 250 ppm
concentration (p 5 0.01), and mosquitocidal activity on fourth instar mosquito larvae (A.
aegyptii) at 250 ppm concentration in 1 h. The MeOH extract from D. X cultorum stem
showed 100 % mortality on fourth instar mosquito larvae (A. aegyptii L.) at 250 ppm

concentration in 4 h and showed antifeedant activity against gypsy moth (L. dispar L.)

117

neonates at 250 ppm concentration (p 5 0.01). However, it was not active on corn
earworm neonates at 250 ppm concentration. The MeOH extract from D. X cultorum
leaves showed antifeedant activity against gypsy moth neonates (L. dispar L.). However,
the hexane and methanol leaf extracts from D. X cultorum did not show any other
insecticidal activity. The hexane and MeOH extracts of the D. X cultorum. stem and leaf
showed no activity against the following organisms at 250 ppm concentration: nematodes
(P. redivivus Goody and C. elegans Dought),and microorganisms (A. parasiticus, C.
albicans, E. coli, S. epidermidis, S. aureus, F. moniliform, F. oxysporum, R. solanii, S.

cerevisiae strain 394, S. cerevisiae strain 394 t_,, S. cerevisiae strain 394 t 2_5).

Separation of the hexane-solublefiactionfi'om D. X cultorum flowers. The hexane-
soluble fraction from solvent -solvent partition of hexane extract from D. X cultorum
flowers as described in chapter IV, showed 100 % mortality on fourth instar mosquito
larvae at 2 h at 250 ppm concentration. This fraction (3.6 g) was purified using vacuum
liquid chromatography (V LC) followed by medium-pressure liquid chromatography
(MPLC) methods to yield seven fractions A-G weighing 38.8, 682.5, 289.0, 225.0, 26.7,
15.7, and 20.5 mg, respectively. Purification of fraction C (115.8 mg) by PTLC on silica
plates yielded seven fiactions A-G weighing 17.9, 18.6, 17.2, 2.1, 2.6, 0.5, 28.8 mg,
respectively. Fractions A, C, and G showed 100 % mortality at 25 ppm in 12, 2, and 4 h,
respectively. While these fractions appeared as only one spot in many TLC analyses, 1H

NMR showed them to be spectroscopically impure.

118

Purification of the Hexane Extract from D. X cultorum Stem. The crude hexane extract
(603 mg) was subjected to fractionation using MPLC on silica. Six fractions, A-F were
collected with weights of 83.2, 103.7, 65.2, 37.4, 39.1, and 136.9 mg, respectively. These
fractions were tested for biological activity against mosquito larvae (A. aegyptii L.) and
nematodes (C. elegans Dought). Fractions A-C showed 100 % mortality on fourth instar
mosquito larva at 100 ppm concentration after 4, 2, and 3 h, respectively. However, these
fractions were not active against C. elegans Dought.

The fraction C (37.8 mg) was purified using PTLC on silica plates. Four bands
were collected, A-D with weights of 21 .9, 10.7, 1.9, 5.5, respectively. Fraction B and C
showed 100 % mortality on fourth instar mosquito larvae at 50 ppm after 3 and 21 h,

respectively.

Separation of the Crude Ethyl Acetate Extract from the Stem. Lyophilized and macerated
stem material (105 g) was extracted sequentially with hexane and ethyl acetate, similar to
the flower material in Chapter V. Crude ethyl acetate extract from the stem (2.29g) was
subjected to solvent-solvent partitioning. As described in chapter V, two fractions yielded
from this separation, were a hexane-soluble fraction (1.26g) and MeOH:H20 (95:5)-
soluble fraction (1.00g). The aqueous MeOH (95:5)-soluble portion was eluted through
C18 sep-pak cartridge, similar to that of the ethyl acetate flower extract in chapter V. The
fraction eluted with MeOHzH20 (672 mg) was subjected to MPLC separation on silica.
Twenty-one fractions A-U were collected based on UV absorbance and they weighed 1.3,
4.3, 6.3, 6.6, 7.3, 21.5, 106.6, 139.2, 57.8, 202.3, 70.3, 72.0, 48.3, 39.0, 46.6, 39.5, 45.3,

5.3, 5.9, 8.7, 4.3 mg, respectively. Some of these fractions were tested against com

119

earworm (H. zea Boddie). Fractions E, G-J, 0 showed excellent antifeedant activity
against corn earworm neonates.

Time constraints and lack of plant material in some of these cases did not allow
the fractions described here to be investigated further for their active constituents. The
work outlined in this appendix indicates that there are additional active constituents in D.
X cultorum stem. Further investigation of the stem fractions to obtain pure and active
compounds, was not conducted, partly because this is not within the scope of this thesis

research.

120

Table A.l. Mosquitocidal assay of MPLC fractions.
Concentration and time at which 100 % mortality was reached in 24 h. n = no activity.

 

p 5 0.1.

Treatment 125 ppm
Fraction A n
Fraction B n
Fraction C 2h
Fraction D 16h
Fraction E n
Fraction F n

Fraction G n

 

121

Table A.2. Average Weight of Com Earworm (H. zea Boddie) after six days when

tested against MPLC fractions.
Treatments were tested at 250 ppm. * p 5 0.01, ns = not significant.

 

Treatment Average Weight (mg)
Control 8.17
Fraction A 8.53 ns
Fraction B 10.95 ns

Fraction G 0.0*
Fraction H 0.03*
Fraction I 0.07 *
Fraction J 003*
Fraction T 9.49 ns
Fraction U 9.34 ns
Control 6.61
Fraction E 2.04”“
Fraction 0 1.18*

 

122

APPENDIX II

RECIPES FOR MEDIA USED IN BIOASSAYS

Table A.3. YMG Agar Media Recipe.
YMG was used to grow Candida albicans.

 

Ingredient Amount
yeast extract 4 g ' L"
malt extract 10 g ' L"
dextrose 4 g ' L"
agar 18 g ' L"

 

Table A.4. Emmons Agar Media Recipe.
Emmons was used to grow, Escherichia coli, Staphylococcus epidermidis and
Streptococcus aureus.

 

Ingredient ' Amount
neopeptone 10 g ' L"
glucose 20 g ' L"
agar 18 g ' L"

 

Table A.5. YPDA Agar Media Recipe.
YPDA was used to grow Saccharomyces cerevisiae strain 394, S. cerevisiae strain 394 t,,,
and S. cerevisiae strain 3940.5-

 

Ingredient Amount
Bacto peptone 20 g ' L"
yeast extract 10 g ' L"
dextrose 20 g ' L"
agar 17 g ' L"
adenine sulfate solution

(0.5%) 1 mL - L"

 

123

Table A.6. PDA Agar Media Recipe.
PDA was used to grow Aspergillus parasiticus, F usarium moniliform, F usarium
oxysporum, and Rhizoctonia solanii.

Ingredient Amount

 

potato dextrose agar 29 g ° L"

 

To prepare agar plates, the ingredients of appropriate medias were weighed and
added to l-L Erlenmeyer flasks. Five-hundred mL of deionized water was added to the
flask, and the solution was mixed well. The flasks were stoppered with a foam plug and
the tops were covered with foil. The media were autoclaved for 20 min at 15 psi at 125°C.
After removal from the autoclave, the flasks were allowed to cool until they could be
handled. Twenty-five mL of agar solution was poured into petri plates in a laminar
airflow hood. The agar was allowed to cool and set. The plates were stored in plastic bags
at room temperature until firrther use.

Table A.7. NG Media Recipe.
NG media was used to culture Caenorhabditis elegans Dought nematodes.

 

Ingredient Amount
NaCl 3 g - L"
Bacto peptone 2.5 g ' L"
Agar 17 g ‘ L'I
Phosphate Buffer 25 mL ' L"
1M CaClZ 1 mL ‘ L'l
1M MgS04 1 mL - L"
cholesterol

5 mg/ mL EtOH lmL - L"

 

124

Table A. 8. Phosphate Buffer Recipe

 

lngedient Amount
KH2P04 11.968 g ' 88 mL" H20
K2HP04 2.088 g ‘ 12 mL" H20

 

Table A.9. Basal Media Recipe.
Basal-heme media was used to culture Panagrellus redivivus Goody.

 

Basal Media
Bacto Soytone 16 g ' L"
Yeast extract 12 g ' L"
Heme solution 8 mL

 

Basal media was mixed and autoclaved for 20 min at 15 psi and 125°C.

Heme solution was prepared by the following method:

In a 50 mL beaker, 0.75 g hemoglobin was mixed with 15 mL of freshly prepared 0.1 N
NaOH. This mixture was stirred on a magnetic stirrer at speed 4.5 for 1 h. After mixing,
this solution was sterile-filtered and added to the basal media. Three mL of this sterile
basal-heme media was dispensed into a sterile scintillation vial. Vials containing this

media were stored at 4°C.

125

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