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9.6 Oé LIBRARY
Michigan State
University

 

 

 

This is to certify that the
dissertation entitled

HEALTH-BENEFICIAL COMPOUNDS IN CORNUS FRUITS

presented by

SHAIJU KAKKANADAN VAREED

has been accepted towards fulfillment
of the requirements for the

PhD degree in HORTICULTURE

 

 

 

 

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HEALTH-BENEFICIAL COMPOUNDS IN CORN US FRUITS

Shaiju Kakkanadan Vareed

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

2005

ABSTRACT
HEALTH-BENEFICIAL COMPOUNDS IN CORNUS FRUITS
By
Shaiju Kakkanadan Vareed

The genus Camus, commonly known as dogwood, is widely distributed in eastern
Asia, and eastern and western parts of North America. Anecdotal reports indicate that
several plants in Camus species are effective for the treatment of various illnesses.
Although, Camus plants are well known for its medicinal properties, very little work has
been done on the isolation and identification of bioactive compounds. A bioassay-
directed investigation of Camus kausa, Camus mas, Camus cantraversa and Camus
alternifalia fruits resulted in the isolation and characterization antioxidant, anti-
inflammatory, anti-cancer and anti-diabetic anthocyanins, delphinidin 3-0-glucoside (1),
delphinidin 3-0-rutinoside (2), delphinidin 3-0-galactoside (3), cyanidin 3-0-galactoside
(4), pelargonidin 3-0-galactoside (5) and cyanidin 3-0-glucoside (6). Acid hydrolysis of
the anthocyanin- enriched fruit extracts resulted in the isolation of anthocyanidins,
delphinidin (7), cyanidin (8), pelargonidin (9), petunidin (10) and malvidin (11). The
anthocyanins in Camus fruits extracts were quantified by HPLC. The amount of
anthocyanins 1, 2 and 6 in C. alternifalia, C. controversa and C. mas were determined to
be 8-10 times higher than other common fruit sources of anthocyanins.

An investigation of the non-pigmented fraction of C. kausa ripened and
unripened fruits resulted in the isolation of ursolic acid (12), fl-sitosterol (13), cornin
(14), kaempherol 3-0-rhamnoside (15), myricetin 3-0-rhamnoside (16), and kaempherol

3-0-glucoside (17), and stenophyllin (18). Both ursolic acid (12) and ,B—sitosterol (13)

were also isolated from the ripened fruits of C. kausa, C. cantraversa and identified in C.
alternifalia.

Anthocyanins and anthocyanidins were tested for lipid peroxidation,
cyclooxygenase (COX-1 and-2) enzymes and tumor cell proliferation inhibitory
activities. Anthocyanins 1 and 2 inhibited lipid peroxidation by 71 and 68%,
respectively, at 50 ,ug/ml. Similarly, they inhibited COX-1 enzymes by 39 and 49% and
COX-2 enzyme by 54 and 48%, respectively, at 100 ,ug/mL. In addition, anthocyanins l
and 2 displayed 50% growth inhibition (ICso) at 21 and 38, 25 and 30, 50 and 76, 60 and
100, and 75 and 100 ,ug/mL, against HCT-116 (colon), MCF-7 (breast), NCI-H460
(lung), SF-268 (Central Nervous System, CNS), and AGS (stomach), human tumor cell
lines, respectively. The most active anthocyanidin malvidin (ll) inhibited colon, breast,
lung, central nervous system and stomach cell grth by 76, 75, 68, 41, and 69%,
respectively, at 200 pg/mL. Anthocyanins and anthocyanidins were also studied for their
ability to induce insulin secretion by rodent pancreatic ,B—cells in vitro. The results
indicated that anthocyanins 1 and 6 were the most effective insulin secretagogues among
the anthocyanins and anthocyanidins tested.

The bioassay guided investigation on Camus fiuits indicated that these plants
could be cultivated as alternate crop to tart cherries to yield fruits for health beneficial
anthocyanins. Ornamental plants in the United States are an untapped and valuable
resource for phytochemicals and functional foods. Our results on the health benefits of
Camus fruits suggest that Camus plants should be an ideal candidate for the

diversification of agricultural crops.

To my parents

ACKNOWLEDGEMENTS

It is a great pleasure and privilege to express my deep sense of gratitude and
profound indebtedness towards my advisor Dr. Muraleedharan G Nair for his valuable
guidance, direction, constructive criticism and inspiring encouragement to me, without
which it would have been impossible to complete the present work.

I express my sincere and heartfelt thanks to the members of my advisory
committee, Dr. Robert E. Schutzki, Dr. Gale M. Strasburg and Dr. Venugopal Gangur for
their valuable suggestions and encouragement. I also acknowledge all past and current
members of the Bioactive Natural Products and Phytoceuticals Laboratory, especially Dr.
Jayaprakasam Bolleddula, for their support and assistance. I would like to thank Dr.
Daniel Holmes and Piera Y. Giroux for their invaluable help with magnetic resonance
and gas chromatographic analyses.

Partial funding of this project was provided by the United States Department of

Agriculture (USDA, NRICGP) grant #2003-35504-13618.

Finally I would like to thank my family-my parents and sisters, a true blessing and
inspiration in my life-for their love, support and continual encouragement. I could not

have done this without you all.

TABLE OF CONTENTS

LIST OF TABLES ................................................................................. viii
LIST OF FIGURES AND SCHEMES ............................................................ ix
KEY TO ABBREVIATIONS ..................................................................... xi
INTRODUCTION .................................................................................... 1
CHAPTER ONE
LITERATURE REVIEW ............................................................................ 4
Introduction ................................................................................... 4
Botany of Camus Species ................................................................... 5
Chemistry of Camus Species ................................................................. 8
Biological Activity of Camus Species .................................................. 35
CHAPTER TWO

ANTHOCYANINS IN THE FRUITS OF CORN US ALT ERNIF OLIA.
CORNUS CONTROVERSA, CORNUS FLORIDA. CORNUS KOUSA.
CORNUSMASAND CORNUS OFFICINALIS... ......40

Abstract ...................................................................................... 40
Introduction ................................................................................... 41
Methods and Materials ....................................................................... 42
Extraction of Camus fruits for anthocyanin quantification. . . . . . . . . . .........43
Quantification of anthocyanins .................................................. 43
Isolation and characterization of anthocyanins ............................... 44
Results ....................................................................................... 46
Discussion .................................................................................... 48
CHAPTER THREE

ANTHOCYAN INS AND ANT HOCYANIDIN S IN CORN US ALT ERNIF OLIA,
CORNUS CONTROVERSA, CORNUS KOUSA, CORNUS FLORIDA, CORNUS

MAS AND CORN US OFFICINALIS FRUITS WITH HEALTH BENEFITS ............. 54
Abstract ..................................................................................... 54
Introduction ................................................................................. 56
Methods and Materials .................................................................... 59

Preparation of Anthocyanins ................................................... 60
Preparation of Anthocyanidins ................................................. 6O
HPLC analysis ..................................................................... 61
Lipid Peroxidation Inhibitory Assay ........................................... 61
Cyclooxygenase Inhibitory Assay .............................................. 62
Tumor Cell Proliferation Assay ................................................. 62
Insulin Secretion Studies ......................................................... 63
Radio Irnmuno Assay (RIA) ..................................................... 64

vi

Results ....................................................................................... 65
Discussion ................................................................................... 68

CHAPTER FOUR
FRUIT MATURITY, FLAVONOID AND ANTHOCYANIN PRODUCTION

Abstract ...................................................................................... 83

Introduction ................................................................................. 84

Methods and Materials ..................................................................... 86

Plant Material ...................................................................... 87

Extraction and Bioassay Guided Isolation of Compounds .................. 87

Results ....................................................................................... 89

Discussion ................................................................................... 90
CHAPTER FIVE

SUMMARY AND CONCLUSIONS ............................................................ 98

REFERENCES .................................................................................... 103

vii

LIST OF TABLES

Table 1.1 Classification of Camus plant ......................................................... 6
Table 1.2. Chemical Constituents found Camus Species ..................................... 11
Table 1.3. Common tannins in Camus species ................................................. 25
Table 1.4. Anthocyanins reported from Camus species ....................................... 33
Table 2.]. Concentration of anthocyanins 1-6 in Camus fruits .............................. 50

viii

LIST OF FIGURES AND SCHEMES

Figure 1.1. Terpenes found in Camus species ................................................... 9
Figure 1.2. Saponins found in Camus species ................................................. 20
Figure 1.3. Iridoids found in Camus species ................................................... 21
Figure 1.4. Sterols found in Camus species ................................................... 23
Figure 1.5. Flavanoids found in Camus species ............................................... 27
Figure 1.6. Furan derivatives found in C. afiicinalis... .28
Figure 1.7. Volatile compounds found in Camus species .................................... 28
Figure 1.8. Aliphatic esters found in Camus species ......................................... 29
Figure 1.9. Cytotoxic compounds found in Camus species ................................. 29
Figure 1.10. Aliphatic compounds found in Camus species ................................. 30
Figure 1.11. Fatty acids found in Camus species ............................................. 31
Figure 1.12. Anthocyanins found in Camus species .......................................... 32
Figure 2.1. Anthocyanins characterized from fruits of various Camus spp ................ 51
Figure 2.2. HPLC profiles of various Camus spp. fruits ................................. 52-53
Figure 3.1. Structures of anthocyanidins 1-6 and anthocyanidins 7-11 ..................... 72
Figure 3.2. Lipid peroxidation inhibitory activities of anthocyanins 1 and 2 ............... 73
Figure 3.3. COX-1 and COX-2 inhibitory activities of anthocyanins 1 and 2 ............ 74
Figure 3.4. In vitro cell proliferation inhibitory results of anthocyanins 1-5 ......... 75-77
Figure 3.5. In vitro cell proliferation inhibitory results of anthocyanidins 7-11 ...... 78-80
Figure 3.6. Insulin secretion activity of compounds 1, 6, 7 and 8 .......................... 81
Figure 3.7. Insulin secretion activity of compounds 4 and 9-11 ............................ 82

ix

Scheme 4.1. Diagrarnmatic representation of flavonoid biosynthesis pathway ........... 95
Figure 4.1. Structures of compounds 12-18 isolated from C. kausa fi'uits ................. 96

Scheme 4.2. Schematic representation of the compounds from C. kausa fruits ........... 97

BHA
BHT
BuOH
CHC13
CHzClz
CH3CN
cv
DMSO
D20
EIMS
EtOH
EtOAc
Gal

Glc
HCl
HCOOH
HOAc
HPLC
H20
H2804

MeOH

KEY TO ABBREVIATIONS

Butylated hydroxylanisole

Butylated hydroxytoluene

Butanol

Chloroforrn

Dichloromethane

Acetonitrile

Cultivar

Dimethyl sulfoxide

Deuterium oxide

Electron impact ionization mass spectrometry
Ethanol

Ethyl acetate

Galactose

Glucose

Hydrochloric acid

Formic acid

Acetic acid

High performance liquid chromatography
Water

Sulphuric acid

Methanol

xi

MPLC

MS

(EDS
PTLC
Si
spp.
TBHQ
TFA

TLC

Medium pressure liquid chromatography
Mass spectrometry

Nuclear magnetic resonance

Octadecyl silica

Preparative thin layer chromatography
Silica

Species

tert-Butylhydroquinone

Trifluroacetic acid

Thin layer chromatography

Ultraviolet

xii

INTRODUCTION

The genus Camus (dogwood) belongs to the family Comaceae that contains about
58 species (Fan and Xiang, 2001). These plants grow mainly in the northern temperate
regions of the world. The name “Camus” is the Latin name for Comelian cherry. The
word ‘cornu’ is for horn (cornu), and refers to the hardiness of its wood
(http://p1ants.usda.gov). Camus mas, Camus aflicinalz’s and Camus kausa bear edible
fruits that are consumed in many parts of Europe and Asia (Seeram et al., 2002, Du et al.,
1974). All Camus plants produce colorful and attractive flowers and fruits. They are
relatively resistant to pest infection compared to many other garden plants and hence are
widely grown as ornamental trees in many landscapes.

Although Camus plants are used only for decorative purposes in the United
States of America, these plants are used in traditional medicines around the world.
Many of the Camus species were reported to have medicinal. use and some were used as
an ingredient in preservatives and sweets (Bailey, 1977). For example, the extracts of C.
mas fruits were used for food and cosmetic preparations in Europe (Polinicencu et a1.,
1980). A decoction from the pulp of C. mas fruits was used for the treatment of arthritis,
fever and a wide range of other ailments (Millspaugh, 1974). The fruits of C. aflicinalis
were used for more than 2000 years in Chinese herbal medicine. C. aflicinalis was used
mainly to reduce menstrual bleeding and unusually active secretions including sweating,
excessive urine, spennatorrheoa, premature ejaculation and various illnesses associated

with liver and kidney (Kim and Kwak., 1998). The fruits of C. aflicinalz’s were also

reported to possess antibacterial, antifungal, hypotensive, antitumor, astringent and
diuretic activities (Kim and Kwak., 1998).

Anthocyanins are the most significant biologically active compounds reported
from Camus species fruits. The brilliant red colors of berries, cherries, vegetables and
fruits are due to anthocyanins. Recent in vitro and in vivo studies indicated that these
compounds possess antioxidant, anti-cancer and anti-inflammatory activities (Seeram et
al., 2002, Kang, et al., 2003, Kamei, et al., 1998). Therefore, the consumption of
anthocyanin-containing foods as dietary ingredient is considered to be highly beneficial
to maintaining health and feeling of wellness.

A detailed literature review of the phytochemicals in Camus spp. revealed that
most of the research has been on the isolation of compounds from C. aflicinalis. Very
little is known about the chemistry of other Camus plants and the biological activities of
the compounds present in them. Moreover, most of the health claims associated with
various Camus plants are anecdotal. Based on the previous research on C. mas in Dr.
Nair’s laboratory at Michigan State University, it is my hypothesis that fruits from native
Camus species have the potential to yield compounds with anti-carcinogenic, anti-
diabetic, anti-inflammatory, and antioxidant activities. In order to test my hypothesis, I
have conducted bioassay-directed isolation and characterization of compounds in Camus
fruits by using cylooxygenase enzymes, lipid peroxidation and cell proliferation
inhibitory and insulin secretion assays. Therefore, the objectives of my research were to
conduct bioassay- directed isolation and identification of compounds in Camus fi'uits
using chromatographic and spectral methods, and determine the anticarcinogenic, anti-

diabetic, anti-inflammatory, and antioxidant efficacies of purified compounds. By taking

into account of the biological activities associated with Camus plants, my proposed
research may lead to planting of Camus trees as an alternate crop for fruit production
with bioactive compounds in addition to its current application as an ornamental tree.
Also, it is expected that the present work should add to the existing knowledge on the
bioactive constituents in Camus fruits.

This dissertation is comprised of a series of chapters detailing the results of this
research. Chapter 1 is a literature review in which the botany, chemical constituents,
traditional use, and pharmacological importance of Camus plants are outlined. In
Chapter 2, the results of characterization and quantification of anthocyanins from C.
kausa, C. flarida, C. controversa and C. alternifolia fruits are presented. Detailed
investigation of insulin secretion, lipid peroxidation, cyclooxygenase, and cell
proliferation inhibitory activities of anthocyanins and anthocyanidins in Camus spp.
fruits are presented in Chapter 3. All non-pigmented compounds isolated from the
unripened and ripened fi'uits of C. kausa are presented in Chapter 4. The data in
Chapter 2 has been accepted for publication in Life Sciences. The data in Chapter 3
were published in Journal of Agricultural and Food Chemistry and Life Sciences.
Chapters 2, 3 and 4 are presented as manuscripts, each with an introduction, material
and methods, results and discussion sections. Finally, the conclusions derived from my

research on Camus fruits are summarized in Chapter 5.

CHAPTER ONE

LITERATURE REVIEW

Introduction

Humankind has benefited from the inherent qualities of over 5000 plants used for
drugs, foods, fibers and dyes, which include more than 14 Camus species and their
varieties. The genus Camus (dogwood) contains about 58 species, mostly
hermaphroditic shrubs or small trees, and is widely grown in North America, Asia,
Europe, South America and Africa (Fan and Xiang, 2001). Many Camus spp. with
medicinal values are used as one of the components in the preparation of preservatives
and sweets (Bailey, 1977). The tree bark of Camus altemifalia and Camus florida has
been chewed by many to release analgesic compounds to treat headaches, toothaches, and
other pains (Moerman, 1998). The bunchberry dogwoods, Camus canadensis, possess
anti-convulsive, anti-fever and analgesic properties in addition to a number of other
medicinal uses. Other species such as Camus rugasa, Camus racemasa, Comusfoemina
and Camus amamum have been used for the treatment of various illnesses associated
with kidney, stomach, throat, and lungs, respectively (Moerman, 1998). Many
ethnobotanical uses of dogwood species in China have also been reported
(www4.ncsu.edu). For example, Camus oflicinalis, "Zhu Yu" or "Zao Pi" in Chinese
medicine, was used as an astringent tonic for impotence, and to treat spennatorrhea,
lumbago, vertigo, and night sweats. Fruits of Camus oblonga have been used as a

substitute for 'Zao Pi.’ Seed oil of several dogwood species including C. oblonga,

Camus alba, Camus hemsleyi, Camus walteri, and Camus wilsoniana have used
commercially in various parts of China. The bark from C. oblonga and Camus capitata
were also used as folk medicines to treat arthritis and injuries. In Japan, several species
of Camus including Camus cantroversa were used to treat the swelling in the body
(www4.ncsu.edu). The fruits of several subspecies of Camus hongkongensis are edible
and used for wine brewing.

Dogwoods carry attractive flowers and fi'uits. They are popular ornamental plants in
a variety of landscapes (Powell, 1997). The dogwood plants beautifully display a wide
range of color in their bracts, foliage and twigs during various seasons of the year.
Generally, dogwood plants grow up to a height of 20 — 30 feet and require little
maintenance.

The flowering dogwood, Camus flarida, is the most popular dogwood in the United
States (http://www.ces.ncsu.edu/fletcher/stafflrbir/comus.html). It is native to the eastern
and central United States. C. flarida flower represents the state flower of North Carolina.
Dogwood industry in Tennessee yields more than $30 million annually to the state
income from the sale of C. florida cultivars (www4.ncsu.edu).

The botany, chemical constituents, and biological activities of Camus plants are

outlined in this chapter.

Botany of Camus plants
The Camus species are deciduous shrubs and occasionally grow as trees. All

Camus species have opposite leaf system except Camus altemifalia, which have an

alternate leaf system. The fruits of all Camus species are berry-like with white, blue, red

or black drupe with one or two seeds.

Dogwood is a member of the genus Camus and it belongs to the family

Comaceae. The family Comaceae has three genera: Camus, Aucuba and Helwingia.

Table 1.1 Classification of Camus plant

Kingdom
Phylum
Subkingdom
Superdivision
Division
Class
Subclass
Order
Family

Genus

Plantae
Embryophyta
Tacheobionta

Sperrnatophyte
Magnoliophyta
Magnoliopsida
Rosidae
Comeals
Comaceae

Camus. L

Bir et al., classified dogwoods according to the season in which they are most

attractive. For example, C. alba (Tartarian dogwood) and C. sericea (Redosier dogwood)

are considered winter dogwoods, which are generally grown for their colorful stems

(http://www.ces.ncsu.edu/fletcher/staff/rbir/cornus.htrnl). Stem color of C. alba and C.

sericea change from creamy yellow, orange and shades of red to deep red during winter.
The spring dogwood, C. flarida and C. kausa (Oriental dogwood) display attractive and
colorful bracts and foliage depending on the cultivar during the spring season.

Among dogwoods, C. florida, C. kausa, and C. nuttallii (Pacific dogwood) are the
three most widely cultivated species in the United States. More than 100 cultivars of C.
flarida have been cultivated in United States (www4.ncsu.edu). Another dogwood, C.
kausa, native to eastern Asia, is a smaller tree with white or pink bracts and dark green
foliage late in the spring season. Popular cultivars of C. kausa include 'Greensleeves,‘
'Milky Way,‘ 'Blue Shadow', 'Rosabellla,' 'Rubra,’ and 'Rosea'. The fi'uits of C. kausa are
red and compound, in contrast to those of C. flarida and C. nuttallii, which are clusters.
The fruits from C. kausa are edible and sweet, and used in wine making in several Asian
countries (Du et al., 1974). C. nuttallii, grows mainly in western regions of North
America. The numbers of C. nuttallii cultivars are much fewer than that of either C.
florida or C. kausa. However, it is still valued as an ornamental dogwood due to its
large, colorful bracts. Hybrid varieties of Camus species are common in many parts of
United States especially in Michigan (www4.ncsu.edu). The natural hybrids of C.
amomum x C. racemasa (C. x amoldiana Rehder) and C. rugosa x C. stalam'fera (C. x
slavinii) are frequently found in Michigan and neighboring states. Wagner reported
another hybrid between C. racemasa (gray dogwood) and C. rugosa (round-leaved
dogwood) from Michigan (Wagner, 1990). A number of arboreta or botanical gardens

have several examples of natural hybrids of other Camus species.

Chemical Constituents of Camus Species

A detailed phytochemical investigation of plants belonging to Camus spp. has
resulted in a diverse group of secondary metabolites. Compounds reported from Camus
spp. to date are summarized in Table 1.2. The secondary metabolites reported from
Camus are divided in to six groups based on structural similarities. These groups include

terpenes, steroids, saponins, iridoids, tannins, flavanoids and related compounds.

Terpenes

Terpenes are important group of plant secondary metabolites and ubiquitous in
several plants. Many of these terpenoids such as menthol, pinene, limonene and camphor
impart characteristic odors and flavors unique to a given plant. The building block of a
terpenoid is isoprene (CH2=C-(CH3)-CH=CH2). Monoterpene, sesquiterpene, diterpene,
triterpene, and tetraterpene are some of the classifications depending on the number of
isoprene units in its molecule. The terpenes reported from Camus species are represented
in Table 1.2.

Most of the reported terpenes in Camus were isolated from its stems. Yan et al.,
developed an HPLC method for the determination of ursolic acid, an important
constituent of Chinese herb, C. aflicinalis, using Hypersil ODS- column with acetic acid-
water-glacial acetic acid mobile system (Yan et al., 2003). This analytical method
showed high accuracy and reproducibility and is employed for the quality control of
various herbal medicines in China. Another terpenoid compound, betulinic acid, was
isolated from the dry roots and bark of C. macrophylla (V enketesh and Merchant, 1984).

Betulinic acid and ursolic acid were also isolated from the ether fraction of dry roots of

C. excelsa (Dominguez et al., 1981). A pentacyclic trihydroxy triterpenic acid, arjunolic
acid, was isolated from the stem of C. capitata and exhibited potent insecticidal activity
(Bhakuni et al., 2002). This terpenoid was isolated from the hexane extract using silica
column and characterized by NMR and mass spectroscopic methods. A detailed
phytochemical investigation of the stems of C. capitata afforded the terpenoid, 3 B-
acetoxy-23-oxo-lup-20 (29)-ene or 3B-acetoxy-24-oxo-lup-20 (29)-ene, in addition to

oleanolic and acetyl oleanolic acids (Bhakuni et al., 1988).

 

Ursolic acid Oleanolic acid

   

R=CH20H Betulin R1=H, R2=CH20H Arjunolic acid

R==COOH Betulinic acid R1=H, R2=CH3 Maslinic acid

Figure 1.1 Terpenes found in Camus species

 

R=OH, 3 ,6 Hydroxy-23-oxo-lup-20 (29)-ene (Lupeol)

R=OAc, 3 ,6 Acetoxy-23-oxo-lup-20 (29)-ene

Figure 1.1. (cont’d). Terpenes found in Camus species

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18

Saponins

Saponins are glycosides of sapogenins. The sapogenin can be a steroid (C-29) or a
triterpene (C-30) and the sugar can be glucose, galactose, pentose, or methylpentose.
They are considered as natural surfactants, or detergents. Some saponins are lmown to
reduce the feed intake and growth rate of nonruminant animals while others are not very
harmful. For example, the saponins found in oats and spinach are implicated to
accelerate the body's ability to absorb calcium and silicon, thus assisting in digestion. On
the other hand, certain pasture weeds contain substantial quantities of dangerous saponins
that are toxic to several animal species. Saponins bind with cholesterol so it cannot be
reabsorbed into the system and is excreted from the body. Saponins are being widely
researched for cancer prevention and cholesterol control. The saponins reported from
Camus species are represented in Table 1.2.

Hostettmann, et al., reported two molluscicidal saponins from C. florida
(Hostettmann, 1978). These compounds were isolated from the methanol extract of the
bark of the plant and characterized as sarsapogenin O-B-D-xylopyranosyH 1—>2)- B-D-
galactopyranoside and sarsapogenin O-B-D-glucopyranosyH1—)2)-B-D-
galactopyranoside by spectroscopic and chemical methods. Another saponin,
arjunglucoside was isolated from C. controversa (Jang et al., 1998). Sequential
extraction of dry roots of C. excelsa with ether and methanol afforded a serious of
phytochemicals including sarsaponin-O-galactosylxyloside (filiferin) (Domoinguez,

1981). Saponins fi‘om various Camus spp. are represented in Fig. 1.2

19

  

l-DH o
HOWW
“9W ”
H

sarsaponin—O-galactosylxyloside (filiferin)

 

arjunglucoside

Figure 1.2 Saponins found in Camus species

Iridoids and Iridoid glycosides

Iridoids are cyclopetanoid monoterpene secondary metabolites of plants. Based
on chemical structures they are classified into iridoid glycoside, nonglycosidic
(aglycones) iridoids, secoiridoids, and bisoridoids. A detailed literature search revealed

that plants in the genus Camus are also potential sources of iridoids and its glucosides.

20

Many iridoids reported from Camus spp. are specifically from C. capitata. The iridoids
detected in Camus spp. are shown in Table 1.2.

J enson et al., characterized two glycosides, cornin and phlorin, from the leaves
and twigs of C. capitata (J enson et al., 1973). Cornin was reported from C. kausa as well
(J enson at al., 1973). The chemical structures of these iridoids were determined by NMR
and mass spectroscopic methods. The iridoid glycoside, dehydromorroniaglycone, along
with several other iridoids, loganin, 7-dehydrologanin, 7-0—methylmorronisde, was
reported fi'om C. afiicinalis (Li et al., 1999). Zhang et a1, developed an HPLC method by
using Kromasil column with 0.05 M NaHzPO4zacetonitrile (6.4:1) isocratic mobile
system, for the simultaneous determination of morroniside and loganin in C. afi‘icinalis

(Zhang et al., 1999). The iridoids found in Camus spp. are represented in Fig 1.3.

0,
”ohm
0,.

   

R=H Morroniside Sweroside

R=CH3 7-0-methyl morroniside

Figure 1.3 Iridoids found in Camus species

21

O O\ O O\
R CH
H 1 HO H 3

C \ C \
HO O H O O

HOH ori‘ HOH H

O O O
H H
93% 83%

H H H H
R1=OH Monotropein Scandoside methyl ester
R1=CH3 Galioside

O O\
CH
HO H 3
C \
O
H H
O O
H
90 o
H H
Dihydrocornin

 

O
H H

7-Dehydrologanin

 

Figure 1.3 (cont’d). Iridoids found in Camus species

22

Sterols

Phytosterols produce a. wide spectrum of biological activities in plants and
animals. In the natural state, they are bound to the fibers of the plant and for this reason,
they are difficult to desorb fiom the fibers. Seeds are the richest source of the sterols and
sterolins. Many animal and human studies showed that phytosterols reduce serum or
plasma cholesterol and low density lipoprotein (LDL) cholesterol levels (Ling and Jones,
1995). The major sterols reported from Camus spp. are shown in Table 1.2.

B-sitosterol and stigmasterol are the two major sterols reported from Camus spp.
Among these sterols, B-sitosterol is very common and present in many Camus spp.,

including C. aflicinalis, C. capitata, C. sangum'ea and C. flarida ( Xu et al., 1995,

Robertson et al., 1939, Yamahara et al., 1981).

 

fl-sitosterol stigmasterol

Figure 1.4 Sterols found in Camus species
Tannins
Tannins are naturally occurring plant polyphenols. They are classified into
hydrolysable and condensed tannins (proanthocyanidins). In hydrolyzable tannins, gallic
acid or ellagic acid is bonded directly to sugar units and is susceptible to hydrolysis. On

the other hand, condensed tannins are oligomers or polymers of flavonoid units and are

23

not susceptible to hydrolysis. Condensed tannins are more widely distributed than
hydrolysable tannins. The tannins found in various Camus plants are represented in
Table 1.2.

Many condensed tannins have been isolated from Camus spp. C. capitata, an
ornamental plant, decorating the mountainsides of Himalayas, is considered as a source
of tannins (Bhakuni et al., 2002). Another species, C. cantraversa, is reported to be a
good source of gallotannins (Lee et al., 1995, Nakaoki and Morita, 1958). A series of
substituted gallotannins including gallic acid, 1-0-galloyl-B-D-glucose, 1,6-di-0-galloyl-
fl-D-glucose, 1,2,3-tri-0-galloyl-B-D-glucose, 1,2,6-tri-0-galloyl-B-D-glucose, 3,4,6—tri-
O-galloyl-B-D-glucose, eugeniin, and gemin D were isolated from the aqueous extract of
the leaves of C. cantraversa. The tannins found in C. cantraversa are represented in

Table 1.3

24

 

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Flavanoids

F lavonoids are polyphenolic compounds that are ubiquitous in plants. They are
grouped into flavonols, flavones, flavanones, isoflavones, catechins, anthocyanins and
chalcones. More than 4,000 flavonoids have been identified, many of which occur in
fruits, vegetables, tea, coffee, and also contain in beverages such as beer, wine and fruit
drinks. The flavonoids have aroused considerable interest recently because of their
beneficial effects to human health. They are known to possess antiviral, anti-allergic,
anti-platelet, anti-inflammatory, anti-tumor and antioxidant activities (Harbone and
Williams, 2001, Conklin, 2000, Kim etal., 2004)

The flavonoids, kaempferol 3-0—galactoside and quercetin 3-0—galactoside, have
been reported from the bracts and leaves of C. flarida (Mudry and Schilling, 1983). It is
known that bracts have higher amount of flavonoids compared to leaves, which indicated
that the role of flavonoids in these plants as pollinator attractants. Quercetin 3-0-
galactoside was reported from the bark of red-osier dogwood, C. stalam'fera (Sterrnitz
and Krull, 1998). Grigorescu et al., identified quercetin 3-0-rhamnosyl glucoside and
kaempferol 3-0- rhamnosyl glucoside from the flowers of C. mas from Rumania
(Grigorescu, 1972). Common flavonoids isolated from Camus species represented in

Fig.1.5

26

 

R1=H R2=ga1 Kaempferol 3-0— galactoside

R1=H R2=glu Kaempferol 3-0— glucoside

R1=H R2: ara Kaempferol 3-0- arabinoside

R1=H R2=glu—>rha Kaempferol 3-0— glucosyl rhamnoside
R1=H R2=H Kaempferol

R1=OH R2=gal Quercetin 3-0-galactoside

R1=OH R2=glu Quercetin 3-0-glucoside

R1=OH R2: ara Quercetin 3-0-arabinoside

R1=OH R2=g1u—>rha Quercetin 3-0- glucosyl rhamnoside
R1=OH R2=glu Quercetin

Figure 1.5 Flavanoids found in Camus species

Kim et al., reported a new furan derivative, Di-methyl-tetrahydrofuran-Z, 5

dicarboxylate along with a known compound, S-hydroxy methyl furfural from the fi'uits

27

of C. aflicinalis (Kim and Kwak, 1998). Furan derivatives isolated from C. aflicinalis are

given below (Fig 1.6).

O
H OH
H3C OCH3
/ \ O
O O O
S-hydroxy methyl furfural Di-methyl-tetrahydrofuran-Z,5dicarboxylate

Figure 1.6 Furan derivatives found in C. aflicinalz‘s

Several volatile flavor compounds have been isolated from the fruits of C.
aflicinalis by gas chromatographic method (Mayazawa and Kameoka, 1989). The major
volatile compounds reported from C. aflicinalis were benzyl cinnamate, isobutyl alcohol,

isoamyl alcohol, furfural, phenethyl alcohol, methyl eugenol, isoasarone and elemicin.

W0

Benzyl cinnarnate R1= R2=H Methyl eugenol
R1=OCH3, R2=H Elemicin

h...

Phenethyl alcohol

 

Figure 1.7 Representative Volatile compounds found in Camus species

28

Two lipid-soluble compounds, ethyl octadecanoate and ethyl heptadecanoate were
reported from Camus cervi (Long et al., 1991) (Fig. 1.8). These compounds were
isolated from C. cervi by chromatographic methods and characterized by GC-MS, IR,

and NMR spectroscopic methods.
0

Ethyl octadecanoate

3

O

CH
HacAO/lKAMMAA/ 3

Ethyl heptadecanoate
Figure 1.8 Aliphatic esters found in Camus species

Dominguez, et al., reported the isolation of 7-hydroxycadelene from C. excelsa
roots (Dominguez et al., 1981). It was isolated from the methanolic extract by using
normal phase chromatography and characterized by NMR spectroscopy.

Scopoletin and a cytotoxic constituent halleridone were isolated from the Korean
and Japanese dogwood, C. cantraversa (Nishino et al., 1988) (Fig 1.9). These
compounds were isolated fiom the methanolic extract of leaves, purified by silica gel

column chromatography, and characterized by NMR and mass spectroscopic methods.

OH
HO 0 o ‘
3 \O / O H
Scopoletin Helleridone

Figure 1.9 Cytotoxic compounds found in Camus species

29

An anti-inflammatory agent, n-hentriacontane, a plant growth promoter,
triacontanol, triacontanoic acid, tetracosanoic acid, pholoroglucinol and gallic acid were
isolated from the stem of C. capitata and characterized by chemical, NMR and mass

spectroscopic methods (Yamahara et al., 1981 and Bhakuni et al., 2002).

CH2
CH3/( )r\ R
R=CH3, r = 29 n-Hentn'acontane
=OH, r = 29 n-Triacontanol

R=COOH, r = 28 n-Triacontanoic acid

R=COOH, r = 22 n-Tetraconsanoic acid

Figure 1.10 Aliphatic compounds found in Camus species

Gas chromatography has been used to analyze various long chain fatty acids,

alcohols, aldehydes and alkenes from flowers and bracts of C. flarida and C. sanguinea

(Delaveau and Paris, 1961). Most of these compounds were unbranched lipophilic

constituents such as lauric, palrnitic, stearic, oleic, linoleic and linolenic acids.

30

bracts of C. flarida (Sando, 1926).

lauric acid
pahnitic acid
H00C/N\//\V/\\/”\v/\\/A\¢/\\/A\//

stearic acid

oleic acid
linoleic acid

linolenic acid

Figure 1.11 Fatty acids found in Camus species

Inositol and scyllitol were reported from the alcoholic extracts of flowers and

pentose phosphate pathways that are responsible for the production of various

compounds including glucose and glucuronolactone.

Anthocyanins

Inositol is involved in the glucuronic acid and

Anthocyanins constitute one of the important groups of biologically active

natural pigments. They are water soluble compounds responsible for the colors of many

fruits and vegetables. Even though they are common in fi'uits and flowers, they may also

31

occur in roots, stems, leaves and bracts. Aglycons of anthocyanins are known as
anthocyanidins. Delphinidin, cyanidin, pelargonidin, petunidin, peonidin and malvidin
are the common anthocyanidins which occur in nature. The sugars that are present
include glucose, galactose, rhamnose, and arabinose (Mazza and Miniati, 2000). The
sugars provide additional sites for modification as they may be acylated with acids such
as p-coumaric, caffeic, ferulic, sinapic, acetic, malonic or p-hydroxybenzoic acid.
Because of the diversity of glycosylation and acylation, there are at least 300 naturally
occurring anthocyanins. The color and stability of anthocyanins in solution depends on

pH. Anthocyanins found in Camus spp. are represented in Table 1.4.

R1
OH
+ O
HO 0\ R2

/ O-glycoside
OH
R1 R2

Delphinidin OH OH
Cyanidin OH H
Pelargonidin H H
Malvidin OCH3 OCH3
Peonidin OCH3 OH

Figure 1.12 Anthocyanins found in Camus species.

32

 

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34

Biological activities of Camus species

The genus Camus is a rich source of biologically active compounds. Several
species in this genus have been used in traditional medicines in many Asian countries.
For example, C. aflicinalis and C. cantraversa have been used in Chinese and Korean
traditional medicines. Numerous bioactive anthocyanins, flavanoids, terpenes, saponins,
iridoids and tannins have been isolated from several of Camus species. The fermented C.
mas fi'uits have been used as a beverage in Turkey (Millspaugh, 1974) and the extracts of
C. mas fruits are used in European cosmetics. The pulp of C. mas was processed for the
treatment of arthritis, fever and a wide range of other ailments. Also, it was used for the
treatment of senility, lumbago, diabetes, cystitis and tinnitus. Limonene and kaurene
were the important odorants found in C. mas (Seeram et a1, 2002). An important Chinese
herb, C. aflicinalis, was used to reduce active secretions including copious sweating,
excessive urine, spennatorrheoa and premature ejaculation, etc. It was also used for the
treatment of various illness associated with kidney and liver. The hits were reported to
possess antibacterial, antifungal, hypotensive, antitumor, astringent and diuretic activities
(Post etal., 1995).

Anthocyanins are one of the major biologically active compounds found in all
Camus species. As in the case of cherries and berries, fruits of Camus species contain
substantial quantities of anthocyanins. Anthocyanins have several beneficial roles in
plants and animals. They protect the plants from the harmful effect of UV radiation
(Burger and Edward, 1996). They enhances fertilization and seed dispersal through
birds and other organisms. Anthocyanins were reported to possess a wide range of

biological activities including antioxidant, anti-inflammatory, anti-cancer and anti-

35

diabetic activities (Wang et al., 1998; Kamei et al., 1995). Therefore, the use of
anthocyanin containing foods as part of diet may be beneficial to human health. This has
resulted in the phytoceutical and botanical supplement industries investigating fruits that
have a high content of anthocyanin for purposes of formulating new commercial
products. Anthocyanins are important ingredients in several herbal folk medicines which

have been used in 12th century to induce menstruation (N ahaishi, 2000).

Antioxidant Activity

Galloylglucosides from the roots of C. capitata were analyzed for antioxidant and
radical scavenging activities (Tanaka et al., 2000). The antioxidant activity of
galloylglucosides was higher than that of tannic acid, used as a common medicine.
Among various extracts used for study, the methanolic extract of C. capitata root showed
highest anti-oxidant activity. These results suggested the existence of polyphenols or
secondary metabolites with strong antioxidant activity in the extracts of Camus species.
The radical scavenging activity of the root extract of C. capitata was found to increase
with the polyphenols concentration. This revealed that polyphenols including
galloylglucose was responsible for the radical scavenging activity in C. capitata. The
phytochemical investigation of polyphenol content of eight Camus species indicated that
mono-galloylglucose, ,B-glucogallin, was the major polyphenol. Similarly C. capitata
leaves contained large amounts (1.46% as dry weight) of hydrolysable tannin l, 2, 3, 4, 6-
penta-O-galloyl-fl-D-glucose. The level of this tannin was 2-10 times higher in C.
capitata than in other Camus species (Tanaka et al., 1998). The methanolic extracts of

100 medicinal plants were screened for antioxidant activity using Fenton’s reagent/ethyl

36

linoleate system as well as for free radical scavenging activity using the 1,1,—diphenyl—2-
picryl hydrazyl (DPPH) free radical generating system and showed that C. afiicinalis
fruits extract has highest activity (Kim et a1, 1997). The methanolic extracts of the peel
and seeds of C. oflicinalis were also analyzed by oven test methods in soybean oil and
lard, for antioxidant properties. Antioxidant activities of both of these extracts were
comparable to common synthetic antioxidants, BHA, BHT and TBHQ. Further studies
of the methanol extract indicated that gallic acid and methyl gallate were responsible for

the antioxidant activity (Sahng et al., 1990).

Antimicrobial Activity

The methanolic extracts of C. aflicz’nalis fruits inhibited the growth of Bacillus
dysentriae, Staphalacacci aureus and Escherichia cali (Mau et al., 2001). An extract
mixture, prepared by mixing equal volumes of methanolic extracts of three Chinese
herbs, C. aflicinalis, Cinnamammum cassia and Chinese chive, showed good
antimicrobial activity against common food borne microorganisms. It inhibited the
growth of E. cali and showed excellent stability to heat, pH and storage conditions (Mau
et al., 2001). Among 21 methanolic extracts of Korean medicinal plants studied for
antimicrobial activity, the extract from C. aflicinalis was the most effective. The
compound from the active fraction identified was ursolic acid (Kim et al., 1996). The
extracts from the fruits of C. drummandii also showed good antimicrobial activity when

tested on E. cali, S. aureus and B. psychraphilus (Post et al., 1995).

37

Antidiabetic Activity

C. aflicinalis is an important medicinal plant used for the antidiabetic
preparations in China. Twelve Chinese herbal drugs used for the treatment of type H
diabetics contained the extracts of C. aflicinalis (Li et al., 1999). Also, the methanolic
extract enhanced the proliferations of islets and increased post prandial secretions of
insulin (Quian et al., 2001). The ether extract of C. aflicinalis also showed antidiabetic
activity against experimental rats with type-1 diabetes induced by streptozotocin.
Fractionation of the ether extract revealed that the activity was due to ursolic acid (Kim

and Oh, 1999).

Anticancer activity

Anthocyanins in fruits and vegetables exhibited a wide spectrum of anticancer
activity. Tart cherry anthocyanins inhibited the growth of human colon cancer cell lines
HT-29 in vitro (Kang, et al., 2003). The anthocyanin fraction from red wine have been
reported to suppress the growth of HCT-l 15 cells, which were derived from human colon
cancer or AGS cells from human gastric cancer (Kamei, et al., 1998).

Many Camus seeds have substantial amount of oil and are used commercially in
China. Camus oil could be a good vegetable oil for patients with coronary heart
diseases. It attenuated aortic atherosclerotic lesions in rabbits and reduced cholesterol
accumulations in the intima of aorta (Huangfu et al., 1984). The triterpene, arjunolic
acid, isolated from the stem of C. capitata was tested for insect growth and as antifeedant

by using 4 ‘h instar larvae of Spliarctz‘a oblique. The effective concentration (ECso)

38

observed to inhibit 50% feeding and growth inhibition was 618 and 667 ppm,
respectively (Bhakuni et al., 2002).

Even though several compounds and their biological activities were reported, many
more is hidden among various Camus spp., especially C. kausa, C. cantraversa, and C.
altemifalia. It is my objective to investigate the therapeutic potential of the compounds
from Michigan grown Camus fruits. I am also looking for the potentials of Camus
plants as an alternative crop in Michigan so that it could generate additional income to
Camus growers and improve the economy of the State of Michigan. My studies will be
focused on the phytochemicals in ripened and unripened Camus fruits for bioactive
compounds. Therefore, the present study deals with the isolation, structure elucidation

and biological activities of phytochemicals in C. kausa, C. cantraversa and C. altemifalia

fruits.

39

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CHAPTER TWO

ANTHOCYANINS IN CORNUS ALTERNIFOLIA, CORNUS CONTROVERSA,

CORNUS KOUSA AND CORNUS FLORIDA FRUITS.

Abstract

The anthocyanins in Camus altemifalia, Camus cantraversa, Camus kausa and Camus
flarida were quantified by HPLC and characterized by spectroscopic methods. The
analyses of C. altemifalia and C. cantraversa revealed that both contained delphinidin 3-
O-glucoside (1), delphinidin 3-0-rutinoside (2) and cyanidin 3-0—glucoside (6),
respectively. Similarly, C. kausa and C. flarida showed identical anthocyanin profiles
with major anthocyanins as cyanidin 3-0-ga1actoside (4) and cyanidin 3-0-glucoside (6),
respectively. The amount of anthocyanins l, 2 and 6 in C. altemifalia and C.
cantraversa were 8.21, 8.44 and 0.02 mg; and 7.74, 5.92, and 0.02 mg/g of fresh fruits,
respectively. The anthocyanins 4 and 6 in C. kausa and C. flarida were 0.02 and 0.16
mg; and 0.62 and 0.03 mg/g fresh fruits, respectively. This is the first report of the
quantification of anthocyanins in C. altemifalia, C. kausa and C. florida in addition to the

anthocyanins not previously quantified in C. cantraversa.

 

' This chapter has been accepted for publication in Life Sciences, 2005 (Shaiju K Vareed, Muntha K Reddy,
Robert E. Schutzki, Muraleedharan G Nair. Anthocyanins in Camus altemifalia, Camus cantraversa,
Camus kausa, and Camus flarida fruits with health benefits)

40

Introduction

The genus Camus (dogwood) belongs to the family Comaceae, which consists of
about 58 species (Fan and Xiang, 2001). The Camus spp. is widely distributed in the
northern hemisphere, eastern Asia, eastern and northern part of the United States. The
dogwood plants in general are characterized by brilliant, colorful and attractive flowers
and fi'uits and hence are widely grown as ornamental plants throughout the United States.
There are several reports of its use in traditional medicine and as a food preservative
(Jianrong and Daozong, 2003; Hwang and Yeon, 2002). For example, C. afiicinalis, a
widely grown Camus spp, has been used in Chinese herbal medicine and known for its
tonic, analgesic and diuretic activities (Kean and Hwan, 1998). Fruits from several
Camus spp. have used for improved liver and kidney functions (Y ongwen et al., 1999).
It is also reported to have anti-bacterial, antihistamine, anti-allergic, anti-microbial and
anti-malarial activities (Zanyin et al., 1949; Man et al., 2001). In Europe, C. mas or
Cornelian cherry fruits were reported to have food and cosmetic applications (Seeram et
al., 2002). Similarly, fruits from C. cantraversa have been used as an astringent and as a
tonic in Korea and China (J ang et al., 1998).

The only Camus spp. with alternate leaves is C. altemifalia and it is native to
eastern United States. It produces attractive, dark-blue and berry-like fruits. Another
common landscape tree, C. kausa, is a small deciduous tree also found in China, Japan
and Korea. It bears sweet and edible fruits and is used for the production of wine in
many parts of China and Korea (Du et al., 1974). The plant C. kausa is highly resistant to
diseases and pests and hence it is widely used as a landscape plant. However, C. flarida,

commonly known as flowering dogwood, is the most popular dogwood in the United

41

States. It is recognized as the state flower of North Carolina and Virginia, the state tree
of Missouri and Virginia and the state memorial tree in New Jersey.

The chemical investigation C. cantraversa has resulted in the isolation of several
compounds including flavonoids, terpenoids and tannins (Dongho et al., 2000; Nakaoki
and Morita", 1958). Fruits from several Camus spp. have been studied for anthocyanin
content. The anthocyanins impart bright colors to several fi'uits and vegetables and
possess antioxidant, anti-inflammatory, anticancer and anti-diabetic activities (Wang et
al., 1999; Kamei et al., 1995; Jayaprakasam et al., 2005; Chandra et al., 1992).
Therefore, the food industry is interested in fiuits and vegetables with high content of
bioactive anthocyanins to manufacture supplements with preventative and therapeutic
uses. Although C. afficinalis and C. mas were studied by us in the past for their
anthocyanin content, very little is known about the phytochemicals in the fruits of other
Camus spp (Seeram et al., 2002). In this manuscript, we have characterized and
quantified the anthocyanins in native C. altemifalia, C. cantraversa, C. flarida and C.
kausa fi'uits. In addition to the anthocyanins not previously quantified in C. cantraversa,
this is the first report of the quantification of anthocyanins in the fruits of C. altemifalia,

C. flarida and C. kausa species.

Material and Methods
All solvents were of ACS reagent grade. 1H- and 13 C NMR spectra were recorded
on Varian 500 and 125 MHz spectrometers using CD3OD/DC1 solution. Chemical shifls

are given in parts per million relative to CD3OD at 3.31ppm for IH NMR.

42

Plant Material. Fruits of C. mas, C. afficinalis, C. cantraversa, C. altemifalia, C. kausa,
and C. flarida were collected from Michigan State University campus in August-
September, 2004. The locations of the trees are recorded in the Michigan State
University Herbarium Plant Database. The fi'uits were collected and analyzed on the
same day to prevent the degradation of anthocyanins due to storage.

Extraction of Camus fruits for anthocyanin quantification. Fresh fi'uits of C. mas, C.
afiicinalis, and C. flarida (25 g each), C. altemifalia and C. cantraversa, (10 g each) and
C. kausa (100 g) were weighed and homogenized separately with methanol (1% HCl, 20
mL) for 3 min using a Kinematica CH-6010 (Roxdale, ON, Canada) homogenizer and
centrifuged (model RCSC, Sorvall Instruments, Hoffman Estates, IL) at 10000g for 20
min at 4°C. The residue was further extracted with acidic methanol (3 x 15 mL) and the
extracts were collected by centrifugation. The combined supematants were made up to
100 mL (C. kausa 200 mL) with acidic methanol.

Quantification of anthocyanins. The fi'uits from Camus spp. were extracted
immediately after the collection. The anthocyanins were quantified by using Waters
2010 HPLC system (Waters Corp.) equipped with Empower Software, Shodex Degasser,
Autosampler (Waters 717), Photodiode Array Detector (Waters 996), according to the
method published from our laboratory (Yunjun et al., 2005). The separation was
performed on a Capcell Pak (Dichrome, Santa Clara, CA) C18 column (150 x 4.6mm
id; 5 mm particle size) maintained at 25°C. The mobile phase used under gradient
conditions consisted of 0.1% trifluroacetic acid /water (v/v; A); 50.4% water/48.5%
acetonitile/1.0% acetic acid/0.1% trifluroacetic acid (v/v/v/v; B). The conditions were 20

% of A to 60% B in 26 min and then to 20 % B in 4 min and maintained for another 10

43

“my

min to a total of 40 min run time. The flow rate was 1 ml/min. The injection volume for
all samples was 50 ,uL and detection of anthocyanins was performed at 520 nm.

Pure anthocyanins 1, 2, 3, 4, 5, and 6 were weighed (2 mg each) and dissolved in
acidic methanol (1% HCl, 2 mL) separately. The stock solutions were diluted with acidic
methanol (1% HCl) to yield 0.50, 0.25, 0.13, 0.063, 0.031, 0.016, 0.0078, 0.0039, and
0.00095 mg/mL concentrations, respectively. The standard solutions of each anthocyanin
were analyzed in triplicate. Calibration curves were obtained by plotting the mean peak
areas of triplicate injections of each standard against concentrations. The Camus fruit
extracts were also analyzed in triplicate at two different concentrations and mean peak
areas were used for the quantification of anthocyanins 1-6 in these extracts.

Isolation and characterization of anthocyanins 1-6: The anthocyanins 1-6 were
isolated according to the method previously published from our laboratory (Seeram et al,
2002). In summary, the fi'uits fiom C. cantraversa plants (1.45 kg) were blended with
acidic methanol ( 1% HCl, 2 x 500 mL) for 2 min at room temperature and centrifuged
(model RC5C, Sorvall Instruments, Hoffman Estates, IL) at 10000g for 20 min at 4°C.
The residue was extracted further with acidic methanol (2 x 600 mL). The combined
supematants were evaporated to dryness at reduced pressure (35 °C) to yield a reddish
gummy extract (111 g). A portion of this extract (43 g) was dissolved in water (250 mL)
and fractionated by an XAD-2 column (500g, amberlite resin, mesh size 20-50; Sigma
Chemical Co., St. Louis, MO). The resin with adsorbed anthocyanins was then washed
with water (6 x 10 L). The water fraction was discarded. The adsorbed anthocyanin was
eluted with methanol (2 x 2 L) and concentrated. The resulting aqueous concentrate was

lyophilized to yield an amorphous red powder (10.3 g). This anthocyanin powder (1 g)

44

was dissolved in waterzmethanol (1:1, 1% HCl, 3 mL) and purified further by 018
MPLC column (350 x 40 m) using waterzmethanol (1% HCl) as the mobile phase under
gradient conditions, starting with 77.5% of water. Fractions I (125 mL), II (150 mL), 111
(150 mL) and IV (200 mL) were collected when water:methanol gradient was at 72:28
(v/v). The HPLC analysis of each fraction revealed that fiactions I and 11 contained pure
anthocyanins 1 (20 mg) and 2 (30 mg), respectively.
Anthocyanin l: 1H NW(CD3OD/DC1) 8 3.46 ( 1H, t, J = 9.3 Hz, H—4"), 3.55 (2H, m, H-
3" and H-S"), 3.70 (1H, dd, J= 9.2, 7.8 Hz, H-2"), 3.73 (1H, dd, J= 12.1, 5.7 Hz, H-6’B),
3.91 (1H, dd, J= 12.1, 2.2 Hz, H-6"A), 5.30 (1H, d, J= 7.5 Hz, H-l"), 6.66 (1H, d, J=
2.0, H-6), 6.89 (1H, br s, H-8), 7.75 (2H, s, H-2' and H-6 ), 8.94 (1H, s, H-4). This 1H
NMR spectral data was identical to the spectral data of delphinidin 3-0-glucoside (Mas ct
aL,2000)
Anthocyanin 2
1H NMR (CD3OD/DC1) 6 1.14 d (1H, d, J = 6.0, H-6'”), 3.32 (1H, t, J = 9.5, H-4'"), 3.43
(1H, t, J= 9.5, H-4"), 3.54 (1H, m, H-5'"), 3.56 (1H, t, J= 9.5, H-3"), 3.59 (1H, dd, J=
1.5, 11.5, H-6"), 3.64 (1H, dd, J = 3.3, 9.5, H-3'"), 3.68 (1H, t, J= 9.5, H-2"), 3.72 (1H,
m, H-5’), 3.80 (1H, m, H-2'"), 4.06 (1H, dd, J= 1.5, 11.3, H-6" ), 4.64 (1H, d, J= 1.5 , H-
1'" ), 5.32 (1H, d, J= 7.8, H-l”), 6.71 (1H, d, J = 2.0, H-6), 6.91 (1H, d, J= 1.5, H-8),
7.76 (2H, br s, H-2' and H-6’), 8.85 (1H, br s, H—4). This 1H NMR spectral data was
identical to the spectral data of delphinidin 3-0— rutinoside (Norbek and Kondo, 1998).
Anthocyanins 3-5 and 6 were isolated from C. mas and C. kausa, respectively, by
the same protocol as mentioned above. The identities of these anthocyanins were

confirmed by co-inj ection with authentic samples (Seeram et al, 2002).

45

 

Results

The Camus plants, known as dogwood, bear attractive flowers and fruits. It
produces flowers during May-June and fruits during August-September. The fruits of C.
mas and C. afiicinalis are morphologically similar. The average weight of ten fruits from
C. mas and C. aflicinalis were 2.03 and 2.06 g, respectively. The smallest fruits among
Camus spp. is from C. cantraversa whereas the largest fruit is from C. kausa. The
average weight of C. cantraversa, C. altemifalia, C. kausa and C. flarida per fruit was
0.07, 0.30, 15.56, and 0.54 g, respectively.

The fruits of C. altemifalia and C. cantraversa showed similar anthocyanin
profiles (Fig. 2C, 2D). The major anthocyanins identified from them were delphinidin 3-
O-glucoside (1), delphinidin 3-0-rutinoside (2) and cyanidin 3-0-glucoside (6),
respectively. The relative amounts of anthocyanins in C. altemifalia were delphinidin 3-
O—rutinoside > delphinidin 3-0-glucoside > cyanidin 3-0—glucoside. Both C. altemifalia
and C. cantraversa contained negligible amount of anthocyanin 6 when compared to
anthocyanins 1 and 2 present in them. The fruits of C. cantraversa gave higher amount
of delphinidin 3-0-glucoside than delphinidin 3-0—rutinoside when compared to the fruits
from C. altemifalia. The relative amounts of anthocyanins 1 and 2 in C. cantraversa
were delphinidin 3-0-glucoside > delphinidin 3-0-rutinoside > cyanidin 3-0-glucoside.
However, fruits from both of these Camus spp. are rich sources of anthocyanins l and 2.

The anthocyanin profiles of C. kausa and C. flarida were also similar (Fig. 2E,
2F). The anthocyanins identified from these species were delphinidin 3-0-glucoside (1)

cyanidin 3-0-glucoside (6) and cyanidin 3-0-galactoside (4), respectively. The

46

L- A"

 

quantities of delphinidin 3-0—glucoside in C. kausa and C. flarida were very small
compared to cyanidin 3-0-glucoside and cyanidin 3-0-galactoside. Also, cyanidin 3-0-
glucoside (6) was very high compared to cyanidin 3-0-galactoside (4) in C.kausa. In C.
flarida, on the other hand, cyanidin 3-0—galactoside was very high compared to cyanidin
3-0-glucoside. The major anthocyanins in C. mas and C. aflicinalz's were delphinidin 3-
O-galactoside (3), cyanidin 3-0—galactoside (4) and pelargonidin 3-0—galactoside (5),
respectively (Fig. 2A, 2B). The concentration of anthocyanins was in the order of
cyanidin 3-0— galactoside > pelargonidin 3-0-galactoside > delphinidin 3-0-galactoside.

The standard solutions of each anthocyanin were analyzed by HPLC in triplicate.
The retention values of anthocyanins 1-6 were 13.50, 14.45, 12.56, 14.79, 16.57, and
15.61 min, respectively. A calibration curve was obtained after plotting the mean peak
area of each anthocyanin against respective concentrations. Calibration curves for
anthocyanins 1-6 were linear with corresponding correlation coefficients of 0.99, 1.0,
0.99, 1.0, 0.99, and 0.99, respectively. The concentration of anthocyanins in each Camus
fruits were then determined from their corresponding calibration curves resulting from
the mean peak area of triplicate injections of each Camus fi'uit extracts.

The contents of anthocyanins 1, 2 and 6 in C. altemifalia and C. cantraversa were
8.21, 8.44 and 0.02 mg; and 7.75, 5.92, and 0.02 mg, respectively, per g of fresh fruits
(Table 1). Similarly, the concentration of anthocyanins 4 and 6 in C. kausa and C.
florida fruits were 0.02 and 0.16; and 0.62 and 0.03, mg/g, respectively (Table 1). The
amount of anthocyanins 3-5 in C. mas and C. aflicinalis were 0.47, 1.66 and 1.62; and

0.15, 0.21, and 0.78 mg/g of fruits, respectively (Table 1).

47

' . uL'I-tu

 

Discussion

The isolation of anthocyanins 1-6 flom C. cantraversa, C. mas and C. kausa was
according to the previously published method (Seeram et al., 2002). Based on the
quantification studies, C. altemifalia fluits showed highest and C. kausa fluits showed
lowest amount of anthocyanins among the Camus fluits studied so far. Anthocyanins 1
and 2 were not reported previously flom Camus spp. Therefore, only these
anthocyanins, isolated flom C. cantraversa, were further characterized by lH-NMR in
this study. As indicated earlier, the anthocyanin profiles of both C. cantraversa and C.
altemifalia were identical. The lH-NMR data of anthocyanins 1 and 2 were in
agreement with the literature data of delphinidin 3—0-glucoside and delphinidin 3-0—
rutinoside, respectively (Mas et al., 2000; Norbek and Kondo, 1998).

This is the first report of the quantification of anthocyanins in the fluits of C.
altemifalia, C. kausa and C. flarida. Both C. altemifalia and C. cantraversa fluits are
excellent sources of bioactive anthocyanins based on the unusually high concentration of
anthocyanins (total anthocyanins in C. altemiflalia and C. cantraversa were 16.67, and
13.68, g/kg of flesh fluits, respectively), detected in them. Also, the higher levels of
delphinidin 3-0-glucoside and delphinidin 3-0-rutinoside could be used as taxonomical
markers for the identification C. altemifalia and C. cantraversa fluits. The anthocyanin
content in these fruits was several times higher than other commonly consumed fluits and
vegetables. For example, total anthocyanins present in ‘Montmorency’, which is the
major commercial tart cherry cultivated in the United States, and ‘Balaton®’ tart cherries
are 0.24, and 0.08, g/kg of flesh fluits, respectively (Wang et al., 1997). Because

anthocyanins are well known for its beneficial activities and non-toxic in nature, it is

48

 

possible that these plants could be cultivated as alternate crops to yield fluits for health

beneficial anthocyanins.

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Table 2.1. The concentration of delphinidin 3-0-glucoside (1), delphinidin 3-0—
rutinoside (2), delphinidin 3-0-galactoside (3), cyanidin 3-0— galactoside (4),
pelargonidin 3-0-ga1actoside (5) and cyanidin 3-0—glucoside (6) in the fruits of Camus

spp.

 

Cornus fi'uits Anthocyanins (mg/g of fresh fruits)

 

 

C. altemifalia 8.21 8.44 ND ND ND 0.02

C. controversa 7.75 5.92 ND ND ND 0.02

C. kousa ND ND ND 0.02 ND 0.16
C. florida ND ND ND 0.62 ND 0.03
C. mas ND ND 0.47 1.66 1.62 ND
C. officinalis ND ND 0.15 0.21 0.78 ND

 

ND = Not detected

50

 

 

Anthocyanin R' R" R’”

 

1 OH OH H
2 OH OH Rha
6 OH H H

 

 

 

 

Anthocyanin R' R" R'"
3 OH OH H
4 OH H H

5 H H H

 

51

Figure 2.1.

Anthocyanins characterized from fruits of various Cornus spp.

 

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Figure 2.2 HPLC profiles of Camus spp. fruits. (2A) C. mas; (ZB) C. officinalis; (2C) C.

altemifalia: Anthocyanins 1. delphinidin 3-0-glucoside, 2. delphinidin 3-0-rutinoside, 3.

delphinidin 3-0-galactoside, 4.

galactoside, 6. cyanidin 3-0-glucoside.

52

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Figure 2.2 (Cont’d). HPLC profiles of Cornus spp. fruits. C. altemifalia; (2D) C.
controversa; (2E) C. kausa, and (2F) C. florida: Anthocyanins 1. delphinidin 3-0-
glucoside, 2. delphinidin 3-0-rutinoside, 3. delphinidin 3-0-galactoside, 4. cyanidin 3-0-

galactoside, 5. pelargonidin 3-0-galactoside, 6. cyanidin 3-0-glucoside

53

CHAPTER THREE

ANTHOCYANINS AND ANTHOCYANIDINS IN CORNUS AL T ERNIF OLIA,
CORNUS CONTROVERSA, CORNUS KOUSA, CORNUS FLORIDA, CORNUS IllAS

AND CORNUS OFFICINALIS FRUITS WITH HEALTH BENEFITS.

Abstract

Anthocyanins are natural pigments widely distributed in plant kingdom. They are
responsible for a variety of colors in fruits, flowers and vegetables. Anthocyanidins are
aglycones of anthocyanins. Both anthocyanins and anthocyanidins are reported as
powerful antioxidant and anti-inflammatory agents. Delphinidin 3-0— glucoside (1) and
Delphinidin 3-0— rutinoside (2) were not studied earlier for their inhibition of lipid
peroxidation and cyclooxygenase enzymes (COX-1 and COX-2) activities. At 50 pg/mL,
anthocyanins 1 and 2 inhibited lipid peroxidation by 71 and 68%, respectively. Similarly,
they inhibited COX-1 enzymes by 39 and 49% and COX-2 enzyme by 54 and 48%,
respectively, at 100 ,ug/mL. Anthocyanins and anthocyanidins were tested for cell

proliferation inhibitory activity against human cancer cell lines, AGS (stomach), HCT-

 

' Results in this Chapter have been

1. Accepted for publication in Life Sciences 2005 (Shaiju K Vareed, Muntha K Reddy, Robert E
Schtzki, Muraleedharan G Nair. Anthocyanins in Camus altemifalia, Camus cantraversa, Camus
kausa and Camus flarida fruits with health benefits).

1. Published in Journal of Agriculture and Food Chemistry (Jayaprakasam Bolleddula, Shaiju K
Vareed, Lawrence K Olson, Muraleedharan G Nair. Insulin secretion by bioactive anthocyanins and
anthocyanidins present in fruits. 2005, 53, 28-31).

2. Published in Life Sciences (Zhang Yunjun, Shaiju K Vareed, Muraleedharan G Nair. Human tumor

cell growth inhibition by nontoxic anthocyanidins, the pigments in fruits and vegetables. 2005, 76,
1465-72).

54

116 (colon), MCF—7 (breast), NCI-H46O (lung), and SF-268 (Central Nervous System,
CNS) at 12.5 - 200 ug/mL concentrations. Anthocyanin 1 displayed 50% growth
inhibition (ICso) at 21, 25, 50, 60, and 75 ,ug/mL, against colon, breast, lung, central
nervous system, and stomach, human tumor cell lines, respectively. Similarly, ICso
values for anthocyanin 2 were 38, 30, 76, 100, and 100 ,ug/mL against colon, breast, lung,
central nervous system, and stomach, respectively. Anthocyanins, delphinidin 3-0-
galactoside (3), cyanidin 3-0— galactoside (4), pelargonidin 3-0-galactoside (5) and
cyanidin 3-0—glucoside (6) did not show activity at a concentration lower than 200
ug/mL. The anthocyanidin malvidin (11) inhibited stomach, colon, lung, breast, and
central nervous system cell growth by 69, 75.7, 67.7, 74.7 and 40.5%, respectively, at
200 ug/mL. Similarly, pelargonidin (9) inhibited stomach, colon, lung, breast, and
central nervous system cell growth by 64, 63, 62, 63 and 34%, respectively, at 200
ug/mL. At 200 ug/mL, cyanidin (8), delphinidin (7) and petunidin (10) inhibited the
breast cancer cell growth by 47, 66 and 53%, respectively. Anthocyanins 1, 4-6 and
anthocyanidins 7-11 were studied for their insulin secretion ability by rodent pancreatic
beta cells (INS-1 813/32) in vitro. For insulin secretion studies, the compounds were
tested in the presence of 4 and 10 mM glucose concentrations. Our results indicated that
cyanidin-3-glucoside (6) and delphindin-3-glucoside (1) were the most effective insulin
secretagogues among the anthocyanins and anthocyanidins tested at 4 and 10 mM
glucose concentrations. Pelargonidin-B-galactoside (5) is one of the major anthocyanins
and its aglycone, pelargonidin, caused a 1.4—fold increase in insulin secretion at 4 mM
glucose concentration. Rest of the anthocyanins and anthocyanidins tested in our assay

had only marginal effects on insulin at 4 and 10 mM glucose concentrations.

55

Introduction

Anthocyanins belong to an important class of plant compounds, known as
flavanoids, that impart colors to flowers, fruits and vegetables (Mazza and Miniati, 1993).
Anthocyanins occur in almost all parts of plants but are more common in fruits and
flowers. They are water soluble pigments derived from the flavylium cation (2-phenyl-
benzopyrylium). The number and position of hydroxyl and methoxyl groups, sugar
moieties with or without aliphatic or aromatic acids bonded to the sugar moiety make
anthocyanins different from one another (Mazza and Miniati, 1993). Anthocyanins with
major aglycons in plants are delphinidin, cyanidin, pelargonidin, peonidin, petunidin and
malvidin. The common sugar moieties bonded to anthocyanidins are glucose, galactose,
rhamnose and arabinose. In plants anthocyanin content depends on various factors
including light intensity, temperature, nutrient stress and pathogen attack (Beckwith et
al., 2004). Intense light and low temperature are the most favorable conditions for
anthocyanin production (Beckwith et al., 2004).

Anthocyanins are reported to have wide range of biological activities including
antioxidant, anti-inflammatory, anti-cancer and anti-diabetic activities (Kim, et al., 2004,
Harbone and Williams.,2000, Conklin, 2000). A recent in vitro study indicated the
absorption of anthocyanins in the small intestine of rats (Talavera et al., 2004). The
health benefits of anthocyanins, for example, prevention of chronic diseases such as
cancer, heart diseases, and aging, may be closely associated with their antioxidant
properties (Omenn, 1995). Today, food industries prefer to use these natural colorants
over synthetic colors may be due to its health benefits and lower risk of side effects

(Chandra et al., 2001).

56

Heart diseases, cancer and stroke cause numerous deaths in the United States. In
2002, 58% of the total mortality in US was due to these diseases (www.cdc.gov.). Even
though good treatments are available now for these deadly diseases, the incidence and the
mortality rate are still quite high. Epidemiological studies suggested that incorporation of
fi'uits and vegetables in diet reduces the incidence of cancer. It is also reported that low
intake of fruits and vegetables may increase the chance of degenerative diseases by two
times. This suggests that phytochemicals in fruits and vegetables have beneficial effects
in preventing many of the diseases. Recent studies show that protective action of fruit
and vegetables against the chronic and degenerative diseases may be due to the
antioxidant activity of compounds present in them (Kenneth, 2000). Many plant
phenolics, tocopherols, carotenoids and ascorbic acid were reported to act as good
antioxidants (Frankel, 1998). Most of these antioxidants exhibit anticarcinogenic and
antimutagenic activities. Oxidative stress in body is strongly associated to the coronary
heart diseases. Reactive oxygen species such as superoxide radical (02'), hydrogen
peroxide (H202), singlet oxygen (102), peroxyl radical (ROO'), alkoxyl radical (OR') and
the hydroxyl radical (-OH') are responsible for the oxygen toxicity in our body. These
reactive species are produced from molecular oxygen due to the action of pollutants,
drugs, activated leucocytes and normal cellular respiration. Various studies suggest that
oxidation has a major role in heart diseases, cancer, diabetes, and Alzheimer’s disease

(Frankel. 1998).

Cyclooxygenase enzymes, COX-1 and COX-2, are mediators of inflammatory
reactions. These enzymes catalyze the conversion of arachidonic acid to PGHz, the

precursor of all prostaglandins and thromboxanes (Smith et al., 1996). COX enzymes

57

mediate two distinct reactions. Firstly, it converts AA to PGGz through cyclooxygenase
activity. Secondly it reduces PGG; to PGHZ via the peroxidase activity. PGH; is further
converted into various prostaglandins (Trifan et al., 1999). COX-1 and COX-2 are
similar in catalytic properties but are different in biological activities. COX-1 is
expressed constitutively in cells and is regarded as housekeeping enzyme where as COX-
2, is induced in response to various growth factors and inflammatory stimuli. Inhibition
of COX-1 may cause several side effects including gastric ulceration. Several studies
using animal model for colon cancers indicate that expression of COX-2 is responsible
for the transformation of colon epithelial cells (Srikant et al., 2003). Recent studies also
showed that selective COX-2 inhibitors possess anticancer activities (Shone et al., 2003).
Diabetes mellitus is a metabolic disorder characterized by high levels of glucose
in the blood resulting from the defects in insulin production, insulin action, or both
(www.cdc.gov.). The major function of insulin is to counter the concerted action of a
number of hyperglycemia-generating hormones and to maintain normal blood glucose
levels. Diabetic patients have a shortage of insulin or decreased ability to use insulin,
which leads to the accumulation of glucose in the blood. Heart disease is the leading
cause of diabetes-related deaths. Diabetes can also cause a number of other diseases
including stroke, blindness, kidney failure, pregnancy complications, lower-extremity
amputations, and deaths related to flu and pneumonia. There are two types of diabetes,
type-1 or insulin-dependent diabetes (IDDM) and type-2 or non-insulin—dependent
diabetes (NIDDM). Type 1 diabetes results from the destruction of pancreatic B-cells of
the body’s immune system. Type-1 diabetes may account for 5 to 10% of all diagnosed

diabetes and common among children and young adults. Type-2 diabetes is linked to

58

obesity and physical inactivity and accounts for 90-95% of diabetic cases. Type-2
diabetes is prevalent among people older than 40 (www.cdc.gov.).

One of the significant health benefits implicated to the consumption of
anthocyanins is the low risk of coronary heart diseases. Several studies have shown that
tart cherry anthocyanins, cyanidin-B-glycosides, exhibited in vitro antioxidant and anti-
inflammatory activities (Wang, et al., 1999, Seeram, et al., 2001). The antioxidant
property of anthocyanins and anthocyanidins suggested that they played an important role
in the prevention of mutagenesis and carcinogenesis (Omenn, 1995). The anthocyanins
in purple colored sweet potato and red cabbage suppressed colon carcinogenesis induced
by l, 2-dimethylhydrazine (DMH) and 2-amino-1-methyl-6-phenylimidazo [4,5-b]

pyridine in rats (Hagiwara et al., 2002).

Although anthocyanins present in fruits and vegetables are known for their health
benefits, the cell proliferation inhibitory activity and insulin secretion activity of pure
anthocyanin and anthocyanidins are not reported. This chapter deals with lipid
peroxidation, cyclooxygenase, tumor cell proliferation inhibitory and insulin secretion

activities of anthocyanins in Camus species and their corresponding anthocyanidins.

Materials and Methods

All solvents were of ACS reagent grade. tert-Butylhydroquinone (TBHQ), butylated
hydroxyanisole (BHA), butylated hydroxytoluene (BHT), aspirin, ibuprofen, naproxen,
and 3-(4,5-dimethyl-2-thiazyl)-2,S-dipheny1-2H-tetrazolium bromide (MTT) were
purchased from Sigma-Aldrich Chemical Company Co (St.Louis, MO). Celebrex "and

Vioxx® were provided by Dr. Subash Gupta, Sparrow Hospital, MI. The COX-1

59

enzyme was prepared from ram seminal vesicles purchased from Oxford Biomedical
research, Inc. (Oxford, MI). The COX-2 enzyme was prepared from prostaglandin
endoperoxide H synthase-2 (PGHS-2) —cloned insect lysate. Fetal bovine serum (FBS)
and Roswell Park Memorial Institute 1640 (RPMI-1640) medium were purchased from
Gibco BRL (Grand Island, NY). HEPES, penicillin- streptomycin, glutarnine, sodium
pyruvate, 2-mercaptoethanol, trypsin-EDTA, BSA (Bovine, Albumin; RIA Grade), Folin-
Ciolatues reagent and chemicals used for the preparation of buffers were purchased from
Sigma-Aldrich Chemical Co. (St. Louis, MO). Human tumor cell lines SF-268 (Central
Nervous System, CNS), NCI H460 (lung), and MCF-7 (breast) were purchased from the
National Cancer Institute (NCI, Bethesda, MD). AGS (stomach) and HCT-l l6 (colon)
were purchased from American Type Culture Collection (ATCC, Rockville, MD). INS-l
832/13 cells were obtained from Dr. Christopher Newgard, Duke University, NC. All
cell lines were maintained in a humidified chamber at 37 °C with 5% CO2 in RPMI-1640
medium supplemented with 10% fetal bovine serum, penicillin (1 unit/ 100 mL), and
streptomycin (1 ,ug/ 100 mL) in the Bioactive Natural Products and Phytoceuticals
Laboratory (BNPP) at Michigan State University.

Preparation of Anthocyanins 1-6. Described in Chapter 2

Preparation of Anthocyanidins 7-11. Anthocyanidins were prepared by the acid
hydrolysis of respective anthocyanins under nitrogen atmosphere. During our insulin
secretagogues studies with grape skin, we have found that one of the red grapes, cabemet
sauvignon, contained substantial quantities of delphinidin, cyanidin, petunidin and
malvidin glycosides (Zhang et al., 2004). Therefore, pure delphinidin (7), cyanidin (8),

petunidin (10) and malvidin (11) aglycones were prepared by the acid hydrolysis of

60

purified grape skin anthocyanins. The purified anthocyanins from grape skin (1.0 g)
were hydrolyzed with HCl (3 M, 3h at 95 °C). The solution containing anthocyanidins
thus obtained was cooled at room temperature and evaporated under reduced pressure
(40°C). The residue from the hydrolysis (0.74 g) was then dissolved in MeOHzH2O (1:1,
2 mL), and purified by medium pressure liquid chromatography (MPLC) using a C13
column (350 x 40 mm). The solvent system used was acidic MeOH:H2O (pH = 3.0)
under gradient conditions starting from 45% to 55% MeOH. The fractions collected from
MPLC were analyzed by high performance liquid chromatography (HPLC) to ensure
purity. Similarly, pure pelargonidin (9) aglycone was prepared from the acid hydrolysis
of C. mas fruit anthocyanins.

HPLC analysis. The purity of anthocyanidins 7-11 was determined by HPLC. (The
HPLC method is described in Chapter 2).

Lipid Peroxidation Inhibitory Assay. The anthocyanins 1 and 2 were tested in vitro for
their ability to inhibit the oxidation of large unilamellar vesicles (LUVs) (Arora et al.,
1998). The assay was conducted in a buffer consisted of HEPES (100 uL), NaCl (200
uL), N2- sparged water (1.64 mL), test sample (20 11L) in water and LUV (20 uL)
suspension. The peroxidation was initiated by the addition of FeCl2 (20 uL, 0.5 mM)
solution and was monitored by observing the fluorescence at O, 1, 3, 6, 9, 12, 15, 18, and
21 min using a Turner model 450 digital fluorometer (Bamstead Thermolyne, Dubuque,
IA) at 384 nm. The decrease of relative fluorescence intensity with time indicated the

rate of lipid peroxidation. All compounds were tested at 50 ug/mL. The antioxidant

standards BHA, BHT and TBHQ were tested at 1.66, 2.2 and 1.8 ug/mL, respectively.

Inhibition of lipid peroxidation by anthocyanins 3-6 and anthocyanidins 7-11 were

61

 

reported earlier from our laboratory and hence was not studied at this time (Seeram et al.,
2002)

Cyclooxygenase Inhibitory Assay. COX activities of anthocyanins 1 and 2 were
assessed by monitoring the initial rate of 02 uptake using a micro oxygen chamber and
electrode (Instech Laboratories, Plymouth Meetings, PA) attached to a YSI model 5300
biological oxygen monitor (Yellow springs Instrument, Inc., Yellow Springs, OH) at 37
°C. The assay was conducted according to the previously reported procedure (Wang et
al., 1999). The test samples and controls were dissolved in water. Each assay mixture

contained Tris buffer (0.6 mL, 0.1 M, pH 7), phenol (1 mM), hemoglobin (85 pg), and

water or test samples (10 uL). COX-1 or COX-2 enzyme (10 uL) was added to the
chamber and incubated for 3 min. The reaction was initiated by the addition of
arachidonic acid (10 pL of a 1 mg/mL solution). Duplicate analysis was performed for
each sample and the standard deviation was calculated for n=2. The data were recorded
using QuickLog for windows data acquisition and control software (Strawberry Tree,
Inc., Sunnyvale, CA). Commercial anti-inflammatory drugs aspirin, ibuprofen, naproxen,
and vioxx were tested at 180, 2.52, 2.06, and 1.67 ug/mL, respectively. COX-1 and -2
inhibitory activities of anthocyanins 3-6 anthocyanidins 7-11 were reported earlier fi'om
our laboratory and hence were not studied again (Seeram et al., 2002).

Tumor Cell Proliferation Assay (MT T). The assay was performed according to the
previously published method (Denizot and Lang, 1986). MCF-7 (beast), SF-268 (CNS),
NCI H460 (lung), HCT-116 (colon) and AGS (gastric) human tumor cells were cultured
in RPMI-1640 medium containing penicillin-streptomycin (10 units/mL for penicillin and

10 ,ug/mL for streptomycin) and 10% fetal bovine serum (PBS). The cells were grown in

62

 

a humidified incubator (37 °C, 5% CO2), counted using hemacytometer, and transferred
in to 96-well microtiter plates and incubated for 24 h. The samples were dissolved in
water/DMSO and fiuther diluted with RPMI medium. After 24 h of incubation, test
samples (100 pL) in appropriate dilution were added to each well containing the
appropriate tumor cells and further incubated 48 h. After incubation, an aliquot (25 ,uL)
of MTT solution (5 mg MTT dissolved in 1 mL of phosphate-buffered saline solution)
was added and the plates were further incubated for 3 h at 37 °C after wrapping it with
aluminum foil. The medium was removed from each well and cells treated with DMSO
(200 pl.) The plates were then shaken and optical density was measured using a
microplate reader at 570 nm. Adriamycin, dissolved in 0.1% DMSO and 0.1% DMSO or
Water in RPMI 1640 media were used as positive control and solvent control,
respectively. The sample was assayed in triplicate, and three independent experiments
were carried out to calculate ICso values.

The assay was conducted in triplicate for each sample concentration, positive and
solvent controls. Three parallel experiments were performed. The cell viability of
samples at each concentration was calculated with respect to solvent control. The cell
viability at each concentration was calculated by dividing the optical density of samples

with the optical density of solvent control.

Insulin Secretion Studies. INS-1 832/ 13 cells were maintained in the Bioactive Natural
Products and Phytoceutical Laboratory at Michigan State University in 5% CO2/air at 37
°C. The cells were cultured in RPMI-1640 medium containing 11.1 mM glucose and
supplemented with 10% FBS (Fetal Bovine Serum), 10 mM HEPES, 100 U/ml penicillin,

100 ug/ml streptomycin, 4 mM glutamine, 1 mM sodium pyruvate, and 50 uM 2-

63

mercaptoethanol. Cells were passed weekly afier trypsin-EDTA detachment. The cells
were counted using a hemacytometer and transferred into 24-well plates at a density of
0.64 x 106 cells per well and incubated for 24 h. The cells were then cultured for an
additional 24 h in RPMI-1640 containing 4 mM glucose and the supplements described
above. Cells were then incubated twice for 30 min in Krebs-Ringer Bicarbonate buffer
(KRBB) containing 4 mM glucose and 0.1% BSA. Cells were rapidly washed with
KRBB and incubated for 60 min KRBB containing 4 or 10 mM glucose with or without
the indicated anthocyanins or anthocyanidins. The medium was then removed for
determination of insulin release. The cells were washed twice with PBS and dissolved in
1 M NaOH. Cellular protein concentration was then determined by Lowry assay.
Anthocyanins and anthocyanidins were dissolved in DMSO to obtain desired
concentrations. Final concentration of DMSO was maintained at 0.1%. The insulin
secreted into the medium by the cells was determined by radioirnmunoassay and
normalized to total cellular protein.

Radioimmuno Assay (RIA). The RIA Kit was purchased from LINCO Research Inc.
(St Charles, MO), and the assay was conducted according to the manufacturer’s
directions. Briefly, insulin standards were prepared in the 0.1-10 ng range and added
(100 p1) to 12 x 75 mm test tubes. An aliquot of samples (25 p1) from the insulin
secretion studies, assay buffer (75 pl) and '25 I labeled insulin (100 pl) was then added to
each test tube. An aliquot of anti-rat insulin antibody (100 pL) was added and the tubes

were incubated at 4°C for 24 h. To the solutions, 1 m1 aliquot of the precipitating reagent

was added and the tubes were further incubated for 20 min at 4°C to precipitate the

64

 

 

insulin bound to the antibody. The tubes were then centrifuged and the radioactivity was
measured using a gamma counter.

Lowry protein Assay. The amount of protein in the assay wells was determined by
Lowry method (19). The Lowry assay solution was prepared by mixing the Lowry
solution, CuSO4.5H2O (1%), and sodium tartarate (1%). The protein sample (100 pl) and
Lowry assay solution (1 mL) were mixed in a test tube (12 x 75). An aliquot of Folin-
Ciolatues reagent (100 pl) was added to these tubes and incubated for 30 min at room
temperature. The optical density of resulting solutions was recorded using UV

spectrophotometer at 700 nm.

Results

The methanol extracts of C. altemifalia (A) and C. cantroversa (B) inhibited lipid
peroxidation by 56 and 53%, respectively, at 250 pg/mL. Inhibition of lipid peroxidation
by anthocyanins 1 and 2 were 71 and 68%, respectively, at 50 pg/mL (Fig. 3.2).
Commercial antioxidants BHA, BHT and TBHQ were used as positive controls in the
lipid peroxidation assay at 1.8, 2.2 and 1.66 yg/mL, respectively, to yield about 80-90%
inhibition and inhibited the lipid peroxidation by 81, 85 and 84%, respectively.

The anthocyanins 1 and 2 inhibited COX-1 enzyme by 39 and 49%, respectively,
at 100 ,ug/mL. Similarly, they inhibited COX-2 enzyme by 54 and 48%, respectively, at
100 ,ug/mL (Fig. 3.3). The positive controls aspirin (180 ,ug/mL,) ibuprofen (2.52
,ug/mL), naproxen (2.06 ,ug/mL) and Vioxx® (1.67 ,ug/mL) inhibited COX-1 and COX-2
enzymes by 61 and 24, 53 and 59, 80 and 96, and 0 and 76%, respectively. The varying

concentrations of positive controls were used to obtain the inhibitions between 50-100 %.

65

Anthocyanins 1-6 and anthocyanidins 7-11 were assayed for their ability to inhibit
the proliferation of colon, breast, lung, central nervous system, and stomach human tumor
cell lines. These compounds were assayed at 200, 100, 50, 25 and 12.5 pg/mL to obtain
their 50% grth inhibitory (ICso) values. The anthocyanin, 1, exhibited 50% growth
inhibition (ICso) at 21, 25, 50, 60 and 75 ,ug/mL, against colon, breast, lung, central
nervous system and stomach, respectively (Fig. 3.4A). The ICso values observed for
anthocyanin 2 were 38, 30, 76, 100 and 100 yg/mL against colon, breast, lung, central
nervous system and stomach, respectively (Fig. 3.4B). Anthocyanins 3-5 showed good
inhibition of tumor cell proliferation at 200 pg/mL. However, inhibitory activities of
anthocyanins 3-5 were marginal below 100 pg/mL. Anthocyanin 3 inhibited the growth
of breast and lung tumor cell lines by 34 and 22%, respectively, at 200 pg/mL (Fig.
3.4C). Anthocyanin 4 displayed 50% inhibition towards breast cancer cell line at 200
pg/mL (Fig. 3.4D). It did not show inhibition to the proliferation of any other cancer
cell line. Anthocyanin 5 inhibited breast, central nervous system, lung, and colon cell

growth by 40, 20, 51 and 76%, respectively, at 200 ppm (Fig. 3.4E).

Anthocyanidins were prepared by the hydrolysis of anthocyanin enriched extract
from Cabernet sauvignan grape skin under acidic condition. The hydrolyzed mixture
was cooled, evaporated under reduced pressure and purified by MPLC afforded
delphinidin (7), cyanidin (8), petunidin (10) and malvidin (11). Similarly, pure
pelargonidin (9) aglycone was prepared from the acid hydrolysis of C. mas fi'uit
anthocyanins.

The anthocyanidin cyanidin (8) inhibited the growth of breast cancer cells by 35

and 47% at 100 and 200 pg/mL (Fig. 3.5B), respectively. However, cyanidin did not

66

 

inhibit the growth of other cancer cell lines at 100 and 200 pg/mL. Similarly, delphinidin
(7) inhibited the growth of breast cancer cell lines by 27 and 64% at 100 and 200 pg/mL,
respectively, but did not affect the growth of stomach, lung, CNS and colon (Fig. 3.5A)
cancer cell lines. The anthocyanidin malvidin (11) was effective against the cell
proliferation on all cell lines tested. It inhibited the cell proliferation of colon, breast,
lung, central nervous system, and stomach cancer cells by 75.7, 74.7, 67.7, 40.5, and 69
%, respectively, at 100 pg/mL (Fig. 3.5E). At 200 pg/mL, pelargonidin (9) inhibited 34,
62, 64, 63, and 65% of colon, breast, lung, central nervous system, and stomach cancer
cells growth, respectively (Fig. 3.5C). One of the less abundant anthocyanidins found in
nature, petunidin (10), showed 53 and 24% cell growth inhibitions of breast and stomach
cancer cells at 200 pg/mL (Fig. 3.5D). Adriamycin (doxorubicin), the positive control,

was tested at 0.181, 0.363, 0.725 and 1.45 pg/mL.

Anthocyanins 1, 4-6, and anthocyanidins 7-11 were assayed for their ability to
stimulate insulin secretion by rodent pancreatic beta cells (INS-1 813/32). Anthocyanins
and anthocyanidins were assayed at 4 and 10 mM glucose levels in the cell growth
medium at 50 pg/mL initial concentration. The insulin secretion at 4 mM and 10 mM
glucose were 27 and 83 ng of insulin/mg of protein, respectively (Fig. 3.6A).
Anthocyanins delphinidin 3-0— glucoside (l) and cyanidin 3-0-glucoside (6) secreted
49.48 and 36.14 ng of insulin/mg of protein, respectively, at 4 mM glucose concentration.
At 10 mM glucose level, anthocyanins l and 6 showed insulin secretion of 113 and 119
ng of insulin/mg of protein, respectively. The anthocyanins cyanidin-3-0—galactoside (4)
and pelargonidin-3-0-galactoside (5) did not impact the insulin secretion at 4 mM

glucose concentration. However, cyanidin-3-0-galactoside showed an increase of 17 ng

67

of insulin/mg of protein at 10 mM glucose concentration (Fig. 3.7). The pelargonidin-3-
O-galactoside (5) was tested only once due to the limited amount of sample. The
anthocyanin cyanidin-3-glucoside (6) was also evaluated for dose dependent insulin
secretion at 5, 10, 50, 100 and 250 pg/mL concentrations. At 4 mM glucose level, the
untreated cells and the cells treated with anthocyanin 6 secreted 33 and 46 ng of
insulin/mg of protein, respectively. However, there was no significant difference in
insulin secretion by compound 6 at 10, 50, 100 and 250 pg/mL concentrations.
Anthocyanidins delphinidin (7) and cyanidin (8) secreted 25 and 29 ng of insulin/mg of
protein, respectively, at 4 mM glucose concentration. At 10 mM glucose level,
anthocyanidins 7 and 8 showed insulin secretion of 48 and 88 ng of insulin/mg of protein,
respectively (Fig 3.6B). Pelargonidin (9) secreted 49 and 91 ng of insulin/mg of protein
at 4 and 10 mM glucose, respectively (Fig 3.7). The aglycone petunidin (10) increased
insulin secretion by 4 ng of insulin/mg protein at 4 mM glucose concentration and no
significant increase in insulin level was observed at 10 mM level. However, malvidin
(11) did not show an increase in insulin secretion with respect to the untreated cells.

Anthocyanin 2 and 3 were not tested in this assay due to the limited supply of samples.

Discussion

Anthocyanins are pigments responsible for the orange, red, purple and blue colors
of many fruits, vegetables, flowers, leaves, and roots. They are found in nature as
polyhydroxylated and or methoxylated heterosides, derived from the flavylium ion or 2-
phenylbenzopyrilium. Anthocyanins are biogenetically produced from

tetrahydroxychalcone, naringenin, a precursor involved in the pivotal step of flavonoid

68

biosynthesis (Strack and Wray, 1993). Out of the 21 anthocyanidins reported, only 6
anthocyanidins are commonly found in fruits and vegetables. They are delphinidin,
cyanidin, pelargonidin, peonidin, petunidin and malvidin. Anthocyanidins could very
well be the immediate metabolite after ingestion of anthocyanins. This is because the [3-
glucosidase enzyme found in intestinal bacteria can easily hydrolyze respective
anthocyanins (glycosides) to anthocyanidins (aglycones) (Miyazawa, et al., 1999).
Although anthocyanins are well known for their antioxidant and anti-inflammatory

activities, very little is known about their insulin secretion and anticancer activities.

Delphinidin 3-0— glucoside (1) showed higher inhibitory activity than delphinidin
3-0- rutinoside (2) in the lipid peroxidation, COX, and cell proliferation inhibitory
assays. This is true because the bioactivity of anthocyanins is proportional to the molar
amount of respective aglycones present in them. The aglycone, delphinidin, was not
active against the cell lines tested except breast (ICso =162.4 ,ug/mL). Also, delphinidin
3-0-galactoside did not show activity towards any cancer cell lines tested even at 200
,ug/mL. However, delphinidin 3-0— glucoside and delphinidin 3-0— rutinoside showed
potent grth inhibitory activity towards all tumor cell lines tested. Also, delphinidin 3-
0- glucoside showed higher grth inhibitory activity than delphinidin 3-0— rutinoside in
all cell lines studied. This indicated that each anthocyanin exhibits different biological
activities with varying potency and the activity is dependent on both the aglycone and the
glycoside substitution on its 3-position.
The cell proliferation inhibitory results obtained in this study for cyanidin,
delphinidin, pelargonidin, petunidin and malvidin on stomach, colon, lung, breast and

CNS cancer cell lines are in agreement with the effects reported for cyanidin on human

69

colon tumor cell lines HT—29 and HCT-l 15 (Kang, et al., 2003 and Kamei, et al., 1998).
The data from the MTT assay indicated that a free hydroxyl group at 3-position in the
flavylium moiety in anthocyanidins contributed to the cell proliferation inhibitory activity
against human cancer cell lines studied. Also, the number of hydroxyl and methoxyl
groups in B ring of anthocyanidin strongly influenced the growth inhibition of cancer cell
lines studied. The highest inhibitory activity was demonstrated by malvidin with
hydroxyl groups at 3 and 4' positions and methoxy groups at 3' and 5' positions.

It is well known that dietary antioxidants protect pancreatic B-cells fiom the
damage due to oxidative stress. Various reports also indicated that consumption of fruits
and vegetables, especially rich in polyphenols, decreased the incidence of type-2 diabetes
(Anderson and Polansky, 2002; Landrault, 2003). The insulin secretion studies in our
laboratory with pancreatic B-cells suggested that both anthocyanins and anthocyanidins
are insulin secretagogues. Among the anthocyanins studied, delphinidin-3-0-glucoside
(1) showed highest activity at lower glucose concentration. Although cyanidin-3-0-
glucoside (6) was less active than delphinidin-3-0-glucoside at lower glucose
concentration, it was more active at higher glucose concentration. Among the
galactosides, pelargonidin-3-galactoside (5) did not induce insulin secretion at 4 and 10
mM glucose concentrations studied where as cyanidin-3-galactoside (4) showed
significant increase in insulin secretion. The ability of anthocyanins studied to secrete
insulin was in the increasing order of delphindin-3-0-glucoside > cyanidin-3-0-glucoside
> pelargonidin-3-0-galactoside. This indicated that the number of hydroxyl groups in
ring-B of anthocyanins played an important role in their ability to secrete insulin. Among

the anthocyanidins tested, pelargonidin was the most active at 4 mM glucose. Other

70

aglycones did not potentiate significant insulin secretion at 4 or 10 mM glucose
concentrations studied.

In summary the in vitro studies with various human tumor cell lines and
pancreatic B-cells suggest that both anthocyanins and anthocyanidins in Camus him
may be effective for the prevention of cancer and type-2 diabetes. However, in vivo
studies and clinical evaluation of these compounds must be carried out to validate the in

vitro results.

71

OH
HO 6 0
\ R"
c

 

 

/ OR
OH

R R' R"
1 Glc OH OH
2 Glc—>Rha OH OH
3 Gal OH OH
4 Gal OH H
5 Gal H H
6 Glc OH H
7 H OH OH
8 H OH H
9 H H H
10 H OCH3 OH
1 1 H OCH3 OCH3

 

Figure 3.1 Structures of anthocyanidins (1-6) and anthocyanidins (7-11).

1. Delphinidin 3-0-glucoside: 2. Delphinidin 3-0- rutinoside; 3. Delphinidin 3-0-
galactoside; 4. Cyanidin-3-0-galactoside; 5. Pelargonidin-3-0-galactoside; 6. Cyanidin-
3-0—glucoside; 7. Delphinidin; 8. Cyanidin; 9. Pelargonidin; 10. Petunidin and 11.

Malvidin.

72

 
    

 

   

 

 

0 o
o
c . - .2
§ 0'9 i“ l—fl—Fe(ll)controll
g —4F—aner
a 0.61 1+1
15 1 ‘
c {+2 t
o i
g l-a—A
_ 0.3 4 I
.: .-4>—B
5 r n
a!
0

 

0 5 10 15 20

Time (min)

Figure 3.2 Lipid peroxidation inhibitory activities of anthocyanins 1 and 2 and methanol
extracts of C. altemifalia (A) and C. cantraversa (B). Synthetic commercial antioxidants
BHA, BHT, and TBHQ were tested at 1.66, 2.2 and 1.8 pg/mL, respectively. Oxidation
of lipid was initiated by the addition of ferrous ions. The rate of peroxidation was
monitored by the measurement of decrease in fluorescence intensity with respect to time.
Anthocyanins and extracts were tested at 50 and 250 ,ug/mL respectively. Antioxidant
standards BHA, BHT and TBHQ inhibited lipid peroxidation by 81, 85 and 84%,

respectively. Vertical bars represent the standard deviation of each data point (n = 2).

73

60-

b
O
I

530117
@1092 ;

°/0 Inhibition

N
O
1

 

t
it
5

 

Figure 3.3. COX-1 and COX—2 inhibitory activities of anthocyanins 1 and 2 and
methanol extracts of C. altemifalia (A) and C. contraversa (B). Anthocyanins and
extracts were tested at 50 and 250 ,ugmL respectively, at pH 7.0. Commercial anti-
inflammatory drugs, aspirin (180 ,ug/mL,) ibuprofen (2.52 ,ug/mL), naproxen (2.06
pg/mL) and vioxx (1.67 ,ug/mL) were used as positive controls. Aspirin, ibuprofen,
naproxen and vioxx inhibited COX-1 activity by 61, 53, 80 and 0% respectively, and
COX-2 activity by 24, 59, 96 and 76%, respectively. Vertical bars represent the standard

deviation of each data point (n=2).

74

3

 

 

 

 

 

 

 

 

 

 

 

 

E 90 - {i—o— SF-268m'
g l—n—mF-r !
E 60 I+Hcr-116 I
8 -°-AGS i
a! 30 ~ j+mmeot
o I 1 fl I
o 50 100 150 200
Con (ppm)
Figure. 3.4A
120 -
g 90 - a—O—SF-268 7
§ I—n— MOP-7 ‘
E 60 - l+ HOT-116
3 —a—AGS
:9 so J j—n— NCI H460J
o I T I I .
o 50 100 150 200
Con (ppm)
Figure. 3.4B

Figure 3.4. In vitro cell proliferation inhibitory results of anthocyanins 1-6 against
human cancer cell lines. Adriamycin was used as positive control. The vertical bars
represent i SD of three individual experiments conducted in triplicate. 3.4A. Delphinidin
3-0—glucoside; 3.4B. Delphinidin 3-0—rutinoside. Anthocyanins were tested at 200, 100,
50, 25, and 6.25 pg/mL, respectively. At 200 pg/mL, all cell lines exposed to

anthocyanins displayed grth characteristics identical to the solvent control.

75

 

+ HOT-116

 

 

 

 

 

 

 

 

200 -i

l —o-—SF-268

l
g 150 1 K1; 3 4 +MCFJ
g 1004 :. +NCl-H46O
> ‘ —o—AGS
0\° 50 —,

l

O -¥—~——~——— —-— “I __,__
0 100 200
Conc (ppm) 3
Figure. 3.4C
-0— SF-268
5": 160 _ I3\D/fl\\fl +MCF-7
13' 120 - A +NCI—H 460
E \ —a-—AGS
75 80 -
0
°‘ 40 -
0 I I I fir
0 50 100 150 200
Gone (ppm)
Figure. 3.4D

Figure 3.4 (cont’d). In vitro cell proliferation inhibitory results of anthocyanins against

human cancer cell lines. 3.4C. Delphinidin 3-0-galactoside; 3.4D. Cyanidin 3-0—

galactoside.

76

+ HCT116

 

 

 

 

150 -
—o—SF-268
E + MCF-7
E 100 ‘ —a— NCl-H46O
2 —o—AGS
8 50 _
s
0 I I I l
0 50 100 150 200
Gone (ppm)
Figure. 3.4E

Figure 3.4 (cont’d). In vitro cell proliferation inhibitory results of anthocyanins against

human cancer cell lines. 3.4E. Pelargonidin 3-0-galactoside.

77

+ AGS

 

 

 

 

+ SF-268
3‘ 150 - +HCT-116
E5 -><— NCI H460
'3 100 -
5 + MCF-7
"23’ _
U 50
°\9
0 I I I I
0 50 100 150 200
Conc (pg/mL)
Figure 3.5A
+AGS
+ SF-268
140 - + HCT-l 16
g 120 - —X—NCI H460
I: 100 - E!!! a i g —X— MCF-7
g 80 ~ 1 \,K
= 60 I \
0
L: 40 " x
e\ 20 .
0 I I I I
O 50 100 150 200
Conc (pg/mL)
Figure 3.5B

Figure 3.5. In vitro cell proliferation inhibitory results of anthocyanidins 7-11 against

human cancer cell lines. DMSO and adriamycin were used as solvent and positive

controls, respectively. Anthocyanidins were tested at 200, 100, 50, 25, and 6.25 pg/mL,

respectively. The vertical bars represent i SD of three individual experiments conducted

in triplicate. 3.5A. Delphinidin; 3.5B. Cyanidin.

78

+ AGS

 

 

 

 

 

 

 

+ SF-268
+ HCT-l 16
100 _ -x— NCI H460

g + MCF-7

.5

.9.

>

3 50 -

°\6

O . 1
0 100 200
Conc (pg/mL)
Figure 3.5C
+ AGS
+ SF -268
+ HCT-l 16
100 ->4— NCI H460

g + MCF-7

.2 I

>

E 50 .

6\"

O I I I I
0 50 100 150 200
Conc (pg/mL)
Fig 3.5D

Figure 3.5 (cont’d). In vitro cell proliferation inhibitory results of anthocyanidins against

human cancer cell lines. 3.5C. Pelargonidin; 3.5D. Petunidin.

79

 

 

+ AGS

 

-l- SF-268
+ HCT-l 16

3* 100 2 -X—NCI H460

E + MCF-7

3.

>

i 50 -

U

6\°

O I I I I
0 50 100 150 200
Conc (pg/mL)
Fig 3.5E

Figure 3.5 (cont’d). In vitro cell proliferation inhibitory results of anthocyanidins against

human cancer cell lines. 3.5E. Malvidin.

80

ng of lneulinlmg of protein

 

 

IM' ** I‘M”

ng of lnsulinlmg of protein

 

 

Fig 3.68

Figure 3.6 (A) The amount of insulin secreted per milligram of protein by compounds 1
and 6 and (B) by compounds 7 and 8 in the presence of 4 and 10 mM glucose. The final
DMSO concentration in the assay wells was 0.1%. The results represented are the
average of three or five independent experiments and each sample was assayed in

duplicate. Insulin secretion by compounds 1, 6, 7 and 8 were significant at * (95% or p S

0.05) or 1"“ (99% or p s 0.01) as determined by LSD using the t-test

81

* tam?

{I10mM‘

J A
o N
O O
1 n

 

ng of insulin/mg of protein

 

 

Figure 3.7 The insulin secreted by compounds 4, 9-11 at 4 and 10 mM glucose
concentrations. The amount of insulin secreted was normalized to milligram protein.
The final DMSO concentration in the assay wells was 0.1%. The results represented are
the average of three independent experiments and each sample was assayed in duplicate.
Insulin secretion by compounds 4, 9-11 was significant at * (95% or p S 0.05) as

determined by LSD using the t-test.

82

CHAPTER FOUR

FRUIT MATURITY, FLAVONOID AND AN THOCYANIN PRODUCTION IN

CORN US FRUITS

Abstract

The genus Camus is well known for its medicinal properties. Several Camus
species are used as traditional medicines in Asian countries including China, India, Korea
and Japan. Camus kausa, a widely grown plant among Camus species, has not been
investigated for its bioactive constituents. Bioassay— guided isolation and characterization
of ripened red fi'uits of C. kausa afforded ursolic acid (12) and B-sitosterol (13) in
addition to cyanidin-3-0-glucoside. The matured green fi'uits of C. kausa afforded
cornin (l4), kaempherol 3-0-rhamnoside (15), myricetin 3-0-rhamnoside (16),
kaempherol 3-0—glucoside (l7) and stenophyllin (18) in addition to compounds 12 and
13. The green fruits were devoid of anthocyanins. These compounds are isolated for the
first time from C. kausa. Also, it is hypothesized that the biosynthetic pathways for the
production of flavonoids in C. kausa fruits during ripening was shut down in favor of the

production of anthocyanins.

83

Introduction

Flavonoids including anthocyanins are responsible for the flower colors in many
species. These compounds account for a variety of flower colors such as red, orange,
pink, purple, blue and yellow (Shirely, 2001). F lavonoids constitute a diverse family of
aromatic molecules that are derived from phenylalanine and malonyl-coenzyme A. The
colorless flavonoids such as flavonols and flavones may affect the flower color through
the formation of molecular complexes with anthocyanins by the process called
copigrnentation (Justesen et al, 1997).

Flavonoids comprise of several groups including chalcones, flavones, flavanols,
flavandiols, anthocyanins, aurones and condensed tannins. Both flavonols and
anthocyanins originate from the same substrate dihydroflavonol during their biosynthesis
(Scheme 4.1). The dihydroflavonols represent as important targets in flavonoid
synthesis. They act as intermediates for the production of anthocyanins through the
action of dihydroflavonol 4- reductase (DFR) enzyme. The colorless flavonols are
produced by the action of flavonol synthase (FLS) enzyme. FLS acts on
dihydroflavonols by introducing an olefinic bond between C-2 and C-3 of the ring C in
presence of cofactors 2—oxoglutarate, ascorbate and Fe” ions (Halto et al, 1993).
However, the conversion of dihydroflavonols into anthocyanins involves at least 3
enzymatic steps beginning with a reduction at the 4-carbon position by dihydroflavonol
4-reductase (DFR). The competition between flavonol synthase (F LS) and DFR for
common substrate may alter the flavonol-anthocyanin ratio in plants. By changing the
flavonol- anthocyanin ratio, it is possible to produce the plants with different flower

colors (Mol et al, 1998). Most plant tissues produce anthocyanins in response to external

84

stresses such as high light intensity and temperature. A low level expression of genes
responsible for anthocyanin production may lead to the accumulation of flavonols by
diverting dihydroflavonol to flavonols with FLS expression (Nielson et al., 2002).
However, the control of F LS gene expression may direct flavonoid biosynthetic pathway
either to anthocyanins or to flavonols in vegetative tissues. For example, in lisianthus
(Eustama grandiflarum Grise), the loss of FLS expression resulted in the loss of flavonol
production and accumulation of dihydroflavonols without the overall increase in
anthocyanin production. In contrast, tobacco plants showed three-fold increase in
anthocyanin accumulation due to the loss of FLS activity (Nielson et al., 2002).

The genus Camus contains many medicinal plants. Many of these species are
used in traditional medicines in Asian countries. For example, Camus aflicinalis, known
as "Zhu Yu" or "Zao Pi" in Chinese medicine, was used as an astringent tonic for
impotence, spermatorrhea, lmnbago, vertigo and night sweats (www.ncsu.edu). The
fruits of C. aflicinalz’s were used for antidiabetic preparations in China for several years.
Fruits of Camus oblonga have been used as a substitute for 'Zao Pi' (www.ncsu.edu).
Camus kausa, native to eastern Asia, commonly known as Korean or kousa dogwood, is
an ornamental tree. It is a smaller tree with white or pink bracts and has dark green
foliage late in the spring season. Its cultivars are increasingly used as landscape plants
compared to the flowering dogwood Camus flarida, the widely used dogwood native to
United States of America. This is mainly due to lack of disease and insect problems
typically associated with C. flarida (Trigiano et al., 2004). C. kausa bears colorful,
attractive and edible fruits and are used for the production of wine in China (Seeram et

al., 2002). Three anthocyanins, cyanidin 3-0—glucoside, delphinidin 3-0—glucoside, and

85

pelargonidin 3-0-glucoside were reported from C. kausa fruits (Du et al., 1974).
Polyphenols B-glucogallin, (+)-catechin, (+)-gallocatechin and procyanidin B-3 were also
identified from the callus of C. kausa (Kanji et al., 1993).

Anthocyanins are the major bioactive compounds reported in Camus species.
Total anthocyanin content of many Camus species is 10-15 times higher than other fruits
used as sources of anthocyanins (Shaiju et al., 2005). Very little is known about the
compounds other than anthocyanins in Camus fruits. Most reports were focused on the
isolation, characterization and biological activities of compounds fi'om C. oflicinalis.
Our preliminary studies with water, methanol, and ethyl acetate extracts of C. kausa fruits
showed promising lipid peroxidation and COX enzymes inhibitory activities. In this
chapter, the bioassay guided isolation and characterization of compounds fiom ripened

and matured green C. kausa fi'uits are presented.

Materials and Methods

All solvents used for isolation and purification were of ACS reagent grade
(Aldrich Chemical Co., Inc., Milwaukee, WI). 1H NMR spectra was recorded at 300
MHz on Varian INOVA and 500 MHz on VRX instruments. 13C NMR spectra were
recorded at 75 and 125 MHz instruments. Compounds were dissolved in CDC13,
CDgOD, and DMSO-d6 and chemical shifts are given in parts per million (ppm) relative
to CDC13, CD301), and DMSO-d6 at 7.24, 3.31, and 2.49 ppm, respectively for 1H NMR,
and 77.0, 49, and 39.5 ppm, respectively, for 13 C NMR. The silica gel used for Medium

Pressure Liquid Chromatography (MPLC) was from Merck (35-70 pm particle size).

86

Thin Layer Chromatography (TLC) and Preparative Thin Layer Chromatography (P-
TLC) plates (20 x 20, 500pm) were purchased from Analtech, Inc. (Newark, DE).

Plant Material: The red ripened fruits of C. kausa were collected on the campus of
Michigan State University in August-September, 2002. The locations of trees were
recorded in the Michigan State University Herbarium Plant Database. The green and
matured C. kausa fruits were collected at 3934 E Sunwind Drive, Okemos, Michigan.
Extraction and Bioassay Guided Isolation of Compounds 12-18. The ripened fruits of
C. kausa (1.6 kg) were pitted and the pulp was blended with water (1000 mL), and
successively extracted with water, methanol, and ethyl acetate (500 mL x 3) to yield 124,
21.5, and 1.4 g of extracts, respectively.

The methanol extract (21.5 g) was stirred successively with ethyl acetate,
methanol and water. The ethyl acetate and methanol soluble portion were combined
based on TLC and evaporated under reduced pressure (1.3 g). This extract was applied to
MPLC (silica) column and fractionated with CHClgzMeOH solvent system under gradient
condition from 100% CHC13 to 100% MeOH. A total of 15 fractions were collected and
each fraction was analyzed and combined based on TLC. Fractions 4 (33 g, 590 mL),
and 5 (760 g, 130 mL) were combined based on TLC and purified by MPLC column
(silica). The column was eluted, under gradient conditions, with 100% hexane to 100%
acetone. A total of 10 fractions were collected. The evaporation of fraction 5 (60 mL)
under reduced pressure afforded compound 12 (41.3 mg). The 1H and '3 C NMR spectral
data of compound 12 were identical to the published spectral data of ursolic acid
(Seebacher et al., 2003). The fi'action 2 resulting from the above column on repeated

purification by MPLC (silica) column with hexane:acetone as the mobile phase afforded

87

 

compound 13 (24.0 mg). The 1H and 13‘C NMR spectral data of compound 13 were
identical to the published spectral data of B-sitosterol (Sakuri and Rahmani, 1995). The
ethyl acetate extract mainly contained compounds 12 and 13, as confirmed by TLC, was
not studied further.

The fresh and unripened green fruits of C. kausa (1.6 kg) were successively
blended with methanol (1000 mL x 2) and ethyl acetate (1000 mL x 2), and centrifuged
(model RC5C, Sorvall Instruments, Hoffman Estates, IL) at 10000g for 20 min at 4°C.
The combined supematants after evaporation under reduced pressure yielded methanol
(80 g) and ethyl acetate (10 g) extracts, respectively. An aliquot of the methanolic
extract (35.0 g) was dissolved in methanol (100 mL) and partitioned with hexane (150
mL x 3) to yield methanol (24.0 g) and hexane-soluble (10.0 g) fractions. A portion (5.1
g) of the methanol-soluble fraction was fractionated by MPLC (C-18) column using
MeOHzH2O as the mobile phase under gradient conditions fi‘om 10% MeOH to 100%
MeOH. The fraction 2 (606 mg) from the above column was purified by C-18 column
using MeOHzH2O mobile system and subsequent purification by preparative thin layer
chromatography afforded compound 14 (8.9 mg). The 1H and 13C NMR spectral data of
compound 14 were identical with that of comin (Tanaka et al., 2001). The fraction 6 was
further fractionated by Prep-HPLC (X-terra® Prep MS C13, 19 x 250, 10 pm) under
isocratic conditions using 0.1% TFA/H2O: CH3CN (75:25) as the mobile phase. The
flow rate was maintained at 3 mL/min and the peaks detected at 275nm. A total of six
fractions were collected and firrther purification of fraction 5 by HPLC using 0.1%
TFA/H2OzCH3CN (80:20) as the mobile phase afforded compounds 15 and 16,

respectively. The 1H and 13C NMR spectral data of compound 15 and 16 were in

88

 

agreement with kaempferol 3-0-rhamnoside and myricetin 3-0-rhamnoside, respectively
(Markhem et al., 1982). The compound 17 was obtained by the purification of fraction 6
using the same mobile system mentioned above. The 1H and 13 C NMR spectral data
indicated that compound 17 was kaempferol 3-O-glucoside (Markhem et al., 1982)
Another portion of the methanolic extract (23.5 g) was dissolved in water (200
mL) and centrifuged. The supernatant was then fractioned by XAD-16 resin. The resin
was washed with water ( 1000 mL x 2) and the adsorbed compounds were eluted with
methanol (500 mL x 2) and the solution evaporated under reduced pressure to yield a
residue (16.5 g). A portion (3.5 gm) of the was residue was further fractionated by
MPLC (silica) using CHCl3zMeOH as the mobile phase afforded six fractions. The
fraction 2 obtained from the silica column was evaporated under reduced pressure and
washed with CHC13 to afford compound 18 (2.4 mg). Compound 18 was identified as
stenophyllin based on comparison of 1H and '3 C NMR spectral data to those reported in

the literature (Tanaka et al., 2001).

Results

The Camus plants belong to the family Comaceae, commonly known as
dogwood, produce colorful and attractive flowers and fruits. The Japanese dogwood, C.
kausa is a deciduous tree and it produces flowers during May-June and fruits during
August-September. All Camus plants including C. kausa are widely grown as
ornamental plants. The edible C. kausa fruits are fleshy, round or oval in shape with an
attractive red color (Seeram et al., 2002). We have compared the fresh weights of C.

kausa, C. mas, C. oflicinalis, C. controversa, C. altemifalia and C. florida ripened fruits.

89

 

Ripened fruit of C. kausa weighed about 15.6 g per fruit and was the highest among all
the Camus fruits studied (Shaiju et al., 2005). Even though C. kausa fruits are edible and
widely used for the production of wine in Asian countries, these fruits are not consumed
in the USA.

The ripened C. kausa fruits were sequentially extracted with water, ethyl acetate
and hexane. The methanol extract showed promising activity in the preliminary lipid
peroxidation and cyclooxygenase enzyme inhibitory assays. The fractionation and
purification of this MeOH extract afforded compounds 12 and 13 in addition to the
anthocyanin, cyanidin 3-0—glucoside. The ethyl acetate extract of C. kausa contained
mainly of compounds 12 and 13 based on TLC and hence was not analyzed further.

Since all Camus fruits studied yielded primarily anthocyanins, we have
investigated bioactive compounds in green fruits. For this, matured green C. kausa fruits
were collected just before the ripening process. The methanol extract of green C. kausa
fruit was partitioned with hexane to remove chlorophylls and fractionated to afford

compounds 12-18 (Fig. 3).

Discussion

The fi'uits of C. kausa, commonly known as kousa dogwood, resemble to
raspberries. The plant C. kausa is a deciduous shrub or a small tree of about 7-10 m in
height. Its cultivars include 'Auturnn Rose', 'Ballerina', 'Beni Fuji' and 'China Girl'.
Many of these cultivars are produced by the hybridization of C. kausa and C. florida.
The anthocyanins are primary bioactive constituents in its ripened fruits. We have

recently reported several anthocyanins and its concentrations in Camus fruits (Shaiju et

90

 

al, 2005). Anthocyanins cyanidin 3—0-glucoside and cyanidin 3-0-galactoside were
characterized and quantified from C. kausa ripened fruits.

Other components isolated from C. kausa ripened fruits were compounds 12 and
13. However, the analysis of unripened fruits afforded compounds 14-18 in addition to
compounds 12 and 13 (Fig. 3). ,The concentration of 12 and 13 in ripened fi'uits was
several times higher than in green fruits. It was interesting to note that C. kausa ripened
fruits did not yield flavonoids but gave primarily anthocyanins cyanidin 3-0—galactoside
and cyanidin 3-0—glucoside. This indicated that the biosynthetic pathway of flavonoids
was regulated to a halt during the ripening process and favored the reaction which
resulted in the accumulation of anthocyanins (Scheme 4.1).

We also have studied the ripened fruits of C. cantraversa and C. altemifalia for
bioactive compounds. Both compounds 12 and 13 were isolated and characterized from
methanol and ethyl acetate extracts of C. cantraversa and identified in C. altemifalia
ripened fruits. The aqueous extracts of C. cantraversa and C. altemifalia are shown to be
excellent sources for delphinidin 3-0-glucoside (1) and delphinidin 3-0-rutinoside (2)
(Shaiju et al, 2005). The total anthocyanin content in these fruits is also shown to be 20—
25 times higher than other known natural sources of anthocyanins. Based on the absence
of flavonoids in the ripened Camus fruits, it is hypothesized that the flavonone-
dihydroflavonol-anthocyanin biosynthetic route was dominant during ripening in Camus
fruits which then lead to the accumulation of anthocyanins (Scheme 1).

The anthocyanin accumulation in leaves and flowers depends on various factors
such as nutrients, temperature, availability of water, and in particular, light (Sharnir and

Nissim, 1997). We have reported anthocyanin production in relation to light quantity in

91

”1'"

 

Pennisetum setaceum Cvs. Rubrum and Red Riding Hood (Beckwith et al., 2004). When
P. setaceum leaves were exposed to different light environmental conditions such as UV
supplemental light in the green house, high-pressure sodium supplemental light in the
green house, cool-white fluorescent light in the growth chamber and outside sunlight, the
production of anthocyanin varied remarkably (Beckwith et al., 2004). The maximum
level of anthocyanin detected was in plants grown under fluorescent light conditions.
This indicated that both light intensity and temperature played a role in anthocyanin
production and accumulation. That is, low temperature generally promoted anthocyanin
production while high temperature inhibited its accumulation (Mazza and Miniati. 2000).
Anthocyanin may acts as a protective shield to the photosynthetic machinery by
absorbing UV radiation. The effect of UV light on anthocyanin accumulation was also
studied in Catinus caggygria (Shamir and Nissim, 1997). The exposure of C. caggygria
to UV light between 300 -400 nm produced a significant accumulation of anthocyanins in
its leaves.

Various enzymes, phenylalanine ammonia-lyase (PAL), chalcone synthase,
flavanone 3- hydroxylase, flavanone 3'-hydroxylase and several glucosyltransferases are
responsible for the biosynthesis of anthocyanins. However, phenylalanine ammonia-
lyase is recognized as a critical enzyme for anthocyanin synthesis (Ebel and Hahlbroch,
1982). Anthocyanin accumulation during ripening of several fruits is directly related to
the PAL activity (Aoki et al., 1970; Hyodo, H, 1971). The studies on the relationship
between PAL and anthocyanin at different temperatures indicated that both anthocyanin
synthesis and PAL activity increased at low temperatures (Tan, 1979). Fruits held at

altemating temperatures of 6 and 18°C produced double the amount of anthocyanins

92

compared to the fruits at 18 °C. The PAL activity was also stimulated by ethylene, low
nutrient level, light, water condition, sugar content and mu ripening. A report by
Faragher and Broheir indicated that anthocyanin accumulation increased with the rise of
ethylene level in ripening of fruits due to the stimulation of PAL activity (Faragher and
Brohier, 1984). It was also hypothesized that low temperature may promote the
anthocyanin production by reducing the activity of gibberellins (Saure, 1990).

Sugars are essential for the synthesis of anthocyanins. Fructose, glucose, lactose,
maltose, and sucrose stimulate the synthesis of anthocyanins (Vestrheim, 1970). The
catabolism of glucose though pentose phosphate pathway (PPP) has been associated with
anthocyanin production in many fruits (Faust, 1965). Many reports indicate that
anthocyanin production increase several times during the ripening of the fruits. For
example, total anthocyanin in ripened raspberry fruits was four fold higher than in
unripened fruits. In the case of ‘Montmorency’ sour cherries, total anthocyanin content
increased from 2 to 43.6 mg per100 g fresh wt during ripening (Gross, 1987).

A recent report of gene expression studies of flavonoid biosynthesis in lisianthus
(Eustama grandiflarum Grise) indicated that phenylalanine ammonia-lyase (PAL),
chalcone synthase (CHS), chalcone isomerase (CH1) and flavonoid 3' hydroxylase (F3'-
OH) were the important enzymes expressed during bud development (Nielson et al.,
2002). During the floral development, two new enzymes, dihydroflavonol 4- reductase
(DFR) and flavonoid 3'5’- hydroxylase (F3’5' —OH), were expressed in addition to PAL,
CHS and CH1 although both CHS and CH1 were expressed in lower levels (N ielson et al.,

2002). This indicated that during the early flower development of lisianthus, there was

93

 

flavonoid accumulation and in later stages the pathway shifted more towards the
accumulation of anthocyanins. A similar scenario is possible in Camus plants as well.
Our studies with C. kausa fruits indicated that flavonoids are the major
compounds in matured green fruits. The flavonoids accumulation in early stages of fi'uit
development could be for protecting the plants from reproductive tissue damage. The
high flavonols production may be due to the shift of biosynthetic pathway in favor of
flavonols due to the over expression FLS gene. Contrary, ripened Camus fi'uits showed
high amount of anthocyanins and no flavonoid was detected in any of its species. This
could be due to the shift of biosynthetic pathway in favor of anthocyanins with the
expression of DHR. Molecular experiments to determine the up or down regulation of
genes responsible for flavonoid and anthocyanin production in Camus fruits should be
carried out. Also, such experiments will be useful to further enhance the production of

anthocyanins in Camus and possibly in other edible fruits.

94

 

 

‘ CARBOHYDRATE W

Phenylalanine

 

Acetvl -CoA +
3 Malonyl-CoA 4-Coumaroyl-CoA
CHS
CHI

 

Fs. Homo
OH O
F lavanones

F3H $0
0I

H0 0:1? 0
OH 0 OH
FLS Isoflavones

Dihydrofiavonols

 

 

FIavonols

Anthocyanins

Condensed tannins

Scheme 1

Scheme 4.1. Reported flavonoid and anthocyanin biosynthetic pathway. CHS Chalcone
synthase, CHI Chalcone isomerase, FSI F lavone synthase, IFS Isoflavone synthase, FLS
Flavanol synthase, F3H Flavanone 3-hydroxylase, and ANS anthocyanidin synthase

(Harbone, 1988., Taiz and Zieger, 1998)

95

13

 

 

Compound R1 R2 R3

 

15 H H Rha
16 OH OH Rha
17 H H Glc

 

 

O O
0 \CH3
\
0 HO
H3C 0 OH
OH
H0
14 18

Figure. 4.1 Structures of compounds 12—18 isolated from C. kausa fruits.

96

 

 

Red ripened fruits C. kausa

DFR activity
No FLS activity

Anthocyanins
Ursolic acid

B-sitosterol

Green matured fruits

 

FLS activity
No DFR activity

Kaempferol 3-O-rhamnoside
Myricetin 3-O-rhamnoside
Kaempferol 3-O-glucoside

Stenophyllin
Ursolic acid
B-sitosteroi

Comin

Scheme 4. 2. Compounds isolated from C. kausa his

97

 

 

CHAPTER FIVE

SUMMARY AND CONCLUSIONS

The genus Camus have been used for thousands of years in many parts of the
world especially in China and Japan. Camus fi'uits are mainly used as a medicinal agent
for the treatment of diseases associated with liver and kidney. The fi'uits of C. afi'icinalis
have been used for antidiabetic preparation in China for centuries. Even though Camus
species has reported several medicinal properties, very little is known about its bioactive
compounds. A detailed investigation of botany, chemistry, biological and
pharmacological activities of Camus spp. conducted earlier studies is presented in
Chapter 1. Based on the detailed literature review it was determined that C. mas, C.
aflicinalis, C. contraversa, C. altemifalia, C. kausa and C. florida fruits should be
investigated further for bioactive compounds with potential human health benefits.

The ripened fruits of C. mas, C. officinalis, C. cantroversa, C. altemifalia, C.
kausa, and C. flarida were collected from Michigan State University campus in August-
September 2002 and 2004. The matured green fruits of C. kausa were collected from
3934 E Sunwind Drive, Okemos, Michigan in August 2004. The anthocyanins in these
fruits were isolated, characterized and quantified by various chromatographic and
spectroscopic techniques. The isolation, characterization and quantification of
anthocyanins in these Camus spp. fruits are presented in Chapter 2. The quantification
results of anthocyanins, delphinidin 3-0—glucoside (1), delphinidin 3-0—rutinoside (2),

delphinidin 3-0-galactoside (3), cyanidin 3-0- galactoside (4), pelargonidin 3-0—

98

galactoside (5) and cyanidin 3-0-glucoside (6), indicated that C. cantraversa, C.
altemifalia and C. mas are the excellent sources for these anthocyanins. The amount of
delphinidin 3-0—glucoside (l), delphinidin 3-0-rutinoside (2), and cyanidin 3-0-
glucoside (6) in C. altemifalia and C. cantraversa were 8.21 , 8.44 and 0.02 mg; and 7.74,
5.92, and 0.02 mg/g of fresh fruits, respectively. Similarly, delphinidin 3-0—galactoside
(3), cyanidin 3-0- galactoside (4), and pelargonidin 3-0—galactoside (5) in C. mas were
0.47, 1.66 and 1.62; and 0.15, 0.21, and 0.78 mg/g of fruits, respectively. Anthocyanin
content in these species are 20-25 times higher than other major fi'uit sources of
anthocyanins.

Although both anthocyanins and anthocyanidins were reported as potent
antioxidant and anti-inflammatory agents, anticancer and insulin secretion activities of
these compounds were not studied earlier. A detailed investigation of biological
activities of anthocyanins and anthocyanidins were discussed in Chapter 3. Based on the
studies conducted as part of my thesis research project, it was determined that both
anythocyanins and anthocyanidins exhibited good anticancer and insulin secretion
activities in addition to lipid peroxidation and cyclooxygenase enzyme inhibitory
activities. At 50 ,ug/mL, delphinidin 3-0— glucoside (l) and delphinidin 3-0— rutinoside
(2) inhibited lipid peroxidation by 71 and 68%, respectively. Similarly, they inhibited
COX-1 enzymes by 39 and 49% and COX-2 enzyme by 54 and 48%, respectively, at 100
,ug/mL. Anthocyanins, delphinidin 3-0— glucoside and delphinidin 3-0-rutinoside, and
the anthocyanidin malvidin showed potent cell proliferation inhibitory activities when
tested against hmnan cancer cell lines, AGS (gastric), CNS (central nervous system, SF-

268), HCT-ll6 (colon), NCI-H460 (lung), and MCF-7 (breast). Delphinidin 3-0-

99

glucoside (1) displayed 50% grth inhibition (ICso) at 21, 25, 50, 60, and 75 pg/mL,
against colon, breast, lung, central nervous system (CNS), and stomach human tumor cell
lines, respectively. Similarly, ICso values for delphinidin 3-0— rutinoside (2) were 38, 30,
76, 100, and 100 pg/mL against colon, breast, lung, central nervous system (CNS), and
stomach cell lines, respectively. Anthocyanins delphinidin 3-0- glucoside (1) and
cyanidin 3-0- glucoside (6), and anthocyanidin pelargonidin (9) also showed promising
insulin secretion activity when tested against rodent pancreatic B cells at 4 and 10 mM
glucose concentrations.

A detailed study of all the non-pigmented fractions of C. kausa, C. cantraversa,
and C. altemifalia fi'uits was discussed in Chapter 4. Bioassay-guided isolation,
purification and characterization of methanol, ethyl acetate and hexane extracts of C.
kausa ripened and unripened fruits afforded ursolic acid (12), ,B-sitosterol (13), corrrin
(14), flavanoids, kaempherol 3-0— rhamnoside (15), myricetin 3-0—rhamnoside (16),
and kaempherol 3-0— glucoside (17), and stenophyllin (18). The compounds ursolic acid
(12) and fl-sitosterol (13) were also identified in the fruits of both C. cantraversa and C.
altemifalia. This is the first report of these compounds from C. kausa fruits.

My research on various Camus fi'uits for bioactive compounds indicated that
anthocyanins are the major active compounds present in all Camus fruits. The evaluation
of several compounds yielded from Camus fruits in my study confirmed that
anthocyanins and anthocyanidins were the most active in inhibiting lipid peroxidation,
cyclooxygenase enzymes and the grth of human tumor cell lines. Also, it suggested

that Camus fruits containing high levels of these anthocyanins are powerful insulin

100

secretagogues and may be useful for the prevention of type-2 diabetes. The bioassay
results also explained the anecdotal health claims associated with Camus fi'uits.

The in vitro cell proliferation studies with various human cancer cell lines suggest
that anthocyanins in C. altemiflalia and C. cantraversa fruits may be useful in the
prevention of certain tumor progression. Because C. altemiflalia and C. cantraversa
fruits have high amount of health beneficial anthocyanins, it is possible that these plants
could be cultivated as alternate crops to yield fruits for tumor cell growth inhibitory and
insulin secretion anthocyanins and anthocyanidins. However, in vivo studies and clinical
evaluation of these compounds must be carried out to further validate the in vitro results.

Ornamental plants, both indigenous and introduced, provide a tremendous
resource for value added products and functional foods. Anecdotal information on many
of these species suggests the application of these plants not only in human health but also
in the diversification of agricultural production. The Camus spp. is already in
mainstream horticulture production as landscape omamentals. They also have similar
botanical traits to horticultural crops in food production. For example, C. mas and C.
aflicinalis can be easily integrated into tart cherry (‘Montmorency’ and ‘Balaton®’)
production systems. Trees can be managed the same, C. mas and C. afficinalis require
minimal chemical inputs, and hits can be harvested with the same equipment in the fall
after the demands of cherry production has ceased. Production of C. mas while providing
an economically valued crop can extend a production season without disrupting the
current production system. The same is true for C. altemifalia and C. cantraversa; these
species could be integrated into small fruit production systems. Ornamental plants

production in the United States is an untapped resource for phytochenricals and

101

functional foods. Ornamental species can make a seamless transition into cropping
systems with the value added benefits. Camus spp. is one example where investigation,

examination, and analysis offer therapeutic options in human health.

102

REFERENCES

Anderson, R. A.; Polansky, M. M. Tea enhances insulin. activity. J. Agric. Food Chem.
2002, 50, 7183-7186. '

Aneuville, 0.; Breuer, D. K.; De Witt, D. L.; Hla, T.; Funk, C. D.; Smith, W. L.
Differential inhibition of human prostaglandin endoperoxide H synthase -1 and —2
by nonsteroidal anti-inflammatory drugs. J. Pharmacol. Exp. Ther. 1994, 271,
927. '

Aoki, S.; Araki, C.; Kaneo, K.; Katayama, 0. L-Phenylalanine ammonia-lyase activities
in Japanese chestnuts, strawberries, apple fruit and brackens. Nippon Shakuhin,
Kagyo Gakkaishi. 1970, 17, 507-511.

Arora, A.; Nair, M. G.; Strasburg, G. M. Structure activity relationships for antioxidant
activities of a series of flavonoids in a liposomal system. Free Rad. Bio. Med.
1998, 9, 1355-1363.

Bailey, L.H.; In manual of cultivated plants. Macmillan, New York, 1977, p 77.

Bain, J. F .; Denford, K. E. The flavonoid glycosides of Camus canadensis L. and its
allies in northwestern North America. Experimentia 1979, 35, 863-864.

Beckwith, A. G., Zhang, Y., Seeram, N. R, Cameron N, P., Nair. M. G. Relationship of
light quantity and anthocyanin production in Pennisetum setaceum ‘Rubrum’
and ‘Red Riding Hood’. J. Agric. Food Chem. 2004, 52, 456-461.

Bhakuni, R. S.; Shukla, Y. N.; Tripathi, A. K.; Prajapati, V.; Kumar, S. Insect inhibitory
activity of arjunolic acid isolated from Camus Capitata. Phytotherapy Reser.
2002, 16, 68-70.

Bhakuni, R. S.; Shukla, Y. N.; Thakur, R. S. New triterpenoid from Camus capitata.
Phytochemistry. 1987, 26, 2607-2610.

Chandra, A; Rana, J; Li, Y. Separation, identification, quantification, and method
validation of anthocyanins in botanical supplement raw materials by HPLC and
HPLC-MS. J. Agric. Food. Chem 2001, 49, 3515-3521.

Chandra, A.; Nair, M. G. lezzoni A. Evaluation and characterization of the anthocyanins
pigments in tart cherries (Prunus cerasus L). J. Agri. Food Chem. 1992, 40, 967-
969.

Chang, J. C.; Nair, M. G.; Nittiss, J. L. Metabolites of daidzein and genisten and their
biological activities. J. Nat. Prod. 1995, 58, 1901.

103

Chester, W.; Stone, C. Comparative studies of the anthocyanins in the red-fruited and
yellow fruited flowering dogwood. Bull. Torrey Batan.Club. 1964, 91, 506-507.

Choi, W. H.; Park, W. Y.; Hwang, B. Y.; 0h, G. J.; Kang, S. J.; Lee, K. S. Phenolic
compounds from stem bark of Camus walteri Wanger. Saengyak Hakhoechi.
1998, 29, 217-224.

Conklin, K. A. Dietary antioxidants during cancer and chemotherapy: Impact on
chemotherapeutic effectiveness and development of side effects. Nutrition and
Cancer, 2000, 37.

Delaveau, P.; Paris, R. R. The presence of rutoside and gallic acid derivatives in the
flowers of Cornus mas. Preliminary studies in C. sanguinea. Bull. Soc. Chim. Bio.
1961, 43, 661-666.

Denizot F .; Lang R. Rapid colorimetric assay for cell growth and survival. Modification
to the tetrazolium dye procedure giving improved sensitivity and reliability. J.
Immunol. Methods. 1986, 89, 271-277.

Dolezal, M.; Valisek, J.; Famfulikova, P. Chemical composition of less-known wild
fruits. Royal. Socie. Chem. 2001, 269, 241- 244.

Dominguez, X. A.; Franco, R.; Cano, G.; Garcia, S.; Gracian, P.; Sanchez, S. Mexican
Medicinal plants XLV. Chemical study of tepeacuilote roots (Camus excelsa
H.B.). Rev. Latinaom. Quim 1981, 12, 35-37.

Dongho, L.; Shin—Jung, K.; Seung-Ho, L.; Jaiseup, R.; Kyongsoon, L.; Kinghom, A. D.
Phenolic compounds from the leaves of Camus cantraversa. Phytochemistry

2000, 53, 405-407.

Du. C. T.; Wang, P. L.; Francis, F. J. Anthocyanins of Camus altemifalia and C. alba.
Hart Science, 1975, 10, 35-37.

Du, C. T.; Wang, P. L.; Francis, F. J. Anthocyanins of Comaceae, Camus kausa Hance
and Cornusflarida L. Hort Science. 1974, 9, 243-244.

Du, C. T.; Francis, P. J. Anthocyanins from Camus mas. Phytochemistry 1973, 12, 2487-
2489.

Du, C. T.; Francis, F. J. New anthocyanin from Camus mas. HartScience 1973, 8, 29-30.

Du. C. T.; Wang, P. L.; Francis, F. J. Anthocyanins of Comaceae, Camus canadensis.
Phytochemistry, 1974, 13, 2000.

Ebel, J .; Hahlbroch, K. The Flavanoids: Advances in research. Harbone, J. B, Marby, T.
J ., Eds., Chapman & Hall, London, 1982, 641.

104

Egger, K.; Keil, M. Flavanol glycosides in flowers of Camus mas. Z. Pflanzenphysial.
1969, 61, 346-347.

Endo, T.; Taguchi, H. Constituents of Camus officinalis. Yakugaku Zasshi. 1973, 93, 30-
32.

Fan, 0, Xiang, Q. Y. Phylogenetic relationship with Camus (Comaceae) based on 26S R
DNA sequences. Am. .1. Bot. 2001, 88, 1131-1138.

F aragher, J. D.; Brohier, R. L. Anthocyanins accumulation in apple skin during ripening:
Regulation by ethylene and phenylalanine ammonia-lyase. Sci. Hart. 1984, 22, 89.

Faust, M. Physiology of anthocyanin development in McIntosh apple. 1. Participation of
pentose phosphate pathway in anthocyanin development. Prac. Am. Soc. Hart.
Sci. 1965, 87, 1.

Frankel E. N. Lipid Oxidation. The Oily Press LTD, 1998, Dunn Otter Place, West Ferry,
Dundee, Scotland.

Fu, S.; Li, Z.; Wang, Z. Study on the gross saponins in natural beverages of Camus
officinalis. Zhejiang Linxueyuan Xuebao 1998, 15, 105-107.

Goiffon, J. P., Mouly, P. P., Gaydou, E. M. Anthocyanin pigment determination in red
fruit juices, concentrated juices and syrups using liquid chromatography. Anal.
Chem. Acta 1999, 382, 39-50.

Grigorescu, E.; Selmiciu, 1.; Stanescu, U. Biochemical study of flavanoids in blossoms of
Camus mas. Wyglozane Symp 1972, 83-89.

Gross, J. Pigments in fruits. Academic Press Inc (London) Ltd, 24-28 Oval Road,
London, NW1 7 DX. 1987.

Hagiwara, A.; Yoshino, H.; Ichihara, T.; Kawabe, M.; Tamano, S.; Aoki, H.; Koda, T.;
Nakarnura, M.; Irnaida, K.; Ito, N. and Shirai, T. Prevention by natural food
anthocyanins, purple sweet potato color and red cabbage color, of 2-amino-1-methyl-
6-phenylimidazo [4,5-b] pyridine (PhIP)-associated colorectal carcinogenesis in rats
initiated with 1,2-dimethylhydrazine. J. T axical. Sci. 2002, 27, 57—68.

Hann, R. M.; Sando, C. E. Scyllitol from flowering dogwood (Camus flarida). J. Biol.
Chem. 1926, 68, 399-402.

Harbome, J. B, Williams, C. A. Anthocyanins and other flavonoids. Nat. Prod. Rep.,
2001, 18, 310-333.

105

Hatano, T.; Ogawa, N.; Kira, R.; Yasuhara, T.; Okuda, T. Tannins of comaceous plants
I. Comusiins A, B, C, dimeric, monomeric and trimeric hydrolysable tannins from
Cajficinalis, and orientation of valoneoyl group in related tannins. Chem.
Pharm. Bull. 1989, 37, 2083-2090.

Hatano, T.; Yasujara, T .; Okuda, T. Tannins of comaceous plants 11 Comusiins D, E, and
F, new dimeric and trimeric hydrolysable tannins from Camus aflicinalis. Chem.
Parm. Bull. 1989, 3 7, 2665-2669.

Hatano, T.; Yasuhara, T.; Ahe, R.;Okuda, T. Tannins of comaceous plants. Part 3. A. A
galloylated monoterpene glucoside and dimeric hydrolysable tannin from Camus
officinalis. Phytochemistry 1990, 29, 2975-2978.

Havsteen, B. F lavonoids A class of natural products of high pharmacological potency.
Biochem. Phamacol. 1998, 32, 1141-1148.

Hitoshi, M.; Yuko, N.; Masao, H.; Yumiko,Y.; Kazuypshi, O. Antioxidant activity of
black currant anthocyanin aglycons and their glycosides measured by
chemiluminescence in a neutral pH region and in human plasma. J. Agric. Food
Chem. 2002, 50, 5034-5037.

Hostettmann, K.; Hostettmann K, M.; Nakanishi, K. Molluscicidal saponins from Camus
florida L. Helv. Chim. Acta. 1978, 61 , 1990-1995.

Huangfu, Y., Wu, W., Sun, J ., Effect of Camus oil from the fruit of Camus macrophylla
wall on experimental atherosclerosis. Wuhan Yixueyuan Xuebaa, 1984, 13, 30-34.

http://www.gardenbed.com/source/20/1934_med.as_p

http://p1ants.usda.gov/classification/classification.cgi. USDA Natural resources
conservation services, Plants classification

http://www2.fpl.fs.fed.us/TechSheets/HardwoodNA/htmlDocs/comus.html. Technology
Transfer Fact Sheet, Center for Wood Anatomy Research, USDA, Forest Service-
Forest Product Laboratory

http://www.ces.ncsu.edu/fletcher/stgff/rbir/cornus.html

http://www4.ncsu.edu:8030/~quxiang/comushorticulture.html

Hrazdina, G. Anthocyanins, T he flavonoids: Advances in Research, J. B Harbone and T. J
Marby Eds 1982 , 135-188, Chapman & Hall, London.

Hwang; Yeon, S. Method for extraction of Chinese herbal medicine with restorative and
tonic action. Kangkae Taeho Kongbo. 2002.

106

Hyodo, H. Phenylalanine ammonia-lyase in strawberry fruits. Plant Cell Physiology.
1971, 12, 989.

lshimuaru, K.; Arakawa, H.; Neera, S. Polyphenol production in cell cultures of C. kausa.
Phytochemistry 1993, 32, 1193-1197.

Jang H. M.; Yeon H. B.;Soo K. M.; Ho L. D.; Jung K. S.; Seup R. J.; Soon L. K.
Chemical components from the stem bark of Camus cantraversa HEMSL.
Saengyak Hakhoechi 1998, 29, 225-230.

J ayaprakasam, B.; Vareed, S. K.; Olson, L. K.; Nair, M. G. Insulin secretion by bioactive
anthocyanins and anthocyanidins present in fi'uits. J. Agric. Food Chem. 2005, 53,
28-31.

Jenson, S. R.; Kjaer, A.; Juhl, N. B. Glucosides in Camus capitata and C. kausa.
Phytochemistry, 1973, 12, 2301.

Jensen, S. R.; Kjer, A.; Juhl, N. B. Dihydrocornin, a novel natural iridoid glucoside. Acta
Chem. Scand. 1973, 27, 2581-2585.

Jianrong, L.; Daozong X. Research development of Camus afiicinalis Sieb. et Zucc.
Functional compounds and its application in food industry. Shipin Kexue, 2003,
24, 161-163.

Justesen, H.; Anderson A. S.; Brandt, K. Accumulation of anthocyanins and flavones
during bud and flower development in Campanula isaphylla Moretti. Anna. Bat.
1997, 79, 355-360.

Kamei, H.; Hashimoto, Y.; Koide, T.; Kojima, T.; and Hasegawa, M. Anti-tumor effect
of methanol extracts from red and white wines. Cancer Biatherapy and
Radiapharmacology 1998, 13, 447-452.

Kamei, H.; Kojima,T.; Hasegama,M.; Koide, T.; Umeda, T.; Yukawa, T.; Terabe, K.
Suppression of tumor cell grth by anthocyanins in vitro. Cancer Invest. 1995,
13, 590-594.

Kang, S. Y.; Seeram, N. P.; Nair, M. G.; Bourquin, L. D. Tart cherry anthocyanins inhibit
tumor development in ApcM'" mice and reduce proliferation of human colon cancer

cells. Cancer Letters, 2003, 194, 13-19.

Kanji I.; Hiroko A.; Sumana N. Polyphenol production in cell cultures of Camus kausa.
Phytochemistry, 1993, 32, 1193-1197.

Kean K. D.; Hwan K. J. A furan derivative from Camus officinalis. Arch. Pharm. Res.
1998, 21, 787-789.

107

Kelly, F. J. Use of antioxidants in the prevention and treatment of diseases. J. Int. Fed.
Clin. Chem Chem. Lab. Med. 1998, 10, 21-23.

Kenneth A. C. Dietary antioxidants during cancer chemotherapy: Impact on
chemotherapeutic effectiveness and development of side effects. Nutr. Cancer,
2000, 37, 1-18.

Kim, D. K; Kwak, J. H.; Ryu, J. H.; Kwon, H. 0; Song, K. W. A component fi'om
Camus aflicinalis enhances hydrogen peroxide generation from macrophages
Saegyak Hakhoechi 1996, 27, 101-104.

Kim, H. P., Son, K. H., Chang, H. W., Kanr, S. S. Anti-inflammatory plant flavonoids
and cellular action mechanisms. J. Pharmacol. Sci. 2004, 96, 229—245

Kim, H. Y.; Oh, J. H. Screening of Korean forest plants for c lens aldose reductase
inhibition. Bioscience, Biotechnology, and Biochemistry. 1999, 63, 181-188.

Kim, B. J .; Kim, J. M.; Kim, H. P.; Heo, M. Y. Biological screening of 100 plant extracts
for cosmetic use (11): anti-oxidative activity and free radical scavenging activity.
Intern]. J. Cosmetic Sci. 1997, 19, 299-307.

Kim, D. K.; Kwak, J. H. A firran derivative from Camus aficinalis. Archi. of Pharmacal.
Res. 1998, 21 , 787-789.

Kurihara, T.; Kikuchi, M. Studies on the constituents of flowers. IX. The components of
the flowers of Camus cantraversa Hemsl. Yakugaku Zasshi 1978, 98, 969-972.

Landrault, N.; Poucheret, P.; Azay, J.; Krosniak, M.; Gasc, F.; Jenin, C.; Cros, G.;
Teissedre, P.L. Effect of a polyphenols-enriched chardonnay white wine in
diabetic rats. J. Agric. Food. Chem. 2003, 51, 311-318.

Lee, D.; Kang, S. J.; Lee, S. H.; Ro, J .; Lee, K.; Kinghom, A. D. Phenolic compounds
from the leaves of Camus cantraversa. Phytochemistry, 2000, 53, 405-407.

Lee, S. H.; Tanaka, N.; Noneka, G.; Nishioka, I. Tannins and related compounds. Part 86.
Sedoheptulose digallate from Camus aflicinalis. Phytochemistry, 1989, 28, 3469-
3472.

Li, J .; Pan, Y.; Qin, X.; Zhang, X. Determination of loganin in Jiantangqing capsules by
RP-HPLC. Zhangcaayao, 1999, 30, 820-822.

Ling W, H.; Jones P. J. Dietary phytosterols: A review of metabolism, benefits and side
effects. Life Sciences 1995, 57, 195-206.

Mamedove, N.; Craker, L. E. Cornelian Cherry: A prospective source for phytomedicine.
Acta Hart. 2004, 629, 83-84.

108

Marchant, C. A. Environmental health perspective supplements. 1996, 104, 1065-1073.

Markham, K. R.; Chari, V. M.; Mabry, T. J. Carbon-13 NMR spectroscopy of flavonoids.
In The Flavanoids: Advances in Research: Harbone, J. B.; Marby, T. J., Eds;
Chapman and Hall; New York, 1982, 19-134.

Mas, T.; Susperregui, J .; Berke, B.; Chez, C.; Moreau, S.; Nuhrich, A.; Vercauteren J.
DNA triplex stabilization property of natural anthocyanins. Phytochemistry,
2000, 53, 679-687.

Man, J. L.; Chen, C. P.; Hsieh, P.C. Antimicrobial effect of Chinese chive cinnamon, and
corni fructus. J. Agric Food Chem. 2001, 49, 183-188.

Mazza, G.; Miniati, E. Anthocyanins in Fruits, Vegetables and Grains; CRC press, 2000.

Miller, E. R. Comin, a glucoside from Carnusflarida L. Am. Pharm. Assacn. 1928, 17,
744-750.

Millspaugh, C. F. In American Medicinal Plants. Dovar Publications. New York, 1974,
282.

Min, J. H.; Yeon, H. B.; S00, M. K.; Ho, L. D; Jung, K. S; Seup, R. J; Soon, L. K.
Chemical components from the stem bark of Camus cantraversa HEMSL.
Saengyak Hakhoechi. 1998, 29,225-230.

Miyazawa, M.; Kameoka, H. Volatile flavor components of crude drugs. PartVII.
Volatile flavor components of Cami Fructus (Caflicinalis Sieb.Et Zuee.). Agric.
Biol. Chem. 1999, 53, 3337-3340.

Moerman, D. E. Native American Ethnobotany. Timber Press, Inc. 1998, 176-180.

Mudry, P.; Schilling, E. E. Flavonoids of Camus flarida. Bull. T arrey. Bot. Club. 1983,
110, 226-227.

Nahaishi, H. Effects of black current anthocyanins intake on dark adaptation and VDT
work-induced transient refractive alteration in healthy humans. Alt. Med. Rev
2000, 5, 553-562.

Nakaoki, T.; Morita, N. Medicinal Resources XII. Components of the leaves of Camus
cantraversa, Ailanthusaltissima, and Ricinus communis. Yakugaku Zasshi 1978,

78, 558-559.

Nishino, C.; Kobayashi, K.; Fukushima, M. Helleridone, a cytotoxic constituent from
Camus cantraversa. J. Nat. Prod. 1998, 51 , 1281-1282.

109

Nielsen, I. L.; Haren, G. R.; Magnussen, E. L.; Dragsted, L. 0. Rasmussen, S. E.
Quantification of anthocyanins in commercial black currant juice by simple high
performance liquid chromatography. Investigation of their pH stability and
antioxidative potency. J. Agric. Food Chem. 2003, 51 , 5861-5866.

Nielson, K.; Deroles, S. 0; Markham, K. R.; Bradley, M. J .; Podivinsky, E.; Manson, D.
Molecular Breeding. 2002, 9, 217-229.

Norbek, R.; Kondo, T. Anthocyanins from flowers of Crocus (Iridaceae).
Phytochemistry, 1998, 4 7, 861-864. ’

Omenn, G. S. What accounts for the association of vegetables and fruits with lower
incidence of cancers and coronary heart diseases? Ann. Epidemiol. 1995, 5, 333—
335.

Polinicencu, C.; Popescu, H.; Nistror, C. Vegetal extracts for cosmetic use. 1. Extracts
from fi'uits of Camus mas. Preparation and characterization. Clujul Med 1980, 53,
160-163.

Post, D. M.; Urban; James, E. Antimicrobial activity of dogwood fruits (Camus
drummandii) from winter food caches of eastern wood rafts (Neotama floridana).
J. Chem. Eco. 1995, 21 , 419-425.

Powell, MA. The flowering dogwood. Horticulture Information Leaflet, 600. North
Carolina Cooperative Extension Service 1997.

Robertson, A.; Soliman, G.; Owen, E. C. Polyterpenoid compounds. I. Betulic acid from
Cornusflarida L. J. Chem. Soc. 1939, 1267-1273.

Sando, C. E. Inositol from blackberry (Rubus argutus Link) and flowering dogwood
(Cornusflarida). J. Biol. Chem. 1926, 68, 403-406.

Sando, C. E.; Markley, K. S.; Matlack, M. B. Some chemical constituents of flowering
dogwood (Camusflorida). J. Biol. Chem. 1936, 114, 39-45.

Sahng, S.; Liu, Y.; Xiao, X.; Sun, 2.; Zhang, J.; Tian, S.; Jiang, X. Antioxidant
properties of extracts from the stone of Camus aflicinalis. Linchan Huaxue Yu

Gongye 1990, 10, 217-225.

Saure, M. C. External control of anthocyanin formation in apple, Sci. Hart. 1990, 42,
181.

Seebacher, W.; Simic, N.; Weis, R.; Sat, R.; Kunert, 0. Spectral assignments and
reference data. Mag. Resa. Chem. 2003, 42, 636-638.

110

Seeram, N. P.; Schutzki, R.; Chandra, A.; Nair, M. G. Characterization, quantification,
and bioactivities of anthocyanins in Camus species. J. Agric. Food. Chem. 2002,
50, 2519-2523.

Seeram, N. P.; Bourquin, L. D.; Nair, M. G. 2001. Degradation products of cyanidin
glycosides fi'om tart cherries and their derivatives. J. Agric. F aod Chem. 2001, 49,
4924-4929.

Shamir, M. 0.; Nissim, A. L. UV-light effect on the leaf pigmentation of Catinus
caggygria ‘Royal Purple’. Sci. Harti. 1997, 71, 59-66.

Shirley, B. W. Flavonoid biosynthesis. A colorful model for genetics, biochemistry, cell
biology and biotechnology. Plant Physio. 2001, 126, 485-493.

Shone, C. W; Hwang, L. J; Jen, W. S; Ming, L. J; Yuan, H. C.; Wen, K. Y. Colon cancer
cells with high invasive potential are susceptible to induction of apoptosis by a
selective COX-2 inhibitor. Can. Sci. 2003, 94, 253-258.

Shrikant, A.; Debnath, M.; Jesse, J. Cyclooxygenase-2 and colon cancer. Prac. Ind.
Nat. Aca. Sci. Part B. Biological Sciences. 2003, 69, 569-585.

Slimestad, R.; Anderson, Q. M. Cyanidin 3-(2-glucosylgalactoside) and other
anthocyanins from fruits of Camus suecica. Phytochemistry 1998, 49, 2163-2166.

Smith W. L.; Garavito R. M.; DeWitt D. L. Prostaglandin endoperoxide H synthases
(cyclooxygenenases)-1 and -2. J. Bio Chem. 1996, 271 , 33157-33160.

Sterrnitz, F. R.; Krull, R. E. Iridoid glycosides of Camus canadansis. A comparison with
some other Camus species. Biache. Sys. Ecol. 1998, 26, 845-849.

Steyn, W. J .; Wand, S. J, Holcrofi, D. M.; Jacobs, G. Anthocyanins in vegetative tissues:
A proposed unified function in photoprotection. New Phytol. 2002,155, 349-361.

Strack, D.; Wray, V. The Anthocyanins. The Flavanoids. Advances in research since
1986. Harbome, J B Ed London: Chapman and Hall, 1993, 1-19.

Taiz, L.; Zeiger, E. Plant Physiology. Sinauer Associates, Inc., 1998, Publishers.
Sundarland, Masachesetts.

Talavera, S; Felgines, C; Texier, O; Besson, C; Manach, C; Lamaison, L. J; Remesy, C.
Anthocyanins are efficiently absorbed from the small intestine in rats. J. Nutr.
2004, 134, 2275-2279.

Tan, S.C. Relationship and interactions between phenylalanine ammonia-lyase,

Phenylalanine ammonia-lyase inactivating system and anthocyanins in apples. J
Amer Saci. Hart. 1979, 104, 581, 1979.

111

Tanaka, N.; F amiya, T.; Shimomuru,K.; lshimuaru, K. Micropropagation and polyphenol
production in Camus plants. Nippon Shokuhin, Kagaku Gakkaishi, 1998, 5, 170-
177.

Tanaka, N.; Shimomuru, K.; lshimuaru, K. Bioactivities of Camus capitata adventitious
root producing galloyl glucoses Phytochemistry 2000, 4, 15-21.

Tanaka, T.; Fujioka, T.; Fujii, H.; Mihashi, K.; Shirrromuru, K.; lshimuaru, K. An ellagic
acid compound and iridoids from Camus capitata root cultures. Phytochemistry
2001, 57, 1287-1291.

Tian, G.; Zhang, T.; Yang, F. Preparative isolation of gallic acid from Camus aflicinalis
Sieb et Zucc by high speed counter currant chromatography. T ianran Chanwu
Yanjiu Kaifa 2000, 12, 52-55.

Tian, G.; Zhang, T.; Yang, F .; Itro, Y. Separation of gallic acid from Camus ofi‘icinalis
Sieb. et. Zucc by hi gh-speed counter—currant chromatography. J. Chromatography
A. 2000, 886, 309-312.

Trifan O. C.; Smith R. M.; Thompson B. D.; Hla T. Over expression of cyclooxygenase-2
induces cell cycle arrest. .1. Bio. Chem. 1999, 274, 34141-34147.

Trigiano, R. N.; Ament, M. H.; Windham, M. T.; Moulton, J. K. Genetic profiling of red-
bracted Camus kausa cultivars indicates significant cultivar synonymy. Hort. Sci.
2004, 39, 489-492.

Vareed, S. K.; Reddy, M. K.; Schutzki, R. E.; Nair, M. G. Anthocyanins in Camus
altemifalia, Camus cantraversa, Camus kausa, and Camus florida fruits with
health benefits. Life Sciences, 2005 (in Press).

Venkatesh, M.; Merchant, J. R. Chemical investigation of Camus macrophylla Wall.
Current Sci. 1984, 53, 35.

Vestrheim, S. Effects of chemical compounds on anthocyanin formation in ‘Mclnto’
apple skin. J. Am. Soc. Hort. Sci. 1970, 95, 712.

Viano, J .; Gaydou, E. M. Composition of fatty acids and sterols of oils extracted from
fruits of three species harvested in the Massif du Luberon: Aphyllantes
monspeliensis L., Ranunculus gramineusL., and Camus sanguinea L. Corps Gras
1984, 31, 195-197.

Wagner, W. H. A natural hybrid of gray dogwood, Camus racemasa, and round-leaved
dogwood, C. rugosa, from Michigan. The Michigan Batanist 1990, 29, 131-137.

112

Wang H, Nair G.M, Strasburg Gale, Chang Y. C, Booren A. M, Gary J I, Dewitt D. L.
Antioxidant and anti-inflammatory activities of anthocyanins and their aglycon,
cyanidin, from tart cherries. J. Nat. Prod. 1999, 62, 294-296.

Wang, H.; Nair, M. G.; Iezzoni, A. F.; Strasburg, G. M.; Booren, A. M.; Gray, J. I.
Quantification and characterization of anthocyanins in ‘Balaton’ tart cherries. J.
Agri. Food Chem. 1997, 45, 2556-2560.

Xu, L.; Li, H.; Tian, L.; Li, K.; Li, B.; Qian, T.; Sun, N. Chemical constituents of
common Macrocarpium (Camus afficinalis). Zhangcaoyaa 1995, 26, 62-65.

Yamahara, J .; Mibu, H.; Sawada, T.; Fujimura, H.; Takino, S.; Yoshikawa, M.;
Kitagawa, I. Biologically active principles of crude drugs. Antidiabetic principles
of Cornus fructus in experimental diabetes induced by streptozotocin. Yakugaku
Zasshi 1981, 101, 86-90.

Yang, T. H.; Liu, S.C.; Sen, M. H. Constituents of the fruits of Camus oflicinalis. Tai-
Wan Yaa Hsueh Tsa Chih 1971, 22, 1-4

Yan, L; Tang, W; J i, Y. Determination of ursolic acid in the pulp of Camus afficinalis
by HPLC-ELSD. Yaowu Fenxi Zazhi. 2003, 23, 358-359.

Yongwen Z.; Yuwu C.; Shiping Z. A. Sedoheptulose gallate from the fruits of Camus
ofiicinalis. Yaaxue Xuebao 1999, 34, 153-155.

Zanyin G. H.; Wang H. P. Survey of Chinese drugs for presence of antibacterial
substances. Science 1949,110, 11-12.

Zhang, L.; Wang, C.; Li, 2.; He, Y.; Zhou, Y.; Wang, F.; Liu, W.; Zhang, Z.; Zhang, A.
Determination of loganin and morroniside in Camus affrcinalis injection by
HPLC. T ianran Chanwu Yanjiu Yu Kafia, 1999, 11, 49-52.

Zhang, Y.; Vareed, S. K.; Nair, M. G. Human tumor cell grth inhibition by nontoxic
anthocyanidins, the pigments in fruits and vegetables. Life Sciences 2005, 76,
1465-72.

Zhang, Y.; Jayaprakasam, B.; Seeram, N. P.; Olson, L. K.; DeWitt, D. L.; Nair, M.
G. Insulin production and cyclooxygenase enzyme inhibition by Cabernet
sauvignon grape skin compounds. J. Agric. Food. Chem. 2004, 52, 228-233.

Zhao, W.; Chang, Y.; Li, J.; Hu, H.; Zhao, S.; Xue, Z. Study on the immuno
pharmacological effect of Japanese comel dogwood (Camus aflicinalis)
Zhangcaoyaa 1990, 21 , 113-116.

Zhao, S. P.; Xue, Z. Chemical constituents of Camus oflicinalis Sieb et Zucc. Yaoxue
Xuebao 1992, 27, 845-848.

113

Zorina, A. D.; Matyukhina, L. G.; Ryabinin, A. A. Triterpenes in some plant species
Khimiya Priradnykh Soedinenii, 1966, 2, 291.

114